Symposium: Proteins in Vivo: From the Ribosome Through the Chaperone to the ... - Cell · 2017. 1....

Sunday, February 12, 2017 15a

Symposium: Proteins in Vivo: From theRibosome Through the Chaperone to the NativeState

80-SympChanneling Nascent Proteins Towards the Native State: Role of theRibosome and Molecular ChaperonesSilvia Cavagnero, Rayna M. Addabbo, Matthew D. Dalphin, Yue Liu,Miranda F. Mecha.Chemistry, University of Wisconsin-Madison, Madison, WI, USA.Despite much progress over the last six decades, we are still far from understand-ing the mechanism of protein folding and aggregation in the cell. This lack of in-formation poses tremendous challenges to progress inmany areas of life sciences,and it severely impedes key efforts in biomedical research. Learning more aboutthe interplay between protein folding and aggregation in bacterial cells has a directimpact on the development of strategies to treat microbial infection and on theoptimization of protein-based drug production in the pharmaceutical industry.In bacteria, the majority of soluble cellular proteins fold or aggregate co-translationally and immediately post-translationally. The ribosome andmolecularchaperones play a key role in this process. This presentation will report progress,failures and surprises on our molecular-level understanding of how the ribosome,ribosomal proteins and cotranslationally active molecular chaperones modulatethe balance between protein folding and aggregation in the cell. By probing thestructure and dynamics of nascent proteins by fluorescence depolarization in thefrequency domain, multidimensional NMR, biochemical tools and kinetic simu-lations, wewill attempt to recapitulate some fundamental concepts of general sig-nificance, and suggest how these concepts can be specifically exploited tounderstand and reprogram the bacterial translation machinery to maximize theyield and timely production of correctly folded proteins.

81-SympRibosomes in Motion: The Dynamics of Nature’s Protein SynthesisMachineryRuben L. Gonzalez Jr.Department of Chemistry, Columbia University, New York, NY, USA.The ribosome can be regarded as a molecular machine that converts chemical andthermal energy into productive mechanical work. This chemo- and thermome-chanical view of ribosome function continues to fuel efforts to identify themobilecomponents of the ribosomal machine, characterize the structural dynamics ofthese components, and develop an understanding of how these dynamics are regu-lated in order to direct mechanical processes during protein synthesis. It is withinthis context that our research group has developed a fully reconstituted, fluores-cently labeled in vitro translation system and used it for single-molecule fluores-cence imaging studies of protein synthesis. Together with ensemble biochemicalinvestigations of protein synthesis by the ribosome and structural studies of func-tional ribosomal complexes, single-molecule fluorescence imaging ofprotein syn-thesis continues to provide unique and powerful mechanistic insights into thisfundamental biological process. In this talk, I will present recent and ongoingwork from our research group aimed at developing an ever-deeper understandingof the function of the ribosome during protein synthesis. In addition, I will discusswhat we envision lies ahead as single-molecule fluorescence imaging approachescontinue to evolve and expand to address increasingly complex mechanistic andregulatory aspects of this fundamental biological process.

82-SympFrom Native to Amyloid in the Test Tube and in Cells: A Journeyof Misbehaving AntibodiesMarina Ramirez-Alvarado1, Marta Marin-Argany1, Christopher J. Dick1,Luis M. Blancas-Mejia1, Pinaki Misra1, Yi Lin2, Angela Williams3,Jonathan S. Wall3.1Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA,2Medicine-Division of Hematology, Mayo Clinic, Rochester, MN, USA,3Medicine and Radiology, The University of Tennessee Graduate School ofMedicine, Knoxville, TN, USA.Light chain (AL) amyloidosis is an incurable disease characterized by themisfolding, aggregation, and systemic deposition of amyloid composed ofimmunoglobulin light chains. We have conducted thermodynamic and fibrilformation studies of both variable domain and full length immunoglobulin pro-teins involved in AL amyloidosis. We have found that there is thermodynamicrange -under physiological conditions- in which these proteins populatepartially folded states that favor amyloid formation.We have studied the internalization of these same AL proteins in soluble formand as amyloid fibrils into human cardiomyocytes. Our results show how

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external amyloid aggregates rapidly surround the cells and act as a recruitmentpoint for soluble protein, triggering amyloid fibril elongation. A fraction ofaggregates surrounding the cardiomyocytes is internalized via micropinocyto-sis (same mechanism followed by soluble proteins). AL amyloid fibrils areshown to cause cell growth arrest at low concentration. Soluble proteins induceapoptosis, demonstrating different cytotoxic mechanisms between soluble pro-tein and amyloid aggregates.In vitro amyloid formation experiments show that heterologous recruitment oflight chains is kinetically determined by the conformation of the amyloidogenicconformational precursor and modulated by the differential ability of each pro-tein to either nucleate or elongate fibrils.Studies on the early events of fibril formation followed by dynamic light scat-tering, chromatography and electron microscopy have shown differences in thespecies formed by amyloidogenic proteins (stable small oligomers) comparedto control proteins (large oligomers observed).Overall, our studies emphasize the complex interactions between light chainand cells that result in fibril internalization, protein recruitment, and cytotox-icity that may occur in AL amyloidosis.

Symposium: Biophysics of the Cytoskeletal-Membrane Interface

83-SympModeling Membrane Tubules with Lipid Droplets and MigrasomesMichael M. Kozlov.Physiology and Pharmacology, Sackler Faculty of Medicine, Tel AvivUniversity, Tel Aviv, Israel.Membrane tubules of few tens of nanometer cross-sectional diameters andmicron-scale lengths represent a basic structural component of intra-cellularorganelles, such as endoplasmic reticulum and Golgi Complex, and emergefrom plasma membranes in the course of cell crawling on extra-cellularmatrices and substrates. Besides barrier functions, the tubular membranesserve as platforms for formation of peculiar cell organelles, Lipid Dropletsand Migrasomes, whose properties are to be understood in terms of simplephysics. Lipid Droplet can be regarded as lenses of hydrophobic substance(triacylglycerol, sterol esters) growing up between the two membrane leafletsinto micron-large buds, which, possibly, detach from the membrane to formemulsion-like droplets. Migrasomes are micron-size spherical bodies discov-ered, recently, to form on membrane tethers pulled out of the cell body inthe course of cell migration. We address the micromechanics of the two organ-elles to gain understanding of physics behind their formation and evolution.We analyze the shape and energy of a membrane tubule containing a lipiddroplet in dependence of the droplet size and the elastic properties of thetubular membrane and the lipid monolayers covering the droplet surface.We determine the conditions of the droplet detachment from the tubule. Wesuggest a physical model of Migrasome as a rigid membrane domain, whichswells within a membrane tubule into a sphere-like body as a result of interplaybetween the membrane bending rigidity and lateral tension. By computing thedomain shapes for varying membrane elastic properties and the domain sizesand comparing the results with the available images, we suggest the criteriaof Migrasome formation.

84-SympMultiscale Simulation of Proteins at the Membrane-Cytoskeleton InterfaceGregory A. Voth.University of Chicago, Chicago, IL, USA.Protein-protein interactions at the cell membrane play a crucial role in dictatingcell morphology, adhesion, motility, and cytokinesis. Examples include thecoupling of transmembrane integrins with the cytoskeleton as well asformin-driven polymerization of actin filaments in fission yeast ring assembly.The intrinsic mechanosensitivity of these proteins is an important determinantof emergent large-scale effects from collections of membrane-associated com-plexes such as focal adhesions and cytokinetic nodes. Whether integrin andformin-nodes clustering respond to mechanical cues remains controversial inthe field, due to inherent limitations of experimental approaches in isolatingspecific protein contributions. Coarse-grained (CG) simulations of such sys-tems can be indispensable tools for distinguishing between such competing sce-narios. In this talk, I will discuss how we apply CG simulations to recapitulatethe complex behavior observed by our experimental collaborators and placethem into a rigorous framework to learn more about the microscopic behaviorat play. First, using a newmodel of single-point, two state integrin particles on aquasi-2D membrane, we demonstrate how cell spreading on soft substrates isregulated by integrin activation and how multiple integrin subtypes differen-tially transmit traction stress. By further including explicit coupling to a

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fluctuating cytoskeleton, we have studied the effect of single actin filamentpolymerization on integrin clustering and ligand binding. Second, I will discussrecent work on the mechanosensitivity of formin proteins in yeast cytokineticnodes. I will show results from our computational CG model explaining howmodulating formin activity by myosin generated forces on actin filamentscan increase the efficiency of cell division.

85-SympHow Actin Cytoskeleton Dynamics Induce Membrane TubulationsCecile Sykes1,2.1Curie Institute, Paris Cedex 05, France, 2CNRS, Paris, France.E-mail: [email protected]: http://umr168.curie.fr/en/Sykes-groupCell deformations are part of many vital processes such as division, motility orintracellular transport. They result from the continuous re-organization of theactin cytoskeleton underneath the membrane. In order to unveil how biochem-ical assembly can change membrane shape, we develop a minimal systembased on purified components to control all the relevant parameters.We reconstruct a finely tuned actin network on cell-sized liposomes toaddress the respective role of membrane and actin network in membrane tu-bulation. The actin network is reconstituted at the outer surface of fluores-cently labeled liposomes with purified proteins that allow to followsimultaneously membrane and actin dynamics. The system is tuned togenerate a branched cortical network next to the membrane. We demonstratethat the actin cytoskeleton induces the formation of membrane tubes withdifferent morphologies that are reminiscent of endocytosis and finger-likeprotrusions in cells: tubes are spontaneously pulled outside the liposomesand spikes grow towards liposome centers. By decreasing membrane tensionvia osmotic shock, we show that spike formation is promoted withoutaffecting tubes pulling. These results suggest that these structures are twoindependent deformations relying on different mechanisms, both based onmembrane deformation induced by cytoskeletal dynamics. This investiga-tion highlights how membrane tubulation depends on a mechanical balancebetween the membrane and the actin network.

86-SympRedefining the Role of the Arp2/3 Complex: Regulation of Morphology atthe Leading EdgeKaren L. Anderson1, Christopher Page1, Mark F. Swift1, Praveen Suraneni2,Mandy EW Janssen1, Thomas D. Pollard3, Rong Li2, Niels Volkmann1,Dorit Hanein1.1Sanford Burnham, La Jolla, CA, USA, 2Department of Cell Biology, JohnsHopkins School of Medicine, Baltimore, MD, USA, 3Molecular Cellular &Developmental Biology and of Cell Biology and of Molecular Biophysics &Bi, Yale University, New Haven, CT, USA.Arp2/3 complex is thought to be the primary protrusive force generator in cellmigration by controlling the assembly and turnover of the branched filamentnetwork that pushes the leading edge of moving cells forward. However,mouse fibroblasts without functional Arp2/3 complex migrate at rates similarto wild-type cells. Correlative fluorescence and large-scale cellular cryo-tomography studies combined with automated actin-network analysis allowexamining the effect of the absence of functional Arp2/3 complex on the ar-chitecture of actin networks in fibroblast cells at the single-filament scale.Because Arp2/3 complex can only fulfill its function in the cell when allseven subunits are assembled, genetic elimination of the ARPC3 subunit en-sures that functional Arp2/3 complex is absent in the ARPC3-/- cells versusthe wild type. Quantitative analysis at the single filament scale of theseisogenic cell lines suggests that the role of Arp2/3 complex in cell motilitycontains, in addition to generating dendritic actin networks at the leadingedge and filopodia regulation, an element that is responsible for the fine-tuning of actin-bundle morphology at the cell periphery as well as inprotrusions.

Platform: Membrane Pumps, Transporters, andExchangers I

87-PlatPseudomonas Aeruginosa CDF Transporters CzcD and YiiP are Involvedin Zn2D Efflux, Outer Membrane Permeability and Antibiotic ResistanceAgostina Salusso, Daniel Raimunda.INIMEC-CONICET-UNC, Cordoba, Argentina.Transporters of the cation diffusion facilitator (CDF) family form dimersthat export transition metals from the cytosol. The opportunistic pathogenPseudomonas aeruginosa presents three homologous CDF genes, czcD, cotP

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and yiiP. CotP appears to have a role in Co2þ homeostasis as the deletionmutant strain DcotP is more sensitive and accumulate this ion. In inhibitionhalo experiments, DczcD and DyiiP strains showed low Zn2þ sensitivity. How-ever, in iron-rich media and in the presence of Zn2þ the strains secreted higherlevels of the iron chelator pyoverdine. The non-pyoverdine producer triple mu-tants,DzntA-DpvdA-DczcD andDzntA-DpvdA-DyiiP, displayed increased Zn2þ

sensitivity and increased intracellular Zn2þ accumulation compared to thedouble mutant DzntA-DpvdA. Most importantly, the strains DczcD, DyiiP,DpvdA-DczcD and DpvdA-DyiiP showed a 4- to 8-fold increased sensitivityto imipenem and ciprofloxacin and a 2-fold increased sensitivity to chloram-phenicol and gentamycin. Complemented strains attained similar levels of anti-biotic resistance as the PAO1 parental strain. These observations correlatedwith increased sensitivity to EDTA-lysozyme treatment and overexpressionof OprN and OprD porins in DczcD and DyiiP vs. WTand DcotP. We hypoth-esize that while CotP is a Co2þ efflux transporter CzcD and YiiP export Zn2þ tothe periplasm and that their function is required for regulation of OprD synthe-sis, outer membrane stability and therefore antibiotic tolerance.

88-PlatFunctional Characterization of the Urea Transporter UreIfrom Helicobactor PyloriAndreas Horner1, Christine Siligan1, Johannes Preiner2,Sergey A. Akimov3, Peter Pohl1.1Institute of Biophysics, Johannes Kepler University Linz, Linz, Austria,2Center for Advanced Bioanalysis GmbH, Linz, Austria, 3Laboratory ofBioelectrochemistry, A.N. Frumkin Institute of Physical Chemistry andElectrochemistry, Russian Academy of Sciences, Moscow, RussianFederation.More than 50% of the world’s population is infected with Helicobactor pylori, apathogenic bacterium responsible for numerous gastroduodenal disorders such aschronic gastritis, peptic ulcer disease, and gastric cancer. A small proton-gatedinner membrane channel HpUreI represents H.pylori’s life insurance. It ensuressurvival in the acidic gastric juice, by means of urea transport from the periplasmto the cytoplasm, where urea is hydrolyzed by urease. In turn, the hydrolysisproducts ammonia and carbon dioxide buffer the periplasm to pH 6.1. MD sim-ulations suggested that water is transported along with urea through the channel.We tested that hypothesis by estimating the unitary transport rates for bothmolecules. To this end we overexpressed HpUreI in E.coli, purified the trans-porter and reconstituted the protein into large unilamellar vesicles (LUVs).We observed water flux by osmotically deflating the vesicles or inducing ureatransport under isosmotic conditions at neutral and acidic conditions. Weextracted the unitary transport rates from the intensity of scattered light by (i) ex-tending our recent treatment of the Rayleigh-Gans-Debye equation for solventtransport (1) across vesicular membranes to solute transport and (ii) determiningthe number of reconstituted transporters (2) and their oligomeric state (1)with fluorescence correlation spectroscopy before and after detergent-mediatedvesicle solubilization. In addition we confirmed the hexameric arrangement byacquiring high-speed AFM images of the proteoliposomes spread on mica.(1) Horner A, et al. (2015) The mobility of single-file water molecules is gov-erned by the number of H-bonds they may form with channel-lining residues.Science Advances 1(2):e1400083. (2) Hoomann T, Jahnke N, Horner A, KellerS, & Pohl P (2013) Filter gate closure inhibits ion but not water transportthrough potassium channels. Proc. Natl. Acad. Sci. U. S. A. 110(26):10842-10847.

89-PlatUnderstanding the Conformational Diversity of Proton-CoupledOligopeptide Transporter (POT) FamilyBalaji Selvam, Diwakar Shukla.Department of Chemical and Biomolecular Engineering, University ofIllinois, Urbana, IL, USA.Proton-coupled oligopeptide transporters (POTs) are members of the majorfacilitator superfamily (MFS) proteins involved in uptake of nutrients acrossof the cell membrane. These proteins undergo intrinsic conformational changesbetween outward-facing (OF), occluded (OC) and inward-facing (IF) states totransport substrate molecules. AtNRT1.1 is a plant nitrate (NO3

-) transporteridentified in Arabidopsis thaliana, belonging to the POT family. It sharesclose structural and functional homology to the bacterial di/tri-peptide trans-porter PepTSo. In our study, we investigated the functional mechanism ofAtNRT1.1 and PepTSo. Both transporter protein crystal structures are obtainedin the IF state and dynamics between functionally important intermediate statesremains elusive. We performed all atom molecular dynamics simulations overmicrosecond timescales to explore the conformational landscape of AtNRT1.1

and PepTSo. Since the conformational changes of these proteins occur at large

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timescales, an adaptive sampling approach has been employed to efficientlysample the free energy landscapes. MD results reveal key gating residuesthat act as transition switches for intracellular and extracellular opening ofthe transporter. In AtNRT1.1, the residues Lys164-Tyr480 of helices 4 and 10stabilize the OC state and helices 1 and 7 move ~15A to reach the OF state.In PepTSo, Ser131-Tyr431 of helices 4 and 10 act as gating residues to stabilizethe OC state while the OF state is obtained by breaking the interaction betweenArg32-Asp316 of helices 1 and 7. Our finding sheds light on the conformationaldynamics and functional mechanisms of AtNRT1.1 and PepTSo leading tobetter understanding of the role of hidden intermediate states in membranetransporters.

90-PlatInsights into the Cation Selectivity and Cooperativity with Sugarin Salmonella Typhimurium Melibiose PermeaseHariharan Parameswaran, Lan Guan.Cell Physiology & Molecular Biophysics, Texas Tech University HealthSciences Center, LUBBOCK, TX, USA.The melibiose permease of Salmonella typhimurium (MelBSt), a member ofMajor Facilitator Superfamily, utilizes the electrochemical gradient of Naþ,Liþ or Hþ for accumulation of galactosides. Based on crystal structures andfunctional studies, it has been proposed that MelBSt has a single galactoside-binding site and all three cations compete for a single cation site. Using purifiedwild-type or mutants MelBSt, direct measurement of Naþ or melibiose bindingwere performed with isothermal titration calorimetry. The results show that thebinding affinity for Naþ or melibiose is mutually increased by about 6-8 fold,exhibiting a positive cooperativity. To reveal the cation selectivity betweenNaþ and Hþ, the dissociation constant (Kd) of Na

þ binding was measured asa function of pH. The absolute KD value for Naþ of 600mM and for Hþof0.4 mM (i.e. pKa = 6.4) are determined, showing that MelBSt selects for H

þ

over Naþ. With a pKa value slightly lower than the neutral pH, MelB is ableto use either Hþ or Naþ as the coupling cation. Furthermore, Hþ release duringNaþ binding was analyzed by measuring the overall heat changes in three re-action buffers with different ionization enthalpies. While Kd for Na

þ is similar,the enthalpy changes obtained in the three buffers fit to a linear function with aslope of �0.3 and �0.03 at pH 7.45 or 8.2, respectively. It is likely that about30% MelBSt proteins engage Hþ release during Naþ binding at pH 7.45 andnearly all MelBSt is deprotonated at pH 8.2. The results also support the notionthat Naþ and Hþ exclusively bind to a single site. This study provides insightsinto the molecular mechanisms operated by MelB to utilize either Naþ or Hþ.

91-PlatModulation of Energy Conversion Through Manipulation of theRetinal Thermal Equilibrium by an Aromatic Residue in the Seven-Transmembrane Receptor BacteriorhodopsinXiaoyan Ding1,2, Yujiao Gao1, Chao Sun1, Haolin Cui1, Juan Wang1,Yanan Yang1, Dinu Iuga3, Fang Tian2, Anthony Watts4, Xin Zhao1.1Department of Physics, East China Normal University, Shanghai, China,2Department of Biochemistry and Molecular Biology, Penn State College ofMedicine, Hershey, PA, USA, 3Department of Physics, University ofWarwick, Coventry, United Kingdom, 4Department of Biochemistry,University of Oxford, Oxford, United Kingdom.Light-driven proton pumps, a number of the microbial rhodopsin family seven-transmembrane receptors, distribute widely among Archaea, Eubacteria, andEukaryota for harvesting and converting light energy in a wider spectral range.The common feature of those proteins is that the retinal chromophore maintainsa cis-trans thermal equilibrium in the dark-adapted state. Absorption of aphoton causes photo isomerization of the chromophore from the all-trans tothe 13-cis, 15-anti configuration and triggers a series of structural rearrange-ments in the protein that initiates the vectorial translocation of a proton outof the cell. However, how the cis-trans thermal equilibrium impacts on the pro-ton translocation photocycle, and further impacts on the ATP formation is stillpoorly understood. Here, as a novel study to address this challenge, Y185Fmutant, an aromatic residue at the retinal binding pocket of bacteriorhodopsin,is identified to shift the cis-trans isomerization thermal equilibrium to a cisdominated state and causes a weakening of the M state, loss of the O stateand elongation of the proton pumping cycle, and further decreases the ATP for-mation rate. Different interactions of Tyrosine 185 with the retinal chromo-phore are observed in the dark-adapted state and the M state, indicating thatTyrosine 185 may serve as a rotamer switch to maintain the proton transloca-tion kinetics and further the energy conversion in bacteriorhodopsin.Reference: Ding, X., et al. 2016. Mediation mechanism of tyrosine 185 onthe retinal isomerization equilibrium and the proton release channel in theseven-transmembrane receptor bacteriorhodopsin. Biochim. Biophys. Acta1857:1786-1795.

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92-PlatDetermination of Ion Channel and Pump Fluxes for Chinese HamsterOvary Cells using Rubidium Uptake MeasurementAzita Fazelkhah.University of Manitoba, Winnipeg, MB, Canada.Cells expend a significant portion of their energy and have considerable appa-ratus to control the flow ions across the cell membrane. Ions concentrationshave also been demonstrated to be important to processes such as apoptosisand implicated in monitoring cells metabolism for biopharmaceutical produc-tion. Accurate quantitative modelling of ion transport is challenging since thedensities of ion channels and pumps are known for relatively few cells. In thiswork the ion channels and pumps fluxes for CHO cells were estimated usingRubidium uptake measurements combined with Ouabain inhibition of pumps.Attached CHO cells were incubated in a buffer containing 5.4 mM Rb and insome cases Ouabain was also added to the buffer in different concentrationsover a period of two hours to inhibit the Naþ-KþATPase pump to study passiveand active ion fluxes across the membrane.Using Inductively Coupled Plasma Optical Emission Spectrometry, the Rbcontent of the lysed cells after two hours of incubation was 10.6 mg/L,which is a result of Rb transfer through the channels and pumps. From this,the Rb flux through the active and passive pathways is estimated to be8.5�10�12 mol.cm�2.s�1.The Rb entry through the diffusion pathway was estimated using Ouabain toinhibit the Naþ-Kþ ATPase activity. With 5 mM concentration of Ouabainthe inhibition was maximized and the Rb uptake is reduced by 80% comparedto the control. From this result the Rb flux through the channels is estimated tobe 2.8�10�12 mol.cm�2.s�1.The obtained results were used in temporal ion concentration simulations usingmethodology of Hernandez 2000. These simulations are in agreement with thework where ion pumps were inhibited via ATP production inhibition in Mito-chondria using Oligomycin (B. Saboktakin Rizi, Biomicrofluidics, 2014).

93-PlatFunctional Effect of Human FXYD2, FXYD4, FXYD6, and FXYD7on Human Alpha 1 Beta 1 Sodium-Potassium ATPaseSharan Bijlani, Natalia Armas Capote, Grace Shim, Dylan Meyer,Pablo Artigas.Cell Physiology, Texas Tech University Health Science Center, Lubbock,TX, USA.The Naþ,Kþ-ATPase builds and maintains the Naþ and Kþ gradient across theplasmalemma. Functional Naþ,Kþ-ATPase requires association of a- and b-sub-units. A third single transmembrane protein of the FXYD family (FXYD1-FXYD7) modulate Naþ,Kþ-ATPase function in a tissue specific manner. Here,we used electrophysiology on oocytes expressing human a1b1 Naþ,Kþ-ATPaseto evaluate the functional effects of associationwith human FXYD2, 4, 6&7.Un-der two-electrode voltage clamp, in the presence of 125 mMNaþo, application ofKþ

o activated outward currents (due to 3Naþ/2Kþ exchange) with voltage-dependent apparent dissociation constant. For a1b1, K0.5,0 = 1.1 50.1 mM andK0.5100= 1.5 50.3 mM (at 0, n=6, and �100 mV, n=6, respectively). TheK0.5,0 for all FXYDs was 1-1.2 mM; the K0.5100 was ~1 mM for FXYD2 andFXYD6 and ~2 mM for FXYD 4 and FXYD7. Thus, these FXYDs inducedchanges in Kþ

o affinity could be relevant at hyperpolarized potentials. Wemeasured the transient charge movement observed in the absence of Kþ

o, whichdescribes the voltage-dependent equilibrium between Naþ-bound and externallyopen Naþ-free states. The steady-state charge-voltage curves were fitted with aBoltzmann distribution. At constant [Naþ]o a shift in the distribution’s center(V1/2) to positive voltages signals an increase in Naþo affinity while a shiftto negative voltages indicates a reduction, with a 25 mV per two fold changein Naþo affinity. The V1/2 were (in mV) �41 50.5 (a1b1, n=6), �16 52(FXYD4, n=4), �26 5 0.3 (FXYD7, n=2), �57 52 (FXYD2, n=7), and�60 53 (FXYD6, n=7). These results suggest similar increase in Naþo affinityby FXYD4 or FXYD7 and identical reduction in Naþo affinity by FXYD2 andFXYD6. Measurement of K0.5 for NaþKþ-ATPase activation by intracellularNaþ in inside-out patches is underway. Supported by R15-NS081570-01A1.

94-PlatThe Energy-Transduction Domain of the SERCA Calcium Pump is aPrime Therapeutic Target in Heart Failure and ObesityJoseph M. Autry1, Michel Espinoza-Fonseca1, Bengt Svensson1,Christine B. Karim1, Stephanie J. Valberg2, John K. Lee1, David D. Thomas1.1Biochemistry, Molecular Biology, and Biophysics, University of Minnesota,Minneapolis, MN, USA, 2Large Animal Clinical Sciences, Michigan StateUniversity, East Lansing, MI, USA.We have used a comprehensive array of techniques to characterize theenergy-transduction domain of the sarco/endoplasmic reticulum calcium

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transport ATPase (SERCA), including x-ray crystallography, solution bio-chemistry, fluorescence spectroscopy, molecular dynamics simulation, andsmall-molecule activation. The energy-transduction domain of SERCA me-diates long-range allosteric coupling of ATP hydrolysis in the cytosolicheadpiece to Ca2þ transport in the transmembrane domain. In heart failure,decreased SERCA activity correlates with disease progression in patients;increasing SERCA activity by drug or gene therapy reverses heart failurein animal models. Here we solved the x-ray crystal structure of SERCA incomplex with a novel small-molecule activator, thus identifying a uniquebinding site on the energy-transduction domain. Conformation-specific pro-teolytic cleavage and intramolecular glutaraldehyde cross-linking was usedto determine activator effect on SERCA headpiece structure. Fluorescencespectroscopy was used to identify kinetic transitions accelerated by the acti-vator. We propose a structural and biochemical mechanism for small-molecule activation of SERCA. Interestingly, the activator site is locatedvicinal to the binding site of sarcolipin (SLN), a muscle phosphoproteinthat regulates SERCA energy expenditure and muscle metabolism to provideresistance against diet-induced obesity and extreme cold through non-shivering thermogenesis. SLN uncoupling of ATP hydrolysis from Ca2þ

transport by SERCA was examined using microsecond MD simulation,finding that the cytosolic domain of SLN induces a salt bridge-mediatedstructural rearrangement of the energy-transduction domain, which allosteri-cally uncouples SERCA by disrupting Ca2þ occlusion at residue E309 intransport site II, and thereby facilitating futile Ca2þ backflux in muscle.Our results demonstrate that the energy-transduction domain is a key controlelement that provides a structural motif for activation of Ca2þ transport bycardiac SERCA2a in heart failure and for uncoupling ATP hydrolysis bymuscle SERCA1a in obesity.

Platform: Voltage-gated Na Channels

95-PlatTetrodotoxin Resistance: Natural Experiments to Understand Voltage-Gated Sodium Channel Structure and FunctionShana L. Geffeney1, Gabriela Toledo2, Charles T. Hanifin1.1Biology, Utah State University-Uintah Basin, Vernal, UT, USA, 2Biology,University of Virginia, Richmond, VA, USA.Here we report data from our long-term studies of naturally occurring tetro-dotoxin (TTX) resistant voltage-gated sodium channels (VGSCs) in TTX-resistant metazoans. These results identify critical biophysical propertiesin individual members of the VGSC gene family as well as differences be-tween members. Members of the VGSC gene family are expressed indifferent excitable tissues and encode proteins with different biophysicalproperties. Characterizing isoform-specific properties is critical for under-standing functional differences between excitable cells because, in nerveand muscle cells, the compliment of VGSCs expressed controls cell excit-ability. TTX is a neurotoxin used by diverse taxa including octopuses, newts,and fishes. The toxin causes paralysis and death by binding to and blockingthe pore of VGSCs. Amino acid substitutions in the channel pore alter TTXbinding as well as change biophysical properties such as voltage-dependenceof activation and slow inactivation. Multiple species have evolved resistanceto TTX through pore substitutions in their VGSCs. We have used thesenatural experiments to identify shared substitutions in TTX-resistantVGSC orthologs (e.g. NaV1.4) and paralogs (e.g. NaV1.5 and NaV1.7) ofmultiple TTX-resistant animals. By characterizing the properties of channelsencoded by synthesized snake NaV1.4 genes, our work identifies interactionsbetween pore residues and demonstrates that multiple convergent amino acidsubstitutions are compensatory. Specific amino acid changes rescue channelfunction when they occur in combination with substitutions that dramaticallyincrease TTX resistance. Additionally, our work confirms that TTX resis-tance evolved prior to ecological exposure in snake and salamander lineages.These patterns suggest that selective pressures on channel function ratherthan a requirement for TTX resistance supported alterations in channel struc-ture in our study taxa. By comparing convergent and divergent substitutions,we identify critical biophysical differences between members of the VGSCgene family.

96-PlatUsing Specific Blockers to Identify TTX-S NaV Channels Subtypes in RatMuscle Afferent NeuronsRenuka Ramachandra, Keith S. Elmslie.Pharmacology, AT Still Univ/Kirksville College of Osteopathic Medicine,Kirksville, MO, USA.Muscle pain is transmitted by group III and IV afferents, but our knowledge ofthe NaV channel types that generate the action potentials in these neurons is

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incomplete. Our previous investigations have demonstrated the dominance oftetradotoxin-resistant (TTX-R) NaV1.8 channels in muscle afferent somata,but the TTX-sensitive (TTX-S) channel types have not yet been investigated.Using immunohistochemistry, we have recently demonstrated expression ofTTX-S NaV1.6 and NaV1.7 channels in the somata and axons of group IV neu-rons, but the contribution of these channels to NaV current is unknown. Wehave tested three NaV1.7 channel-selective blockers on group IV neurons(diameter < 33 mm) to determine the contribution of NaV1.7 current. Theblockers were ProToxin-II (100 nM; IC50 = 0.3 nM), Ssm6a (100 nM; IC50 =25 nM) and ICA-121431 (ICA, 1 mM, IC50 = 19 nM). To our surprise,ProTx-II and Ssm6a failed to block NaV current in group IV neurons. Theaverage affect on the TTX-S current was �1.2 5 5.0% for ProTx-II andþ17.2 5 38% for Ssm6a (both NS). For ICA, we found a 60 5 23% blockof the TTX-S current (p < 0.05). Based on the published IC50s we expectedall three blockers to produce similar effects, and do not yet understand reasonfor the absence of block by ProTx-II and Ssm6a. It is interesting that human andrat NaV channels can respond differently to blockers. For human channels, ICApotently blocks hNaV1.3, but not hNaV1.7, whereas the block is opposite for ratchannels, rNaV1.3 is insensitive and hNaV1.7 is potently blocked (McCormacket al. (2013) PNAS 110: E2724). Based on the ICA result, we tentativelyconclude that ~60% of the TTX-S current in group IV neurons is generatedby NaV1.7 channels.

97-PlatMultiple Mechanisms of Propofol Inhibition of the Voltage-Gated SodiumChannel Nachbac: A 19F NMR InvestigationYali Wang1, Marta M. Wells1, William Dailey2, Roderic Eckenhoff2,Pei Tang1, Yan Xu1.1University of Pittsburgh, Pittsburgh, PA, USA, 2University of Pennsylvania,Philidelphia, PA, USA.Voltage-gated sodium channels are important molecular targets for anes-thetics. The intravenous general anesthetic propofol was found to inhibitNaChBac, a prokaryotic sodium channel from Bacillus halodurans, but littleis known about where propofol binds and how propofol binding inhibits thefunction of this channel. Here, we used 19F-NMR saturation transfer differ-ence (STD) spectroscopy to quantify propofol interactions with NaChBacand to understand the inhibitory mechanisms by propofol. 19F probes wereintroduced, one at a time, by tagging 1,1,1-trifluoro-3-bromo acetone tosite-directed single cysteine mutations that had been computationally pre-dicted to be involved in propofol binding. Quantitative 19F-NMR STD be-tween the 19F probes and 4-fluoropropofol, a fluorinated propofol analogshow that propofol binds with different affinities to the sites in different re-gions of NaChBac. The order of fluoropropofol binding strength, reflectedin the cross relaxation rate constant (s), from the strongest to the weakest,is as follows: the channel activation gate at the cytoplasmic end of thepore, the voltage sensing domain near the gating charge-carrying residues,the ion selectivity filter at the end of the P1 loop, the S4-S5 linker, and thecentral cavity of the pore. No STD was detectable in the extracellular inter-face of channel. The finding of multiple binding sites is consistent with theprevious observations with the volatile anesthetics isoflurane and sevoflurane.Our results suggest that propofol inhibits NaChBac through multiple sites withdistinct mechanisms. As a channel blocker, propofol interferes with the acti-vation gate and selectivity filter and obstructs conductance by binding to theinner pore. As an inhibitory modulator, propofol binding interrupts chargemovement in the voltage-sensing domain and restricts the pivot motion ofthe S4-S5 linker. The study provides a molecular basis for the understandingof propofol inhibitory action on voltage-gated sodium channels. This researchwas supported by grants from the NIH.

98-PlatThe Complete Crystal Structure of an Open Activated Sodium ChannelJennifer Booker, Altin Sula, Claire E. Naylor, B.A. Wallace.Insitute of Structural andMolecular Biology, Birkbeck College, University ofLondon, London, United Kingdom.Voltage-gated sodium channels (Navs) play essential roles in excitable tis-sues, with the activation and opening of these channels resulting in the initialphase of the action potential. A new high resolution (2.45 A) crystal struc-ture of the NavMs prokaryotic sodium channel, has provided the first view ofa complete sodium channel structure, enabling visualisation of the interac-tions of the voltage sensor (VS), S4-S5 linker, pore, and C-terminal domains.The structure has a canonical activated conformation for its VS S4 helix,with a novel S4-S5 linker conformation that leads to an open selectivity filterwhich in turn leads to an open activation gate at the intracellular membranesurface. This ends in a C-terminal domain (CTD) that acts as a ‘‘glue’’ be-tween the monomers that comprise the tetrameric channel. The activated VS

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conformation produces a heretofore unseen extensively-hydrogen bondedand salt-bridged interaction motif involving a sodium-channel specific S3Trp, the S4-S5 linker, the end of the S6 transmembrane region and thetop of the CTD. The interaction motif involves a number of residues whichare conserved across eukaryotic and prokaryotic sodium channels, butnot other ion channels. This structure demonstrates for the first time thephysical linkage between channel activation and opening, enabling a com-plete model for the mechanism of sodium channel activation/opening andclosing. (*= these authors contributed equally; supported by grants fromthe U.K. BBSRC)

99-PlatThe Cardiac Channel NaV1.5 Inactivation is Modulated by its C-TerminalEF-hand DomainBernd R. Gardill, Ching-Chieh Tung, Ricardo E. Rivera-Acevedo, Filip VanPetegem.Biochemistry and Molecular Biology, University of British Columbia,Vancouver, BC, Canada.The cardiac isoform of voltage-gated sodium channels Nav1.5 is responsiblefor the rapid upstroke of the action potential after excitation. The underlyinggene of the alpha-subunit SCN5A is mostly expressed in the myocardiumalthough some expression in other tissue has been reported. Auxiliary pro-teins like calmodulin have been identified to modify channel gating. Muta-tions in SCN5A and calmodulin have been linked to several arrhythmias,including Brugada syndrome and Long-QT syndrome type 3. The channelarchitecture is organized in four homologous transmembrane repeats and aC-terminal EF-hand domain. After channel opening Nav1.5 undergoes inac-tivation within milliseconds which leads to a non-conducting state of thechannel. The cytoplasmic DIII-IV linker is known as inactivation gate. Itcontains the hydrophobic IFM motif which has been shown to stabilizethe inactivated state. A previous study suggested a direct interaction of theC-terminal part of the DIII-IV linker and the C-terminal EF-hand domainof the channel stabilizing the inactivation gate in the inactivated state.Here, we present an alternative model where the EF-hand destabilizes the in-activated state through binding to the DIII-IV linker. Isothermal titrationcalorimetry was utilized to characterize binding of WT and mutant inactiva-tion gate peptides to the EF-hand domain. NMR spectroscopy enabled usto map the exact interaction site on the EF-hand domain. Two-electrodevoltage clamp measurements in Xenopus laevis oocytes provided data oninactivation gate mutants and EF-hand disease mutants in the context offull length channel. Mutations that abolished EF-hand binding lead to a pro-found left-shift of steady-state inactivation, suggesting a role of the EF-handdomain in destabilizing the inactivated state.

100-PlatTransgenic Approach to Exploration of Calmodulin’s Role on SodiumChannel Function within CardiomyocytesJeffrey Abrams, Alexander Katchman, Lin Yang, Steven Marx.Columbia University Medical Center, New York, NY, USA.Many disease-causing mutations within Nav1.5 are localized to its C-terminaldomain. This region interacts with numerous auxiliary proteins. Amongstthe most controversial of these interactions is that with calmodulin. To clarifycalmodulin’s role on sodium channels expressed within cardiomyocytes,we employed a transgenic approach.We generated transgenic mice withdoxycycline-inducible, cardiac-specific expression of either FLAG-epitope-tagged C374Y-Nav1.5 (WT), or C374Y / IQ1908-1909AA-Nav1.5 (IQ/AA).The C374Y mutation increases tetrodotoxin sensitivity, enabling electrophys-iologic distinction between endogenous and transgenic Nav1.5. The IQ/AAmutation is known to decrease binding affinity with calmodulin. Limb-leadECG’s were measured under anesthesia. Biophysical properties of Nav1.5were measured in isolated cardiomycoytes. Quantification of late currentwas determined by normalizing persistent current detected with 100 mM[Na]o/3mM [Na]i, to peak current measured with 3 mM [Na]o/3mM [Na]i.Transgenic sodium channels trafficked to the sarcolemma and were detectedusing FLAG-specific antibodies. Electrophysiologic characteristics of WTNav1.5 were comparable to Nav1.5 in non-transgenic mice. The IQ/AA trans-genic channel also trafficked to the membrane and demonstrated comparableelectrophysiologic properties. The amount of late current determined by theratio between persistent current and peak sodium current was not differentin non-transgenic, transgenic WT, and transgenic IQ/AA mice. QT intervalwas not prolonged in IQ/AA mice recorded using limb-lead ECG under iso-flurane anesthesia. In contrast, mice expressing transgenic FLAG-epitope-tagged F1759A-Nav1.5, a lidocaine resistant sodium channel that also hasincreased persistent current, showed prolonged QT intervals. These datademonstrate a novel approach to study mutant human Nav1.5. In contrast

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to reports on heterologously expressed Nav1.5, mutation of the calmodulinbinding site did not perturb electrophysiologic properties of Nav1.5 incardiomyocytes.

101-PlatInvestigating Ca2D-Dependent Regulation of Sodium Channels viaThermodynamic and Structural Analysis of Nav1.4 and Nav1.5 CarboxyTail Interactions with CalmodulinJesse B. Yoder1, Sandra B. Gabelli2, L. Mario Amzel1.1Biophysics & Biophysical Chemistry, Johns Hopkins University Schoolof Medicine, Baltimore, MD, USA, 2Medicine, Biophysics & BiophysicalChemistry, Johns Hopkins University School of Medicine, Baltimore,MD, USA.Voltage-gated sodium channels (Nav) are essential for cardiac and skeletalmuscle function. Channelopathic mutations in cardiac sodium channel(Nav1.5) and skeletal muscle sodium channel (Nav1.4), particularly in theircytoplasmic carboxy tails (CTNav), give rise to numerous arrhythmias andmyotonias. In Nav regulation, CTNav partners with the Ca2þ-sensing proteincalmodulin (CaM). We aim to understand how CaM regulates channel func-tion. Ca2þ-free CaM (apoCaM) is a regulatory modulator of Nav, andapoCaM bound to the CTNav increases the channel’s open probability.CaM also participates in the Ca2þ-dependent inactivation (CDI) of Nav1.4.In contrast, Nav1.5 does not show CDI and no role of Ca2þ-CaM is knownin Nav1.5. To understand the Ca2þ-control of CaM regulation of these sodiumchannels we have collected binding data of CTNavs with CaM, in the pres-ence and absence of Ca2þ. Binding data of CTNavs with CaM mutantswith Ca2þ-binding knocked out in either of CaM’s functional domains (lobes)have also been collected to understand the distinct roles of CaM’s lobes.Collectively, these binding data have allowed us to predict CTNav andCaM populations as a function of Ca2þ concentration. To gain informationon the structural changes induced by Ca2þ on the CTNav-CaM complexes,we conducted small angle scattering (SAXS) experiments on CTNav1.4-CaM and CTNav1.5-CaM in the presence and absence of Ca2þ. The distancedistribution function, P(r), shows changes in both complexes upon addition ofCa2þ. Computational modeling of flexible forms of the complexes revealsconformations of CTNav-CaM, in the presence and absence of Ca2þ, thatare compatible with our SAXS data. The molecular envelope of theCTNav1.5-apoCaM matches well with the apoCaM-CTNav1.5 crystal struc-ture previously determined by our lab.

102-PlatTargeting the Cardiac Sodium Channel to Increase Excitabilityof Stem-Cell Derived CardiomyocytesValentin Sottas, Cristian Mihnea Trache, Nina D. Ullrich.Heidelberg University, Heidelberg, Germany.The cardiogenic potential of stem-cell derived cardiomyocytes (iPSC-CM) andtheir prospective use for cardiac cell therapy crucially depends on their excit-ability and functional integration in myocardial tissue. Indeed, previous studiesfrom our group have shown that cell excitability and intercellular coupling arestrongly reduced in iPSC-CMs compared to primary cardiomyocytes. For clin-ical aspects, impaired excitability and electrical signal propagation may lead toconduction slowing and the development of arrhythmia. In this project, wefocus on the idea that cardiomyocyte excitability and conduction propertiesare interrelated and depend on the expression of the cardiac sodium channelNav1.5 and the major gap junction forming protein connexin-43 (Cx43). Wetested the hypothesis that molecular remodeling of both proteins enhancescell excitability as reflected in Nav1.5 activity and action potential (AP) prop-erties with the aim to approach native cardiomyocyte function. Using a combi-nation of molecular modulation and electrophysiological evaluation in voltageand current clamp modes, our data demonstrate that enhanced Nav1.5 expres-sion in iPSC-CMs significantly increased sodium current (INa in pA/pF: control40.5510.5, Nav1.5 118.3527.2) and upstroke velocity (dv/dtmax, in V/s:156518 vs. 276529, respectively) of the AP, a critical determinant of cellexcitability. Typically, a fraction of iPSC-CMs also exhibited spontaneousAPs with low dv/dtmax (<50 V/s) driven without Nav1.5, a hallmark of imma-turity. However, after Nav1.5 overexpression, all recorded APs showed fastdv/dtmax. Furthermore, INa was also increased in Cx43-overexpressing iPSC-CMs (INa 66.2519.8 pA/pF) suggesting that Cx43 may influence Nav1.5expression and thereby cell excitability. This notion was further confirmed inimmunostainings of Cx43-overexpressing iPSC-CMs demonstrating increasedNav1.5 expression at the plasma membrane, which suggests a common regula-tion pathway between both proteins. In conclusion, modulation of Nav1.5 andCx43 expression greatly enhances the excitability of iPSC-CMs and mayrepresent a powerful new target for improving the functional maturation ofiPSC-CMs.

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Platform: Optical Microscopy and Super-resolution Imaging: Novel Approaches andAnalysis I

103-PlatSuper-Resolution Imaging of Unlabeled Proteins on DNAAnna EC Meijering, Andreas S. Biebricher, Erwin JG Peterman,Gijs JL Wuite, Iddo Heller.Physics and Astronomy, Vrije Universiteit Amsterdam, Amsterdam,Netherlands.Direct visualization of DNA-protein interactions at the single-molecule level isof ever increasing importance to unravel the salient details of a wide range ofDNA-associated processes. Recent emergence of super-resolution imaging hasthe potential to further boost the impact of such investigations. However, cur-rent imaging methods heavily rely on (fluorescent) labeling strategies, whichcan be challenging and potentially even interfere with the molecular interac-tions under scrutiny. Here, we introduce a new label-free method to imagethe presence and location of DNA-bound proteins with super-resolution onoptically manipulated DNA. The method is based on localization microscopyof DNA-intercalating dyes that locally bind to bare DNA but not to protein-bound DNA sections, yielding an inverted image that reveals the ‘shadows’of the proteins on the DNA: inverse binding-activated localization microscopy(iBALM). We present the proof of principle of iBALM as well as of functionalvariations to this method and provide experimental and theoretical data thatdescribe the spatial and temporal resolution that can be obtained. iBALMhas the potential to become a valuable addition to the single-molecule toolkitand enable direct visualization of processes that were previously not possibledue to limitations posed by labeling.

104-PlatVarying Label Density to Probe Membrane Protein Nanoclusters inSTORM/PALMFlorian Baumgart1, Andreas Arnold1, Konrad Leskovar1, Kaj Staszek1,Martin Foelser1, Julian Weghuber2, Hannes Stockinger3,Gerhard J. Schuetz1.1Institute of Applied Physics, TU Wien, Vienna, Austria, 2University ofApplied Sciences Upper Austria, Wels, Austria, 3Medical University ofVienna, Vienna, Austria.Superresolution microscopy has facilitated the investigation of cellular struc-tures at length scales far below the optical diffraction limit. When applied tothe plasma membrane, the presence of a variety of protein nanoclusters wasrevealed, which lead to speculations whether nanoclustering was a generalfeature of plasma membrane proteins. Particularly in T lymphocytes, clusteringof signaling proteins has been proposed to represent a fundamental mechanismfor cell activation. Recently, however, doubts were raised whether imagingartifacts inherent to PALM/STORM might have influenced or even causedthe observation of some of those protein clusters. To approach these con-cerns, we developed a method to robustly discriminate clustered fromrandom distributions of molecules detected with single molecule localizationmicroscopy-based techniques like PALM and STORM.1 The approach is basedon deliberate variations of the labeling density of the samples and quantitativecluster analysis. Our method circumvents the problem of clustering artifactsgenerated by the blinking statistics of the fluorophores used. It can be readilyapplied to PALM and STORM experiments where either overexpressed pro-teins are present over a broad range of expression levels or antibody concentra-tions are titrated to achieve different degrees of labeling.1 Baumgart et al., Nat. Meth. 13: 661-664 (2016)

105-PlatTracking Oligomeric Transcription Factor Dynamics by Pair Correlationof Molecular Brightness (pCOMB)Elizabeth Hinde1, Elvis Pandzic1, Zhengmin Yang1, Ivan Ng2,Marie Bogoyevitch3, David Jans4, Enrico Gratton5, Katharina Gaus1.1EMBL AUSTRALIA NODE IN SINGLE MOLECULE SCIENCE,University of New South Wales, Sydney, Australia, 2Program in EmergingInfectious Diseases, Duke-NUS Medical School, Singapore, Singapore,3Department of Biochemistry and Molecular Biology, University ofMelbourne, Melbourne, Australia, 4Department of Biochemistry andMolecular Biology, Monash University, Melbourne, Australia, 5Laboratoryfor Fluorescence Dynamics, Biomedical Engineering, University ofCalifornia Irvine, Irvine, CA, USA.Oligomerisation of transcription factors controls their translocation into thenucleus and DNA binding activity. Here we present a fluorescence micro-scopy method termed pCOMB (pair correlation of molecular brightness)

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that tracks the mobility of different oligomeric species within live cell nucleararchitecture.1 pCOMB amplifies the signal from the brightest species presentand filters the dynamics of the extracted oligomeric population based onarrival time between two locations. Here we use this method to demonstratea dependence of signal transducer and activator of transcription 3 (STAT3)mobility on oligomeric state. We find that upon entering the nucleusSTAT3 dimers must first bind DNA to form STAT3 tetramers, which arealso DNA-bound but exhibit a different mobility signature. Examining thedimer-to-tetramer transition by a cross pair correlation analysis (cpCOMB)reveals chromatin accessibility to modulate STAT3 tetramer formation.Thus the pCOMB approach is suitable for mapping the impact oligomerisa-tion has on transcription factor dynamics.1. Hinde, E.*, E. Pandzic, Z. Yang, I. H. W. Ng, D. A. Jans, M. A. Bogoye-vitch, E. Gratton and K. Gaus*. 2016. Quantifying the dynamics of theoligomeric transcription factor STAT3 by pair correlation of molecularbrightness. Nature Communications. 7(11047).

106-PlatReconstructing Spatial Features of Nucleocytoplasmic Transport usingProjected Cargo LocalizationsLi-Chun Tu1, Maximiliaan Huisman1, Yu-Chieh Chung2, Carlas Smith1,David Grunwald1.1RNA Therapeutics Institute, University of Massachusetts Medical School,Worcester, MA, USA, 2No affiliation, Worcester, MA, USA.Time-resolved single molecule experiments have provided tremendous infor-mation pertaining to molecular dynamics and localization over the last decade.Biological processes take place in a light sensitive environment, on time scalesfrom sub-millisecond (ms) to hours and length scales from nanometer to milli-meter, presenting a number of experimental challenges. One such challenge isthe need for speed in image acquisition to correctly follow single moleculemovements during translocation through the nuclear pore complex (NPC).Translocation through the central channel has repeatedly been reported to befaster than 20 ms.Biochemical and structural data of the components that make up the NPC haveled to the question if specific spatial transport routes exist within the NPC invivo. Millisecond time resolutions and three-dimensional spatial resolution inthe range of only a few nm are needed in order to resolve the path traveledby transport receptors and cargos by single molecule real-time microscopy.These imaging requirements are challenging and are not met by current tech-nology; however, it was suggested by Ma & Yang that highly time-resolved2D tracking data can be interpreted as projected cargo densities and subse-quently transformed into a 3D cargo distribution. Such distributions would pro-vide valuable insights into the function of NPC mediate transport in cells. Herewe present a thorough analysis of the conditions needed for this method to workfor the nuclear pore complex and the limits to which the results may beinterpreted.

107-PlatFollowing a Giant’s Footsteps: Single-Particle and Super-ResolutionApproaches to Decipher the Nuclear Transport of Hepatitis B Virus CapsidsGiulia Paci, Niccolo Banterle, Christine Koehler, Edward A. Lemke.EMBL (European Molecular Biology Laboratory), Heidelberg, Germany.Nucleocytoplasmic transport occurs through nuclear pore complexes (NPCs),and is a tightly regulated process. The Hepatitis B Virus (HBV) capsid isone of the largest cargoes ever shown to be imported intact to the nuclearside of the NPC. The import of such large cargoes - close in dimension tothe expected maximum size of the NPC central channel - is largely unknown,as well as to what extent plasticity of the NPC transport conduit is required toaccommodate the transport. To decipher the molecular details of this host-pathogen interaction, we developed a dual strategy that allows us to visualizewith high spatiotemporal resolution the viral capsid, as well as the elusivepermeability barrier of the NPC. We follow nuclear import of fluorescentlylabelled HBV capsids in mammalian cells using single-particle tracking on apurpose optimized self-designed HILO microscope. We observe an averagetransport time in the hundreds of milliseconds range, orders of magnitude fasterthan what expected for such a large cargo. In addition, slower interactions occurat the cytoplasmic filaments and nuclear basket, pointing to docking and un-docking interactions being the rate limiting factor. Complementarily, to deci-pher the changes occurring in the NPC during viral transport, we developeda method to image site-specifically labelled nucleoporins in the cell usingsuper-resolution microscopy (SRM). We combine site-specific tagging ofproteins, through an enhanced Amber suppression system, with DNA-PAINTmicroscopy, enabling the visualization of even low abundance proteins suchas nucleoporins. This synergistic approach gives us the potential to resolvethe plasticity of the permeability barrier of NPCs, allowing us a dual view on

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the viral transport from both the cargo and the ‘‘gate’’ (NPC) level in an unprec-edented manner.

108-PlatDevelopment of a Simultaneous Six-Color Fluorescence Microscope withSingle-Molecule SensitivityJingyu Wang, Jamie Barnett, Luke Springall, Neil M. Kad.School of Biosciences, University of Kent, Canterbury, United Kingdom.Prokaryotic Nucleotide excision repair (NER) is a complex mechanisminvolving six proteins: UvrA, UvrB, UvrC, UvrD, DNA polymerase 1 andDNA ligase. To holistically study the kinetics of NER, multiple labelling andimaging channels are required to differentiate the parties involved in the dam-age search and repair activities. Using Quantum dot (Qdot)-conjugated UvrA,UvrB, UvrC, UvrD, DNA polymerase 1 and a Qdot labelled lesion we aim todetect and determine the significance of all the complexes that contribute torepair. For this purpose, we have adapted an existing fluorescence microscopeinto a multi-color simultaneous fluorescence microscope by spectrally sepa-rating 6 emission channels based on available Qdots: 525 nm, 565 nm,585 nm, 625 nm, 655 nm and 705 nm. These six channels were created usingparallel optical splitting devices distributed into two paths and imaged on twoidentical state-of-the-art Hamamatsu, ORCA-Flash4.0 V2 scientific CMOScameras. Each low noise, high QE (>80%) camera sensor has 2048 x 2048pixels, therefore every spectral channel has 682 x 2048 pixels; sufficiently largeto image elongated DNA tightropes. Although the Qdots can be illuminated bya single 488 nm source, the fluorescence microscope also uses four excitationlasers: 405 nm, 488 nm, 561 nm and 637 nm combined into a single path. Thisallows for numerous combinations of channels to be excited and separatelymodulated using an Arduino-based control element linking the cameras tothe lasers. Using this system, we present data obtained on the heterogeneityof the complexes formed during NER.

109-PlatIntracellular Delivery ofMembrane Impermeable Photostable FluorescentProbes into Living Cells for Super-Resolution MicroscopyYuji Ishitsuka1, Kai Wen Teng1, Pin Ren1, Yeoan Youn1, Xiang Deng2,Pinghua Ge1, Andrew Belmont2, Paul R. Selvin1.1Department of Physics, University of Illinois Urbana-Champaign, Urbana,IL, USA, 2Department of Cell and Developmental Biology, University ofIllinois Urbana-Champaign, Urbana, IL, USA.Specific labeling of the cellular target with fluorophore is one of the funda-mental requirement in all fluorescence imaging techniques including singlemolecule and super-resolution microscopy techniques. While extracellular tar-gets may be tagged with virtually any kind of probes, intracellular labeling ofliving cells is limited to the use of fluorescent proteins and limited selection ofmembrane permeable dyes. Here we show that pore forming proteins can beused to temporarily permeabilize the cells and allow delivery of various fluo-rescent probes, ranging from organic dyes (<1 kDa) to fluorescent immuno-globulin antibody (~150 kDa), for specific labeling of intracellular targets forlive fluorescence cell imaging. We demonstrate that permeabilized cells canefficiently be recovered to carry on normal cellular processes shown by nucleartranslocation of nanobody-labeled p65 proteins in response to chemical stimu-lation. One of the most photostable but membrane impermeable organic fluo-rophore, Atto 647N, has been delivered into cells to label actin fibers andkinesin dimers and to perform super-resolution fluorescence microscopy imag-ing dSTORM and single molecule tracking, respectively. This technique opensup a large number of stable, but otherwise membrane impermeable fluores-cence labeling probes that are available for investigating transfected and endog-enous intracellular targets.

110-PlatBright and Photostable Fluorophores for Advanced FluorescenceMicroscopyQinsi Zheng1, Jonathan B. Grimm1, Anand K. Muthusamy1,Robert H. Singer1,2, Luke D. Lavis1.1Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA,USA, 2Albert Einstein College of Medicine, New York, NY, USA.Advanced fluorescence microscopy, including single-molecule and super-resolution imaging, demands bright and photostable fluorophores. We haverecently reported a general approach to improve fluorophores brightness inliving cells by substituting the N,N-dimethylamino groups found in classicdyes with four-membered azetidine rings (Nature Methods 12, 244250(2015)). In an unpublished work we have synthesized new derivatives contain-ing substituents on the azetidine ring. Using this approach we were able to finetune the wavelength and fluorogenecity of the fluorophore without affectingbrightness. Here, we report that several of these novel substituted-azetidinefluorophores, as well as the substituted-xanthene ones, exhibit substantial

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improvements in photostability in living cells. These fluorophores enablerobust multi-color, wash-free imaging with a large photon budget. We areinvestigating their phototoxicity and mechanism in order to maximize the pho-tostability, to generalize this approach to different fluorophores, and to applythese fluorophores to diverse biological settings, including living cells, tissues,and animals. We freely share our fluorophores with the academic community.This work is supported by HHMI and NIH (U01 EB 021236).

Platform: Membrane Protein Structures I

111-PlatStructure Inhibition and Regulation of a Two-Pore Channel TPC1Alexander F. Kintzer, Robert M. Stroud.Biochemistry and Biophysics, University of California at San Francisco, SanFrancisco, CA, USA.Membrane transport serves vital functions in the cell, providing nutrients forgrowth, relaying electrical signals, evading pathogens, and maintaininghomeostasis. Voltage- and ligand-gated ion channels propagate cellular elec-trical signals by coupling changes in membrane potential and the binding ofmolecules to channel opening and selective passage of ions through themembrane barrier. Two-pore channels (TPCs) are intracellular ion channelsthat integrate changes in membrane potential, second messengers, and phos-phorylation to control endolysosomal trafficking, autophagy, cellular ion andamino acid homeostasis, and ultra-long action potential-like signals. Theybroadly impact human diseases related to trafficking including filoviral in-fections, Parkinson’s disease, obesity, fatty liver disease, and Alzheimer’sdisease. The response of TPCs to multiple cellular inputs suggests a multi-state gating mechanism. Nevertheless, the mechanisms that govern cyclesof activation and deactivation or ‘gating’ in the channel remain poorly un-derstood. To understand the bases for ion permeation, channel activation,the location of voltage-sensing domains and regulatory ion-binding sites,and phosphoregulation, we determined the crystal structure of TPC1 fromArabidopsis thaliana to 2.87A resolution. This reveals for the first timehow TPC channels assemble as ‘quasi-tetramers’ from two non-equivalenttandem pore-forming subunits. We determined sites of phosphorylation inthe N-terminal and C-terminal domains that are positioned to allostericallymodulate channel activation by cytoplasmic calcium. One of the two voltagesensing domains (VSD2) encodes voltage sensitivity and inhibition byluminal calcium locks VSD2 in a ‘resting’ conformation, distinct from theactivated VSDs observed in structures of other voltage-gated ion channels.The structure shows how potent pharmacophore trans-Ned-19 allostericallyacts to inhibit channel opening. In animals, trans-Ned-19 prevents infectionby Ebola virus and Filoviruses by blocking fusion of the viral and endolyso-somal membranes, thereby preventing delivery of their RNA into the hostcytoplasm. The structure of TPC1 paves the way for understanding the com-plex function of these channels and may aid the development of antiviralcompounds.

112-PlatNovel Mechanism of Gating in the TrkH-TrkA ComplexHanzhi Zhang1, Zhao Wang1,2, Mingqiang Rong1,3, Yaping Pan1,Wah Chiu1,2, Ming Zhou1.1Baylor College of Medicine, Houston, TX, USA, 2National Center forMacromolecular Imaging, Houston, TX, USA, 3Kunming Institute ofZoology, China Academic of Science, Kunming, China.The superfamily of Kþ transporters (SKT) is ubiquitous in bacteria, fungi andplants. SKT proteins are required for survival of bacteria in low Kþ conditionsand are involved in salt regulation in fungi and plants. Bacterial SKTs have twocomponents, a membrane embedded protein that resembles an ion channel anda cytosolic protein that regulates channel gating [1]. Crystal structures of twobacterial SKT systems were reported recently [2,3]. In both structures, themembrane embedded component forms a homodimer onto which a homotetra-meric ring of the cytosolic protein docks. Single-channel activities of one of thecomplexes, the TrkH (membrane embedded) -TrkA (cytosolic) complex, wererecorded and analyzed: ATP or ATP analogs such as AMPPNP activates thechannel while ADP closes it [2]. The structure of the TrkH-TrkA complex islikely in a closed conformation because it was crystallized in the presence ofNADH, a ligand that does not activates the channel. In order to understandhow ATP or its analogs induces channel opening, we solved the structure ofthe TrkH-TrkA complex in the presence of AMPPNP to 3.29 A by x-ray crys-tallography. When compared to the previous structures, the new structureshows that each TrkA protomer binds to two AMPPNP molecules, and thatthe TrkA tetramer assumes an elongated conformation that likely induces achange in the TrkH. Conformational changes in the TrkH involve significantchanges in the dimer interface and are different from any other channels of

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known structure. These new observations and hypotheses will be validated andtested by mutational and functional analyses.[1] Levin EJ, Zhou M. Recent progress on the structure and function of theTrkH/KtrB ion channel. Curr Opin Struct Biol. 2014;27:95-101.[2] Cao Y, Pan Y, Huang H, et al. Gating of the TrkH ion channel by its asso-ciated RCK protein TrkA. Nature. 2013;496(7445):317-22.[3] Vieira-pires RS, Szollosi A, Morais-cabral JH. The structure of the KtrABpotassium transporter. Nature. 2013;496(7445):323-8.

113-PlatStructural and Functional Characterization of a Calcium-ActivatedCation Channel From Tsukamurella PaurometabolaBala Dhakshnamoorthy, Ahmed Rohaim, Huan Rui, Lydia Blachowicz,Benoit Roux.University of Chicago, Chicago, IL, USA.The selectivity filter is an essential functional element of Kþ channels that ishighly conserved both in terms of its primary sequence and its three-dimensional structure. Here, we investigate the properties of an ion channelfrom the Gram-positive bacterium Tsukamurella paurometabola with a selec-tivity filter formed by an uncommon proline-rich sequence. Electrophysiolog-ical recordings show that it is a non-selective cation channel and that its activitydepends on Ca2þ concentration. In the crystal structure, the selectivity filteradopts a novel conformation with Ca2þ ions bound within the filter near thepore helix where they are coordinated by backbone oxygen atoms, a recurrentmotif found in multiple proteins. The binding of Ca2þ ion in the selectivity filtercontrols the widening of the pore as shown in crystal structures and in molec-ular dynamics simulations. The structural, functional and computational dataprovide a preliminary characterization of this calcium-gated cation channel.

114-PlatCrystal Structure of a Low CO2-Inducible Protein,LCI1 in Chlamydomonas ReinhardtiiTsung-Han Chou.Physics and Astronomy, Iowa State University, Ames, IA, USA.The assimilation of atmospheric carbon dioxide (CO2) by microalgae andplants for photosynthesis has not been fully understood. Gaining insight intothe intricate structural details of the CO2 scavenging mechanism by the photo-synthetic green algae Chlamydomonas reinhardtii can pave the way for utiliz-ing abundant CO2 as a valuable alternative fuel resource. Here, we present acrystal structure of the Chlamydomonas reinhardtii LCI1 channel, which isinvolved in the CO2-concentrating mechanism (CCM) to assimilate inorganiccarbon resources. Combined with X-ray crystallography, mass spectrometryand computational simulation, our data indicate that the LCI1 membrane pro-tein forms a trimeric assembly, in which each protomer conducts unchargedCO2 and shuttles this inorganic carbon species across the cell membrane.

115-PlatStructural Role of ABCG5/ABCG8 in Sterol TransportJyh-Yeuan (Eric) Lee, Daniel Rosenbaum, Helen Hobbs.UT Southwestern Medical Center at Dallas, Dallas, TX, USA.ATP binding cassette (ABC) transporters play critical roles in maintaining sterolhomeostasis in eukaryotic organisms, including yeast, plants and mammals. Inhumans, the heterodimeric ABCG5/ABCG8 (G5G8) mediates the excretion ofcholesterol and dietary plant sterols into bile and into the gut lumen. Mutationsinactivating either ABCG5 or ABCG8 cause sitosterolemia, a rare autosomalrecessive genetic disorder characterized by plant sterol accumulation, hyper-cholesterolemia, and premature coronary atherosclerosis. ABCG5 and ABCG8are half ABC transporters;each subunit consists of an N-terminal nucleotide-binding domain (NBD) and a C-terminal transmembrane domain (TMD). TheNBDs dimerize to form two catalytically asymmetric nucleotide-binding sites(NBS), one that is catalytically active (NBS2) and the other inactive (NBS1).To understand the structural basis for G5G8-mediated sterol transport we devel-oped a large-scale purification of human G5G8 by exploiting Pichia patorisyeast. We crystallized the transporter in lipid bilayers, solved its structure in anucleotide-free state at 3.9 A resolution, and generated the first atomic modelof an ABC sterol transporter. G5G8 presents a new structural configuration forthe TMD of ABC transporters, which is present in a large and functionallydiverse ABC2 superfamily. We discover that the TMD and the NBS are coupledthrough networks of interactions that differ between NBS1 and NBS2, reflectingthe catalytic asymmetry of the transporter. A series of conserved polar residues inthe TMD form polar networks that we proposed play a role in transmitting sig-nals from the ATPase catalysis in the NBS to sterol transport on the TMD.Molecular dynamic simulation and long-range coevolution analysis revealedan inward-upward TMD movement that predicts a significant conformationalchange between the TMD subunits. Thus, the G5G8 structure provides amolecular framework that allows us to propose a mechanistic model for ABC

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transporter-mediated sterol transport and to analyze the disruptive effects of mu-tations causing sitosterolemia. The structure will serve as a structural templatefor homology modelling to a wide range of transport system that is regulatedby ABCG transporters and by ABC2 superfamily.

116-PlatStructural and Mechanistic Basis of Proton-Coupled Metal Ion Transportin the SLC11/NRAMP FamilyCristina Manatschal, Ines A. Ehrnstorfer, Raimund Dutzler.Department of Biochemistry, University of Zurich, Zurich, Switzerland.Divalent metal ion transporters (DMTs) of the SLC11/NRAMP family trans-port iron and manganese across cellular membranes. These proteins are highlyconserved across all kingdoms of life and thus likely share a common transportmechanism. Our previous crystal structure of Staphylococcus capitis DMT(ScaDMT) has established the structural relationship of the SLC11 familywith the amino acid transporter LeuT and it revealed the location of a conservedtransition-metal ion binding site in the center of the transporter. In this struc-ture, ScaDMT adopts an inward-facing conformation. Recently, we have deter-mined the crystal structure of Eremococcus coleocola DMT (EcoDMT) in anoutward-facing conformation. Together these structures define the endpointsof the transport cycle. Functional assays with proteoliposomes establishedEcoDMT as secondary active transporter that couples the symport of Mn2þ

and protons with a KM in the low micromolar range. Mutants of residues ofthe metal ion binding site severely affected both, Mn2þ and proton transportthus suggesting that the transport of protons requires conformational changesof the transporter. Inspection of both structures revealed two protonatable res-idues close to the metal ion binding site that have changed their accessibility toeither side of the membrane as potential candidates for proton acceptors.Mutation of one of these residues, a conserved histidine on a-helix 6b, resultedin metal ion transport that appears to be no longer coupled to protons, whichimplies that this residue likely plays a central role in proton transport. Takentogether, our studies have revealed the conformational changes underlyingtransition-metal ion transport in the SLC11 family by the alternate accessmechanism and they provide important insights into the determinants of itscoupling to protons.

117-PlatStructural Basis of Concentrative Nucleoside TransportMarsha M. Hirschi, Zachary L. Johnson, Seok-Yong Lee.Duke University, Durham, NC, USA.Nucleosides are essential molecules for the living cell. As precursors to nucle-otides they serve to fuel the salvage pathway of DNA and RNA synthesis.Certain tissues, such as the brain and bone marrow, lack the capacity for denovo synthesis and therefore rely completely on the influx of nucleosides.Concentrative nucleoside transporters (CNTs) utilize sodium or proton gradi-ents to transport nucleosides across the cell membrane. These secondary activetransporters also play an essential role in the termination of adenosinesignaling, which controls important cellular processes such as neuromodulationand cardiovascular function. In addition to natural substrates CNTs are also theconduit for many anti-cancer and anti-viral drugs, making them of special in-terest from a pharmacological point of view. Notably, CNTs are the main trans-port route for a popular pancreatic cancer drug, gemcitabine. We previouslyreported on the structure of CNT from Vibrio cholerae in complex with varioussubstrates and nucleoside-like drugs. Each of these structures captured thetransporter in the inward-facing occluded conformation. In order to describethe transport mechanism in further detail we performed crystallization studieswith CNT from Neisseria wadsworthii (CNTNW). CNTNW is highly homolo-gous to human CNT3, sharing 38% sequence identity and nearly identical sub-strate binding sites. Here we present crystal structures of CNTNW captured inalternative conformations. We confirmed the physiological relevance of theconformations by crosslinking experiments. Our structural analyses and func-tional studies provide new insights into the mechanism of CNTs and cellularnucleoside uptake in molecular detail.

118-PlatInvestigating the Structure of the Drug Transporter EmrEMaureen Leninger, James R. Banigan, Geliana Abramov,Nathaniel J. Traaseth.Chemistry, New York University, New York, NY, USA.Multidrug resistance in bacteria is a critical challenge in public health and drugdiscovery. One of the primary mechanisms of resistance is efflux pumps, whichcouple an energetically favorable process with the export of a drug moleculeagainst its concentration gradient.1 Efflux pumps from the small multidrugresistance protein family are ubiquitous among bacteria. These secondaryactive transporters couple the efflux of a wide variety of toxic compoundswith the proton gradient of the inner membrane.2 To gain insight into this

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transport process we carried out a series of biophysical experiments, includingsolution and solid-state NMR spectroscopy using the multidrug transporterEmrE. EmrE is an asymmetric and antiparallel homodimer, which couplesthe efflux of cationic drugs with the import of protons. Previously, we revealeda large change in structure and dynamics due to the acid/base chemistry at aconserved glutamic acid residue.3 In this work we focus on describing thesite-specific structural changes within the substrate binding pocket that drivethe large conformational change in EmrE. Specifically, our oriented solid-state NMR and solution NMR experiments reveal backbone and side chainstructural perturbations upon binding protons or drugs that reveal a specificmechanism involving aromatic residues in the binding pocket. Finally, we builton our previous findings 3 to show how conformationally biased mutants allowfor the unambiguous determination of monomer-specific restraints for struc-tural elucidation of EmrE in lipid bilayers.References1. Pu, Y.; Zhao, Z.; Li, Y.; Zou, J.; Ma, Q.; Zhao, Y.; Ke, Y.; Zhu, Y.; Chen, H.;Baker, Matthew A. B.; Ge, H.; Sun, Y.; Xie, Xiaoliang S.; Bai, F., EnhancedEfflux Activity Facilitates Drug Tolerance in Dormant Bacterial Cells. Molec-ular Cell 2016, 62 (2), 284-294.2. Nikaido, H.; Pages, J. M., Broad-specificity efflux pumps and their role inmultidrug resistance of Gram-negative bacteria. FEMS Microbiol Rev 2012,36 (2), 340-63.3. Gayen, A.; Leninger, M.; Traaseth, N. J., Protonation of a glutamate residuemodulates the dynamics of the drug transporter EmrE. Nat Chem Biol 2016, 12(3), 141-145.

Platform: Membrane Active Peptidesand Toxins I

119-PlatAn In-Cell Solid-State NMR Portrayal of the Action Mechanismof Antimicrobial Peptides with Intact BacteriaMarwa Laadhari1, Alexandre A. Arnold1, Andree E. Gravel1,Frances Separovic2, Isabelle Marcotte1.1Chemistry, Universite du Quebec a Montreal, Montreal, QC, Canada,2Chemistry, University of Melbourne, Melbourne, Australia.Antimicrobial peptides (AMPs) are promising candidates to act against drug-resistant bacteria since they can disrupt the bacterial lipid barrier, leading tocell death. 2H solid-state NMR is a valuable tool to study at a molecular levelthe action of AMPs on the lipid organization and dynamics within the mem-brane bilayer. While these studies are usually performed with model phospho-lipid membranes, the complexity of the bacterial cell wall prompts thedevelopment of in-cell NMR techniques to take into account interactions ofall constituents. In particular, Gram(�) bacteria are protected by lipopolysac-charides in their outer membrane while a thick peptidoglycan covers the lipidbilayer of Gram(þ) bacteria. We have thus developed novel 2H solid-stateNMR tools to study in vivo the bactericidal action mechanism of aurein 1.2and caerin 1.1, AMPs excreted by frog skin. Using deuterated palmitic acid,we have established a protocol to 2H-label phospholipids in Gram(þ) Bacillussubtilis, and optimized the deuteration protocol of Gram(�) Escherichia coli.Via a combination of static and magic-angle spinning (MAS) experiments,our results support the interaction of the AMPs with membrane lipids leadingto decrease in lipid chain order. This effect is, however, observed at higher pep-tide concentration with B. subtilis and most likely attributable to an interactionwith negatively-charged components in its cell walls, such as teichoic acids.Our study shows that the nature of the cell wall plays a role upon the actual con-centration of AMPs on the bacterial membrane and highlights the importance ofstudying membrane interactions with intact bacteria.

120-PlatAction of Antimicrobial Peptides on Bacterial and Lipid Membranes: ADirect ComparisonJoseph E. Faust, Pei-Yin Yang, Huey W. Huang.Physics & Astronomy, Rice University, Houston, TX, USA.Are lipid bilayers a good model for studying bacterial membranes? We studythis problem by comparing the action of antimicrobial peptides (AMPs) onE. coli spheroplasts and on giant unilamellar vesicles (GUVs) made frommodel lipid bilayers. We observed AMPs inducing membrane permeabilityin spheroplasts or GUVs under well defined concentrations of AMPs and dyemolecules. The permeability allowed the dye molecules to diffuse in and outof the cells or vesicles, from which we were able to measure the instantaneousmembrane permeability by fluorescence recovery after photobleaching(FRAP). We also compared the effect of AMPs with that of metabolic poisonCCCP and daptomycin that kills bacteria by leaking potassium ions. The actionof AMPs on spheroplasts is unique in producing an intracellular fluorescence

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intensity time curve that increases in a sigmoidal fashion to a steady state.This regular pattern is reproducible by melittin, LL37 and alamethicin, butnot by CCCP or daptomycin. Remarkably, a similar regular pattern was repro-duced in GUVs. Indeed the steady-state membrane permeabilization inducedby AMPs is quantitatively the same in spheroplasts and GUVs. There are how-ever interesting dissimilarities in details that reveal differences between bacte-rial and lipid membranes. Spheroplast studies reveal a steady state membranepermeability independent of AMP concentration from 0.05 to 5.0 mM, that isdifficult to reproduce by lipid vesicles due of the fragility of pure lipid bilayers.Tension in GUVs also influences the action of AMPs, whereas the spheroplastmembranes are tensionless. Despite these differences, our results provide astrong support for using model lipid bilayers to study the molecular interactionsof AMPs with bacterial membranes.

121-PlatCharacterization of Anti-BiofilmPeptideActivity: ABiophysical ApproachLi-av T. Segev-Zarko1, Ron Saar-Dover1, Vlad Brumfeld2, Maria LuisaMangoni3, Yechiel Shai1.1Biomolecular sciences, Weizmann institute of science, Rehovot, Israel,2Chemical Research Support, Weizmann institute of science, Rehovot, Israel,3Biochemical Sciences, La Sapienza University, Rome, Italy.The increasing number of multidrug resistant bacteria to available antibioticsis a growing problem worldwide. One major strategy for resistance and animportant reason for failure of therapy in the clinic is biofilm formation. Tocope with unfavorable surroundings many bacteria live as biofilms, sessilemicro-colonies adherent to surfaces that secrete an extracellular polymericsubstance. An attractive alternative to conventional antibiotics are antimicro-bial peptides (AMPs), innate immune system molecules that target the bacte-rial cytoplasmic membrane, causing membrane disruption and cell death.AMPs biophysical properties are extensively studied regarding planktonicbacteria but much less so regarding biofilm formation. By designing and syn-thesizing a series of model peptides that share the same amino acids compo-sition but differ in their biophysical properties, we investigated how differentsteps of biofilm formation are affected by the AMP’s features. We modifiedthe peptides characteristics using different approaches of charge segregationand amino acids D enantiomers. We used Pseudomonas aeruginosa, an oppor-tunistic Gram-negative bacteria, which is a leading cause of severe pulmonaryinfections in cystic fibrosis patients and medical device contamination. Ourwork demonstrates that the peptides combat biofilm at different stages of itsformation and maintenance: (1) killing bacteria at their planktonic stage (2)preventing bacterial adhesion to biomaterials and (3) degrading pre-formedbiofilm. We show that substitution of L-to-D amino acids alters the peptidesbiophysical properties and improves their activity against each stage in thebiofilm life cycle. By investigating which biophysical properties are essentialfor anti-biofilm activity we also discovered new mechanisms of peptidesactivity.

122-PlatEffects of Lipid Composition, Peptide Charge, and Molecularity on theStructure of Antimicrobial Peptide Transmembrane PoresAlmudena Pino Angeles, Themis Lazaridis.Chemistry, City College of New York, New York, NY, USA.The structure of transmembrane pores formed by antimicrobial and cell-penetrating peptides depends on the properties of the membrane and the pep-tide. To examine the effect of lipid composition, we simulated magainin 2(MAG2) antiparallel tetramers in DMPC, DMPG and DMPC/PG 1:1 bilayers.Together with the results from our previous work (1), we observe that theMAG2 pore size initially decreases as the anionic content of the membrane in-creases. However, in pure DMPG the observed pore is similar in size to that inDMPC, but with considerably more unfolded peptides. We also simulated aPGLa parallel tetramer in POPE/PG 3:1 and observed that 1-2 monomers eitheradopt or tend to adopt a surface orientation during the 5 ms of the simulation, inpartial agreement with NMR data (2).We explored the effect of molecularity by simulations of hexamers and octamersof melittin and octamers of MAG2 and comparison with previous tetramer re-sults (1,3). The results show a stable wide pore for the MAG2 octamer, withall the monomers involved in the formation and support of the water channel.On the contrary, in both melittin oligomers the number of peptides in contactwith the pore is reduced during the simulation. Finally, we explored the effectof peptide charge by comparing simulations of hexamers of melittin (chargeþ5) and its gain-of-function variant MELP5 (charge þ2) (4). Whereas the me-littin hexamer broke up into two trimers, one surrounding a pore and one dry, theMELP5 hexamer simulation resulted in a stable wide pore.(1) Pino-Angeles A, Leveritt JM 3rd and Lazaridis T. PloS Comput Biol (2016)12(1):e1004570

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(2) Stranberg E, Zerweck J et al. Biophys J (2013) 104(6):L9-11(3) Leveritt JM 3rd, Pino-Angeles A and Lazaridis T. Biophys J (2015)108(10):2424-6(4) Wiedman G, Fuselier T et al. J Am Chem Soc (2014) 136(12):4724-31

123-PlatSpontaneous Formation of an Ensemble of Structurally DiverseMembraneChannel Architectures from a Single Antimicrobial Peptide MaculatinYukun Wang1, Charles Chen1, Dan Hu2, Jakob Ulmschneider2,Martin Ulmschneider1.1Johns Hopkins University, Baltimore, MD, USA, 2Shanghai JiaotongUniversity, Shanghai, China.Antimicrobial peptides (AMPs) are an ancient, powerful, and ubiquitouscomponent of the innate immune defence in all domains of life and play akey role in controlling the human microbiome. Many AMPs are known toselectively target and form pores in microbial membranes, killing a wide vari-ety of pathogens at low micromolar concentrations. Fundamental questionsremain, however, regarding the molecular mechanisms of membrane targeting,pore formation and function, and the extent to which poration contributestowards antimicrobial activity. Here we report an experimentally guided andvalidated unbiased long-timescale simulation methodology that yields the com-plete mechanism of spontaneous pore assembly in the membrane for the amphi-philic pore-forming AMP maculatin at atomic resolution. Rather than a singlewell defined pore, maculatin was found to form an ensemble of well definedtemporarily functional channels that continuously form and dissociate in themembrane. All channels are formed from highly symmetric low-oligomeric as-semblies of up to 8 membrane-spanning peptides that mimic integral membraneprotein channels in structure. Each channel has a different architecture, func-tional lifetime, and ion conductivity and overall membrane permeabilizationis dominated by higher order oligomers, which form ion channels that alsoconduct water at high rates. The highest order oligomers were also found toefficiently conduct small dyes. Channel architectures as well as their relativeweighting in the ensemble are sensitive to minor mutations as well as variationof lipid tail length. All pores are formed spontaneously by the consecutive addi-tion of individual helices to a transmembrane helix or helix bundle, in contrastto current poration models postulated for AMPs. The diversity of channel archi-tectures formed by a single sequence is remarkable and could explain why nosequence-function relationships in AMP sequences have been discovered todate. Structural ensembles formed by AMP may be the key to preventing bac-terial resistance.

124-PlatAn Exact Model of Daptomycin Binding to Lipid BilayersAntje Pokorny, Tala O. Khatib.Chemistry and Biochemistry, Univ. North Carolina Wilmington,Wilmington, NC, USA.Daptomycin is a cyclic lipopeptide of clinical importance in the treatment ofmulti-drug resistant infections, including those caused by methicillin-resistant S. aureus (MRSA) strains. Similar to other antimicrobial peptides,daptomycin binds with preference to the anionic cytoplasmic membranes typi-cally found in prokaryotes. However, in contrast to most linear, alpha-helicalpeptides, daptomycin binds to lipid bilayers only in the presence of calciumions and its activity is absolutely calcium-dependent. We measured the interac-tion of daptomycin with anionic lipid membranes using kinetic binding exper-iments and equilibrium titrations. The data were analyzed using an exact modeldescribing the interactions of daptomycin with the lipid bilayer that includessolution and membrane-bound states, and the influence of calcium ions ondaptomycin-lipid interactions.

125-PlatThe Metallopeptides Piscidin 1 and Piscidin 3 Employ Membrane andNuclease Activity to Eradicate Planktonic, Biofilm, and Persister CellsMyriam L. Cotten1, M. Daben J. Libardo2, Ali Adem Bahar3, Riqiang Fu4,Vitalii I. Silin5, Dacheng Ren3, Mihaela Mihailescu5, Alfredo Angeles-Boza2.1Applied Science, College of William and Mary, Williamsburg, VA, USA,2Chemistry, University of Connecticut, Storrs, CT, USA, 3Biomedical andChemical Engineering, Syracuse University, Syracuse, NY, USA, 4NationalHigh Magnetic Field Laboratory, Tallahassee, FL, USA, 5Institute forBioscience and Biotechnology Research, University of Maryland, Rockville,MD, USA.Piscidin 1 (P1) and piscidin 3 (P3) are highly potent host-defense peptides(HDPs) active against drug resistant bacteria, and therefore important for thedesign of novel anti-infective therapeutics. Both peptides are cationic andinteract with the anionic lipid membranes of bacteria. They also translocateacross these membranes and colocalize with intracellular DNA. Whileboth peptides have similar three-dimensional structures, P1 is generally more

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biologically active than P3 for reasons that are not understood. Here, wecompare the disruptive effects of P1 and P3 on lipid bilayers and DNA, andinvestigate the role of their amino terminal Cu and Ni (ATCUN) binding motifin their mechanism of action. We rely on a combination of complementarymethods, including CD- and NMR-monitored titrations, structural solid-stateNMR, neutron diffraction with deuterium labeling, surface plasmon resonance,microscopy, biological activity assays, and voltammetry. We find by solid-stateNMR that both peptides use an a-helical structure to interact with bacterial cellmembrane mimics and isolated DNA. Interestingly, while P1 is more mem-brane active than P3, the latter is more disruptive to DNA. Through neutrondiffraction studies, we demonstrate that P1 inserts more deeply in lipid mem-branes and induces a major conformational rearrangement of the lipid head-groups, which leads to bilayer defects and significant water penetration intothe interstices of the membrane. Remarkably, both peptides bound to Cu2þ

act as nucleases that damage isolated DNA within minutes and P3 is signifi-cantly more effective than P1. In the context of bacterial cells, we show thatcopper plays an essential role in planktonic, biofilm, and persister cell eradica-tion. Furthermore, we find that the more membrane-active P1 kills bacteriarapidly while the more DNA-damaging P3 results in slower cell death. Takentogether, these results identify host defense metallopeptides with complemen-tary and multi-pronged mechanisms of action as valuable templates to considerfor the design of novel strategies against drug resistant bacteria.

126-Plat20D Years and no End in Sight: Histidine-Rich Designer Peptideswith pH-Dependent Membrane Topology and with Multifacet BiomedicalPotentialChristopher Aisenbrey1, Philippe Bertani1, David Fenard2, Anne Galy3,Elise Glattard1, Martin Gotthardt4, Antoine Kichler5, Nan Liu4,Arnaud Marquette1, Regine S€uss4, Louic Vermeer1, Dennis Wilkins-Juhl1,Justine Wolf1, Burkhard Bechinger1.1Chemistry, University of Strasbourg/CNRS, Strasbourg, France, 2InnovationTechnologique Lentivirus, Genethon, Evry, France, 3Genethon/InsermUMR951, Evry, France, 4Pharmazeutische Technologie und Biopharmazie,University of Freiburg, Freiburg, Germany, 5Laboratoire ‘‘Vecteurs:Synthese et Applications Therapeutiques’’ (V-SAT), CNRS, Strasbourg,France.The synthetic LAH4 peptides were designed to investigate the interactions thatdetermine the membrane topology of helical peptides (1). Their core consists ofalanines, leucine and four histidines arranged to form an amphipathic helix, aswell as two lysines at each terminus. Through protonation of its histidines (pKsbetween 5.4 and 6.0) the alignment of the helices is transmembrane at neutralpH and in-plane at pH <5.5 (1). The LAH4 peptides exhibit membrane pore-formation and antimicrobial action at both neutral and at acidic pH includingagainst clinical isolates where the low pH configuration is more active (2). TheLAH4 peptides have been found to also exhibit potent DNA and siRNA transfec-tion activities (3). Therefore they can act as a non-viral vector and has indeed beenused for the delivery of quantum dots or protein-based vaccines. Furthermore,transduction by adeno-associated viruses or lentiviruses is enhanced by LAH4(4) or non-peptidic mimetics of this family of peptides (5). Recent and ongoingbiophysical, structural and cell biological investigations will be reported whichaim to understand these activities at atomic resolution (3, 6-8).(1) B. Bechinger, J.Mol.Biol. 263, 768 (1996).(2) A. J. Mason, et al., J. Biol. Chem. 284, 119 (2009).(3) B. Bechinger, et al. J Pept Sci (2017, in prep.).(4) S. Majdoul, Seye, A.K., Kichler, A., Holic, N., Galy, A., Bechinger, B.,Fenard, D., J. Biol. Chem., 291, 2161 (2016)(5) C. Douat, C. et al. Angew. Chem Int. Ed 54, 11133 (2015)(6) C. Aisenbrey, B. Bechinger, Langmuir 30, 10374 (2014).(7) R. Macha�n, P. Jurkiewicz, T. Steinberger, B. Bechinger, M. Hof, Langmuir30, 6171 (2014).(8) A. Farrotti, G. Bocchinfuso, A. Palleschi, N. Rosato, E. S. Salnikov, N.Voievoda, B. Bechinger, L. Stella, BBA 1848, 581 (2015).

Platform: Micro- and Nanotechnology

127-PlatImproving the Temporal Resolution of Nanopore RecordingsSiddharth Shekar1, Chen-Chi Chien2, David Niedzwiecki2, Marija Drndi�c2,Kenneth Shepard1.1Electrical Engineering, Columbia University, New York, NY, USA,2Physics and Astronomy, University of Pennsylvania, Philadelphia,PA, USA.Solid-state nanopores are being pursued for a number of applicationsincluding, most notably, DNA sequencing. One of the challenges that

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nanopores present is the fast rate at which molecules translocate. Significantimprovements in the measurement bandwidth can be obtained throughthe optimization of detection electronics and reduction in nanopore membranecapacitance. We present a low-noise, custom-designed complementary metal-oxide-semiconductor (CMOS) amplifier chip capable of recording transloca-tion dynamics in nanopores at bandwidths up to 10 MHz. We integratestate-of-the-art silicon nitride nanopores with this amplifier to achieve signalto noise ratios (SNRs) of better than 10 at 5 MHz bandwidth in ssDNA trans-location experiments. We observe transient features with durations as short as200 ns in some translocation events, features that would have been hidden atlower recording bandwidths. We also use our platform to record ssDNA trans-location through glass-passivated silicon-nitride nanopores with membranecapacitances of less than 1 pF, further extending the achievable recordingbandwidth. At these speeds, the potential exists to realize free-running DNAsequencing using solid-state nanopores.

128-PlatIdentification of Single Nucleotides in SiN NanoporeHaojie Yang, Zhongwu Li, Yunfei Chen, Wei Si.Jiangsu Key Laboratory for Design and Manufacture of Micro-NanoBiomedical Instruments, Southeast university, Nanjing, China.A nanopore with 1.8 nm in diameter is fabricated on a reduced SiN membraneby focused ion beam and high energy electron beam. Through measuring theblockade ionic current for single nucleotides passing through the solid-statenanopore, it is found that the current blockade can be used to statisticallydetect and identify all four types of nucleotides. The current blockade fittedby Gauss function share the same trend as the volume of four type of nucle-otide (dGTP > dATP > dTTP > dCTP). We find the dwell time distributionsare asymmetric in shape. They are characterized by sharply increasing peaksat shorter times followed by broader decays at longer times. The distributionshapes were fitted to first-passage time distributions obtained from the 1DFokker-Planck equation. The velocity and diffusion constant can be obtainedfrom the fitting function. It is found that larger single nucleotide mass has asmaller diffusion coefficient, the same as the Einstein relation. The larger sin-gle nucleotide volume has a faster velocity. In order to explain the experi-mental phenomena, molecular dynamics (MD) simulations were conductedusing a SiN pore with 1 M KCl. Results show that anions dominate the ionicmigration in the nanopore, namely the concentration and velocity of anions inthe nanopore are much higher than that of cations. The electroosmotic flowwhich generated by anions has the same moving direction as single nucleo-tides. We consider singe nucleotides can be driven by both electroosmoticflow and electric field through the nanopore. Because the four types of nucle-otides have the same surface charge, the single nucleotide with larger volumehas a higher velocity provided by the electroomotic flow.In our experimentresult shows single nucleotide can be statistically identified by the SiN nano-pore, and the MD simulation result shows the single nucleotide transmissionmechanism in the 1.8 nm SiN nanopore.

129-PlatFormation of Synthetic Nanopores with Diameters from 20-50 nm byLaser-Assisted Dielectric BreakdownCuifeng Ying, Jared Houghtaling, Bodo Wilts, Michael Mayer.Adolphe Merkle Institute, Fribourg, Switzerland.Fabrication of nanopores in synthetic substrated by controlled breakdown(CBD)[1] is a robust and well characterized method that allows sub-2 nm nano-pore fabrication, which is a challenge for traditional fabrication methodssuch as transmission electron microscopy (TEM). Due to its stochastic pore for-mation process, however, attempts to widen a nanopore using CBD can result inrandom formation of multiple nanopores instead of increasing the size of asingle pore. Since CBD is a broadly-accessible, tuneable, and cost-effectivemethod, it would be useful to fabricate nanopores with diameters from 20 to50 nm for applications beyond DNA sequencing. This work focuses on fabri-cating such large nanopores by optically localizing the formation and enlarge-ment of defects in the SiN membrane[2]. We show that the breakdown timeof SiNmembranes (i.e. time for dielectric breakdown at constant applied poten-tial difference across the membranes) can be lowered significantly by focusinga laser beam on the membrane. To avoid forming multiple nanopores duringthe enlargement process, low voltage pulses were applied. In this study,differences of parameters between nanopores fabricated by standard CBDand laser-assisted CBD are discussed. The size, number and shape of the result-ing nanopores are assessed by various techniques, including protein transloca-tion experiments.[1] Kwok, H.; Briggs, K.; Tabard-Cossa, V. PLoS One 2014, 9, (3), e92880. [2]Pud, S.; Verschueren, D.; Vukovic, N.; Plesa, C.; Jonsson, M. P.; Dekker, C.Nano letters 2015, 15, (10), 7112-7.

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130-PlatSalt Rejection using Conically Shaped Pores with Patterned SurfaceChargesCrystal Yang1, James Boyd2, Yinghua Qiu2, Zuzanna S. Siwy2.1Dept. of Chemistry, University of California, Irvine, Irvine, CA, USA,2Dept. of Physics and Astronomy, University of California, Irvine, Irvine,CA, USA.In light of global potable water scarcity, whether it be due to increasing agri-cultural demands, higher standards of living, or overdrawing from under-ground sources, the need for efficient desalination techniques is of highimportance. Reverse osmosis membranes offer excellent rejection of saltbut the throughput of the technology is very low due to the sub-1nm effec-tive size of the membrane voids. We propose a different approach to achievedesalination using membranes containing pores with opening diameter offew nm, and walls with high density of surface charges and surface chargepatterns. We found that even 5 nm in diameter nanopores and containing ajunction between a zone with positive surface charges and a zone with nega-tive surface charges could offer rejection of salt up to 80% from 100 mMKCl. Increasing the pore diameter can increase the flux of water even twoorders of magnitude compared to fluxes achievable with reverse osmosismembranes. Experiments were performed with track-etch membranes withdensities of pores up 109 per cm2. Membranes in polyethylene terephthalateand polyimide were tested. Experimental observations were supported by thenumerical modeling performed by solving Poisson-Nernst-Planck andNavier-Stokes equations. Dependence of salt rejection on salt concentration,applied pressure and surface charge pattern is investigated in detail. Discus-sion on the range of electrostatic interactions in aqueous solutions as a func-tion of surface charge density and surface charge arrangement will also beprovided.

131-PlatGenetically Encoded DNA-Protein Hybrid OrigamiFlorian Praetorius, Hendrik Dietz.Technische Universit€at M€unchen, Garching near Munich, Germany.Here we describe an approach to bottom-up fabrication with nanometer-precision that allows integrating the functional diversity of proteins indesigned three-dimensional structural frameworks. We reimagined the suc-cessful DNA origami design principle using a set of custom staple proteinsto fold a double-stranded DNA template into a user-defined shape. Each sta-ple protein recognizes two distinct double-helical DNA sequences and cancarry additional functionalities. The staple proteins we present here arebased on the transcription activator-like (TAL) effector proteins. Due to theirrepetitive structure these proteins offer a unique programmability that en-ables us to construct numerous staple proteins targeting any desired DNAsequence. Our approach is general, meaning that many different objectsmay be created using the same set of rules, and it is modular, because com-ponents can be modified or exchanged individually. We present rules forconstructing megadalton-scale DNA-protein hybrid nanostructures; intro-duce important structural motifs, such as curvature, corners, and vertices;describe principles for creating multi-layer DNA-protein objects withenhanced rigidity; and demonstrate the possibility to combine our DNA-protein hybrid origami with conventional DNA nanotechnology. Since allcomponents can be encoded genetically, our structures should be amenableto biotechnological mass-production. Moreover, since the target objects canself-assemble at room temperature in near-physiological buffer, our hybridorigami may also provide an attractive method to realize positioning andscaffolding tasks in vivo. We expect our method to find application bothin scaffolding protein functionalities and in manipulating the spatial arrange-ment of genomic DNA.

132-PlatDNA Nanoparticles Programmed from the Top Down with VariableDesign MotifsSakul Ratanalert, Remi Veneziano, Mark Bathe.MIT, Cambridge, MA, USA.A hallmark of naturally evolved RNA assemblies, such as the ribosome,ribonuclease-P, and tRNAs, is their ability to fold from a single nucleic acidstrand into complex, functional 3D structures with diverse biological functionsin the cell. A long-standing aim of synthetic structural biology has been to mimicthese properties of self-folding and functionality using a single strand of DNA, asa stepping stone to RNA, because of its fewer secondary structure conformationsand increased preference to hybridize in solution. Deriving the rules for suchself-assembly also allows for the ability to extend the design space to structuresbeyond the set of naturally evolved molecules. Toward this end, we have devel-oped a top-down sequence design approach based on the principle of scaffolded

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DNA origami. First, we present a fully autonomous algorithm to produce asingle-stranded DNA scaffold and complementary staple strands, which foldinto nearly arbitrary target 3D objects, from Platonic solids to non-spherical to-pologies, with near quantitative synthetic yield, demonstrated for a dozen struc-tures experimentally (Veneziano, Ratanalert, et al., Science, 2016). Second, wepresent a powerful approach that eliminates staple strands entirely, offering theability to program a single DNA molecule to fold into these arbitrary 3D shapeson its own, similar to natural RNA assemblies. We examine the folding pathwaysof each of these design modalities using quantitative PCR and present a thermo-dynamic model to optimize sequence design, folding temperature, and yield(Ratanalert et al., in prep, 2016). Together, these algorithms solve a long-standing challenge of synthetic structural biology to program nearly arbitrary3D geometries using synthetic nucleic acids.

133-PlatpH-Responsive Reversible Regulation of Enzyme Activity by DNA-BasedNanostructureSeong Ho Kim1,2, So Yeon Kim1,2.1University of Science and Technology, Seoul, Korea, Republic of, 2KoreaInstitute of Science and technology, Seoul, Korea, Republic of.Here, we present a novel method for reversibly regulating enzyme activity bycaging an enzyme into a pH-responsive DNA-based tetrahedron nanostructure.pH-dependent opening/closing of the DNA cage was verified by measuringfluorescence resonance energy transfer between two vertex corners of thetetrahedron. The position of the covalent enzyme attachment in DNA was care-fully chosen such that the attached enzyme faced inward the DNA cage. Bothproteinase K protection assay and single-molecule based pull-down assayshowed that the encapsulated enzyme were exposed to either proteinase Kor target antibody by pH-dependent opening of DNA cage. Remarkably, wefound that the caging/uncaging process were reversible, implying that enzymeactivity toward relatively larger substrates than DNA cage can be reversiblyregulated. Considering that the DNA cage is widely used as a delivery carrier,our method can be further extended to reversibly regulate cell function bypH-dependent activity control of delivered enzyme.

134-PlatEco-Friendly Processing for Engineering Bio-Safe Quantum Dots andtheir Interaction with Biological SystemsMarta d’Amora1, Marina Rodio1, Alberto Diaspro1,2, Romuald Intartaglia1.1Istituto Italiano di Tecnologia, genova, Italy, 2Department of Physics,University of Genoa, Genova, Italy.Inorganic nanomaterials have gained attention for delivery vehicles, genedetection systems, labeling and therapeutic applications. Many efforts havebeen reported in the synthesis of heavy metal quantum dots (QDs), for long-term, real-time cell labeling applications. Exposure to these QDs in living tis-sue endanger several issues due to their chemical composition, artificial ligandand/or the employed solution routes. In particular, surface coating/stabilizationof nanomaterials by chemical organic molecules, such as citrate have shown totrigger different interaction at cellular level [1]. However, it is still difficult todraw a definite conclusion. Therefore, other alternatives, taking into accountthe nanoparticles fabrication strategy and the minimum toxicity of the carrieritself, are crucial for potential success of nanomaterials in the clinical setting.[2,3] Owing to its biocompatibility and biodegrability, silicon based nanoma-terials are ideal candidates for in vivo applications [4]. Here, we will report asafe engineering approach based on liquid phase pulsed laser ablation tech-nique enabling the generation of photoemissive, highly pure (i.e. free of chem-ical ligand at the surface) silicon quantum dots. Furthermore, we will presentthe physicochemical interactions of these non-toxic nanotools having uniquesurface chemistry with biological systems.(1) Mu et al., Chem Rev., 114, 7740. (2014)(2) Intartaglia et al., Nanoscale, 4, 1271, (2012)(3) Rodio et al., J. Colloid Interface Sci. 465, 242 (2016)(4) Park et al, Nature Materials, 8, 331 (2009)

Symposium: RNA Structures and Dynamics

135-SympCleaving Fast and Slow: Strategies for Self-Assembly of Catalytic RNASarah A. Woodson1, Subrata Panja1, Boyang Hua2, Krishnarjun Sarkar1,Taekjip Ha3.1Dept Biophysics, Johns Hopkins Univ, Baltimore, MD, USA, 2DeptBiophysics & Biochemistry, Johns Hopkins Medical School, Baltimore, MD,USA, 3Dept Biophysics & Biochemistry, HHMI & Johns Hopkins MedicalSchool, Baltimore, MD, USA.Ribozyme RNA motifs are widespread in nature and their adaptation tovaried growth conditions has been sparsely investigated. Ribozymes fold

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into specific three-dimensional structures to promote self-cleavage of specificphosphodiester bonds. Metal cations stabilize the folded RNA, which usuallycorrelates with biochemical activity. We used ensemble and single moleculeFRET to compare the folding dynamics of a 195 nt bacterial group I ribozymeand a 54 nt Twister ribozyme from Oryza sativa. Whereas the group I ribo-zyme remains stably folded in 2 mM MgCl2 that is sufficient for its activity,Twister ribozyme folds and unfolds on the second timescale even in 100 mMMgCl2. Nevertheless, Twister still self-cleaves in 50 mM MgCl2, althoughvisits to the folded state are transient and infrequent. Surprisingly, transitionmetals activated Twister ribozyme even more efficiently than Mg2þ. Ongoingefforts to understand how folding dynamics tune RNA activity will bediscussed.

136-SympAdventures with RNA GraphsTamar Schlick.Courant Inst, New York Univ, HHMI, New York, NY, USA.RNA’s modular, hierarchical and versatile structure makes possible diverse,essential regulatory and catalytic roles in the cell. It also invites systematicmodeling and simulation approaches. Among the diverse computational andtheoretical approaches to model RNA structures, graph theory has been appliedin various contexts to study RNA structure and function. I will present an over-view of recent graph theoretical approaches for predicting and designing RNAtopologies using graphical representations of RNA secondary structure, data-mining tools for junction topology prediction, and hierarchical sampling ofgraphs based on statistical potentials. As evident from the work of many groupsin the mathematical and biological sciences, graph theoretical approaches offera fruitful avenue for designing novel RNA topologies and predicting tertiarystructures from given secondary structures.Of possible interest- H.H. Gan, S. Pasquali and T. Schlick, Nucl. Acids Res. 31:2926 (2003)- N. Kim et al., J. Mol. Biol. 341:1129 (2004)- G. Quarta and K. Sin and T. Schlick, PLoS Comput. Biol. 8: e1002368 (2012).- C. Laing, S. Jung, N. Kim, S. Elmetwaly, M. Zharan, and T. Schlick, PLOSOne 8(8): e71947 (2013).- N. Kim, C. Laing, S. Elmetwaly, S. Jung, J. Curuksu, and T. Schlick, Proc.Natl. Acad. Sci. USA 111: 4079 (2014).- M. Zharan, C. S. Bayrak, S. Elmetwaly, and T. Schlick, Nuc. Acids Res. 43:9474 (2015).- N. Baba, S. Elmetwaly, N. Kim, and T. Schlick, J. Mol. Biol. 428: 811 (2016).- L. Hua, Y. Song, N. Kim, C. Laing, J. T. L. Wang, and T. Schlick, PlOS One11: e0147097 (2016).

137-SympThe Structural and Mechanistic Origins of Catalysis in NucleolyticRibozymesDavid M. Lilley, Timothy J. Wilson, Yijin Liu.Life Sciences, University of Dundee, Dundee, United Kingdom.The nucleolytic ribozymes are a structurally diverse and widespread group ofcatalytic RNA species. They accelerate transesterification reactions around amillion fold, resulting in the site-specific cleavage or ligation of RNA. Thepotential entities that can participate are the nucleobases, 20-hydroxyl groupsand hydrated metal ions. Probable catalytic strategies are the facilitation ofin-line attack, stabilization of the phosphorane transition state, deprotonationof the nucleophile and protonation of the oxyanion leaving group (these lasttwo being general base-acid catalysis).While much of the above was studied in ribozymes such as hammerhead,hairpin and VS, these principles are well illustrated by newer ribozymes. Wehave solved the crystal structure of the twister ribozyme, which adopts a doublepseudoknot fold with a central active site. This well illustrates the four strate-gies summarized above. We have recently solved the structure of a new ribo-zyme, that appears mechanistically very different, with a key role for abound metal ion where an inner-sphere water molecule acts as a general base.

Platform: Membrane Receptors and SignalTransduction I

138-PlatOligomerization of the Epidermal Growth Factor Receptor OrganizesKinase-Active Dimers into Competent Signaling PlatformsSarah R. Needham1, Laura C. Zanetti-Domigues1, Anton Arkhipov2,Venkatesh P. Mysore3, Dimitrios Korovesis1, Selene K. Roberts1,Christopher J. Tynan1, Daniel J. Rolfe1, Michael Hirsch1,Alireza Lajevardipour4, Andrew H.A Clayton4, Peter J. Parker5,6,Yibing Shan3, David E. Shaw3,7, Marisa L. Martin-Fernandez1.

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1Central Laser Facility, Science and Technology Facilities Council,Oxfordshire, United Kingdom, 2Allen Institute for Brain Science, Seattle,WA, USA, 3D. E. Shaw Research, New York, NY, USA, 4Faculty of Science,Engineering and Technology, Swinburne University of Technology, Victoria,Australia, 5Division of Cancer Studies, King’s College London, London,United Kingdom, 6Francis Crick Institute, London, United Kingdom,7Department of Biochemistry and Molecular Biophysics, ColumbiaUniversity, New York, NY, USA.Epidermal growth factor receptor (EGFR, or HER1/ErbB1) is a cell-surfacereceptor tyrosine kinase that plays a fundamental role in regulation ofcellular metabolism, growth and differentiation. Dysregulation of EGFR orother members of the human epidermal growth factor receptor (HER)family (HER2/ErbB2/Neu, HER3/ErbB3, and HER4/ErbB4) can lead to thedevelopment of various cancers. EGFR signaling is activated by ligand-induced receptor dimerization. Ligand binding also induces EGFR oligomeri-zation, but the structures and functions of the oligomers are poorly understood.Although the majority of EGFR research has largely focused on adimerization-dependent activation mechanism, recent analyses suggest thatoligomerization also plays a crucial role in EGFR signaling. Here, we use flu-orophore localization imaging with photobleaching (FLImP) to probe thestructure of EGFR oligomers. We find that at physiological EGF concentra-tions, EGFR assembles into oligomers, as indicated by pairwise distances ofreceptor-bound fluorophore-conjugated EGF ligands. The pairwise liganddistances correspond well with predictions from our structural model of theoligomers constructed from molecular dynamics simulations. The model sug-gests that oligomerization is mediated extracellularly by unoccupied ligand-binding sites and that oligomerization organizes kinase-active dimers inways optimal for auto-phosphorylation in trans between neighboring dimers.We argue that ligand-induced oligomerization is essential to the regulationof EGFR signaling.

139-PlatKinetics of G Protein-Coupled Receptor Dimerization from Markov StateModel Analysis of Coarse-Grained SimulationsDiego Prada-Gracia, Kristen Marino, Davide Provasi, Marta Filizola.Department of Pharmacological Sciences, Icahn School of Medicine atMount Sinai, New York, NY, USA.Experimental and computational studies published over the past decade havemostly focused on the structural and thermodynamic properties of putativeG Protein-Coupled Receptor(GPCR) dimers. Although equally crucial foraddressing the role of dimerization in GPCR function, a thorough descriptionof the timescales required to form different dimeric interfaces has yet to be pro-vided. In this study, we approach this question by applyingMarkov State Modelanalysis to coarse grained simulations of a prototypic GPCR, the mu-opioidreceptor (MOR), carried out in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-choline/cholesterol membrane model using an adaptive sampling strategy.We identify several dimerization interfaces of MOR grouped in four macro-states with significantly different dimerization rates, and characterize the roleof lipid dynamics, protein-lipid and specific protein-protein interactions inmodulating the kinetic properties of receptor dimerization. Furthermore, wediscuss the interplay between receptor activation and dimer formation bycomparing the results obtained for the activated and inactive receptor con-formations of MOR, and observe substantial differences in the dimerizationkinetics for the two conformations.

140-PlatDNA Nanotechnology for Understanding Ephrin Receptor ClusteringAlessandro Bosco, Erik Benson, Bjorn Hogberg, Ana Teixeira.Medical Biochemistry and Biophysics (MBB), Karolinska Institutet,Stockholm, Sweden.Membrane protein biophysical context has been proved to be fundamental inthe modulation of the cell-cell contact signalling generated by ligand receptorrecognition, however tools and techniques that are able to control membranemicroenvironment at the nanoscale and then interpret the molecular mecha-nisms are still scarce. DNA nanotechnology offers a powerful tool to target re-ceptor macromolecular assembly at the nanoscale.In recent years it has been shown that in breast cancer cell lines, the spatial or-ganization of ephrin controls the formation of cluster of Eph receptors and thisphenomenon has been linked to tumor aggressiveness. In our lab, we recentlydesigned and charachterized rod-like DNA structures decorated with ephrinswith nanometric precision that can be used to target Eph receptors. In partic-ular, we showed that the nanoscale spacing of pre-dimerized Fc-fusedephrin-A5 directs the levels of EphA2 receptor phosphorilation and activationin human breast cancer cells and modulates their invasive properties. More-over, we are currently developing substrates that recreate the intramembrane

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signaling geometry at cell-cell contact. These are designed and produced byemploying sheet-like ligand decorated DNA nanostructures anchored on artifi-cial supported lipid bilayers. Such structures are rendered from polyhedral flatsheet meshes that allow high modularity in design of shapes and protein spatialdistributions. The DNA flat sheets are functionalized with monomeric ephrin-A5 conjugates with spatial distributions that allow or not dimerization of thereceptor. These substrates will allow an unparalleled control of the dimerizationof the receptor and will elucidate the role of cytoskeleton during the formationof the signaling assembly.Together with biochemical techniques, high-resolution microscopies, andcomputational tools, this approach will give insights into molecular mecha-nisms of spatial organization of ligands and receptors during clustering andtheir effects on receptor-mediated signaling.

141-PlatSAM Domain Inhibits Oligomerization and Auto-Activationof EphA2 KinaseXiaojun Shi1, Deanna M. Bowman1, Vera M. Hapiak2, Ryan W. Lingerak3,Ji Zheng2, Matthias Buck4, Bingcheng Wang2, Adam W. Smith1.1Department of Chemistry, University of Akron, Akron, OH, USA,2Rammelkamp Center for Research, MetroHealth Medical Center,Cleveland, OH, USA, 3Department of Biology, University of Akron, Akron,OH, USA, 4Department of Physiology & Biophysics, Case Western ReserveUniversity, Clevenland, OH, USA.Eph receptors are the largest family of receptor tyrosine kinases (RTKs) andbind to membrane-tethered ligands called ephrins. Eph-Ephrin interactionsare involved in various biological processes, such as neural development, tis-sue patterning and vascular growth. Several structural studies have shownthat the extracellular domain (ECD) of EphA2 binds ephrin and forms anoligomer that promotes kinase activation. However, the role of the intracel-lular domain (ICD) in this process remains largely unexplored. Here, wereport on the unique role of the sterile a motif (SAM) domain in regulatingEphA2 kinase activation. First, western blots and cell function assays indi-cated that deletion of the SAM domain leads to constitutive activation ofthe kinase. We then carried out fluorescence correlation spectroscopy(FCS) measurements to investigate the dynamics and lateral organizationof EphA2 receptors in live cancer cell membranes. The FCS results showedthat deletion of the SAM domain led to receptor oligomerization. This sug-gested that constitutive activation of the SAM-deletion construct observed inthe cell functional assays were a result of receptor oligomerization. Ligandactivation with ephrinA1 (EA1) induces oligomerization of the receptors, butdeletion of the SAM domain led to larger oligomers than those with the fulllength receptor. FCS measurements were performed on cancer cells treatedwith two kinds of soluble EA1 ligand, dimeric EA1-Fc and monomeric EA1(mEA1), were used. While inconsistencies exist in the literature, our FCS re-sults demonstrated that both dimeric and monomeric EA1 activation led tooligomerization and activation of EphA2 through induction of the distinctclusters. Together, our work clarifies the unique function of SAM domainin EphA2 signaling.

142-PlatSingle-Molecule Analysis of the Supramolecular Organization of the M2Muscarinic Receptor and the Gai1 ProteinClaudiu Gradinaru1, Dennis D. Fernandes1, Rabindra Shivnaraine2,James Wells3.1Physics, University of Toronto, Mississauga, ON, Canada, 2Molecularand Cellular Physiology, Stanford University, Palo Alto, CA, USA,3Pharmaceutical Sciences, University of Toronto, Toronto, ON, Canada.G Protein-coupled receptors constitute the largest family of transmembranesignaling proteins and the largest pool of drug targets, yet their mechanismof action remains obscure. That uncertainty relates to unresolved questionsregarding the supramolecular nature of the signaling complex formed by re-ceptor and G protein. We therefore have characterized the oligomeric statusof eGFP-tagged M2 muscarinic receptor (M2R) and Gi1 by single-particle pho-tobleaching of immobilized complexes (Shivnaraine et al, J. Am. Chem. Soc.,2016, 138, pp 11583-11598). The method was calibrated with multiplexedcontrols comprising 1-4 copies of fused eGFP proteins. The photobleachingpatterns of eGFP-M2R were indicative of a tetramer and unaffected by musca-rinic ligands; those of eGFP-Gi1 were indicative of a hexamer and unaffectedby GTPg S. A complex of M2R and Gi1 was tetrameric in both, and activationby a full agonist plus GTPg S reduced the oligomeric size of Gi1 withoutaffecting that of the receptor. A similar reduction was observed upon activa-tion of eGFP-Gai1 by the receptor-mimic mastoparan plus GTPgS; a consti-tutively active eGFP-Gai1 mutant was predominantly dimeric. The oligomericnature of Gi1 in live CHO cells was demonstrated by means of Forster

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resonance energy transfer and dual-color fluorescence correlation spectros-copy in studies with eGFP- and mCherry-labeled Gai1; stochastic FRETwas ruled out by means of non-interacting pairs. Our results suggest thatthe complex between M2R and holo-Gi1 is an octamer comprising four copiesof each, and that activation is accompanied by a decrease in the oligomericsize of Gi1. The structural feasibility of such a complex was demonstratedin molecular dynamics simulations.

143-PlatLive Cell Super-Resolution Microscopy Measures Membrane-DrivenSorting of B Cell Receptor Signaling PartnersSarah A. Shelby, Sarah L. Veatch, Matthew B. Stone.University of Michigan, Ann Arbor, MI, USA.B cells are acutely sensitive to stimulation through antigen-induced clus-tering of the B cell receptor (BCR). Transduction of the activation signal,which is key for the adaptive immune response, involves rearrangementof membrane-resident signaling molecules relative to clustered BCR. Thedetails and dynamics of this re-organization have been difficult to directlyobserve due to the small dimensions of signaling complexes and the poten-tially weak or transient interactions that drive their formation. To overcomethese obstacles, we have utilized live-cell super-resolution localization mi-croscopy to simultaneously image BCR and other membrane species duringantigen-induced cell activation. The sensitivity of this quantitative tech-nique has allowed us to measure the effects of subtle forces, such as thoseoriginating from lipid compositional heterogeneity, that influence the inter-actions of BCR with its signaling partners. We found that minimalmembrane-anchored probes that partition into ordered domains of modelmembranes co-localize with BCR clusters, while anchors that partitioninto disordered domains are excluded from clusters, suggesting that BCRclusters can nucleate local enrichment of a specific lipid composition. Weare exploring the effects of membrane-driven sorting on recruitment ofBCR signaling proteins to receptor clusters and how it contributes to theirspecific role in signal transduction. In particular, we have found that thepalmitoylated transmembrane adapter proteins LAT2, LIME, and PAG,which serve distinct adapter functions in BCR signaling, are diferentiallyrecruited to BCR clusters. This differential recruitment is due to a combi-nation of interactions between the transmembrane domains of these proteinswith the membrane and to specific protein-protein interactions. Our resultssuggest that the membrane influences local sorting of downstream signalingproteins around BCR clusters and provide insight on the forces drivingregulation of BCR signaling.

144-PlatMechanism of CD36 Signal Transduction by F-actin and LipidNanodomainsSwai Mon Khaing, Nicolas Touret.Biochemistry, University of Alberta, Edmonton, AB, Canada.CD36, a multi-ligand plasma membrane receptor, has been implicated in im-munity, metabolism and angiogenesis. We have recently demonstrated thatCD36 nanoclustering at the plasma membrane is key to the initiation ofCD36 signaling. In endothelial cells (ECs), the binding of thrombospondin-1(TSP-1, an endogenous extracellular matrix anti-angiogenic factor) to CD36nanoclusters activates an associated Src family kinase, Fyn, leading to ECsapoptosis, hence, inhibiting angiogenesis. We are interested in elucidatingthe mechanisms of CD36-Fyn enrichment and the role of lipid nanodomainsand actin cytoskeleton during TSP-1 induced signaling in ECs. We hypothesizethat lipid nanodomains play a role in bringing together CD36-Fyn to F-actinregions through adaptor molecules which forms a signaling platform. Usingmicroscopy methods on HeLa cells co-transfected with Fyn and various fluo-rescent lipid biosensors and stained for F-actin (Phalloidin-AF647), we deter-mined that Fyn is enriched on F-actin area at sites of phosphatidylinositol4,5-bisphosphate enrichment (PIP2). During TSP-1 stimulation on HumanMicrovascular Endothelial Cells (HMEC), the CD36-Fyn-F-actin enrichmentshift to domains containing PI(3,4,5)P3, suggesting a role for the phosphoino-sitide 3-kinase in signaling. The role of this kinase is further investigated usinginhibitor targeting PI3-Kinase subunits for signal transduction. Additionally,we will be using rapamycin dimerization system to understand the role ofthese lipid nanodomains in CD36-Fyn signaling. Furthermore, to characterizethe adaptor molecules involved in connecting F-actin to lipid nanodomainsand/or CD36 nanoclusters, we are conducting APEX2 proximity labellingand fractionation approaches followed by mass spectrometry (MS) analysis.The MS screen will further narrow down proteins that are biotinylated, adjacentto CD36 and enriched in F-actin. Altogether, our investigation will provide in-sights into understanding the activity of plasma membrane receptor nanoclus-tering and signaling.

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145-PlatPlasma Membrane Diffusion Modes of FcεRI Receptor for ImmunoglobinE Measured with Imaging Fluorescence Correlation SpectroscopyNirmalya Bag, David Holowka, Barbara Baird.Department of Chemistry and Chemical Biology, Cornell University, Ithaca,NY, USA.Spatio-temporal organization of the plasma membrane plays a significant rolein cell signaling. A prevailing view is that the plasma membrane spatially seg-regates into ordered lipid (Lo-like) nanodomains co-existing with disorderedlipid (Ld-like) regions. Our laboratory, using super-resolution fluorescence im-aging, previously showed that immunoglobin E bound to its receptor, FcεRI,(IgE-FcεRI complex) undergoes time-dependent redistribution on the mem-brane surface after multivalent ligand stimulation, upon which the receptor isphosphorylated by membrane-anchored Lyn kinase. A large body of evidenceshows that this interaction of IgE-FcεRI and Lyn occurs in the ordered regionsof the plasma membrane enriched in cholesterol. We are examining the natureand dynamics of plasma membrane organization as experienced by IgE-FcεRIin resting state and after antigen stimulation. We employ imaging total internalreflection fluorescence correlation spectroscopy (ITIR-FCS), which mapslateral diffusion with pixel resolution, to investigate the diffusion distributionof IgE-FcεRI complexes on the ventral surface of live RBL cells in nativeand experimentally modulated (e.g., cholesterol depleted) conditions. Wealso conduct spot variation FCS (svFCS), which analyzes space-dependenceof diffusion coefficients from the same set of ITIR-FCS data, to reveal the ex-istence of nanoscopic obstacles (such as nanodomains) that affect the diffusionof IgE-FcεRI complexes. These observations are compared with the diffusionbehavior of protein markers that are known to prefer ordered or disordered re-gions of plasma membrane. These results, in conjunction with super-resolutionimaging, which determines the size and density of these complexes, will pro-vide a new level of insight into plasma membrane organization and its dynamicremodeling upon ligand-receptor stimulation.

Platform: Cell Mechanics, Mechanosensing,and Motility I

146-PlatIntegrin Catch Bond Kinetics Mediate Mechanosensing during CellSpreadingTamara C. Bidone1, Patrick W. Oakes2, Yvonne Beckham3,Margaret L. Gardel3, Gregory A. Voth1.1Chemistry, University of Chicago, Chicago, IL, USA, 2RochesterUniversity, Rochester, NY, USA, 3University of Chicago, Chicago, IL, USA.Cell spreading and polarization are morphogenetic responses to extracellularmatrix adhesion. Cultured fibroblasts polarize when plated on rigid, butnot compliant substrates. In this study, we demonstrate that fibroblasts haveincreased spread area on compliant substrates when integrins are directly acti-vated via manganese (Mn2þ), even upon inhibition of myosin motor activity.Integrin activation is primarily regulated by changes in tertiary and quaternarystructure and Mn2þ treatments enhance the lifetime of integrin/ligand bonds.The decrease of integrin/ligand unbinding rate under tension suggests amechanosensing role of activated integrins in regulating adhesion formation.Using a combination of atomistic molecular dynamics and coarse grainBrownian dynamics simulations, we identify integrin intermediates along theactivation pathway and show that modulation of integrin catch bond kineticsupon Mn2þ-activation promotes integrin binding on compliant substrates.Combining experiments with simulations, our results support the idea thatdifferent regimes of ligand binding and cell spreading result from variationsin integrin catch bond kinetics. This suggests an alternative mechanosensingpathway based on integrin activity and independent of myosin activity.

147-PlatMolecular Tension Sensors Reveal a Minimally Tensioned Integrin Statein Living CellsSteven J. Tan, Chang C. Alice, Armen H. Mekhdjian, Alexander R. Dunn.Stanford University, Stanford, CA, USA.Integrins mediate cell adhesion to the extracellular matrix and enable the con-struction of complex, multicellular organisms. Despite this biological pro-minence, fundamental aspects of integrin-based adhesion remain poorlyunderstood. Notably, the magnitude of the mechanical load experienced by in-dividual integrins within living cells is unclear, due principally to limitationsinherent to existing techniques. We used Forster resonance energy transfer(FRET)-based molecular tension sensors (MTSs) to measure the distributionof forces exerted by individual integrin heterodimers in living cells. Takingadvantage of the sensors’ modular nature, we engineered MTS variants thatdisplay a minimal RGD sequence derived from fibronectin (MTSRGD) or the

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9th and 10th type III domains of fibronectin (MTSFN), and are sensitive to low(0-7 pN) or intermediate (8-11 pN) loads. Using these sensors, we found that alarge fraction of integrins in living cells exert forces <3 pN, while a minoritysubpopulation experiencing substantially higher loads was enriched in largeadhesion complexes. Treatment with the filamentous (F)-actin disruptor cyto-chalasin D revealed that only the high-load integrin population required anintact actin cytoskeleton, and that the low-load (<3 pN) population was suffi-cient to mediate cell adhesion on short, ~15 minute timescales. Integrin engage-ment with the fibronectin synergy site, a secondary binding site specifically fora5b1 integrin, led to increased recruitment of a5b1 integrin to adhesions, but notto an increase in overall cellular traction generation. Consistent with previousreports, the presence of the synergy site did, however, increase the resistance ofcells to detachment via externally applied load. Based on these data, we suggestthat a large pool of engaged, but minimally tensioned integrins may provide asynergy site-dependent adhesive reserve that imparts cells and tissues with me-chanical integrity in the presence of widely varying mechanical loads. Inongoing work, we take advantage of the unique capabilities of MTSs to deter-mine how subpopulations of load-bearing integrins are altered in response toexternal mechanical perturbations, and how (and whether) distinct integrin sub-types bear differing levels of mechanical load.

148-PlatNascent Adhesoins that Form on all Substrates by Recruiting UnligandedIntegrins and are Important for MechanotransductionRishita Changede1, Haogang Cai2, Michael P. Sheetz1.1National University of Singapore, Mechanobiology Institute, Singapore,Singapore, 2Engineering, Columbia University, New York, NY, USA.Integrin adhesions assemble and mature in response to ligand binding and me-chanical factors, but the molecular-level organization is not known. We reportthat ~100-nm clusters of ~50 b3-activated integrins form very early adhesionsunder a wide variety of conditions on RGD surfaces. These adhesions formsimilarly on fluid and rigid substrates, but most adhesions are transient on rigidsubstrates. Without talin or actin polymerization, few early adhesions form, butexpression of either the talin head or rod domain in talin-depleted cells restoresearly adhesion formation. Mutation of the integrin binding site in the talin roddecreases cluster size. We suggest that the integrin clusters constitute universalearly adhesions and that they are the modular units of cell matrix adhesions.They require the association of activated integrins with cytoplasmic proteins,in particular talin and actin, and cytoskeletal contraction on them causes adhe-sion maturation for cell motility and growth. Using gold nano-patterning weobserve that these clusters grow using unliganded but activated integrins.This indicates a mechanism to assemble the clusters rapidly around an activatedand liganded integrin. Integrins are not enzymes hence they would need the aidof other enzymes for mechanotransduction. Nascent adhesions have distinctfunctions on compliant and rigid substrates wherein, the epidermal growth fac-tor receptor is recruited to these clusters only on the rigid substrates, in absenceof EGF. This receptor acts as a mechanoenzyme that is required for mechano-transduction within fibroblasts. Taken together our studies show that nascentadhesions form on substrates of vastly varying rigidities. These modular adhe-sions are formed by a cohort of unliganded integrins recruited to adhesions sitesby cytoplasmic factors and they are central to bring about different mechano-transduction on different substrates.

149-PlatHigh-Resolution Integrin Molecular Tension Dynamics during PlateletAdhesion and ActivationXuefeng Wang1, Yongliang Wang1, Dana N. LeVine2.1Physics and Astronomy, Iowa State University, Ames, IA, USA,2Department of Veterinary Clinical Sciences, Iowa State University, Ames,IA, USA.Platelets are small disc-shaped cell fragments circulating in the bloodstream.Upon injury, platelets adhere and aggregate on injured subendothelium toform blood clots to stop bleeding. Irregular platelet adhesion and aggregationmay cause acute coronary syndrome or other cardiovascular diseases. Membraneprotein integrins mediate platelet adhesion and transmit tensions to activateplatelets, therefore playing important roles in platelet functions. Nevertheless,cellular force study in platelets is very limited and integrin molecular tensionswere never calibrated in platelets, presumably due to the 2~3 mm size of plateletswhich is at the resolution limit of conventional cell traction force microscopy.Here we applied a novel molecular tension sensor and modulator named TGT(tension gauge tether) to study integrin tensions in platelets. TGT is a rupturablemolecular linker with a programmable tension tolerance (Ttol). As a tensionmodulator, TGT globally restricts integrin tensions under the designed level ofTtol, enabling the study of integrin-tension dependency for a certain cellular func-tion. As a tension sensor, TGT maps integrin tensions by fluorescence with a

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spatial resolution of 0.2 mm, ten times higher than conventional methods. UsingTGT, we determined that platelet adhesion and spreading requires integrin ten-sion lower than 12 pN, a force much less than the ~40 pN integrin tensionrequired by regular mammalian cell adhesion. This surprising result suggeststhat platelets have extraordinary adhesion and spreading ability which mightbe beneficial for the rapid response of platelets to injuries. We also monitoredcalcium oscillation in platelets to confirm that platelets are activated at thislow integrin tension level. Next, using TGT as a tension sensor, we providedthe high-resolution (0.2 mm) integrin tension map for platelet adhesion for thefirst time. Real-time tension mapping shows that integrin tensions at a high level(>54 pN) are initially concentrated in one or two micron-sized punctate regionsupon platelet adhesion. After full platelet spreading, these integrin tensionsspread out to the entire platelet-substrate interface and the tension level decreasesinto the range of 12~54 pN. These high integrin tensions are not required forplatelet adhesion and activation, but may be required for platelet contraction,as these tensions were abolished by myosin II inhibition without compromisingplatelet adhesion and activation. Overall, our research has initiated the biome-chanical study of platelets at the molecular tension level, and revealed a richdynamics of integrin tensions in platelets.

150-PlatReceptor Nucleation and Clustering in Cellular Adhesion and MechanicalSignal TransductionKabir H. Biswas1, Kevin L. Hartman1, Ronen Zaidel-Bar1,2,Jay T. Groves1,3.1Mechanobiology Institute, National University of Singapore, Singapore,Singapore, 2Department of Biomedical Engineering, National University ofSingapore, Singapore, Singapore, 3Department of Chemistry, University ofCalifornia, Berkeley, Berkeley, CA, USA.E-cadherin-based cell-cell adhesions are key to development and maintenance ofthe epithelial tissue, and a loss of these adhesions may contribute to cancer devel-opment. These are mechanosensitive structures in that they are strengthened un-der tension. Mechanotransduction in these adhesions has been postulated to bemediated, in part, by a force-dependent conformational activation of a-catenin,which allows it to interact with vinculin, in addition to F-actin, resulting instrengthening of junctions. Here, using E-cadherin adhesions reconstituted onsynthetic, nanopatterned membranes, we show that activation of a-catenin isdependent on E-cadherin clustering, and is sustained in the absence of mechan-ical force or association with F-actin or vinculin. Adhesions are formed byfilopodia-mediated nucleation and micron-scale assembly of E-cadherin clusters,which could be distinguished as either peripheral or central depending on theirrelative location at the cell-bilayer adhesion. While F-actin, vinculin and phos-phorylated myosin light chain associate only with the peripheral assemblies, acti-vated a-catenin is present in both peripheral and central assemblies, and persistedin the central assemblies in the absence of actomyosin tension. Impedingfilopodia-mediated nucleation and micron-scale assembly of E-cadherin adhe-sion complexes, by confining bilayer bound E-cadherin extracellular domainmovement on nanopatterned substrates, reduced levels of activated a-catenin.Taken together, although the initial activation of a-catenin requires micron-scale clustering that may allow development of mechanical forces, sustainedforce is not required for maintaining a-catenin in the active state.

151-PlatTau Like Proteins Reduce Torque Generation in Microtubule BundlesMichael Krieg1, Jan Stuehmer2, Juan G. Cueva1, Richard Fetter1,Kerri Spilker1, Daniel Cremers2, Kang Shen1, Alex R. Dunn3,Miriam B. Goodman1.1Stanford University, Stanford, CA, USA, 2TU Munich, Munich, Germany,3Stanford, Stanford, CA, USA.All animals and plants, even protozoa, have evolved specialized molecular sen-sors that convert mechanical stress into behavioral responses. The touch recep-tor neurons (TRNs) in Caenorhabditis elegans respond to gentle body touch andare especially arguably better characterized on a physiological and ultrastruc-tural level than somatosensory neurons in other animals. C. elegans is a uniquemodel organism in which to study the mechanics of neurons due to their simpleshapes, the known wiring diagram, transparent body, and a rich repertoire ofsimple behaviors. As in other animals, neuron morphology is critical for func-tion in C. elegans. We have previously shown that a functional, pre-stressedspectrin network is critical for mechanosensation and neuron stability underbody-evoked forces (Krieg, Nat Cell Bio, 2014). How the constituent mole-cules of these different neurons establish a functional organization and hownanometer sized molecules can determine cell shape in the millimeter scaleand enable axons to resist external forces is still not understood. We addressedthis question using light, electron and STED microscopy and found that TRNsdefects in the organization of the axonal spectrin lattice and microtubule

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bundles undergo deformations highly similar to a twisted rod under compres-sion. Our data suggests that tau-like proteins minimize microtubule lattice in-teractions and the prevent torque generation that leads to extreme neurondeformations. These experimental results, together with mechanical modelingof the neuron, suggest that spectrin tension and microtubule bundle mechanicsare crucial for stabilizing chiral cytoskeletal networks and produce a special-ized cell shape that we propose is critical for neuronal function.

152-PlatTorque Generation in the Bacterial Flagellar MotorJasmine A. Nirody1, Richard M. Berry2, George Oster1.1University of California, Berkeley, Berkeley, CA, USA, 2University ofOxford, Oxford, United Kingdom.The bacterial flagellar motor (BFM) drives swimming in a wide variety ofbacterial species. Its fundamental role in a variety of biological processes,including chemotaxis and biofilm formation, has made understanding its dy-namics an important question in biophysics. We put forward a mechanically-specific model for motor rotation, pinpointing critical residues and structuresfor motor function. We implicate a steric interaction between the rotor andthe torque-generating complexes (stators). Two surprising predictions of ourmodel are: (1) the duty ratio of the motor is not close to unity as previouslybelieved; and (2) motor rotation is loosely coupled to ion flux. We show thatthese predictions, while contrary to previous reports, are consistent with currentexperimental evidence. We also put forward several further experiments andmeasurements designed to directly test the validity of this model and itsimplications.

153-PlatFission Yeast Contractile Ring Tension Increases ~2-FoldThroughout Constriction and Regulates Septum Closurebut does not Set the Constriction RateSathish Thiyagarajan1, Harvey Chin2, Erdem Karatekin3,Thomas D. Pollard4, Ben O’Shaughnessy2.1Physics, Columbia University, New York, NY, USA, 2ChemicalEngineering, Columbia University, New York, NY, USA, 3Cellular andMolecular Physiology, Yale University, New Haven, CT, USA, 4Molecular,Cellular, and Developmental Biology, Yale University, New Haven, CT,USA.What is the role of the cytokinetic contractile ring? The tensile actomyosin ringis central to cytokinesis, and widely thought to drive cell cleavage as it con-stricts. Here we directly addressed this question by measurements of cytoki-netic ring tension and mathematical modeling. In the model organism fissionyeast, as in other fungi, ring constriction is accompanied by septation, the in-ward growth of cell wall in the wake of the constricting ring that seals daughtercells in new cell wall.We measured ring tensions in live fission yeast protoplasts using a novelmethod based on measuring membrane tension and the furrow geometry. Asconstricted progressed, ring tension increased from ~ 400 pN to ~ 800 pN.To our knowledge these are the first measurements of the evolving ring tensionthroughout constriction.We used these tension values in a mathematical model of septum growth, medi-ated by beta-glucan synthases (Bgs) at the septum edge, hypothesized mecha-nosensitive (Thiyagarajan et al., 2015). The stochastic septum growth producedfaceting, defects and edge roughness. In simulations, ring tension modulatedBgs growth rates in a curvature-dependent fashion, suppressing defects androughness so septum edges were nearly circular. Simulated edges had lowroughnesses (~5%) and a roughness exponent ~0.5, consistent with septumedges we measured in live cells. Our model revealed a mechanosensitivity~15% per pN per Bgs complex.Thus, ring tension regulates septum growth to ensure the septum closes as ashrinking circle (not a slit) and daughter cells are properly sealed by newcell wall. However, the model showed constriction rates are set by the septumgrowth machinery, while ring tension had little effect on the mean rate, explain-ing why experimentally the rate is constant in time.

Platform: Protein Structure and Conformation I

154-PlatThe HIV-1 Pre-Integration Complexes: Structures, Functionsand DynamicsNicolas Levy, Karine Pradeau-aubreton, Sylvia Eiler, Julien Batisse,Oyindamola Oladosu, Benoit Maillot, Marc Ruff.IGBMC, Illkirch, France.After retroviral infection of a target cell, during the early phase of replication,the HIV-1 genomic viral RNA is reverse transcribed by the viral reverse tran-scriptase to generate the double-stranded viral DNA that interact with viral and

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cellular proteins to form the pre-integration complex (PIC). Viral integrase (IN)is a key component of the PIC and is involved in several steps of replicationnotably in reverse transcription, nuclear import, chromatin targeting and inte-gration. Viral components such as IN cannot perform these functions on theirown and need to recruit host cell proteins to efficiently carry out the differentprocesses. IN is a flexible protein, property allowing its interaction with multi-ple partners and enabling its multiple functions in viral replication. The molec-ular mechanisms and dynamics of these processes remain largely unknown.Purification of proteins that participate in these large transient complexes isimpeded by low amounts, heterogeneity, instability and poor solubility. Tocircumvent these difficulties we develop methodologies that enable the produc-tion of stable complexes for structural and functional studies [1] as well as sys-tem for the production of multi-protein complexes from mammalian cellsenabling assembly of entire complexes within cells. Using these strategieswe reconstruct in vitro stable and soluble complexes around IN. We usecryo-EM combined with X-ray crystallography to solve structures of the IN/LEDGF/DNA [2] and IN/LEDGF/INI1/DNA [3] complexes. Other IN com-plexes involved in the PIC nuclear translocation and integration as well asIN post-translational modifications (phosphorylation and acetylation) havebeen characterized and are under study.[1] Levy et al. (2016) Nature comm. 7: 10932[2] Michel et al. (2009) EMBO J., 28, 980-991[3] Maillot et al. (2013) PLoS ONE 8(4): e60734

155-PlatMechanisms of Sequence Dependent Translational StallingLars V. Bock1, Paul PH Huter2, Stefan Arenz2, Michael Graf2,Helmut Grubmueller3, Daniel Wilson2, Andrea C. Vaiana3.1Theoretical and Computational Biophysics, Lars Bock, Gottingen,Germany, 2Gene Center, Munich, Germany, 3Theoretical and ComputationalBiophysics, Max Planck Institute for Biophysical Chemistry, Gottingen,Germany.Ribosomal stalling during protein synthesis in bacteria occurs in different waysand under different conditions. Stalling of specific peptide sequences can be apre-programmed means of detecting the presence of potentially lethal antibi-otics and constitute the initial step of a complex resistance pathway. Anexample of this is the stalling of ErmBL peptide synthesis in the presence ofthe antibiotic erythromycin. In other cases, stalling seems to be an effect of un-usually slow, sequence dependent, rates of amino acid incorporation. This is thecase for translation of proteins containing poly-proline stretches. Poly-prolinesequences are known to stall ribosomes, normal translation rates are achievedonly by recruiting a special elongation factor (EF-P in bacteria). Here, weinvestigate the stalling mechanisms in the two scenarios described above byexplicit-solvent, all-atom molecular dynamics simulations of the ribosome.The simulations are started from high-resolution cryo-EM structures and per-formed under stalling and non-stalling conditions. We find networks of allo-steric interactions between the nascent peptide chain and the ribosome thatdifferently affect the positioning and the dynamics of the peptidyl tRNA rela-tive to the A-site tRNA in such a way as to hinder peptide bond formation de-pending on the presence of the antibiotic (in the first scenario) or the absence ofthe elongation factor (second scenario). The simulation results not only explainthe stalling mechanism, but can also predict the effect of mutations on stalling.In the case of erythromycin induced stalling, these predictions have been exper-imentally confirmed by a toe-printing assay. Our results illustrate the fine de-tails of how the efficiency of peptide bond formation can be modulated byexternal factors in a way that depends on the specific sequence being translated.

156-PlatQuantitative Analysis and Modeling of Translation using RibosomeProfiling Data: How Biophysical Properties of the Ribosome Exit Tunneland the Nascent Polypeptide Modulate the Elongation RateKhanh Dao Duc1, Zain H. Saleem2, Yun S. Song1.1Mathematics & Biology, University of Pennsylvania, Philadelphia, PA,USA, 2Physics, University of Pennsylvania, Philadelphia, PA, USA.Ribosome profiling provides a detailed view into the complex dynamics oftranslation. Although the precise relation between the observed ribosome foot-print densities and the actual translation elongation rates remains elusive, thedata clearly suggest that elongation speed is quite heterogeneous along the tran-script. Previous studies have shown that elongation is locally regulated by mul-tiple factors, but the observed heterogeneity remains only partially explained.To dissect quantitatively the different determinants of translation speed, wehere use a probabilistic model of the translation dynamics to estimatetranscript-specific initiation and local elongation rates from ribosome profilingdata. Using this model-based approach, we infer the extent of interference be-tween ribosomes on the same transcript (which cannot be observed directly

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from current ribosome profiling data), and show that it varies substantiallyacross different genes and different positions. However, we show that neitherribosomal interference nor the distribution of slow codons is sufficient toexplain the observed variation in the mean elongation rate along the transcript.Surprisingly, by optimizing the fit of statistical linear models, we find that thehydropathy of the nascent polypeptide segment within the ribosome plays a ma-jor role in governing the variation of the mean elongation rate. In addition, wefind that positively and negatively charged amino acid residues near the begin-ning and end of the ribosomal exit tunnel, respectively, are important determi-nants of translation speed. This result is consistent with the electrostaticproperties of the ribosomal exit tunnel, which we study by exploring the geom-etry of the tunnel and solving the Poisson Boltzmann equation.

157-PlatIs Protein Single Molecule Dynamics under Force Described by Two orMore States?Jagannath Mondal1, Ronen Berkovich2, Bruce Berne3.1TIFR Center for Interdisciplinary Sciences, Hyderabad, India, 2Ben GurionUniversity, Beer Sheeva, Israel, 3chemistry, Columbia University, New York,NY, USA.Single molecule force spectroscopy is a useful technique for investigating me-chanically induced protein unfolding and refolding under reduced forces bymonitoring the end-to-end distance of the protein. The data is often interpretedvia a ‘‘two-state’’ model based on the assumption that the end-to-end distancealone is a good reaction coordinate and the thermodynamic behavior is thenascribed to the free energy as a function of this one reaction coordinate. Inthis paper, we determined the free energy surface (PMF) of GB1 protein fromatomistic simulations in explicit solvent under different applied forces as a func-tion of two collective variables (the end-to-end-distance, and the fraction ofnative contacts r). The calculated 2-d free energy surfaces exhibited severaldistinct states, or basins, mostly visible along the r coordinate. Brownian dy-namics (BD) simulations on the smoothed free energy surface show that the pro-tein visits a metastable molten globule state and is thus a three state folder, notthe two state folder inferred using the end-to-end distance as the sole reaction co-ordinate. These BD simulations reproduce the unfolding and collapse-refoldingpatterns observed in the force-clamp experiments. This study lends support torecent experiments that suggest that GB1 is not a two-state folder.

158-PlatFinding Protein Folding Funnels in Random NetworksMacoto Kikuchi.Cybermedia center, Osaka University, Toyonaka, Japan.Many attempts have been made for understanding protein folding dynamics us-ing network representations of folding pathway. For example, the Markov statemodel has been widely used in analyzing the transitions between protein con-formations of trajectories obtained by molecular dynamics simulations.In the present work, I also focus on the network structures of protein foldingdynamics, but from a somewhat different perspective. Currently most widelyaccepted theoretical framework of the protein folding is so-called funnel pic-ture, which states that the number of conformations decreases as the energylowers from the denatured state towards the native state. In other words, the en-ergy landscapes of proteins have been designed through Darwinian evolution sothat proteins readily fold to their native states. A question I would like to askhere is ‘‘How rare are such folding-friendly funnel structures?’’.As a first attempt to answer this question, I introduce a random network modelwith each nodes assigned a random energy. Nodes represent the metastableconformation ensembles. One of the nodes is considered as denatured stateand another the folded state. Other nodes represent the intermediate conforma-tion ensembles between them. Edge connecting the nodes are possible transitionsbetween the nodes. The network structure is assumed to be determined by thenative state conformation, and the energy assignment is considered to reflectsthe amino-acid sequence.We then define the ideal funnel structure for this modelas follows: Starting from the denatured state, if all the paths connecting the nodesin energy-lowering direction reach the folded state, then such a network is in aideal funnel structure. The task now is to compute the probability that the idealfunnel is realized for a given network by changing the assignment of energy.

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We generated 1000 random networks for number of nodes from 8 to 16. Theprobability is widely distributed from network to network, and close to thelog-normal distribution . This fact means that there are a small number of net-works that are robust against the mutation. The funnel structure becomes rarerexponentially as the protein becomes longer.

159-PlatMechanistic Insights into NSF Mediated SNARE Complex DisassemblyMinglei Zhao, Ucheor Choi, Axel Brunger.Stanford University, Stanford, CA, USA.Evolutionarily conserved SNARE (soluble N-ethylmaleimide sensitive factorattachment protein receptors) proteins form a complex that drives membranefusion in eukaryotes. The ATPase NSF (N-ethylmaleimide sensitive factor),together with the adaptor protein SNAP (soluble NSF attachment protein), dis-assembles the SNARE complex, making individual SNAREs available for sub-sequent rounds of fusion. We determined structures of ATP- and ADP-boundNSF, and the NSF/SNAP/SNARE supercomplex (known as 20S particle) usingsingle-particle cryo-electron microscopy (cryo-EM). We further dissected themechanism of NSF mediated SNARE complex disassembly using single-molecule fluorescence microscopy based on the cryo-EM structures.

160-PlatCell-Free Synthesis of Site-Specifically Double-Labeled Proteins for MoreAccurate Single-Molecule FRET StudiesMayuri Sadoine1, Michele Cerminara1, Noemie Kempf1,Alexandros Katranidis1, Jorg Fitter1,2.1Institute of Complex Systems ICS-5, Forschungszentrum Juelich, Juelich,Germany, 21. Physikalisches Institut (IA), RWTH Aachen, Aachen,Germany.Single molecule FRET (smFRET) is a powerful tool for looking at proteinfolding and conformational dynamics (1). The application of smFRET to inter-esting proteins involved in pathologies or drug targeting is limited when theprotein of interest is difficult to express in vivo due to toxicity and wherecell-free expression is required. Moreover, site-specific labeling is an importantissue for smFRET (2), but there is still a lack in simple and robust methods forsite-specific labeling of in vitro synthesized proteins for smFRET studies. Theconventional method is to double label the protein via cysteine residues (3), butas a consequence of a single chemistry, labeling specificity is limited and wouldlead to an inaccurate picture of the studied population (4). Incorporation of un-natural amino acids carrying an orthogonal chemistry can increase labelingspecificity as it was reported (4). However, described methods were so farmainly considered for smFRET studies performed on in vivo synthesizedproteins.By using human calmodulin (hCaM) as a model protein we have developed analternative method for site-specific labeling of cell-free synthesized proteins.Site-specificity of our system allowed us to obtain sharper FRET histogramscompared to the conventional labeling method. In addition, by demonstratingfunctionality of the labeled hCaM through binding experiments performed inpresence of ligands and partners, we have shown the biological relevance ofour method.In summary, we have developed a robust and simple method that enables theaccurate study of cell-free synthesized proteins in smFRET. This method canbe used for studying proteins that cannot be expressed in vivo, as it would befor toxic or membrane proteins.1. Tan, Y. W., J. A. Hanson, J. W. Chu, and H. Yang. 2014. Confocal single-molecule FRET for protein conformational dynamics. Methods Mol. Biol.1084:51-62.2. Joo, C., and T. Ha. 2012. Labeling proteins for single-molecule FRET. ColdSpring Harb. Protoc. 2012:1009-1012.3. Kim, Y., S. O. Ho, N. R. Gassman, Y. Korlann, E. V. Landorf, F. R. Collart,and S. Weiss. 2008. Efficient site-specific labeling of proteins via cysteines.Bioconjug. Chem. 19:786-791.4. Seo, M. H., T. S. Lee, E. Kim, Y. L. Cho, H. S. Park, T. Y. Yoon, and H. S.Kim. 2011. Efficient single-molecule fluorescence resonance energy transferanalysis by site-specific dual-labeling of protein using an unnatural aminoacid. Anal. Chem. 83:8849-8854.

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Platform: Protein Dynamics and Allostery I

161-PlatMolecular Simulations to Unravel the Allosteric Interplay between theSH2 Domain and A-loop Plasticity in Protein KinasesGiuseppina La Sala1, Laura Riccardi1, Roberto Gaspari2, Andrea Cavalli2,3,Oliver Hantschel4, Matteo Dal Peraro5,6, Marco De Vivo1,7.1Laboratory of Molecular Modeling & Drug Discovery, Istituto Italiano diTecnologia, Genova, Italy, 2CompuNet, Istituto Italiano di Tecnologia,Genova, Italy, 3Department of Pharmacy and Biotechnology, University ofBologna, Bologna, Italy, 4Swiss Institute for Experimental Cancer Research(ISREC), EPFL, Lausanne, Switzerland, 5Institute of Bioengineering, EPFL,Lausanne, Switzerland, 6Swiss Institute of Bioinformatics (SIB), Lausanne,Switzerland, 7IAS-S/INM-9 Computational Biomedicine,Forschungszentrum J€ulich, J€ulich, Germany.The deregulation of protein kinases is often related with the development ofseveral malignancies such as cancer. Therefore, inhibition of protein kinasesis an established and often effective pharmacological strategy. However,point mutations in kinases are frequently the cause of drug resistance. Toovercome this issue, many efforts are directed towards the design of allostericdrugs, with the goal to inhibit the mutated forms of kinases. To understandthe molecular basis of the allosteric control of protein kinases is essentialfor the design of novel drugs. In this work, we focus on the prototype Ablkinase. Experimental studies have demonstrated that the binding of theSH2 domain on the kinase domain enhances the A-loop trans-autophosphorylation, a required step for full kinase activity. Hence, theSH2 domain acts as an allosteric modulator. However, it is yet poorly under-stood how the SH2 domain affects the A-loop accessibility. Here, we coupledMD simulations with free energy calculations to investigate possible mecha-nisms for such allosteric control in Abl. We found that the A-loop plasticity isaffected by a complex network of interactions that involve several Abl struc-tural features, such as the DFG motif, the HRD motif and the SH2 domain.Hence, we hypothesize that such elements might constitute additional layersthat control the Abl allosteric machinery. These results provide importanthints for deciphering the complex signaling network for Abl activation.Moreover, these findings may help the rational design of Abl binders capableof interfering with such activation mechanism. Finally, since the SH2 domainis found in several other protein kinases, we propose that such a mechanismmay be extended to several kinases which are currently under investigationsin our labs.

162-PlatSubunit Exchange and Activation of Human CaMKII VariantsAna P. Torres OCampo, Brendan Page, Margaret Stratton.UMass Amherst, Amherst, MA, USA.Ca2þ-calmodulin dependent protein kinase II (CaMKII) assembles into anoligomeric ring in which the kinase domains are organized around a centralhub. Notably, the stimulated activity of CaMKII persists even after the with-drawal of a calcium stimulus. CaMKII acquires this Ca2þ-independent activ-ity at a threshold frequency and this property is implicated in long-termpotentiation (LTP). Indeed, transgenic mice expressing mutant versions ofCaMKII have limited LTP and defects in learning and memory. We have pre-viously shown that CaMKII has a remarkable property, which is that activa-tion of CaMKII triggers the exchange of subunits between holoenzymes,including inactive ones, enabling the Ca2þ-independent activation of neigh-boring subunits. Our results have implications for an earlier idea that subunitexchange in CaMKII may have relevance for long-term memory formation.These studies were done using primarily human CaMKIIa, isoform 2. Thereare four human CaMKII genes, CaMKIIa and b are found in the brain, CaM-KIId is in the heart, and CaMKIIg is found throughout the body. Each of thesegenes has several splice variants encoding ~20 different isoforms. The pri-mary difference between these isoforms is in the composition and length ofthe variable linker domain that connects the kinase to the hub. Previousstudies have shown that the length of this linker determines the threshold fre-quency for activation. A comprehensive biochemical study of existing humanCaMKII isoforms has not been completed. We have expanded our study offrequency activation and subunit exchange to the remaining isoforms ofCaMKII in order to investigate whether these properties are ubiquitous andwhy specific isoforms are selectively expressed in different cell types. Ournew data show that as the variable linker domain is lengthened, less CaMis needed for activation. However, above a certain linker length, there is noadded effect.

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163-PlatCan we Automatically Detect Biologically Relevant Order Parametersin Molecular Simulation? Comparing Long Timescale Simulationsof Multiple KinasesSonya M. Hanson, Joshua H. Fass, John D. Chodera.Computational Biology Program, Memorial Sloan Kettering Cancer Center,New York, NY, USA.Kinases are a ubiquitous component of important signaling pathways, includingthose frequently involved in cancer. Understanding the conformational hetero-geneity of kinases has been shown to be important for understanding their func-tion and their role as therapeutic targets. While thousands of structures areavailable via the PDB of kinases in various states, understanding how thesestates are kinetically and energetically related is key to making sense of thesestructures. Molecular dynamics has the power to give us this insight, howeverin simulating kinases structures from hundreds of states found in the PDB orfrom a single starting structure, we have found that defining order parametersto understand these dynamics can be far from straightforward. Analyzing oursimulations, we optimized our order parameters to reflect the slowest processes,the starting structure of the simulation, or known kinase order parameters fromthe literature. To better facilitate this comparison, we have built a Pythonlibrary (kinalysis) that can map any kinase structure or set of structures ontopreviously established kinase states (defined by DFG flip, C-helix switch, acti-vation loop conformation, etc.). This can then be used to understand how wellour generalizable methods for defining metastable states captures biologicallyrelevant conformational changes. There is a careful balance between biasing ananalysis with what you expect to see and allowing a simulation to tell yousomething you didn’t know before. In the long term, we hope this study informshow generalized methods of defining simulation-driven coordinates ratherthan coordinates hand-picked for a specific system add to our understandingof protein conformational dynamics.

164-PlatEvolution of Regulatory Diversity in the Kinase SuperfamilyJai Pandey1, Orna Resnekov2, David Pincus1, Kimberly A. Reynolds3.1Whitehead Institute, MIT, Boston, MA, USA, 2Molecular MedicineResearch Institute, Sunnyvale, CA, USA, 3Green Center for SystemsBiology, UT Southwestern Medical Center, Dallas, TX, USA.Allosteric regulation evolves readily in proteins and homologs often displaystructurally and biochemically distinct allosteric mechanisms. This is exempli-fied by the eukaryotic protein kinase superfamily, in which individual kinasefamily members are regulated by various combinations of protein interaction,dimerization, and/or post-translational modification. Given that allostery re-quires the coordinated activity of multiple, spatially distributed amino acids,it is not obvious how such an apparently complex molecular feature evolvesonce, let alone at a multiplicity of sites. We show that allosteric regulationtaken across a diversity of protein kinases preferentially occurs at specific,conserved surface sites that are distinguished by their pattern of amino acidsequence co-evolution. Further, we show that these sites can be used to engi-neer new allosteric control by introducing synthetic phosphorylation sitesinto yeast Kss1 that result in activation of the pheromone response pathwaythrough Ras-dependent Protein kinase A signaling. These findings stronglysupport a model for the evolution of new allosteric regulation in which aconserved cooperative scaffold facilitates the emergence of regulation at spe-cific sites distributed across the protein surface. Thus, the natural architectureof proteins provides a facile route to rewire cell signaling cascades by evolutionor engineering.

165-PlatThe G41S Variant of Human Cytochrome C Enhances Apoptosis viaIncreased DynamicsAndreas Ioannis Karsisiotis1, Oliver M. Deacon1, Michael T. Wilson1,Colin Macdonald2, Tharin M.A. Blumenschein2, Geoffrey R. Moore2,Jonathan A.R. Worrall1.1School of Biological Sciences, University of Essex, Colchester, UnitedKingdom, 2School of Chemistry, University of East Anglia, Norwich, UnitedKingdom.In addition to its well-known role as a component of the electron transport chain,mitochondrial cytochrome c can also act as a peroxidase in the early stages of theintrinsic apoptosis pathway. Ferricytochrome c contains a hexacoordinatedheme iron, for which H18 and M80 are the axial ligands. Residual peroxidaseactivity under native conditions arises from aminor populated pentacoordinatedform, inwhichM80 is not coordinated to the heme iron.At the onset of apoptosis,binding to cardiolipin increases the proportion of the pentacoordinated form,increasing peroxidase activity which eventually leads to apoptosis. Mutations

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in the human gene for cytochrome c, which result in enhanced mitochondrialapoptotic activity, cause thrombocytopenia 4, an inherited autosomal dominantthrombocytopenia, characterised by a deficiency in the number of platelets in theblood and leading to abnormal bleeding. The first such mutation to be reportedwas G41S. Here we use stopped-flow kinetic studies of azide binding to humanferricytochrome c, backbone amide H/D exchange and 15N-relaxation dynamicsmeasured by NMR spectroscopy to compare the wild type and G41S forms ofhuman cytochrome c. We show that alternative conformations are kineticallyand thermodynamically more readily accessible for the G41S variant than forthe wild-type protein. Residue 41 is located in the 40-57 U-loop, and theincreased loop dynamics in the G41S variant promote the dissociation fromthe heme iron of theM80 ligand, revealing a direct conformational link betweenthe loop and the axial ligand to the heme iron. Increased dissociation of M80 in-creases the population of a peroxidase active state, which is a key non-nativeconformational state in apoptosis.

166-PlatGlobal Disordering in Stereo-Specific Protein AssociationArun Gupta1, Ines Reinartz2, Alessandro Spilotros3, Venkateswara R. Jonna1,Anders Hofer1, Dmitri I. Svergun3, Alexander Schug2,MagnusWolf-Watz1.1University of Umea, Umea, Sweden, 2Karlsruhe Institute of Technology,Karlsruhe, Germany, 3European Molecular Biological Laboratory, HamburgOutstation, Hamburg, Germany.Protein-protein recognition is of fundamental importance for a myriad ofbiological processes and is ultimately a prerequisite for life as we know it.There exist several established mechanisms that promote formation ofstereo-specific protein complexes. Many of these mechanism involve confor-mational changes of one or both proteins in dimeric assemblies as observed in‘‘conformational selection’’ and ‘‘coupled folding and binding’’ scenarios. In‘‘coupled folding and binding’’ events, at least one of the proteins undergoes aglobal ordering event. By using an integrated computational and experimentalapproach we have discovered that also global disordering can be a productiveroute for formation of a stereo-specific protein complex. This mechanism wasobserved for the chaperone binding domain of the Yersinia effector proteinYopH upon binding to its specific chaperone SycH. These two proteins arecrucial for type III secretion system mediated infectivity by Yersinia andseveral other gram negative pathogens. NMR relaxation dispersion experi-ments demonstrated that the otherwise well folded YopH protein dynamicallysamples an expanded high-energy state that corresponds to the SycH bindingcompetent conformation. A structure of the protein complex determinedfrom a hybrid SAXS and computational approach revealed that YopH wrapsaround SycH in a horse shoe like conformation. The binding model was vali-dated by site specific YopH mutations that promoted the disordering event andat the same time displayed improved binding affinity towards SycH. Takentogether the data illustrates a tight coupling between a proteins unfoldingand functional free energy landscapes and add valuable mechanistic insightinto protein-protein recognition.

167-PlatRole of Conformational Entropy in Extremely High Affinity ProteinInteractionsJose A. Caro.Biochemistry & Biophysics, University of Pennsylvania, Perelman School ofMedicine, Philadelphia, PA, USA.Interactions of extreme affinity (Kd ~ fM) underlie many biochemical processesnecessary to life. The physical determinants of such large binding energies arenot well understood. Specific interactions at the interface (DHbinding) and therelease of solvating water (TDSsolvation) are usually assumed to dominate thebinding energetics. The role of conformational entropy (TDSconf) in deter-mining binding affinity has remained elusive, in part due to the difficulties inmeasuring such changes in entropy experimentally. Recent developments inthe Wand laboratory have bridged this gap by using solution NMR measure-ments of dynamics to empirically calibrate a ‘‘conformational entropy meter.’’It has enabled quantitative measurements of the change in conformational en-tropy in protein-ligand binding. The toxin-antitoxin system studied here,barnase-barstar, forms a complex with fM affinity (DGbinding ~ 19 kcal/mol)without undergoing any major structural changes and retaining a hydratedinterface. To explore the role of conformational entropy, the fast (ps-ns time-scale) motions of backbone and side chains of the two proteins were measuredin both the free (unbound) and the complexed (bound) states using NMR spec-troscopy. Furthermore, hydration dynamics were measured in water and in theconfined space of a reverse micelle. The dynamic response observed leads to anunfavorable change in TDSconf, with a more rigid, still hydrated interface. Thiscomprehensive study of both protein and ‘‘ligand’’ (in this case, another pro-tein) and the measured site-specific changes in dynamics and hydration sheds

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light on the thermodynamic contributions that enable fM affinities. Supportedby grants from the NIH, The Mathers Foundation and NSF.

168-PlatPressure Effects on Dissociation of CheY-FliM Complex Studiedby Molecular Dynamics SimulationsHiroaki Hata1, Yasutaka Nishihara1, Masayoshi Nishiyama2,Ikuro Kawagishi3, Akio Kitao1.1Institute of Molecular and Cellular Biosciences, The University of Tokyo,Tokyo, Japan, 2The Hakubi Center for Advanced Research, KyotoUniversity, Kyoto, Japan, 3Department of Frontier Bioscience, HoseiUniversity, Tokyo, Japan.The rotational switching of the bacteria flagellamotor is controlled by binding ofthe signalingmolecule CheY onto FliMwhich is a part of motor basal body. Therotational switching plays a central role in the bacterial chemotaxis. Recently, itwas reported that high hydrostatic pressures of >120 MPa can induce the rota-tional switching even in the absence of CheY [1]. It was also suggested that hy-dration of the switch complex at high pressure induces structural changes similarto those caused by the binding of CheY. To gain further insights into the highpressure effect on themotor switching, we investigated differences in conforma-tion ofmonomericCheY and alsoCheY-FliMcomplex at different pressure con-ditions using molecular dynamics (MD) simulations. Then, pressure effects onthe binding stability of the CheY-FliM complex was studied by dissociatingthe complex. The dissociation of the protein complex was observed using anefficient sampling method, PaCS-MD (Parallel Cascade Selection MolecularDynamics) [2]. In PaCS-MD, the cycle of short MD simulations and selectionof the structures close to the product structure for the next cycle are repeated,which enhances the conformational transitions without any additional externalbiases. From the obtained MD trajectories, the dissociation behavior was char-acterized using coordinates such as the center of mass (COM) distance and thenumber of native contacts between CheY and FliM. Moreover, potentials ofmean force along the COM distance were calculated from probability distribu-tions in steady state obtained by Markov state models. Those potentials ofmean force provided binding free energies of the protein complex. Based onthe results, we will also discuss mechanisms underlying influences of high hy-drostatic pressures on the binding. Such insights would provide a further under-standing towards an accurate regulation of protein-protein interactions.[1] Nishiyama, M. et al. 2013. J. Bacteriol. 195:1809-1814. doi: 10.1128/JB.02139-12.[2] Harada, R. and Kitao, A. 2013. J. Chem. Phys. 139:035103. doi: 10.1063/1.4813023.

Platform: General Protein-Lipid Interactions I

169-PlatPredicting Cholesterol Interaction Sites on GPCRs by MolecularSimulationEdward R. Lyman1, Clement Arnarez2, Eric Rouviere2.1Department of Chemistry and Biochemistry, University of Delaware,Newark, DE, USA, 2Department of Physics and Astronomy, University ofDelaware, Newark, DE, USA.G-protein coupled receptor function depends on the lipid environment, inparticular on cholesterol. Given that brute force mutagenesis of the entiremembrane-facing surface is not practical, an approach is presented to identifyputative cholesterol interaction sites on the surface of GPCRs. In unbiased sim-ulations in the presence of cholesterol, specific residues are identified as loci ofcholesterol interaction, identified on the basis of long-lived, reproduciblecholesterol binding. Results will be presented for several GPCRs, with a specialfocus on the A2A adenosine receptor.

170-PlatMembrane Cholesterol and the Adenosine A2a ReceptorClaire McGraw, Anne S. Robinson.Tulane University, New Orleans, LA, USA.G-protein coupled receptors (GPCRs) represent the largest family of receptorproteins in the living world, having approximately 800 human genes predicted;however the high-resolution crystal structures of only 26 GPCRs have been re-ported (Ghosh et al., 2015). The first human GPCR to be crystallized was theb2-adrenergic receptor (b2AR) in 2007 (Cherezov et al., 2007; Rasmussen etal., 2007; Rosenbaum et al., 2007). Shortly thereafter an alternate crystalform of the b2AR revealed a specific cholesterol binding site between helicesI, II, III and IV. From this work a cholesterol consensus motif (CCM) was es-tablished, which defined specific interactions between cholesterol and the re-ceptor. Utilization of this CCM predicted that as many as 25% of all class AGPCRs could have a specific interaction with cholesterol (Hanson et al., 2008).

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Our focus is the effects of cholesterol on the activity of the Adenosine A2a re-ceptor (A2aR), a class A GPCR. Membrane cholesterol concentrations werevaried in Human embryonic kidney (HEK-293) cells by the use of methyl-b-cyclodextrin (MbCD), which is capable of capturing cholesterol in its innercavity (Pucadyil et al., 2006). We tested the role that bulk cholesterol depletionplayed in expression, ligand binding and downstream synthesis of cyclic AMP(cAMP) in HEK-293 cells.Continuing work from our lab will study the specific interaction betweencholesterol and A2aR by making point mutations in the residues of the CCM.In A2aR those residues include Tyr43(2.41), Ser47(2.45), Lys122(4.43),Ile125(4.46) and Trp129(4.50) (Lee et al., 2013). Our lab will investigatewhether these point mutations effect the interaction between cholesterol andpurified receptor, as well as the receptors activity in both yeast and mammaliancell model systems.

171-PlatProbing Cholesterol-Dependence of Integrin-Urokinase ReceptorComplex Formation using Confocal Dual-Color Fluorescence IntensityAnalysisYifan Ge1, Jiayun Gao2, Rainer Jordan3, Christoph A. Naumann1.1Chemistry and Chemical Bology, Indiana University-Purdue UniversityIndianapolis, Indianapolis, IN, USA, 2Chemistry and Chemical Biology,Indiana University-Purdue University Indianapolis, Indianapolis, IN, USA,3Macromolecular Chemistry, Technical University Dresden, Dresden,Germany.The modern view of a plasma membrane is that of a complex supramolecularassembly of notable lipid/protein heterogeneity, in which the local lipid envi-ronment may have a profound impact on membrane protein distribution andfunction. Yet, the underlying mechanisms of lipid-mediated regulation of mem-brane protein functionality remain elusive, in part due to the limitations ofexisting methodologies and the often small size and transient nature of lipid/protein heterogeneities in cellular membranes. Exemplary, we still have ratherlimited knowledge about the significance of lipid composition in hetero-proteincomplex formation in such membrane systems. To address this fascinating,but poorly understood topic, here we introduce a single molecule-sensitiveconfocal experimental strategy, comprised of dual-color fluorescence peakanalysis and photon counting histogram (PCH) method, which not only allowsthe detection of hetero-protein complexes in well-defined lipid environments,but also provides accurate insight into the composition of such complexes inthe membrane. By applying the described methodology, here we demonstratethe formation of complexes of avb3 integrins and GPI-anchored urokinaseplasminogen activator receptors (uPAR) in cholesterol-containing model lipidmixtures. PCH analysis of fluorescence intensity signals of complexes confirmsfurthermore that avb3-uPAR complexes are comprised of one integrin andtwo uPAR proteins. Remarkably, formation of comparable hetero-protein com-plexes is largely suppressed in a cholesterol-free lipid composition, high-lighting the importance of cholesterol in avb3-uPAR complex formation. Ourresults are intriguing in light of the already established significance of choles-terol in integrin function in cellular membranes.

172-PlatPartitioning of g-Secretase and its Substrates in Lipid MicrodomainsMarilia Barros1, William Houlihan2, Lane Gilchrist2, Yueming Li1.1Memorial Sloan-Kettering Cancer Center, New York, NY, USA, 2The CityCollege of the City University of New York, New York, NY, USA.g-Secretase is a multiprotein complex that catalyzes intramembranous cleav-age of mutiple transmembrane I proteins. The sequential proteolysis of theamyloid precursor protein (APP) by g-Secretase generates amyloid beta pep-tides that form plaques, one of the main pathological hallmarks of Alzheimer’sdisease. g-Secretase also cleaves Notch the abnormal signaling of which canlead to cancer. The lipid environment regulates the location and activity ofmany transmembrane proteins and defines membrane micro or nanodomains,which serve as spatio-temporal platforms for proteins to function properly,and therefore have a modulatory impact on the intramembrane proteolysis.Mechanistically, the lipids may affect substrate recognition by binding tog-secretase and substrates and by excluding or enriching both partners withina microdomain. Despite substantial advances in elucidating how this enzymecomplex functions, the effect of the local membrane lipid microenvironmenton g-secretase cleavage of substrates is still poorly understood. Here, we char-acterize the partitioning of APP and Notch1 substrate and g-secretase usingsolid supported membranes to determine whether the function and activity ofg-Secretase and its substrates r is influenced by the membrane lateral organiza-tion. As a membrane model we chose a canonical raft mixture containing phos-phatidylcholine, sphingomyelin and cholesterol. Atomic force microscopy(AFM) data revealed membrane heterogeneities, ordered (lo) and disordered

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(ld) domains, and the selective localization and clustering of membrane pro-teins. Notch substrate is uniformly distributed in the ld phase while APP sub-strate clusters prefer the boundary between ld and lo domains in agreementwith fluorescence microscopy measurements carried out on proteolipobeads,suggesting that proteolysis and specificity of these substrates are modulatedby membrane microenvironement. In addition, AFM time-course measure-ments suggests that g-secretase recruits specific membrane components andthus creates a favorable lipidic environment for its assembly and activity.

173-PlatLipid/Polydiacetylene Vesicle Composition Alters Mutant Beta-AmyloidPeptide InteractionElizabeth A. Yates1, Michael P. Dorsey1, Brice M. Nguelifack2.1Chemistry, United States Naval Academy, Annapolis, MD, USA,2Mathematics, United States Naval Academy, Annapolis, MD, USA.Alzheimer’s disease (AD) is commonly identified by the formation of proteina-ceous fibrillar aggregate deposits known as amyloids, and the presence ofneuritic amyloid plaques containing aggregated b-amyloid (Ab). Point muta-tions grouped in the hydrophobic core of Ab (positions 21–23) are linked to fa-milial forms of AD. Understanding Ab peptide interactions with lipid surfaceswill provide detailed information into how surface interactions can drive proteinaggregation. We hypothesized changes in the lipid composition of a vesicle willresult in variedmutant Ab peptide aggregationmorphology and kinetics, furthermodulating peptide-lipid interactions. To test our hypothesis we utilized a color-imetric, lipid vesicle-binding assay exposed to specific Ab peptides. The assayintegrates a lipid within a polydiacetylene (PDA) matrix forming a blue vesiclewhen polymerized by UV irradiation. Lipid/PDA vesicles are able to detectpeptide-lipid interaction by quantitatively measuring a blue-to-red conversionas a percent colorimetric response (%CR). We investigated the interaction ofWild Type (Ab1-40) and five mutant Ab peptides (E22G Arctic, E22Q Dutch,A21G Flemish, D23N Iowa, and E22K Italian) with lipid/PDA vesicle systemscomprised of total brain lipid extract (TBLE), dimyristoylphosphatideylcholine(DMPC), and dimyristoylphosphatidylglycerol (DMPG). Time-resolved absor-bance measurements of the ‘‘blue’’ (630 nm) and ‘‘red’’ (490 nm) wavelengthswere obtained. Statistical analysis (ANOVA and t-tests) was performed to testfor significant differences between peptides among the lipid/PDA compositions,as well as compare each mutant Ab peptide to Wild Type within the context ofeach lipid. The most peptide-lipid interaction was observed on the negativelycharged DMPG/PDA vesicle system, and the least on the neutral DMPC/PDAsystem.Dutch andFlemish induced the smallest,whileArctic induced the largest%CR on all lipid/PDA vesicles, respectively. These studies illustrate the role oflipid composition and Ab interactions in the onset of familial AD.

174-PlatSynaptotagmin Interactions with Membranes: Measuring the Force ofCalcium Triggering of NeurotransmissionClemence Gruget1, Jeff Coleman2, Shyam Krishankumar3,James E. Rothman2, Frederic Pincet1, Stephen Donaldson1.1Laboratoire de Physique Statistique, Ecole Normale Superieure, Paris,France, 2School of medicine, Yale University, New Haven, CT, USA,3University College London, London, United Kingdom.A critical step of neurotransmission is the rapid and synchronized fusion of syn-aptic vesicles with the pre-synaptic plasma membrane of the neuron upon cal-cium entry, allowing the release of neurotransmitters immediately upon arrivalof an action potential. A calcium binding protein anchored in the synapticvesicle, the Synaptotagmin-1 (Syt1), has been identified as the calcium sensorof this process, being able to accelerate fusion by more than four orders ofmagnitude in presence of calcium. Despite this crucial role, the molecularmechanism involved remains unclear.The cytosolic domain of Syt1 consists of tandem Ca2þ- binding C2 domains(C2A and C2B) attached to the membrane via a juxtamembrane linker domain.Different sites of the protein are responsible for its specific roles. First, theinteraction of the polybasic region of the C2B domain with the anionic lipidPIP2 is needed for the initial docking of the synaptic vesicle at the plasma mem-brane. Second, upon calcium binding, the aliphatic loops on each C2 domainspartially insert into the membrane, enabling the SNARE proteins to completemembrane fusion.While the mechanistic details described above are mostly well-accepted,importantly, the binding energies of Syt1 with membranes have never been re-ported. Therefore, the aim of our work is to measure the energetics of the majormembrane binding sites of Syt1, i.e. the polybasic motif and the calcium-loopinsertions of C2B and C2A. For this we use a Surface Force Apparatus (SFA), adevice that provides a direct measurement of the interaction force between twosurfaces as a function of their separation distance, with nanoscale resolution.This system has successfully been used to measure the energy of the SNARE

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proteins assembly, but the previous experiments did not include Syt1. In our setup, both surfaces are coated with a lipid bilayer. One of them mimics the syn-aptic vesicle membrane, on which we bind Syt1. The opposing bilayer mimicsthe inner leaflet of the plasma membrane and contains PIP2 and PS lipids. Wewill present results obtained with various lipid compositions and relevant mu-tations of the protein and how these interactions are impacted by the presenceof calcium. Ultimately, we plan to provide a complete mapping of the ener-getics of the critical membrane interaction sites of Syt1.

175-PlatStudy of Insertion of Dengue E into Lipid Bilayers by Neutron Reflectivityand Molecular Dynamics SimulationsJuan M. Vanegas1, Frank Heinrich2, David M. Rogers3, Bryan D. Carson4,Sadie La Bauve4, Brianna C. Vernon5, Bulent Akgun6,7, Sushil Satija8,Aihua Zheng9, Margaret Kielian10, Susan B. Rempe5, Michael S. Kent5.1Dept. of Physics, University of Vermont, Burlington, VT, USA, 2Physics,Carnegie Mellon University, Pittsburgh, PA, USA, 3Chemistry, University ofSouth Florida, Tampa, FL, USA, 4Bioenergy and Defense Technology,Sandia National Labs, Albuquerque, NM, USA, 5Nanobiology, SandiaNational Labs, Albuquerque, NM, USA, 6Bogazici University, Istanbul,Turkey, 7National Institute of Stsndards and Technology, Gaithersburg, MD,USA, 8National Institute of Standards and Technology, Gaithersburg, MD,USA, 9Institute of Zoology, Chinese Academy of Sciences, Beijing, China,10Dept of Cell Biology, Albert Einstein College of Medicine, Bronx,NY, USA.The envelope (E) protein of Dengue virus rearranges to a trimeric hairpin tomediate fusion of the viral and host membranes. Insertion of E into host mem-branes is essential to the process, serving to anchor E into the target membraneand possibly also to disrupt local order within the membrane. Both aspects arelikely to be affected by the depth of insertion, the orientation of the trimer withrespect to the membrane normal, and the interactions that form between thetrimer and the membrane. In the present work, we resolved the depth of inser-tion, the tilt angle, and the fundamental interactions for the soluble portion ofDengue E trimers (sE) associated with planar lipid bilayer membranes ofvarious combinations of POPC with POPG, POPE, and cholesterol by neutronreflectivity (NR) and by molecular dynamics (MD) simulations. The tip of Econtaining the fusion loop (FL) is located at the interface of the headgroupsand acyl chains of the outer leaflet of the lipid bilayers, in good agreementwith prior predictions. The NR measurements and the MD simulations bothindicate that E tilts with respect to the membrane normal upon insertion, pro-moted by either the anionic lipid POPG or cholesterol. The simulations showthat tilting of the protein correlates with hydrogen bond formation between ly-sines located on the sides of the trimer close to the tip (K246 and K247) andnearby lipid headgroups. These hydrogen bonds provide the majority of theinteraction energy whereas interactions involving the FL are a minor contribu-tion. POPG promotes formation of these hydrogen bonds through direct inter-actions with K246, K247, and other polar residues whereas cholesterolindirectly facilitates formation of these hydrogen bonds as a result of a greaterhydrated volume in the headgroups. Simulations in which the protein was heldin a vertical orientation with respect to the membrane show that these stronghydrogen bonding interactions of K246 and K247 with lipid headgroups causessignificant local membrane deformation of a 70:30 POPC:POPG bilayer as thelipids wrap around the periphery of the E trimer. We propose that these inter-actions play the dominant role in membrane anchoring and may also play a rolein initiating mixing of the outer leaflets during the fusion process.

176-PlatThe Great Nuclear Escape: Structure-Based Mechanism of MembraneBudding during Nuclear Egress of HerpesvirusesEkaterina E. Heldwein1, Janna M. Bigalke2.1Department of Molecular Biology and Microbiology, Tufts UniversitySchool of Medicine, Boston, MA, USA, 2Tufts University School ofMedicine, Boston, MA, USA.Herpesviruses are unusual among enveloped viruses because they bud twiceyet acquire a single envelope. They are also the only known mammalian vi-ruses that bud into the nuclear envelope. Recently, we discovered that theherpesvirus nuclear egress complex (NEC) could bud membranes withoutthe help of other proteins and that it formed a coat-like hexagonal scaffold in-side the budding membrane. This discovery established the NEC as the firstvirally encoded budding machine that operates at the nuclear, as opposed tocytoplasmic, membrane but left unknown the structure of the NEC coat andits role in the budding process. To bridge this gap in our knowledge, we deter-mined the 2.8-A crystal structure of the NEC from Herpes Simplex virus(HSV). In crystals, NEC packs into a hexagonal lattice that mimics the hex-agonal NEC coats within budded vesicles. The crystal structure of the NEC

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lattice thus reveals molecular interactions that generate the hexagonal coat.To determine the role of the hexagonal NEC lattice in budding, we mutatedresidues at the oligomeric interfaces observed in the crystals with mutagen-esis. Perturbation of the oligomeric interfaces through mutagenesis blockedNEC-mediated budding in vitro confirming that formation of the hexagonalNEC lattice drives budding. The NEC structure provide a three-dimensionalblueprint for further dissection of its unique budding mechanism. Moreover,the structure represents the first atomic-level view of an oligomeric arrayformed by a membrane-deforming protein.

Platform: Cardiac, Smooth, and Skeletal MuscleElectrophysiology

177-PlatAction Potential Heterogeneity in Murine Sinoatrial Node MyocytesChristian Rickert, Catherine Proenza.Physiology & Biophysics, University of Colorado, Denver, CO, USA.Sinoatrial nodemyocytes (SAMs) act as cardiac pacemakers by generating spon-taneous action potentials (APs). SAMs are characterized by the expression of the‘‘funny current’’ (If) that contributes to the spontaneous diastolic depolarizationphase of the sinoatrial AP. SAMs exhibit a large heterogeneity of morphologicaland electrophysiological properties. However, limited information is availableabout the intrinsic variability in AP waveforms in SAMs. In this study, we co-recorded APs and If from acutely isolated SAMs from mice using voltage andcurrent clamp recordings in the same cells. We refined the definitions ofcommonly used AP waveform parameters to document the heterogeneity ofAPs in both perforated patch (PP) and whole-cell (WC) recordings. These defi-nitions were then implemented in an open-source data analysis program(‘‘paramAP’’) written in Python 3 using NumPy, SciPy, and Matplotlib. Para-mAP was utilized to characterize APs from more than 100 SAMs. Correlationanalysis was used to evaluate relationships among AP waveform parametersand to identify parameters that are strongly correlated with If and AP firingrate. Furthermore, we document time-dependent changes in AP waveformparameters during both PP and WC recordings. In summary, paramAP is apowerful tool to help standardize AP waveform analysis and parameter defini-tions in murine SAMs. Our data suggest that commonly-held assumptions aboutsinoatrial node APs should be extended to accommodate a wider range of APwaveforms. In addition, the time-dependent changes in AP waveform parame-ters should be considered when comparing data from different studies.

178-PlatSK4 Ca2D-Activated KD Channels Regulate Sinoatrial Node Firing Rateand Cardiac Pacing In VivoBernard Attali1, David Weisbrod1, Hanna Bueno1, Joachim Behar2,Shiraz Haron-Khun1, Dor Yadin3, Asher Peretz1, Michael Arad3,Yael Yaniv2.1Physiology, Sackler Medical School, Tel Aviv University, TelAviv, Israel,2Biomedical Engineering Faculty, Technion-IIT, Haifa, Israel, 3Leviev HeartCenter, Sheba Medical Center, Tel Hashomer, TelAviv, Israel.The sinoatrial node (SAN) controls the heart rhythm under physiological con-ditions. We recently showed that SK4 calcium-activated potassium channels(SK4) are important for automaticity of cardiomyocytes derived from humanembryonic stem cells. Here we tested whether SK4 are expressed in adultmouse SAN and play a role in pacemaker function. TRAM-34, a selectiveblocker of SK4, significantly reduced the firing rate and depolarized themaximal diastolic potential in SAN cells. Western blots revealed the presenceof an SK4 protein in mouse SAN. In vivo ECG recording in mice showed thatintraperitoneal injection of SK4 blockers produced bradycardia and prolongedthe PR interval. Mathematical modeling predicted that addition of SK4 to themodel increases SAN firing rate, while its removal decreases pacemaker fre-quency. This work shows that SK4 play a role in SAN pacemaker functionby acting at late repolarization and that they are potential therapeutic targetsfor treating cardiac arrhythmias.

179-PlatCharacteristics of Ivabradine-Sensitive Currents inMouse Sinoatrial NodeMyocytesEmily J. Sharpe1, Stephanie C. Gantz2, Pin Liu2, Bruce P. Bean2,Catherine Proenza1.1University of Colorado - Denver, Aurora, CO, USA, 2Harvard MedicalSchool, Boston, MA, USA.Cardiac pacemaking is driven by spontaneous action potentials (APs) in sino-atrial node myocytes (SAMs). The funny current (If) is thought play a role inthe generation of pacemaker activity in SAMs, but the degree to which itcontributes is incompletely understood. In this study, we used AP clamp

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experiments and the If blocker, ivabradine, to investigate the role of If inpacemaking. Spontaneous APs were recorded from acutely-isolated SAMsfrom mice, and ~10 s of the AP recording was incorporated into a voltageclamp command. This AP clamp protocol elicited both inward and outwardivabradine-sensitive currents in SAMs. If was defined as the ivabradine-sensitive current remaining in the presence of a pharmacological cocktail ofsodium, calcium and potassium channel blockers. Measurable inwardivabradine-sensitive current was evident during diastole in 57% of all cellsand was generally larger in cells with the most negative diastolic voltages.Although we found that 30 mM ivabradine also blocked both L- and T-type cal-cium currents in SAMs, the majority of the ivabradine-sensitive inward currentin the AP clamp experiments appeared to be If because control experimentsshowed that the drug cocktail blocked ~85% of the calcium currents. Ivabra-dine also blocked a substantial outward current during the AP upstroke andrepolarization phases. This outward ivabradine-sensitive current likely includesa contribution of If, which results from its relatively slow deactivation. How-ever some of the outward ivabradine-sensitive current may also reflect off-target block of potassium currents by ivabradine. We are currently assayingthe contribution of potassium currents to the outward ivabradine-sensitive cur-rent. Our results suggest that If is active not only during diastole, but alsothroughout the AP upstroke and repolarization phases of the mouse SAM AP.

180-PlatCalciumActivated Chloride Current inMammalian VentricularMyocytesJanos Magyar1, Balazs Horvath1, Krisztina Vaczi1, Bence Hegyi1,Monika Gonczi2, Beatrix Dienes1, Kornel Kistamas1, Tamas Banyasz1,Istvan Baczko3, Andras Varro3, Gyorgy Seprenyi4, Laszlo Csernoch1,Peter P. Nanasi5, Norbert Szentandrassy5.1Dept. of Physiology, University of Debrecen Faculty of Medicine,Debrecen, Hungary, 2Dept. of Biochemistry and Molecular Biology,University of Debrecen Faculty of Medicine, Debrecen, Hungary, 3Dept. ofPharmacology and Pharmacotherapy, University of Szeged Faculty ofMedicine, Szeged, Hungary, 4Dept. of Medical Biology, University ofSzeged Faculty of Medicine, Szeged, Hungary, 5Dept. of Physiology,University of Debrecen Faculty of Dentistry, Debrecen, Hungary.Background: Calcium activated Cl� current (ICl(Ca)) mediated by TMEM16Aand/or Bestrophin-3 may contribute to cardiac arrhythmias. The true profileof ICl(Ca) during an actual ventricular action potential (AP), however, is poorlyunderstood. Our goal was to study the current profile under normal calciumcycling and AP voltage-clamp condition as well as in case of altered intracel-lular calcium concentration ([Ca2þ]i). The expression of TMEM16A and/orBestrophin-3 in canine and human left ventricular myocytes was examined.The possible spatial distribution of these proteins and their co-localizationwith Cav1.2 was also studied.Methods: Whole-cell configuration of the patch-clamp technique and actionpotential voltage-clamp were used to monitor ICl(Ca), detected as 9-anthracenecarboxylic acid (9-AC)-sensitive current. FURA-2-AMdyewas used tomeasure[Ca2þ]i. Expression and cellular localization of Cav1.2, Bestrophin-3 andTMEM16Awas analyzedwith immunocytochemistry and confocalmicroscopy.Results: Under AP voltage-clamp conditions the profile of ICl(Ca) containedan early fast outward (1.6250.06 A/F) and a late inward component(�0.1650.02 A/F), overlapping early and terminal repolarizations, respectively.Both components were reduced by ryanodine (1.0550.03 A/F;�0.0750.03 A/F), while fully abolished byBAPTA (0.1350.10A/F;�0.0850.02 A/F), but notEGTA (1.1750.09 A/F; �0.1350.02 A/F). Setting [Ca2þ]i to the systolic level(1.1 mM) decreased ICl(Ca), while application of Bay K8644, isoproterenol, andfaster stimulation rates increased the amplitude of ICl(Ca). Both L-type Ca

2þ cur-rent and ICl(Ca) were eliminated by nisoldipine. TMEM16A and Bestrophin-3showed strong co-localization with one another and also with Cav1.2 channelsboth canine myocytes and human ventricular myocardium.Conclusions: Activation of ICl(Ca) in canine ventricular cells requires calciumentry through neighboring L-type Ca2þ channels and is only augmented bySR Ca2þ-release. Substantial activation of ICl(Ca) requires high Ca2þ in thedyadic clefts which can be effectively buffered by BAPTA, but not EGTA.

181-PlatProperties of New Voltage Sensitive Dyes in Cardiac FieldNdeye Rokhaya Faye1, Sushmitha Raja1, Richard Walton1,Phillipe Pasdois1, Fabien Brette1, Alan Urban2, Alexandre Hentz3,Gihad Dargazanli3, Olivier Bernus1.1Electrophysiology and Heart Modelling Institute (IHU-LIRYC), Pessac,France, 2Neuro-Electronics Research Flanders, Imec, Leuven, Belgium,3Sanofi, Chilly-Mazarin, Paris, France.These last years, combination of high spatiotemporal resolution techniqueswith the development of fluorescent voltage sensitive dyes (VSDs) have signif-

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icantly improved our understandings of electrophysiological functions ofelectrically excitable organs (heart, brain). These dyes allow to measure simul-taneously membrane potential changes from multiple sites of biologicalpreparations in a non-invasive way. However, conventional dyes have excita-tion spectra in the blue/green range, which limits the depth of penetrationdue to high absorbance and photon scattering properties. Therefore, the devel-opment of longer wavelength VSDs could have many advantages namely: ac-tion potentials recording from tissue’s deeper layers, enhanced voltagesensitivity, and improved spectral properties such that simultaneous monitoringof multiple parameters (i.e. ion transient, pH).The goal of our study is to develop and characterize novel VSDs with largeStoke shifts and near-infrared spectral properties. Three families of VSDswere thus synthesized from two heterocyclic fluorophores and their properties(spectral properties, membrane staining, and dye phototoxicity) assessed in iso-lated ventricular cells and in Langendorff perfused rat hearts. In addition, spe-cial attention is paid to Signal:background ratios and signal kinetics of thesenew dyes by epifluorescence imaging.Optimal peak emission wavelengths for all dyes was found between 700 nmand 750 nm. The largest observed Stoke shift was found with fluorophore 1family (about 250 nm). Signal:background ratios ranged from 3% (fluorophore2) to 7% (fluorophore 3), compared to 5.5% for the usual conventional dye,Di-4-ANEPPS.Rapid signal decay indicative of internalization was observed for one VSDfamily. However, in other two, signal stability was improved compared toDi-4-ANEPPS. Overall, dyes made with fluorophore 3 show improved voltagesensitivity and potential for applications.

182-PlatDynamic Blood Flow Control in HeartGuiling Zhao, Humberto Joca, W. Jonathan Lederer.BioMET and Physiology, University of Maryland School of Medicine,Baltimore, MD, USA.Perfusion of the heart by blood is essential for the maintenance of physiologicalfunction. Published experiments and investigations by others reveal that themaximum AV O2 difference that can be achieved in heart is always observedbetween the coronary arteries and the venous outflow at the coronary sinusover the full range of blood flow (~five fold) that is achieved. If so, exquisitematching of blood flow and O2 consumption must exist even at the smallestunit of perfusion. How does such tight control occur? We have examined thehypothesis put forward for the brain to determine if, in principle, it couldwork in heart. Nelson and colleagues have suggested that electrical activitycould be a primary regulator of blood flow in the brain. Increased neuronal ac-tivity would lead to an increase in Kþ efflux that would bathe endothelial cellswhich possess inward rectifier potassium channels (Kir). Activation of Kir couldlead to hyperpolarization given the N-shaped Kir current-voltage (IV) relation-ship that we have observed. In the current study, we examined human microvas-cular endothelial cells from heart (HMVEC-C) in culture to determine how theyresponded to elevated [Kþ]o. We found that extracellular increases of Kþ (from5 mM to 15 mM) cause HMVEC-C hyperpolarization. This Kþ-induced mem-brane hyperpolarization is dependent on Kir activation as evidenced by itsblockade by extracellular Ba2þ. These findings suggest that cardiac electricalactivity could contribute to Kþ-dependent hyperpolarization of the endothelialcells that could hyperpolarize the pre-capillary sphincter smooth muscle cellslocally and thereby contribute to activity-dependent blood flow control.

183-PlatKChIP2 Serves Multiple Functions in Cardiac Myocytes in SpliceIsoform-Dependent MannerRandolph Bettinger1, Rostam Panjshiri1, Drew M. Nassal2,Isabelle Deschenes2, Min Jiang3, Gea-Ny Tseng1.1Physiology & Biophysics, Virginia Commonwealth University, Richmond,VA, USA, 2Medicine and Physiology & Biophysics, Case WesternUniversity, Cleveland, OH, USA, 3Institute of Medicine Biotechnology,Chinese Academy of Medical Sciences, Beijing, China.K-channel-interacting-proteins (KChIPs) are EF-hand-motif-bearingCa sensorsthat serve diverse functions. KChIP2 is the major KChIP expressed in the heart,that canmodulate key cardiac channels (Itof, Nav, CaV and IKur) by directly inter-actingwith their pore-forming subunits (Kv4, Nav1.5, Cav1.2, andKv1.5, respec-tively) on the cell surface. More recent data suggest that KChIP2 can traffic tonuclei and exert transcriptional activity. Nine KChIP2 splice variants havebeen identified, that share C-terminal EF-hand-motifs but diverge in N-termini.Based on N-terminal sequences, KChIP2 splice variants can be broadly dividedas palmitoylation-capable and palmitoylation-incapable. Questions: (1) Doespalmitoylation impact on KChIP2 distribution and function?(2) Is KChIP2splicing pattern altered in diseased heart? Methods: (1) Tag KChIP2a and 2c

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(palmitoylation-capable and -incapable)withmCherry, (2) ExpressKChIP2 iso-forms in COS-7 cells alone or with GFP-Kv4.3, (3) Track KChIP2a/c distribu-tion and relationship with Kv4.3 with confocal microscopy, (4) Quantify Itofchannel function and subcellular distribution with patch clamp and subcellularfractionation/immunoblots, (5) Quantify native KChIP2 and Kv4 proteins andItof channel function in ventricular myocytes from young and old spontaneouslyhypertensive rats (SY and SO, 4-6 and 22-24 months respectively; the latter suf-fers from severe hypertrophy/heart failure).Results: (1)When expressed alone,KChIP2a is strong in vesicles, Golgi and plasma membrane (PM), whileKChIP2c is strong in nuclei and weak in PM. Pharmacological inhibition ofpalmitoylation directs KChIP2a from PM to nuclei, but does not alter nucleilocalization of KChIP2c. (2) Kv4.3 coexpression leads to Kv4.3/KChIP2a over-lap in Golgi, post-Golgi vesicles and PM. KChIP2c and Kv4.3 overlap on PM.While bothKChIP2a andKChIP2c accelerateKv4.3 recovery from inactivation,KChIP2a but not KChIP2c increases the current amplitude. (3) Relative to SY,Itof density is markedly reduced in epicardial myocytes (Epi VMs) of SOhearts. Kv4.2 and Kv4.3 proteins are modestly or not lowered in Epi VMs.While KChIP2a protein is markedly reduced, KChIP2c protein is significantlyincreased. Conclusions: Reversible palmitoylation confers dynamic KChIP2alocalization between PM and nuclei, while KChIP2c has a more static distribu-tion. Chronic hypertension/hypertrophy differentially impacts the expression ofKChIP2a and 2c.

184-PlatEffect of Drugs on Repolarization of IPSCD-CardiomyocytesMark Nowak1, Aidan Coon2, Sanjot Singh2, Shimin Wang2,Randall Rasmusson3, G. Bett4.1Cytocybernetics, Buffalo, NY, USA, 2SUNY, University at Buffalo,Buffalo, NY, USA, 3Physiology and Biophysics, SUNY, University atBuffalo, Buffalo, NY, USA, 4Obstetrics and Gynecology, SUNY, Universityat Buffalo, Buffalo, NY, USA.The FDA has proposed a Comprehensive in Vitro Pro-Arrhythmia Assay (CiPAInitiative) to address the clinical potential for cardiotoxicity during pre-clinicaldrug development in part, this involves the use of human-induced pluripotentadult stem cell-derived cardiac myocytes (hiPSC-CM) as a model system tomeasure the effects of drugs on the cardiac action potential (AP). hiPSC-CMs, however, have little or no IK1, resulting in unstable APs and anomalousbehavior in the presence of some drugs. We stabilized APs from hiPSC-CMsthrough the electronic addition of IK1 via dynamic clamp. hiPSC-CMs (iCell,Inc., WI) were cultured according to manufacturer’s instructions, and studiedusing the Amphotericin B perforated patch technique. Cardiac APs (1.5-2.0 nA stimulus pulse for 1.5 ms, 0.5 Hz) were measured in the absence andpresence of increasing drug concentrations. As expected, quinidine, dofetilide,cisapride and sotalol (known IKR blockers) caused dose-dependent increases inAPD. In 10 mM quinidine APD90 increased 144512%; 1420 nM dofetilideincreased APD90 12853%; 103 nM cisapride increased, APD90 17056%;324 mM sotalol increased APD90 139514%). Further, qualitative differencesin potentiation were observed in that cisapride and sotalol, but not dofetilide,caused a pronounced flattening in Phase 2 of the AP. Quinidine (10 mM) alsodecreased dV/dtmax by 68518%, which is consistent with Naþ channel block.Nifedipine (300nM), a Ca2þ channel blocker, significantly decreased APD90 by3551%. The ability to measure stable cardiac APs by the electronic addition ofIK1 allows for more accurate comprehensive evaluation of drug effects on theion channel components comprising the cardiac AP, particularly in the caseof sodium channel block, and may aid in the identification of pro-arrhythmicchanges in AP morphology.

Symposium: Single-Molecule Membrane ProteinDynamics

185-SympSubmillisecond Dynamics of the NMDA ReceptorHugo Sanabria.Clemson University, Central, SC, USA.Observation of single molecules has impacted the way we look at bio-molecular machines. Particularly, because it gave us a tool to understandhow biomachines move as they carry out specific functions. Single moleculefluorescence experiments, done in surface immobilized conditions or freelydiffusing, take advantage of the fact that fluorescence occurs in the nano-second timescale to map a wide range of biologically relevant dynamics,covering over 10 orders of magnitude in time without gaps. Single moleculeForster Resonance Energy Transfer (smFRET) experiments have evolvedto the point where it is possible to work with complex systems includingmembrane proteins. However, many challenges have limited the impact onthe field, mostly because there is the common conception that the obtained

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structural information is very limited. In addition, multiple experimental arti-facts can complicate the interpretation of data. However, recent advances insmFRET experiments, particularly using Multiparameter Fluorescence Detec-tion (MFD), have improved its accuracy and precision, to the point that it ispossible to overcome this mindset. As an example, we use MFD to identifylow populated conformational states of the ligand-binding domain of theN-methyl-D-aspartate (NMDA) receptors, quantify molecular dynamics inthe sub millisecond regime and identify how conformational dynamics shedslight into the mechanism of partial agonism in the GluN1 subunit of theNMDA receptor.

186-SympMeasuring the Free Energy of ClC-ec1 Dimerization in Membranes usingSingle Molecule Photobleaching AnalysisJanice L. Robertson.Molecular Physiology and Biophysics, University of Iowa, Iowa City,IA, USA.Why do greasy membrane proteins interact with their greasy protein partnersinstead of the similarly greasy lipid bilayer? This is a question at the root ofmembrane protein oligomerization and folding in membranes. Recently, weturned to the homodimeric ClC-ec1 Cl-/Hþ antiporter, in order to develop anew model system that can be used to investigate this question. The ClC-ec1dimerization interface is large (1200 A2) and lined by ~20 non-polar residues,forming an interaction surface exhibiting high shape complementarity. Previ-ous studies showed that ClC-ec1 is folded and functional in both monomericand dimeric states. Using a single-molecule microscopy approach, wemeasured the photobleaching distributions across a wide range of subunit/lipiddilutions, with the lowest representing a sub-biological condition of 1 subunitper 40 E. coli membranes. The data shows that wild-type ClC-ec1 follows amonomer to dimer reaction that is reversible and fits to an equilibriumisotherm, allowing for determination of the dimerization free energy in 2:1POPE/POPG lipid bilayers (Chadda et al., eLife 2016). Next, two approacheswere used to investigate how residues at the interaction interface influencethe free energy of dimerization. First, bulky TRP residues were introduced re-sulting in a destabilization of the dimer by 2 kcal/mole per TRP. Second, sub-tractive substitutions to ALA were constructed introducing cavities at theinterface. While most of the ALA substitutions appear to have no effect,L194A destabilizes the dimer by 2 kcal/mole in lipid bilayers while maintain-ing both fold and function. These studies present ClC-ec1 as a robust modelsystem for probing the physical forces driving protein association inmembranes.

187-SympT Cell Receptor Clustering - A Mechanism of Signal TransductionKatharina Gaus.Centre for Vascular Research, University of New South Wales, Sydney,Australia.Antigen recognition by the T cell receptor (TCR) is a hallmark of the adaptiveimmune system. When the TCR engages a peptide bound to the restricting ma-jor histocompatibility complex molecule (pMHC), it transmits a signal via theassociated CD3 complex. How the extracellular antigen recognition event leadsto intracellular phosphorylation remains unclear. We use single-molecule local-ization microscopy and novel analysis to quantify the organization of TCR-CD3 complexes into nanoscale clusters and to distinguish between triggeredand non-triggered TCR-CD3 complexes. For example, we found that onlyTCR-CD3 complexes in dense clusters were phosphorylated and associatedwith downstream signaling proteins, demonstrating that the molecular densitywithin clusters dictates signal initiation. Both pMHC dose and TCR-pMHCaffinity determined the density of TCR-CD3 clusters, which scaled with overallphosphorylation levels. In summary, we propose a model in whcih TCR-CD3clustering translates antigen recognition by the TCR into signal initiation bythe CD3 complex and the formation of dense signaling-competent clusters isa process of antigen discrimination.

Symposium: Mechanotransduction to Physiology

188-SympThe Mechanism of Activation of Piezo Ion ChannelsJorg Grandl.Duke University, Durham, NC, USA.Piezo ion channels mediate the conversion of mechanical touch into electricalsignals and are critical for the organism’s responsiveness to mechanical forces.How Piezos detect mechanical stimuli is unknown.The apparent mechanical sensitivity of Piezo1 varies substantially acrosscellular environments, stimulating methods and protocols, raising the funda-mental questions of what precise physical stimulus activates the channel and

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how its stimulus sensitivity is regulated. Here, we measured Piezo1 currentsevoked by membrane stretch, while simultaneously visualizing and measuringmembrane geometry. Building on this approach, we developed protocols tominimize resting membrane curvature and tension prior to probing Piezo activ-ity. We find that Piezo1 responds to lateral membrane tension with high sensi-tivity as compared to other mechanically activated channels and that restingtension can drive channel inactivation, thereby tuning overall mechanical sensi-tivity of Piezo1. Our results explain how Piezo1 can function efficiently andwith adaptable sensitivity as a sensor of mechanical stimulation in diversecellular contexts.The next step in understanding the mechanism of Piezo activation would be theidentification of structures within Piezos that detect a mechanical stimulus andthat mediate channel inactivation. We hypothesized that specific structureswithin Piezos are highly sensitive to localized application of force, whereasothers are less sensitive in comparison. To test this hypothesis, we developeda novel method, where we apply highly localized force to specific protein do-mains and simultaneously measure ion channel activity upon pressure-clampstimulation. For this, we label specific domains within Piezos with magneticnanoparticles and use an external magnetic field to generate a precise mechan-ical force that is highly localized. Simultaneously, we measure Piezo-activationelectrophysiologically. With this approach we have identified domains that aresensitive to application of localized magnetic pulling force and other domainsthat are completely insensitive to the same force.

189-SympMechanosensing at the SurfaceEllen Lumpkin.Physiology & Cellular Biophysics, Columbia University, New York,NY, USA.Skin is innervated by a rich variety of mechanosensitive neurons that triggerdistinct sensations such as pressure, flutter and pain. A growing body of evi-dence indicates that epithelial cells actively partipate in sensation by modu-lating, and even directly exciting, mechanosensitive neurons in healthy skinand pathophysiological conditions. This research aims to unveil how epithelialMerkel cells work in concert with the nervous system to generate discrimina-tive touch sensation. Using mouse genetics and optogenetics, we previouslydemonstrated that Merkel cells have dual roles: they transduce sustained firingthat signals pressure, and boost firing rates of tactile afferents during dynamictouch. Our current studies focus on defining mechanisms of neurotransmissionbetweenMerkel cells and tactile afferents, and elucidating how connections be-tween Merkel cells and mechanosensory neurons remodeled during healthyskin renewal.

190-SympRole of Piezo Ion Channels in MechanosensationArdem Patapoutian1,2.1Molecular & Cellular Neuroscience, The Scripps Research Institute, LaJolla, CA, USA, 2Howard Hughes Medical Institute, La Jolla, CA, USA.Mechanotransduction is perhaps the last sensory modality not understood at themolecular level. Proteins/ion channels that sense mechanical force are postu-lated to play critical roles in sensing touch/pain (somatosensation), sound (hear-ing), sheer stress (cardiovascular function), etc.; however, the identity of ionchannels involved in sensing mechanical force has remained elusive. We iden-tified Piezo1 and Piezo2, mechanically-activated cation channels that are ex-pressed in many mechanosensitive cell types. We demonstrated that Piezosconstitute an evolutionarily conserved family of mechanically-activated cationchannels. We further showed that Piezo1 is a shear stress sensor in vascularendothelial cells, while Piezo2 is the major transducer of mechanical forcesfor touch sensation in mice. Current efforts focus on understanding structure-function relationship of Piezo proteins, and elucidating their physiological rolesin various biological processes and diseases that depend on mechanotransduc-tion. Finally, we are searching for novel mechanosensors.

191-SympMscS-like Mechanosensitive Ion Channels: Modular Sensorsand Reporters of Membrane TensionElizabeth Haswell, Debarati Basu, Eric S. Hamilton, Grigory Maksaev,Matthew Mixdorf, Ivan Radin, Ryan Richardson, Angela M. Schlegel,Eric Schultz, Yanbing Wang.Biology, Washington University, Saint Louis, MO, USA.A long-standing question is how biological systems sense and perceivemechanical signals such as osmotic pressure, gravity, and touch. One well-established molecular mechanism for force sensing is the activation of mecha-nosensitive (MS) ion channels. The Mechanosensitive channel of Smallconductance (MscS) from E. coli functions as a hypo-osmotic safety valve,opening in response to increased membrane tension and preventing cellular

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rupture. Genes predicted to encode MscS homologs are found in genomesfrom all three kingdoms of life. We have been characterizing the structure,function, and regulation of ten MscS-Like (MSL) proteins in the model plantArabidopsis thaliana. Based on their modest homology to MscS and high topo-logical diversity, we have proposed that MSLs might (1) sense and respond tosources of membrane tension other than environmental hypo-osmotic shock;(2) be regulated by mechanisms in addition to membrane tension; and (3) signalin ways that are separable from ion flux. Evidence in support of all three ofthese hypotheses will be presented. I will also discuss our current effort toengineer novel biosensors based on MSL proteins.

Symposium: Cancer Cell Biophysics

192-SympAutocrine Role of Exosomes in Cellular Adhesion, Migration, and InvasionAlissa Weaver.Vanderbilt University School of Medicine, Nashville, TN, USA.Exosomes are late endosome-derived extracellular vesicles that carry multiplemotility-promoting cargos. The exact role of exosomes in promoting motilityunder diverse circumstances is unclear. Likewise, whether the process of exo-some secretion is essential for cell migration is an open question. We havefound that exosome secretion promotes directionally persistent migration ofcancer cells through tissues. Intravital live imaging reveals that exosome secre-tion promotes a polarized cell morphology and stabilization of lamellipodialprotrusions. Mechanistic in vitro experiments demonstrate that extracellularmatrix (ECM) is an important exosome cargo that enhances motility speed.Consistent with a major role for ECM as a critical motility-promoting exosomecargo, live imaging experiments reveal that exosomes are secreted at the sitesof nascent adhesions and promote their assembly. Additional experiments indiverse in vitro environments indicate a role for additional exosome cargoesin directional sensing. Altogether, our results indicate that autocrine secretionof exosomes is a critical feedback mechanism that promotes cancer cellmotility.

193-SympQuantitative Systems Biology Studies Reveal a Nested Relay Mechanismfor TGF-Beta SignalingJianhua Xing.Computational and Systems Biology, University of Pittsburgh, Pittsburgh,PA, USA.Cells need to reliably sense and transmit information about extracellular stim-uli, and cancer cells typically have dysregulated signal transduction. Inthis talk I will discuss TGF-b induced expression of transcription factorSnail1, and the latter regulates many processes such as the epithelial-to-mesenchymal transition. A widely assumed mechanism states that TGF-bleads to phosphorylation of Smad2 and Smad3 (pSmad2/3), which enter thenucleus and activate Snail1 expression. However, both our quantitative mea-surements and thorough parameter space analysis show that the mechanismdoes not hold. Instead we proposed and confirmed a novel nested relay mech-anism that a relay from pSmad2/3 to Gli1 initializes and maintains Snail1expression, while another relay from active to inhibitory phosphorylation ofGSK3 smoothens the pSmad2/3-Gli1 transition. Our model analysis suggeststhat the network motif allows a cell to respond differentially to differentstrength and duration of TGF-b. To characterize this motif, we generated cellslabeled with fluorescence protein reporters using the CRISPR technique, andare performing live cell imaging studies. This work demonstrates the impor-tance of integrated and iterative computational and quantitative experimentalstudies on elucidating the regulatory mechanism of a complex biologicalprocess.

194-SympModels for Cancer Cell Motility: Regulation and SignalingLeah Edelstein-Keshet.Department of Mathematics, University of British Columbia, Vancouver,BC, Canada.A common feature of cancer cells is aberrant motility, leading to invasion andmetastasis. I will survey several modeling projects in which we have exploredthis aspect of malignant cells. First, I will describe the burst of actin-based pro-trusion that is facilitated by cofilin in mammary carcinoma cells (joint workwith N. Tania and J. Condeelis). Next, I will discuss the intracellular signalingbased on Rho GTPases in melanoma cells that orchestrates their motilityphenotype (joint with WR Holmes, JS Park, and A Levchenko). Finally, Iwill describe some recent efforts at understanding cell-cell signaling that coor-dinates the migration of mammary carcinoma cells towards blood vessels (jointwith H Knutsdottir and J Condeelis).

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195-SympPhysical Interactions in Ductal Microinvasions: Integrating Histology withComputational ModelingKatarzyna A. Rejniak1,2.1Mathematical Oncology, Moffit Cancer Center & Research Institute, Tampa,FL, USA, 2Oncologic Sciences, University of South Florida, Tampa,FL, USA.Progression from a ductal carcinoma in situ (DCIS) to an invasive tumor is amajor step initiating a devastating and often lethal metastatic cascade. Oneof the first steps in this process is the development of ductal microinvasions,i.e., small cohorts of tumor cells that breach the basement membrane surround-ing the duct, and migrate through the extracellular matrix (ECM). At this point,for the first time, the epithelially-derived tumor cells engage in a direct physicalcontact with the extracellular matrix and the stroma. We combined single cell-based model of the tumor-ECM interactions and ECM remodeling, and image-based analysis of the cellular biophysico-chemical features as determined frompatients’ histology samples of DCIS. Using this model we showed how changesin the local microenvironmental niche near the DCIS edge enable initiation andprogression of ductal microinvasions. Of particular interest are the biomechan-ical interactions between the cells and the ECM fiber structure, and microenvi-ronmental features that define niche prone to microinvasions. These findingscan be compared to the patient histology samples and help define criteria forfuture development of new prognostic methods and therapeutic interventionsby targeting the tumor niche.

Platform: Voltage-gated K Channels andMechanisms of Voltage Sensing and Gating I

196-PlatProbing theMovement of the Ball and Chain during N-type Inactivation inKv ChannelsTanja Kalstrup, Roshan Pandey, Rikard Blunck.University of Montreal, Montreal, QC, Canada.N-type inactivation is a mechanism in certain Kv channels where the N-termi-nal peptide occludes the pore upon depolarization resulting in block of ioniccurrents (ball-and-chain). Numerous mutational studies have characterizedN-type inactivation functionally, while X-ray crystal structures have yet toinclude the ball-and-chain structure. It still remains unknown how far the N-ter-minus travels during N-type inactivation and where it is located in the restingstate. Does it reside in a fixed position near the T1 window or is it randomlyfloating in the cytosol? By incorporating a fluorescent unnatural amino acid(Anap) into the N-terminus and into receptor sites (T1 window and S4-S5linker) we directly tracked the movement of the ball peptide using voltageclamp fluorometry in Xenopus oocytes. Voltage dependency and kinetics ofball-peptide movement correlated with N-type inactivating currents. Further-more, we address the question as to whether the fluorescence changes arecaused by polarity changes or quenching, by measuring the Anap emissionspectra at both resting and activated state.

197-PlatMolecular Mechanisms of the Voltage-Dependent Potentiation of KCNHPotassium ChannelsGucan Dai, William Zagotta.Physiology and Biophysics, University of Washington, Seattle, WA, USA.EAG-like (ELK, Kv12) voltage-gated potassium channels in the KCNH channelfamily are primarily and abundantly expressed in the brain. Deletion of the ELKgene leads to hyperexcitability of hippocampal neurons, epilepsy and alteredcognitive functions. One structural feature of KCNHchannels important for chan-nel function is an interaction between the N-terminal EAG domain and the C-ter-minal cyclic nucleotide-bindinghomologydomain (CNBHD).Herewe studied anorthologous zebrafish ELK channel (zELK) using patch-clamp recording andpatch-clamp fluorometry (PCF). We noticed that a depolarizing prepulse potenti-ated the zELK channels in both the current amplitude and the voltage sensitivity.We named this phenomenon voltage-dependent potentiation (VDP) and found itdeveloped and recovered within hundreds of milliseconds. Using direct applica-tion of diC8-PI(4,5)P2 and specific hydrolysis of PI(4,5)P2 by rapamycin-recruitable lipid 5-phosphatase, we demonstrated that, PI(4,5)P2 inhibits zELKchannels, and its depletion suppresses the VDP. In addition, the VDP can bemanipulated by multiple structural perturbations including removing, mutatingor replacing the intracellular N-terminal EAG domain as well as the C-terminalCNBHD. Further, combining transition metal ion FRET and incorporation of afluorescent noncanonical amino acid L-Anap, wemeasured the distances betweenpositions in theEAGdomain andCNBHDduringvoltage-dependent channel acti-vation and deactivation. We found there was an apparent change in the distancebetween the EAG domain and CNBHD with kinetics that match the on- and

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off-rates of VDP. This change in the distance between the N- and C-terminal re-gions was abolished when PI(4,5)P2 was hydrolyzed, and the simultaneous VDPwas eliminated. Collectively, we propose that the activation of zELK channels in-volves a rearrangement of the direct interaction between theN- andC-terminal re-gions, which transitions the channel to a potentiated state.

198-PlatBK Channel Gating-Ring Voltage Dependence MotionsPablo Miranda1, Teresa Giraldez2, Miguel Holmgren1.1National Institutes of Neurological Disorders and Stroke, National Institutesof Health, Bethesda, MD, USA, 2Biomedical Sciences, Medical School,Center for Biomedical Research of the Canary Islands (CIBICAN),University of la Laguna (ULL), La Laguna, Spain.The open probability of Large Conductance Voltage and Calcium dependentpotassium (BK) channels is regulated allosterically by change in the transmem-brane voltage and intracellular concentration of divalent ions (Ca2þ and Mg2þ).BK channels are formed as tetramers of alpha subunits. The ‘‘divalent sensor’’resides within the gating-ring, a large tetrameric structure formed by eightRegulator of Conductance of Potassium (RCK) domains, two from each subunit.EachRCKdomain (RCK1 andRCK2) contains a high affinity-Ca2þbinding site.Using Patch-clamp fluorometry, we have shown large changes in FRET signalswithin the gating-ring in response to Ca2þ and voltage. Here we investigate theorigins of the voltage dependent motions. We tested manipulations that mostlychange the BK channels’ relative probability of opening and in all cases thevoltage dependence of the FRET signals remained within the voltage range ofwild-type. However, mutations in the voltage sensor that are known to shiftthe charge displacement currents have a parallel shift of the gating ring’s mo-tions. These result suggest that the conformational changes of the gating ringare coupled to the voltage sensor, and could be relevant in the allosteric modu-lation of the BK channels.

199-PlatInhibition of BK Channels by Strong Extracellular AcidificationYu Zhou, Xiaoming Xia, Christopher J. Lingle.Washington University School of Medicine, St. Louis, MO, USA.Voltage- and Ca2þ-dependent BK-type large conductance Kþ channels regulatemembrane excitability and homeostasis by providing negative-feedback controlfor local [Ca2þ] in a wide variety of tissues and organelles. Some of these tissuesor organelles, such as distal nephron and lysosome, are exposed to extreme acid-ity (pH<5) at physiological or pathological conditions. Such strong extracellularacidificationmay interfere with BK channel function by neutralizing the negativecharges of key acidic residues on the extracellular side of BK channels. How-ever, little is known about the behavior of BK channels at such acidic conditions.Our results show that BK activation is strongly inhibited at extracellular pHlower than 5. At pH 4 the G-V relationship of BK is positively shifted bymore than 100 mV. The inhibition is largely caused by neutralization of severalacidic residues in the BK voltage sensor domain (VSD), including D133, D147and D153, some of which have been identified to be important for the normalfunction of BK VSD. Since the transmembrane domain of BK channels sharessimilar overall organization with many other voltage-gated ion channels suchas the voltage-dependent Kþ channels, the principle underlying strong extracel-lular acidification-induced inhibition in BK channels may also be applicable tothese channels. Supported by NIH GM118114.

200-PlatSingle-Molecule Fluorescence Imaging of low Affinity Binding Interactionsin Pacemaker ion ChannelsMarcel P. Goldschen-Ohm1, David S. White1,2, Vadim A. Klenchin1,Randall H. Goldsmith2, Baron Chanda1.1Neuroscience, University of Wisconsin, Madison, WI, USA, 2Chemistry,University of Wisconsin, Madison, WI, USA.Molecular recognition is a cornerstone of cellular signaling processes. How-ever, most observations of binding rely on ensemble measurements thataverage over and thereby obscure the individual underlying steps. Single-molecule fluorescence can reveal these dynamic events, but is traditionallylimited to low concentrations of fluorophores only suitable for studying highaffinity reactions. Here, we show how a combination of fluorescence resonanceenergy transfer (FRET) and zero-mode waveguide nanofabricated devices canbe used to study single-molecule association dynamics at the micromolar tomillimolar concentrations necessary for many relevant signaling pathways.As an exemplar system, we apply this approach to cyclic nucleotide bindingat its receptor domain from hyperpolarization cyclic nucleotide-gated (HCN)ion channels. Our observations reveal the underlying dynamic events duringcyclic nucleotide binding: an initial encounter complex when the binding siteis in its receptive state, followed by an isomerization of the bound complexthat traps the ligand.

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201-PlatInvestigating Ligand Binding to HCN Channels by Surface PlasmonResonancePurushottam Tiwari1, Aykut Uren1, Tinatin I. Brelidze2.1Oncology, Georgetown University School of Medicine, Washington, DC,USA, 2Pharmacology and Physiology, Georgetown University School ofMedicine, Washington, DC, USA.Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channelscontrol cardiac and neuronal rhythmicity. HCN channels contain cyclic nucle-otide-binding (CNB) domain in their C-terminal region linked to the pore-forming transmembrane segment with a C-linker. The C-linker couples theconformational changes caused by the direct binding of cyclic nucleotides inthe CNB domain to the pore opening. Surface plasmon resonance (SPR) is apowerful biophysical tool for quantitatively investigating ligand-protein andprotein-protein interactions. Here we used SPR to detect ligand binding to theisolated C-linker/CNB domain of HCN channels. The isolated C-linker/CNBdomains of wild-type (WT) and L586W mutant HCN2 channels were immobi-lized on a NTA sensor chip and cAMP was injected over the protein coated sur-face. The mutant C-linker/CNB domain has been used before for ligand bindingstudies based on the changes in the fluorescence of the introduced tryptophanupon ligand binding. From the cyclic nucleotide concentration dependent SPRresponses, we determined the binding affinity for cAMP to be 6.3 þ 2.6 mMfor theWT and 10.8þ 2.3mM for the mutant C-linker/CNB domains. The bind-ing affinity determined for the mutant L586W C-linker-CNB domains deter-mined with SPR is in agreement with the binding affinity of 13 þ 2 mMdetermined with the fluorescence-based method. These results indicate thatSPR is well suited for the detection of binding of known HCN channel ligands.Therefore, SPR can be used to identify novel HCN channel regulators for treat-ment of diseases associated with abnormal functions of these channels, such asepilepsy and cardiac arrhythmias.

202-PlatDrug Interaction at the Lipid Bilayer-Potassium Channel InterfaceNina Ottosson1, Malin Silvera Ejneby1, Xiongyu Wu1, Samira Yazdi1,Peter Konradsson1, Erik Lindahl2, Fredrik Elinder1.1Linkoping University, Linkoping, Sweden, 2Stockholm University,Stockholm, Sweden.The naturally occurring resin acid dehydroabietic acid (DHAA) opens avoltage-gated K channel by shifting the voltage dependence of the channel acti-vation to more negative voltages. We synthesized 140 DHAA-derivatives andfound that subtle alterations in the chemical structure of DHAA can have alarge effect on the channel opening. Several of the channel-opening DHAA de-rivatives reduced excitability in dorsal root ganglion neurons, suggesting a pos-sibility to act against diseases with increased cellular excitability. Here wesearched for the mechanism and site of action for the DHAA derivatives. Weexpressed the non-inactivating Shaker K channel in Xenopus oocytes and stud-ied the ion currents with a two-electrode voltage clamp technique. By utilizingthe ILT-mutant, which separates the final channel-opening transition from theearlier transitions, we found that the DHAA derivatives had an almost isolatedeffect on the channel-opening transition. 100 mM of one DHAA derivativeaffected this last transition by �65 mV. We also found that the charge profileof the voltage sensor S4 was critical for the effect. Addition of positivelycharged arginines in specific positions increased the channel opening effectsof the negatively charged DHAA derivatives, while addition of arginines onthe opposite side of the S4 helix had opposing effects, suggesting that theDHAA derivatives support or prevent rotation of S4 to support or prevent chan-nel opening. In general, the DHAA derivatives affected channel closing kineticbut not channel opening kinetic suggesting a rather specific effect and site ofaction. In-silico docking, molecular dynamics, and mutational analyses sug-gested a binding site in the cleft between the transmembrane segments S3and S4. We conclude that certain resin-acid derivatives bind at the interface be-tween the lipid bilayer and S3 and S4 of the channel’s voltage-sensor domain,to electrostatically facilitate K channel opening.

203-PlatSpectroscopic Studies of a Bacterial Cyclic Nucleotide-Gated Ion ChannelZachary M. James1, Eric G. Evans2, William N. Zagotta1.1Physiology & Biophysics, University of Washington, Seattle, WA, USA,2Chemistry, University of Washington, Seattle, WA, USA.Cyclic nucleotide-gated (CNG) channels are members of the Kv channel super-family and play a crucial role in the phototransduction and olfactory transductionpathways, where they generate the initial electrical signal following sensorytransduction. CNG channels are directly regulated by cyclic nucleotides, whichbind to a C-terminal cyclic nucleotide-binding domain (CNBD) connectedto the pore by an intervening C-linker domain. Mutagenesis and crosslinking

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experiments indicate that the C-terminal region (C-linker/CNBD) undergoes aconformation change to transduce cyclic nucleotide binding into channel activa-tion, though the exact nature of this change remains unclear. To address thisquestion, we first sought a stable and well-behaved bacterial CNG channel suit-able for spectroscopic studies. We used fluorescence-detection size-exclusionchromatography to screen a library of bacterial orthologs, which identifiedseveral candidates that could be expressed and purified at high levels. Fluxstudies of liposome-reconstituted channels demonstrated that these orthologsare fully functional and are activated by cyclic nucleotides. We then mutatedthe endogenous Cys residues in these channels before introducing Cys residuesthroughout the C-terminal region for spin label and fluorophore attachment.Inter-subunit double electron-electron resonance (DEER) measurements willbe used to detect large-scale structural changes in these channels induced bycyclic nucleotide binding, while transition metal FRET (tmFRET) will be usedto characterize subtle, localized changes to channel structure following activa-tion. Our ultimate goal is to use these two spectroscopic techniques to charac-terize small- and large-scale rearrangements induced by cyclic nucleotidebinding, allowing us to generate a complete structural model for CNG channelactivation.

Platform: Protein Stability, Folding, andChaperones I

204-PlatIntegrated In Vivo and In Silico Studies of Cotranslational Protein Foldingas a Function of Translation RateIan M. Walsh1, Shuxiang Li2, Adrian H. Elcock2, Patricia L. Clark1.1Department of Chemistry & Biochemistry, University of Notre Dame, NotreDame, IN, USA, 2Carver College of Medicine, University of Iowa, Iowa City,IA, USA.In order for a protein to exhibit its proper function, it must first fold correctly.Most of what we know about protein folding comes from in vitro studies ofsmall, single-domain proteins. However, larger and more complex proteinsoften fail to fold efficiently in vitro, yet fold to high yield in cells. One strikingdifference between protein folding in vitro and in vivo is how folding begins.In vitro, protein folding begins from an enormous ensemble of random confor-mations of full length polypeptide chains, while in vivo a protein can begin tofold as it is synthesized by the ribosome. Vectorial appearance of the nascentprotein chain during translation is a universal feature of every protein in the pro-teome, yet its effect on protein folding is poorly understood. Our lab has createdYKB, a protein construct with two mutually exclusive native states (YK-B andY-KB), to study the effects of translation on folding mechanisms. Synonymousmutations to YKB alter the ratio of the YK-B and Y-KB native structures, yethave identical protein sequences. From this we can infer that vectorial appear-ance affects YKB folding, but the ratios provide little molecular-level detail.We have developed a coarse-grain computational model to simulate YKBcotranslational folding. This model accurately reproduces experimental resultsfor YKB refolding in vitro and co-translational folding in vivo. These simula-tions provide us with details that cannot be learned through experiments,including specific molecular trajectories and the effects of altering translationrate on the YKB folding mechanism. Integrating information gleaned from sim-ulations with targeted experimental studies will enable unprecedented insightinto in vivo folding mechanisms, particularly the effects of varying translationrate on cotranslational folding and overall folding efficiency.

205-PlatThe Ribosome Alters the Folding of a Multidomain Nascent ProteinLisa Alexander, Daniel Goldman, Ignacio Tinoco, Carlos J. Bustamante.UC Berkeley, Berkeley, CA, USA.Protein folding is often studied in the context of full-length polypeptides in solu-tion. However, the post-translational folding pathway may not recreate thepathway of folding when a protein is being synthesized by the ribosome. The ef-fects of the ribosome and the rate of translation on folding are notwell understood,partially due to the difficulty of probing the nascent chain without also affectingthe ribosome. Using optical tweezers, we can look in detail at the nascent chainat the single-molecule level with minimal perturbation to the ribosome. Previousstudies have shown that the ribosomal surface can act electrostatically to slow thekineticsof folding.Those studies useda linker tovary the distance fromthe surfaceand did not look at changes in sequence availability or multidomain proteins. Tofurther understand cotranslational folding, we are studying the folding pathway ofa two-domain calcium-binding protein calerythrin. Our results show that the full-length protein in solution folds robustly through a C-domain intermediate, buttruncated versions of the protein can fold to an N domain or a misfolded state.During translation, the C-terminal residues required for native folding are notyet available, but the other observed intermediates could fold, in principle. By

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observing the folding at various sequence positions, and thus nascent chainlengths, we found that the ribosome can not only slow folding, but can also affectthe unfolding rates, particularly increasing the unfolding rate of the misfoldedstate. The ribosome thus prevents the formation of this unproductive state viatwomechanisms: decreasing the probability of folding and decreasing the stabilityof the folded state. This study gives further insight into the importance of the ribo-some to regulate protein structure, and opens up new questions about the interplaybetween elongation and folding.

206-PlatStructural Investigation of an Immunoglobulin Domain on the Ribosomeusing NMR SpectroscopyAnais M.E. Cassaignau1, Helene M.M. Launay1, Christopher A. Waudby1,Tomasz Wlodarski1, Maria-Evangelia Karyadi1, Amy L. Robertson1,Xiaolin Wang1, Carlo Camilloni2, Michele Vendruscolo2,Cheryl A. Woolhead3, Lisa Cabrita1, John Christodoulou1.1Institute of Structural and Molecular Biology, University College London,London, United Kingdom, 2Department of Chemistry, University ofCambridge, Cambridge, United Kingdom, 3Institute of Molecular, Cell andSystems Biology, College of Medical, Veterinary and Life Sciences,University of Glasgow, Glasgow, United Kingdom.Successful protein folding is central to all biological cellular processes with alarge portion of the proteome able to begin to acquire its three-dimensionalstructure in a co-translational manner during its biosynthesis on the ribosome.The vectorial emergence of the nascent polypeptide from the exit tunnel and itsattachment to its parent ribosome results in differences between the details ofthe folding process of isolated polypeptides and that on the ribosome.We have developed a strategy to enable the study of co-translational folding us-ing solution-state NMR spectroscopy, the only technique able to characterize thisdynamic process at atomic resolution. Using isotopically-labelled ribosome-nascent chain complexes (RNCs), we have determined a high-resolution, struc-tural description of protein folding on the ribosome via snapshots that mimic theemergence of an immunoglobulin-like domain within a multidomain protein.Our NMR results reveal the structure and dynamic features of how conforma-tional space is sampled by a fledgling nascent polypeptide as it converts intoits folded state and demonstrate that the entire immunoglobulin domain has tobe emerged from the tunnel before native folding is possible. These findingscontrast with analogous studies of C-terminal truncations of this domain, indi-cating significant differences between folding on the ribosome and that in bulksolution. The ribosome itself is shown to influence the process of folding; weuse a combination of protein engineering and NMR dynamics to describeresidue-specific sites of nascent-chain-ribosome interactions of different magni-tude of strength, and chemical shift-restrained MD simulations of RNCs to un-derpin our spectral observations. The studies presented are providing the firsthigh-resolution insights of these fundamental processes and begin to shape ourunderstanding of the energy landscape sampled by a nascent chain on the cuspof initiation of folding on the ribosome.

207-PlatAccurate Prediction of Cellular Co-Translational Folding IndicatesProteins can Switch from Post- to Co-Translational FoldingDaniel A. Nissley.Chemistry, The Pennsylvania State University, University Park, PA, USA.The rates at which domains fold and codons are translated are important factorsin determining whether a nascent protein will co-translationally fold and func-tion or misfold and malfunction. Here we develop a chemical kinetic model thatcalculates a protein domain’s co-translational folding curve during synthesisusing only the domain’s bulk folding and unfolding rates and codon translationrates. We show that this model accurately predicts the course of co-translationalfolding measured in vivo for four different protein molecules. We then makepredictions for a number of different proteins in yeast and find that synonymouscodon substitutions, which change translation-elongation rates, can switchsome protein domains from folding post-translationally to folding co-transla-tionally—a result consistent with previous experimental studies. Our approachexplains essential features of co-translational folding curves and predictshow varying the translation rate at different codon positions along a transcript’scoding sequence affects this self-assembly process.

208-PlatProtein Sculpting: Probing the Interplay between the Ribosomeand Molecular Chaperones in Protein Folding in the CellRayna M. Addabbo1,2, Matthew D. Dalphin1,2, Yue Liu2,Miranda F. Mecha1,2, Silvia Cavagnero1,2.1Biophysics Graduate Program, University of Wisconsin-Madison, Madison,WI, USA, 2Department of Chemistry, University of Wisconsin-Madison,Madison, WI, USA.

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Understanding the way in which proteins fold inside the cell is of fundamentalimportance to both biology and medicine. In the E. Coli cell, proteins are syn-thesized on the ribosome in the presence of molecular chaperones, TriggerFactor and DnaK. While both the ribosome and molecular chaperones areknown to be important to protein folding in the cell, it has been nearly impos-sible to isolate their independent roles in the folding process since cells die attemperatures over 30C when Trigger Factor and DnaK are deleted simulta-neously. In this work, we are able to access this challenging experimental con-dition and study the interplay between the ribosome and chaperones bysynthesizing a model globin protein in a bacterial cell-free system derivedfrom a Trigger Factor- deleted cell strain in the presence of an in-house-designed peptide inhibitor of DnaK. Surprisingly, we find that translationthrough the ribosome is sufficient to grant solubility to the full-length newlysynthesized protein. Ongoing studies based on time-resolved fluorescenceanisotropy in the frequency-domain and multidimensional nuclear magneticresonance yield additional details on the quality of the de novo-produced pro-tein, and on the intriguing interplay between the ribosome and chaperones inensuring its correct folding.

209-PlatTrigger Factor Boosts the Work Done by Protein Folding under ForceShubhasis Haldar, Rafael Tapia-Rojo, Julio M. Fernandez.biological science, Columbia University, New York, NY, USA.Protein folding under force generates mechanical work, which is integral toseveral biological processes including muscle contraction, proteasomal degra-dation, and protein translation. As molecular chaperones affect protein folding,we hypothesized that chaperones would modulate the mechanical work gener-ated from protein folding. Trigger Factor (TF) is a model chaperone from E.coli, which is found intracellularly in both ribosome-associated and free cyto-solic states. Here, we apply Magnetic Tweezers-based single molecule forcespectroscopy to investigate the effect of TF in the mechanical folding of a sub-strate, protein L (PL). We find that TF increases the folding probability by up to40% at ~8 pN. The half-maximal TF activity, K1/2, is highly force dependent,ranging from 0.6 mM at 6.2 pN to 27.5 mM at 8.9 pN. The functional conse-quence of this TF-assisted mechanical folding is an increase in the effectivework generated, calculated as the product of the force, the PL folding contrac-tion length, and the TF-dependent folding probability. The maximal activity ofTF (at 10 mM and 8.4 pN) contributes 40 zJ to the work done by protein folding,comparable to the energy delivered by myosin motors. 85% of the mechanicalcontribution from TF is achieved between 500 nM and 2 mMTF, in the range ofthe chaperon’s cellular concentration. This is the first study of the influence of amolecular chaperone in the work done by protein folding, which could haveimportant consequences in the translation process, helping polypeptide synthe-sis and assisting protein folding under force.

210-PlatResolution of the Time Sequence of Fast Folding Transitionby the ‘‘Transfer-Quench’’ MethodGil Rahamim, Dan Amir, Elisha Haas.Life Sciences, Bar Ilan University, Ramat Gan, Israel.The rates and efficiency of the folding transition of globular protein might beenhanced by early formation of few sub-domain structures. Ultrafast Forsterresonance energy transfer (FRET) based methods are ideal for characteriza-tion of the transient ensembles of refolding molecules. However, each sitespecific labeling modification might affect rates of folding of near neighborstructural elements and thus limit the ability to resolve fine differences in ratesof folding of these elements. Therefore, it is highly desirable to be able tostudy the rates of folding of two or more neighboring sub-domain structuresusing a single mutant in order to facilitate resolution of the order and interde-pendence of such steps. Here we report the development of the ‘‘Transfer-Quench’’ method for measuring the rate of formation of two structuralelements using a single triple-labeled mutant. This method is based onFRET combined with fluorescence quenching. We placed the donor andacceptor at the loop ends’ and a quencher at an a-helical element involvedin the node forming the loop. The folding of the triple labeled mutant is moni-tored at the acceptor emission. The formation of non-local contact (loopclosure) increases the time dependent acceptor emission while the closureof the labeled helix turn reduces this emission. The method was applied ina study of the folding mechanism of the B domain of staphylococcal proteinA. Only natural amino acids were used as probes and thus possible structuralperturbations were minimized (Tyr and Trp residues as donor and acceptor atthe ends of a long loop between helices I and II, and Cys residue as a quencherfor the acceptor). We found that the closure of the loop is formed with thesame rate constant as the nucleation of helix II in line with the nucleation-condensation model.

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211-PlatMechanically Unfolded Titin Immunoglobulin Domains Refold Faster andMore Accurately in Presence of Chaperone Alpha-B-CrystallinYong Li, Wolfgang A. Linke.Institute of Physiology, Ruhr Univ. Bochum, Bochum, Germany.The spring region of the giant muscle protein titin contains manyimmunoglobulin-like (Ig) domains, which unfold and refold under low (physio-logical) stretch forces. Small heat shock proteins such as alphaB-crystallin(aBC) translocate under physiological or pathological stress to the titin springs.To better understand this protective function we studied the unfolding-refoldingbehavior of an 8-Ig-domain titin construct (I91)8 by single-molecule AFM forcespectroscopy, in the absence/presence of recombinant aBC (pH7; pH6; or pH5)or ‘control’ protein of similar size. Titin Ig domains were unfolded at 175 pN con-stant force applied for a variable ‘‘denature’’ time (tD), then the force was set tozero for a variable ‘‘quench’’ time (tQ) to allow for domain refolding, and finallya ‘‘probe’’ pulse (175 pN; tP = 5 s) was applied to test how many domains had re-folded. Interestingly, Ig domain unfolding kinetics were little affected by aBC.However, upon lowering pH from7 to 6, the refolded fraction (number of refoldedIg domains during tQ indexed to number of unfolded Ig domains during tD)decreased slightly, indicating domain misfolding. At pH5, the refolded fractiondropped by half and ~50%of titin Ig domains showedmisfolding events, an effectindependentof tD (variation, 2 - 40 s). Importantly, aBC (10mMor20mM)normal-ized the refolded fraction to values observed at pH7, whereas control protein hadno suchprotective effect. The refolded fractiondepended stronglyon tQ (variation,0.5 - 10 s), under all experimental conditions. Ig domain refolding kinetics weregreatly slowed by lowering pH from 7 to 5, as quantified on refolded fractionvs. tQ plots, on whichmeans were fit by simple exponentials. Again, aBC normal-ized the refolding kinetics to those observed at pH7 in the absenceof aBC,whereascontrol protein had no such effect.We conclude that aBCspeedsup titin Igdomainrefolding (novel foldase activity!) and protects from domain misfolding, espe-cially under acidic stress, which is frequently encountered in muscle cells.

Platform: Membrane Physical Chemistry I

212-PlatGM1 Softens the Membrane, Induces Domains and Causes SpontaneousTubulation in Giant VesiclesRumiana Dimova1, Tripta Bhatia1, Raktim Dasgupta1,2, Nico Fricke1,Jaime Agudo-Canalejo1, Reinhard Lipowsky1.1Theory & Bio-Systems, Max Planck Institute of Colloids and Interfaces,Potsdam, Germany, 2Raja Ramanna Centre for Advanced Technology,Indore, India.The ganglioside GM1 is present in neuronal membranes at elevated concentra-tions with an asymmetric spatial distribution. It is known to generate curvatureand can be expected to strongly influence the neuron morphology. To elucidatethese effects, we incorporated GM1 into giant unilamellar vesicles (GUVs)made of POPC.We found that even a few mol% of GM1 soften the fluid bilayersignificantly as measured using fluctuation analysis, vesicle electrodeformationand micropipette aspiration. At room temperature and for GM1 fractions at andabove ~5 mol%, we detect the formation of GM1-rich gel-like domains. We usefluorescence microscopy to build a partial phase diagram of the binary mixture(Fricke and Dimova, Biophys. J. 2016, in press). After diluting the vesicle sus-pension with the native buffer, we observe spontaneous formation of nanotubessuggesting desorption of GM1 from the outer membrane leaflet and generationof spontaneous curvature stabilizing the tubes. Employing electroporation ofGUVs, we assess the GM1 asymmetry. The associated spontaneous curvatureis measured using two approaches: micropipette aspiration of GUVs and pull-ing of inward and outward membrane tubes via bead manipulation with opticaltweezers (Dasgupta and Dimova, J. Phys. D. Appl. Phys. 47:282001, 2014).GM1 plays a crucial role in connection with receptor proteins. However, ourresults that GM1 decreases bending rigidity, causes spontaneous curvatureand induces phase separation in the membrane point to an additional importantrole of this ganglioside, namely shaping neuronal membranes. This work is partof the MaxSynBio consortium, which is jointly funded by the Federal Ministryof Education and Research of Germany and the Max Planck Society.

213-PlatInduced Mixing of Phase-Separated Lipid Bilayers by Steric Pressurebetween Adsorbed ProteinsWade Zeno1, Kaitlin E. Johnson2, Darryl Y. Sasaki3, Marjorie L. Longo2.1Biomedical Engineering, University of Texas at Austin, Austin, TX, USA,2Chemical Engineering, University of California, Davis, Davis, CA, USA,3Sandia National Laboratories, Livermore, CA, USA.Our study examined the steric pressure-induced mixing and reversibility ofphase-separated phospholipid regions. It has been previously demonstrated

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that steric pressure among crowded proteins can induce mixing of lipid do-mains. However, prior to our work, the dynamics of this process had yet tobe investigated. The influence of protein steric pressure on dynamics is impor-tant for understanding time-dependent processes in cell membrane-derived bio-materials and highly transient lipid rafts in cells. Therefore we designedexperiments that allowed us to examine the process by which protein stericpressure dissolved lipid membranes. Specifically, fluorescence microscopywas used to examine the targeted binding of proteinaceous particles tophase-separated, supported lipid bilayers. Highly localized binding of theseparticles within the liquid ordered (Lo) domains resulted in a highly crowdedenvironment, leading to a build-up of steric pressure. The alleviation of this ste-ric pressure induced mixing of the Lo domains with the surrounding liquiddisordered (Ld) phase. With the development of a first-principles mass transfermodel, the dynamics of this mixing transition were observed as a functionof the steric pressure among proteins. At sufficiently high steric pressure,rapid dissolution of Lo domains occurred. As steric pressure was decreased,mixing became more gradual and occurred in a step-wise process where smalllipid clusters were initially ejected from Lo domains. Time-dependent dissolu-tion data for Lo domains was in quantitative agreement with the mass-transfermodel. Diffusion coefficients derived from the model indicate that lipids un-dergo diffusion as clusters, rather than single lipids. We also examined systemsin which proteins bound to the Ld phase. Our results strongly suggest thatthe degree of protein crowding and the phase to which proteins are targetedcontrol the rate and mechanism of steric pressure-induced mixing. This phe-nomena is applicable to the development of many biologically derived mate-rials, including high-density arrays, microfluidic networks, and biosensors.Controlled mixing also provides fundamental insights to the impact of proteinson the stability and dynamics of lipid rafts in cells.

214-PlatUnique Modification of Membrane Structure by Lithium: A MolecularDynamics StudyJames Kruczek1, See-Wing Chiu2, Eric Jakobsson2, Sagar A. Pandit3.1University of Florida, Tampa, FL, USA, 2University of Illinois, Urbana, IL,USA, 3University of South Florida, Urbana, IL, USA.Lithium is profoundly different from other ions in the body in multiple ways.Extracellular concentrations of calcium, sodium, potassium, and hydrogenions must be closely regulated for well-being. On the other hand, the bodydoes not closely regulate lithium but rather adapts reasonably well to lithiumconcentration variation of over a thousandfold, in spite of the fact that lithiuminteracts with many biological molecules. This stability coupled with interac-tiveness implies the existence of myriad mutually compensating lithium-modulated processes, which are almost completely not understood. In this studywe explore the interaction of lithium, sodium, potassium, and rubidium withbilayer membranes comprised of POPC, using molecular dynamics simula-tions. Lithium, but not any of the others, forms a long-lasting noncovalentbond simultaneously with the headgroup phosphate and the CO group of theSn2 chain. This interaction significantly alters the structure and the surfacearea of the membrane.

215-PlatMeasuring Lipid Membrane Properties using a MechanosensitiveFluorescence ProbeAdai Colom Diego, Marta Dal Molin, Saeideh Soleimanpour,Emmanuel Derivery, Marcos Gonzalez Gaitan, Stefan Matile, Aurelien Roux.Universite de Geneve, Geneve, Switzerland.To measure the chemical-mechanic states of lipid membranes, once needsvarious tools, many of which being incompatible with cell biology protocols.Applying lessons from nature, we developed a mechanosensitive fluorescentprobe, the twisted dithienothiophene. This push-pull probe, change planariza-tion state in function of his environment, and we have taken full advantageof this mechano-probe potential and we calibrated based on membrane tension,fluidity and different lipid composition by measuring the push-pull fluores-cence lifetime. Likewise, we are able to use this fluorescent probe on life cells,for visualize differences between organelles, as well as to distinguish lipidsproperties among cells cultured on classic plates or in extracellular matrix.

216-PlatDPPC/cholesterol Revisited: Interaction Models to Explain the ExcessHeat Capacity in Unilamellar VesiclesPaulo F. Almeida, Emmanuel Tejada, Faith Carter, Antje Pokorny.Chemistry, Univ North Carolina Wilmington, Wilmington, NC, USA.The binary lipid system DPPC/cholesterol has been studied since the 1970s.Strangely enough, the nature of the interaction between the phospholipid andcholesterol remains unclear. Also strangely enough, for decades, the thermody-namics of these binary mixture have been investigated mainly in multilamellar

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vesicles (MLVs). More recently, giant unilamellar vesicles (GUVs) have beenused, which are excellent to study lipid phase separation, especially by fluores-cence confocal microscopy, but do not easily lend themselves to calorimetry.However, the heat capacity of DPPC across the main phase transition is similarin GUVs and in large unilamellar vesicles (LUVs) but quite different fromthat in MLVs. Much of the attention in the thermodynamics of the phase transi-tion in DPPC/cholesterol has been concerned with understanding the heat capac-ity in MLVs. Here we turn our attention to the DPPC/cholesterol binary systemin LUVs, which we think is a much more relevant type of vesicle to understandthe molecular interactions between DPPC and cholesterol. We compare theexperimental heat capacity (melting) curves in LUVs with the results of MonteCarlo calculations using various models of the interaction between these lipids,including complex formation and simple pairwise interactions.This work has been supported by NSF grant CHE-1464769.

217-PlatCharacterization of the Physiochemical Interactions between LNPs andthe Endosomal Lipids: A Rational Design of Gene Delivery SystemsNandhitha Subramanian, Yoav Atsmon-Raz, Peter D. Tieleman.Department of Biological Sciences & Centre for Molecular Simualtion,University of Calgary, Calgary, AB, Canada.Lipid nanoparticles (LNPs) are a class of materials, with each interacting in acomplex fashion to form specialized bio-containers carrying therapeutic drugsand strands of nucleic acids such as small interfering RNA (siRNA)1. LNPs areat the forefront of the rapidly developing field of nanotechnology with severalpotential applications in drug delivery, clinical medicine, and research. Despiteholding a significant promise for reaching the goal of controlled and sitespecific drug delivery, the engineering of efficient LNPs is limited by thelack of knowledge about the structure of LNPs, the roles of their individualcomponents and the physical interactions with other biomolecules2. Becauseof this, characteristics such as the encapsulation efficiency, polydispersity,and stability of LNPs are not predictable. One of the biggest problems ofLNP gene therapy is the molecular details of the cellular processes that deter-mine the efficiency of intracellular drug delivery is still unclear. Studies haveshown that LNPs enter cells via the endocytic pathway and are engulfed byendosomes. The delivery of siRNA is substantially reduced, as ~70% of theinternalized siRNA undergoes exocytosis through egress of LNPs from lateendosomes/lysosomes3. In this project, we used advanced molecular dynamicssimulations to understand the molecular basis of the structure of LNPs based ontheir composition. Our simulations also provided the first detailed molecular-level view of the interactions of the LNP components with endosomal lipids.Here, we also studied the change in interactions between the LNPs and endo-somal lipids by varying ion concentration and altering pH. Results of our simu-lation will provide a route to the rational design of new LNPs that addresssafety concerns and ensure effective delivery to accelerate the translation ofengineering lipid-based nanoparticles towards the clinic.References1. Semple, S.C., et al. (2010) Rational design of cationic lipids for siRNAdelivery. Nat.Biotechnol. 28,172-1762. Huang, L. and Liu, Y. (2011) In vivo delivery of RNAi with lipid-basednanoparticles. Annu.Rev.Biomed. Eng. 13, 507-5303. Sahay G., et al. (2013) Efficiency of siRNA delivery by lipid nanoparticles islimited by endocytic recycling. Nat.Biotechnol. 31,653-658.

218-PlatTuning Membrane Asymmetry: Controlled Uptake of Negatively ChargedLipids into the Outer Leaflet of LiposomesMarie Markones1, Carina Zorzin1, Louma Kalie1, Sebastian Fiedler2,Heiko Heerklotz2,3.1Institute for Pharmaceutical Sciences, University of Freiburg, Freiburg,Germany, 2Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto,ON, Canada, 3Institute for Pharmaceutical Sciences/BIOSS Centre forBiological Signalling Studies, University of Freiburg, Freiburg, Germany.The presence of negatively charged lipids in the outer leaflets of cellular mem-branes is a key component of many biological membranes. For example, eukary-otic organisms accumulate negatively charged lipids on their extracellularmembrane leaflets during programmed cell death through apoptosis. As a generalfeature, the outer leaflets of bacterial cellular membranes exhibit high contentsof anionic phosphatidylglycerol (PG). Recently, a cyclodextrin-based lipid ex-change assay has been introduced that transports phospholipids from the outerleaflet of donor liposomes to that of acceptor liposomes, thereby generatingmodel membranes of asymmetric lipid composition. In an effort to master andfurther develop the cyclodextrin method, we utilize the zeta potential of mixed1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) liposomes of symmetric and

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asymmetric composition to quantify the incorporation of negatively chargedPOPG into POPC liposomes. The adjustment of cyclodextrin and lipid concen-trations below the maximum lipid-binding capacity of cyclodextrins allows acontrolled POPG transfer without the need of donor liposomes. Furthermore,the zeta potential clearly differentiates between asymmetric and symmetricPOPG incorporation, quantifies the degree of lipid asymmetry, and determinesthe total yield of lipid exchange. Our approach represents an easy to use methodfor the efficient incorporation of negatively charged lipids into the outer mem-brane leaflet of liposomes at physiologically relevant lipid contents.

219-PlatInvestigating the Effects of the Membrane Dipole Field on the Structureand Function of a Model Membrane ProteinCari M. Anderson, Lauren J. Webb.Chemistry, University of Texas at Austin, Austin, TX, USA.Lipid bilayer membranes are composed of hundreds of lipids, sterols and pro-teins, which organize in to a heterogeneous, structural scaffold that controlscritical elements of biological function. The interactions of all of these mole-cules are mostly non-covalent and electrostatic in nature. These interactionsand the diversity of molecules, along with ordered water molecules at thelipid-water interface gives rise to a large electrostatic field that traverses thelipid bilayer. The whole field can be broken down in to three components-transmembrane field, surface field and dipole field. We have extensively stud-ied the membrane dipole field, which propagates from the interior of the lipidbilayer to the lipid head group-water interface, using vibrational Stark effectspectroscopy paired with molecular dynamics simulations. With our knowl-edge of how the dipole field can be altered due to a change in lipid bilayercomposition, we are exploring what role this field plays in controlling the trans-port of ions through a membrane via transmembrane protein channels (TPCs).Gramicidin has been accepted as a good model for TPCs due to its ability toselectively transport ions with a þ1 charge through the pore it creates whenin its channel conformation. Using this well characterized model TPC wehope to elucidate how the structure and function can be changed via non-covalent interactions with other lipids, water molecules and sterols, specificallyarising from the membrane dipole field. Using VSE spectroscopy, we havebeen able to begin to measure how gramicidin alters the dipole field of apure phospholipid bilayer in the form of small unilamellar vesicles (SUVs).We are studying how the transport of þ1 cations through the gramicidin chan-nel and the structure of the TPC are affected by both small and large perturba-tions of the electrostatic field by intercalating sterols in to the lipid bilayer.

Platform: Kinesins and Dyneins

220-PlatLis1 has Two Distinct Modes of Regulating Dynein’s MechanochemicalCycleMichael A. Cianfrocco, Morgan E. DeSantis, Zaw M. Htet, Phuoc T. Tran,Andres E. Leschziner, Samara L. Reck-Peterson.Cellular and Molecular Medicine, University of California - San Diego,La Jolla, CA, USA.Cytoplasmic dynein-1 (‘‘dynein’’) is a minus-end-directed microtubule-basedmotor that couples ATP hydrolysis to force generation to move diverse cargos.Dynein is a single-chain AAAþATPase that contains 6 AAAþ domains, whereAAA1-4 bind nucleotide and AAA1 drives the motor. Lis1 is a conserved andubiquitous dynein regulator. Previously, we showed that Lis1 binds to dynein atAAA4 and causes dynein to slow down and remain attached to microtubules,even in the presence of ATP, which usually releases dynein from its track. Inter-estingly, while AAA3 is occupied by ADP when dynein is walking, either theabsence of nucleotide or the presence of ATP lead to a motor that behaves likeits Lis1-regulated state. This observation led us to hypothesize that Lis1 actsthrough AAA3. To test this we determined how the nucleotide state at AAA3affects Lis1’s regulation of dynein. When AAA3 is nucleotide-free, Lis1increases dynein’s microtubule binding affinity, as we had previously observed,Surprisingly, however, when AAA3 contains ATP, Lis1 has the opposite effect,leading to dynein’s detachment from microtubules. High-resolution cryo-elec-tron microscopy structures of dynein-Lis1 complexes revealed the basis forthese puzzling effects. While a single Lis1 beta propeller (Lis1 is a dimer) bindsto dynein in the AAA3-(no nucleotide) state, a second Lis1 beta propeller isbound to the motor in the AAA3-ATP state. This novel second site is locatedon dynein’s coiled coil ‘‘stalk’’, which connects dynein’s motor domain to itsmicrotubule binding domain. Importantly, the sequence of this site is conservedonly in those dyneins that are regulated by Lis1. Our work revealed that Lis1can act either as a microtubule anchor or a release factor for dynein, dependingupon the nucleotide occupancy at AAA3. We propose a new model for howLis1 serves as a dual regulator of dynein activity in cells.

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221-PlatThe Structure of Complete Human Dynein-1 and its Mechanismof ActivationKai Zhang, Helen Foster, Andrew Carter.Structural Studies, MRC Laboratory of Molecular Biology, Cambridge,United Kingdom.Cytoplasmic dynein-1 is a 1.4 MDa motor protein, which performs essentialcargo transport to the minus end of microtubules. Long distance movement ofsingle dynein-1molecules is induced by binding to its 23-subunit cofactor dynac-tin and a cargo-adaptor protein. Here we present the cryo-EM structure of iso-lated human dynein-1 in its inhibited, phi-particle conformation. We achieveda resolution of 3.8A for the motor domains and 8.4A in the highly-flexible tailregion. We reveal the architecture of the complete complex; identify all subunits,build helices in the tail and a full atomic model for the human dynein-1 motordomains. Mutagenesis to disrupt the interface between the two motor domainsis not sufficient to activate dynein-1. Instead dynactin is required to orient themotors in a parallel fashion so that they can interact correctly with microtubules,thus explaining the structural mechanism of dynein-1 activation.

222-PlatThe Power Stroke Distance of Human Cytoplasmic DyneinYoshimi Kinoshita1, Taketoshi Kambara2, Kaori Nishikawa1,Motoshi Kaya1, Hideo Higuchi1.1Grad Sch of Sci, The Univ Tokyo, Tokyo, Japan, 2QBiC, RIKEN,Osaka, Japan.Cytoplasmic dynein is a motor protein moving along microtubules, and playsimportant roles in vesicle transport and mitosis. To understand the molecularmechanism of dynein motility, we measured the efficiency of FRET from dyneinring-BFP to linker-GFP, and the displacement driven by single-headed dyneininteracting with microtubules by optical tweezers. The efficiency and theapparent stroke displacement depend on ATP concentration. The low efficiencyand displacement at low ATP concentration indicate no conformational changeof dynein at no ATP binding (apo) state that is predominant at low ATP concen-tration. With increasing ATP concentration, population of apo state decreasesand that of the pre-power stroke state such as dynein ADP-Pi should increase.Dynein at the pre-power stroke state will generate the power stroke at bindingto microtubule. Therefore, the displacement increased with ATP concentration.This is the first obtained interesting result and has not been reported for myosinand kinesin power strokes. High FRET efficiency and distance (8-9 nm) at satu-rated concentration of ATP indicate that the 8-9 nm power stroke distance isgenerated by swing of linker. This is supported by the result that the dyneindeleted at the loops interacted with the linker did not generate the power stroke.The power stroke driven by the conformational change of dynein linker will bethe fundamental mechanism of dynein motility.

223-PlatIntracellular Cargo Transport by Single-Headed Kinesin MonomersKristin I. Schimert1, Breane G. Budaitis2, Kristen J. Verhey1,3.1Biophysics Program, University of Michigan, Ann Arbor, MI, USA,2Cellular and Molecular Biology Program, University of Michigan, AnnArbor, MI, USA, 3Department of Cell and Developmental Biology,University of Michigan, Ann Arbor, MI, USA.Kinesins are motor proteins that transport cargoes along microtubules in eu-karyotic cells. The canonical picture of kinesin motility is that the two motordomains of a dimer step hand-over-hand in a tightly coordinated manner. How-ever, it remains controversial whether kinesin-3 family motors function asmonomers or dimers. We compared truncated, single-headed monomeric mo-tors to their dimeric forms using cargo transport assays in cells. We showthat surprisingly, teams of monomeric motors across different kinesin familiesare able to transport cargoes against high load. Strikingly, monomeric transportcapability varies widely even within a single kinesin family. Future work willinvestigate the structural and mechanical features that enable select monomericmotors to cooperatively transport cargoes. These findings will provide insightinto the minimal requirements for collective kinesin motility and intracellularcargo transport.

224-PlatKinesin-1 Cargo Transport through Dense Microtubule NetworksJoelle A. Labastide1, Reilly K. Curtin2, Jennifer L. Ross1.1Phyiscs, University of Massachusetts Amherst, Amherst, MA, USA,2Biology, University of Massachusetts Amherst, Amherst, MA, USA.Essential motor transport processes occurring in living cells rely heavily on traf-ficking of cargoes by kinesin motors through dense microtubule networks.Kinesin-1 motors in particular have been shown to walk unidirectionally alongsingle filaments, and to fall off their tracks upon encountering obstacles, latticedefects, and network intersections. Knowing that both microtubule network

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density and arrangement are highly controlled in vivo, it is likely that microtu-bule network organization is a key component in the regulation of cargo trans-port. To better understand how network geometry controls multi-motor cargotransport, we have tracked quantum-dot molecular cargoes decorated withdifferent numbers of kinesin-1motors throughmicrotubule networks of differentdensities and intersection geometries. Using single moleculemotility assays andSTORM imaging of the network, we have found that cargoes with greaternumbers of motors travel via highly non-linear pathways, while single motortransport is confined to single filaments. These experiments help to illuminatethemechanisms bywhichmotor number and network geometry are used in com-bination to carefully direct transport of essential cargoes throughout the cell.

225-PlatAn Allosteric Gear Shift Mechanism in Eg5 Enhances MechanochemicalCoupling and Shifts the Force-Velocity LandscapeJoseph Muretta1, Babu J.N. Reddy2, Guido Scarabelli3, David D. Thomas1,Barry Grant3, Steven Gross4, Steven Rosenfeld5.1Biochemistry, Molecular Biology and Biophysics, University of Minnesota,Minneapolis, MN, USA, 2University of California Irvine, Irvine, CA, USA,3University of Michigan, Ann Arbor, MI, USA, 4University of CaliforniaIrvine, Irvine, MI, USA, 5Cleveland Clinic, Cleveland, OH, USA.Microtubule motors control a diverse collection of physiologies, including celldivision, organelle traffic, and microtubule dynamics. Fourteen kinesin familymembers are involved in mitosis. The best studied of these is Eg5, which is apoorly processive motor that can occupy a two head-strongly-bound state onthe microtubule. It works primarily in ensembles of tetramers, cross-linkingparallel and anti-parallel microtubules in the maturing spindle. Chemical abla-tion of Eg5 arrests mitosis. Proteomic screens have identified post-translationalmodifications as potential modifiers of Eg5 activity. We investigated a novelmodification, acetylation of lysine 146 by mutating this residue in Eg5’s a2 he-lix to a glutamine. Molecular dynamics simulations demonstrate that modifica-tions of this lysine disrupts critical ionic interactions between the a2 and a1helices, increasing the allosteric coupling between the nucleotide binding siteand Eg5’s mechanical element, the neck-linker. We tested these predictions us-ing structural kinetics measurements made by transient time-resolved FRETdetection of neck-linker docking and switch-1 closure during ATP binding torigor Eg5 attached to microtubules. We also investigated the mechanical con-sequences of this charge mutant by investigating processive motility of fluores-cently labeled Eg5 dimers in the presence and absence of applied load. Thesetests show that the K146Q acetylation-mimic mutation increases the couplingbetween the neck-linker and nucleotide binding site, accelerates ATP drivenneck-linker docking, tightens the force dependence of stepping, and increasesprocessive run-length and stall force in the presence of load, all indicatingthat the modification enhances mechanochemical coupling in Eg5. After modi-fication of this residue, Eg5 behaves much more like kinesin-1, a processivemotor with head-head gating.We are investigating the physiologic implicationsfor these changes.

226-PlatSingle Molecule Characterization of Mitotic Kif15 Reveals Capability toGenerate Force in Anti-Parallel MicrotubulesDana N. Reinemann1, Emma G. Sturgill2, Dibyendu Kumar Das1,Ryoma Ohi2, Matthew J. Lang1,3.1Chemical and Biomolecular Engineering, Vanderbilt University, Nashville,TN, USA, 2Cell and Developmental Biology, Vanderbilt University,Nashville, TN, USA, 3Molecular Physiology and Biophysics, VanderbiltUniversity, Nashville, TN, USA.Proper formation of the mitotic spindle is essential for chromosome separationand cell division. Dimeric Kinesin-12, or Kif15, has been shown to serve as abackup mechanism for cell division, promoting spindle assembly in the absenceof tetrameric Kinesin-5, or Eg5. The mechanism by which Kif15 aids in the as-sembly of the spindle is unknown. Here, single molecule motility assays wereperformed using optical tweezers to evaluate the mechanistic behavior of Kif15on microtubules (MTs). Constructs containing various sub-components of themotor were utilized to determine the role of each domain, including the motorheads, stalk, MT binding domain (N700) and the full length motor (FL). Stepsize, dwell times, and stall forces were measured. Rupture assays were also per-formed on the MT binding domain (Coil-1) to determine the binding strengthand the nature of the Coil-1/MT interaction. Motility and rupture assayswere repeated on subtilisin-digested MTs (dMT) in which the negatively-charged carboxyl-terminal tail of MTs, called the E-hook, is removed. Wefound that the Coil-1 was stronger than the stall force of the motor due to itsinteraction with the E-hook, revealing that Kif15 has the ability to build up me-chanical strain and slide MTs apart. In order to determine the importance of MTorientation, an in vitro optical trapping and fluorescence assay was developed

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to determine the force generating capabilities of Kif15 in different bundle en-vironments. We determined that Kif15 slides anti-parallel MTs apart while par-allel bundles remain stationary with a small amount of antagonizing forcegeneration. Our studies provide insight regarding how Kif15 is able to rescuebipolar spindle assembly in the absence of Eg5.

227-PlatMeasuring Forces Generated by Ensembles of Kinesin-5 Crosslinking TwoMicrotubulesScott Forth1, Yuta Shimamoto2, Tarun Kapoor3.1Biological Sciences, Rensselaer Polytechnic Institute, Troy, NY, USA,2National Institute of Genetics, Mishima, Japan, 3Laboratory of Chemistryand Cell Biology, Rockefeller University, New York, NY, USA.The proper organization of the microtubule-based mitotic spindle is proposedto depend on nanometer-sized motor proteins generating forces that scalewith a micron-sized geometric feature, such as microtubule overlap length.However, it has been unclear whether such regulation can be achieved byany mitotic motor protein. To address this knowledge gap, we have employedan assay that combines an optical trap and total internal reflection fluorescence(TIRF) to show that ensembles of kinesin-5, a conserved mitotic motor protein,can push apart overlapping antiparallel microtubules to generate a force whosemagnitude scales with filament overlap length. We also find that kinesin-5 canproduce overlap-length-dependent ‘‘brake-like’’ resistance against relativemicrotubule sliding in both parallel and antiparallel geometries, an activitythat has been suggested by cell biological studies but had not been directlymeasured. Finally, we performed numerical simulations which reveal thebio-mechanical properties of this motor protein that allow for efficient forcetransmission within crosslinked microtubule networks. Together, our resultsreveal how a motor protein can function as an analog converter, ‘‘reading’’ sim-ple geometric and dynamic features in cytoskeletal networks to produce regu-lated and scalable force outputs.

Platform: Computational Methods andBioinformatics

228-PlatElucidating the Role of Tumor Microenvironmental Heterogeneity with aComputational Model of 3D Breast SpheroidsAleksandra Karolak1, Branton Huffstutler1, Dmitry A. Markov2,Lisa J. McCawley2, Katarzyna A. Rejniak1.1Integrated Mathematical Oncology, H. Lee Moffitt Cancer Center andResearch Institute, Tampa, FL, USA, 2Biomedical Engineering & CancerBiology, Vanderbilt University, Nashville, TN, USA.Tumor development and its response to therapies are highly affected by hetero-geneous microenvironments, including fluctuations in chemical and physicalcues, as well as in the composition of the surrounding extracellular matrix.For example, the abnormal topology of tumor vasculature and tumor tissue leadsto disruption in oxygen transport and in the emergence of regions of low oxygen(hypoxic conditions), and in accumulation of acidic products and decreased pH(acidic conditions). In addition, the elevated mechanical tension is commonlyobserved in the tumor connective tissue (high tension conditions). All these fac-tors affect tumor growth and efficacy of anticancer drugs. Moreover, the micro-environmental conditions can dynamically evolve after treatment that is difficultto investigate in vivo in a systematic way. To represent the complexity and dy-namics of tumor microenvironment we developed a computational model for thegrowth of 3D organotypic cultures integrated with experimental organ-on-the-chip 3D cultures. The computational model was validated based on the datacollected from cultured breast spheroids, including the non-tumorigenic epithe-lial MCF-10A cell line, mildly tumorigenic c-Ha-ras oncogene transfectedMCF-10AT1 cell line, and a metastatic line MCF-10CA-1a. Based on the differencesin these cell lines genetic profiles, cell mechanical properties, and image-guidedvariations in organotypic morphologies, the computational model was able toreproduce in a quantitative way the dynamics of 3D tumor spheroids’ growth un-der various microenvironmental conditions. This calibrated model was subse-quently applied to predict tumor growth and its response to chemotherapy inheterogeneous microenvironments consisting of dynamically changing gradientsof oxygen and pH, as well as tension of the surrounding microenvironment. Theoverarching aim of our project is to use this integrated (in silico-organ and organ-on-the-chip) approach to target the treatment of tumor 3D organotypic cultureswith drugs that display different mechanism of action, such as cell cycle arrest,initiation of apoptosis, or treatment combinations. Furthermore, this approachwill allow us to propose new treatment schedules and dosages that will be testedin the organ-on-the-chip experiments. The progress on this integrated modeldevelopment will be presented.

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229-PlatSpoton: A Machine-Learning Approach for Hot-Spot DeterminationIrina S. Moreira1,2, Panos Koukos2, Rita Melo1,3, Jose G. Almeida1,Antonio J. Preto1, Jorg Schaarschmidt2, Mikael Trellet2, Zeynep H. Gumus4,Joaquim Costa5, Alexandre M.J.J. Bonvin2.1Center for Neurosciences and Cell Biology (CNC.IBILI), Coimbra,Portugal, 2Bijvoet Center for Biomolecular Research, Utrecht University,Utrecht, Netherlands, 3Centro de Ciencias e Tecnologias Nucleares, Lisbon,Portugal, 4Department of Genetics and Genomics and Icahn Institute forGenomics and Multiscale Biology, Icahn School of Medicine at Mount Sinai,New York, NY, USA, 5CMUP/FCUP, Centro de Matematica daUniversidade do Porto, Porto, Portugal.The identification of protein complexes and interactions is crucial for the un-derstanding of cellular organization and machinery. Due to the high difficultyin attaining experimental data about such an important subject, computationaltools and methodologies are emerging as reliable alternatives. It is especiallytrue that Machine-Learning (ML) algorithms hold an incredible promise forprotein interaction research by identifying biological relevant patterns. In thiswork, we have developed and applied ML techniques that went beyond the cur-rent state-of-the-art, leading to quantitative and reliable molecular-level predic-tions of Hot-Spots at protein-protein complexes. Our model was trained on alarge number of complexes and on a significantly larger number of differentstructural- and evolutionary sequence-based features. We used 54 algorithmsfrom a simple linear-based function to support-vector machine models withdifferent cost functions and compared their performance in 6 different pre-processing sets. The best model was achieved by the use of the C5.0 tree-based algorithm with a dataset pre-processed by the normalization of featuresand with up-sampling of the minor class. The method showed an overall accu-racy of 0.88, a sensitivity of 0.91 and a specificity of 0.84 for the independenttest set. Due to the theme’s relevance to the large scientific community workingon structural biology, we have assembled a freely available web-server that canbe found at: http://milou.science.uu.nl/services/SPOTON/

230-PlatMorphodynamic Profiling of Cell Protrusion Based on SpatiotemporalSpectrum Decomposition and Unsupervised ClusteringXiao Ma1, Onur Dagliyan2, Klaus Hahn2, Gaudenz Danuser1.1Bioinformatics, University of Texas Southwestern Medical Center, Dallas,TX, USA, 2Pharmacology, UNC-Chapel Hill School of Medicine, ChapelHill, NC, USA.Cellular morphogenesis is governed and regulated by complex interactions be-tween the cytoskeleton and molecular pathways. Due to their central roles inthe differentiation, embryogenesis and homeostasis process, cell morphologyand morphodynamics are often used for qualitative and quantitative assessmentsof cell state. Here, we propose a framework to quantitatively profile cellular mor-phodynamics based on an adaptive spectrumdecompositionmethod. Thismethoddecomposes the motion of cell boundary into analytical instantaneous frequencyspectrum through Hilbert-Huang transform (HHT). Subsequently, we computedfor eachdecomposed signal the distributionof instantaneous frequency and ampli-tude, from which we compiled the HHT spectrum. For spontaneously protrusiveCos7 cells, the instantaneous magnitudes of protrusionwere found to greatly varyacross different cells with nearly identical genetic and molecular backgrounds,whereas the frequency spectra were remarkably consistent. Meanwhile we exam-ined Cos7 cells in which the Vav2 Guanine Exchange Factor (GEF) activity wasacutely perturbed by optogenetic tools, and found significant shifts in the fre-quency distribution for specific signals. We thus concluded that edge motion ve-locity is an arbitrary parameter of cell morphogenesis whereas the frequencyspectrum encodes the molecular state of regulation. The features extracted fromthe HHT spectrum could be used as a classifier to group subcellular regions intonumerous characteristic sectors with specific molecular states through StatisticalRegionMerging (SRM) based clustering. Finally, wemonitored the Rac1 activitydownstream of Vav2 expressed in Cos7 cells and found that those clustering sec-tors distinguished by morphodynamic parameters indeed reflect regions withdifferent local signaling.Thus, cellmorphodynamic behavior can beused todetectdifferent modes of operations or perturbations in cellular signaling.

231-PlatMechStiff: A New Tool for Evaluating Stress-Induced Dynamicsand Application to Cell Adhesion ProteinsKarolina Mikulska-Ruminska1,2, Andrzej J. Kulik3, Cihan Kaya1,Carine BenAdiba3, Giovanni Dietler3, Wieslaw Nowak2, Ivet Bahar1.1Computational and Systems Biology, Pittsburgh Univesity, Pittsburgh, PA,USA, 2Physics, Astronomy and Applied Informatics, Niculaus CopernicusUniversiy, Torun, Poland, 3Laboratory of Physics of Living Matter, EcolePolytechnique Federale de Lausanne, Lausanne, Swaziland.

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Based on recent findings, there is growing evidence that many biological systemsin the brain integrate biochemical information with mechanical signals makingcritical decisions about cell differentiation, synaptic regulation, axonal growth,neuronal migration or proliferation. These events seem to be controlled by themechanical behavior of intervening proteins in addition to their well-studiedbiochemistry; and therefore to obtain a quantitative understanding of the me-chanical events implicated in neurosignaling events is becoming increasinglyimportant. We have developed to this aim the MechStiff module that we imple-mented in ProDy API (1), based on an earlier comparison (2) of anisotropicnetwork model predictions with single molecule atomic force microscopy(smAFM) data. We further usedMechStiffmodule to evaluate the effective resis-tance of individual domains in the neuronal adhesion protein, contactin, to uni-axial tension. Contactins play an important role in maintaining the mechanicalintegrity and signaling properties of chemical synapses in the brain. To validatethe predictions on the the molecular-level stress-strain behavior of contactin,we compared MechStiff predictions with smAFM measurements as well as re-sults from steered molecular dynamics simulations. The multiscale approachcombining anisotropic network model with molecular simulations emerges asa useful tool for interpreting experimental data and characterizing contactinnanomechanics. It also helps reveal the stress-induced conformational changesbeing accommodated by cell adhesion proteins and can be readily extended toelucidating the stress-induced dynamics of multicomponent or modular proteins.1.Bakan, A., A. Dutta, W. Mao, Y. Liu, C. Chennubhotla, T. R. Lezon, and I.Bahar. 2014. Evol and ProDy for bridging protein sequence evolution andstructural dynamics. Bioinformatics 30:2681-2683.2.Eyal, E., and I. Bahar. 2008. Toward a molecular understanding of the aniso-tropic response of proteins to external forces: insights from elastic networkmodels. Biophys. J. 94:3424-3435.

232-PlatOptimal Temperature Set for Replica Exchange SamplingDominik Gront.Faculty of Chemistry, University of Warsaw, Warsaw, Poland.Replica Exchange Monte Carlo method has been introduced to improve sam-pling of a rugged energy landscape for such systems as polymers, biopolymersand spin glasses. Efficiency of the method however critically depends on the setof replica temperatures used for simulations. A novel method selecting theseparameters has been recently proposed1, which numerically evaluates the prob-ability of replica swap between temperatures based on estimated density ofstates for a system under study. Here we extend this method and prove it pro-vides the optimal set of temperatures i.e. temperatures that guarantee the fastestflow of replicas from the lowest to the highest temperature.During an initial phase of the protocol, energy distributions are collected atdifferent temperatures. Based on these observations, the density of states forthe system is computed by the multihistogram method. Knowing the density,improved temperature set is established by minimizing the mean first passagetime of replicas in the temperature space. The procedure has been illustratedwith a coarse-grained protein folding simulation and all-atom dynamics inAMBER force field. The method has been implemented in BioShell package.2,31 D. Gront and A. Kolinski ‘‘Efficient scheme for optimization of paralleltempering Monte Carlo method’’ Journal of Physics: Condensed Matter 200719(3) 036225 http://dx.doi.org/10.1088/0953-8984/19/3/0362252 D. Gront and A. Kolinski ‘‘BioShell - a package of tools for structural biologycomputations’’ Bioinformatics 2006 22(5):621-6223D. Gront and A. Kolinski ‘‘Utility library for structural bioinformatics’’ Bio-informatics 2008 24(4):584-585

233-PlatMolecular Design of a Nanoparticle-Polymer Conjugated Drug DeliverySystem for PD-166793 in Cardiovascular RepairMerina Jahan1, Stephen K. Roberts2, Andrew B. Greytak2, Mark J. Uline1.1Chemical Engineering, University of South Carolina, Columbia, SC, USA,2Department of Chemistry and Biochemistry, University of South Carolina,Columbia, SC, USA.Overexpression of matrix metalloproteinases (MMPs) following myocardialinfarction (MI) is linked to deleterious left ventricle remodeling and heart failure.Current research has focused on introducing a therapeutically relevant concentra-tion of effective MMP inhibitor to the MI site to mitigate the harmful tissue re-modeling. Theoretical molecular level studies provide an effective platform fordesigning novel delivery systems for MMP inhibition that can provide valuableinsights for experimental researchers. This study focuses on developing a drugdelivery pathway using PD-166793 that has shown great promise as a broadspectrum MMP inhibitor in recent years. In this system, PD-166793 is boundto poly methyl acrylic acid (PMAA) polymer and one end of the polymer is teth-ered to a spherical silica nanoparticle surface to carry the drug to the desired site.

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Amolecular model using single chain mean field theory (SCMFT) is used to scanthe wide range of possible design parameters. The molecular theory properly ac-counts for the highly non-additive coupling of molecular interactions among allthe species. The size, shape, electrical properties and physical conformations ofthe polymer, drug and solvent are taken into account. The binding of PD-166793with polymer is modeled by a ligand-receptor binding mechanism. The model isused to study the variation of this binding with changing pH, salt concentration,grafting density and length of the polymer. Experimental studies have shown thatthis system is capable of retaining PD-166793 at more than 100 times the inhib-itory concentration against MMP-2 with a particle concentration of 2.5mg/mL.The model is used as a tool for continual improvements in binding of PD-166793 by providing valuable feedback on how the variations of system param-eters affect the binding efficiency.

234-PlatSystematic Analysis of Symmetry in Membrane ProteinsAntoniya A. Aleksandrova.CSB, NINDS - NIH, Bethesda, MD, USA.Membrane proteins are encoded by around one third of a given genome, andplay key roles in transmission of information and chemicals such as neurotrans-mitters into the cell. Available membrane protein structures have revealed anabundance of symmetry and pseudo-symmetry, which are observed not onlyin the formation of multi-subunit assemblies, but also in the repetition of inter-nal structural elements. Secondary active transporters provide striking exam-ples of the functional significance of symmetry. For instance, the structuresof many transporters consist of two pseudo-symmetric repeats with oppositetransmembrane orientations. These repeats can form asymmetric conforma-tions to create a pathway to one side of the membrane, consistent with theso-called alternating access hypothesis. By having one repeat adopt the confor-mation of the second repeat and vice-versa, the protein can create a new asym-metric structure that is open to the opposite side of the membrane. This‘‘asymmetry exchange’’ underlies rocking-bundle or elevator-like movementsthat result in the transport of a substrate. In this context, a systematic study ofsymmetry should provide a framework for a broader understanding of themechanistic principles and evolutionary development of membrane proteins.However, existing analyses lack the detail and breadth required for such a sys-tematic study. Therefore, in this project we aim to quantify both the extent anddiversity of symmetry relationships in known structures of membrane proteins.To achieve this task, we combine the output of two programs for symmetrydetection, namely SymD and CE-Symm, each of which has certain limitations.Our approach also allows us to explore the characteristics that discriminatesymmetric from pseudo-symmetric or asymmetric structures. We anticipatethat this analysis will provide a valuable foundation for addressing a wide rangeof questions relating to the function and evolution of these important proteins.

235-PlatMembrane Recruitment can Increase the Number of Protein Assembliesby Many Folds: Insights from Theory and Reaction-Diffusion SimulationOsman N. Yogurtcu, Margaret E. Johnson.Biophysics, Johns Hopkins University, Baltimore, MD, USA.A significant number of the cellular protein interaction networks, such asreceptor-mediated signaling and vesicle trafficking pathways, includes reac-tions that involve membranes as a molecular assembly platform. Membranesboth reduce the search space and induce a cooperative binding effect for sta-bilizing complexes with multiple membrane recruiter molecule binding sites.Mathematical models and computer simulations provide insight into the dy-namics of complex formation and help identify general principles that governsuccessful recruitment and assembly on membranes. However, sufficientlylong and physically accurate simulations of protein assemblies are quite chal-lenging. In this work, using equilibrium theory and a very efficient in-labdeveloped single-molecule scale stochastic simulation software, we providesimple formulas for quantifying how the ratio of membrane-to-solution,both in vitro and in vivo, can change the observed protein-protein interactionstrengths of peripheral membrane proteins by orders of magnitude. We showthat the magnitude of complex formation enhancement has a simple functionalform that applies whenever membrane recruiter concentrations are sufficientlyhigh, and surprisingly, is independent of the protein binding strength. We pro-pose that membrane localization works as a mechanism that ensures assemblyonly at specific times (after recruitment to surfaces) but does not preciselyregulate the proteins involved since they benefit equally from surface restric-tion. This robust strategy is employed by adaptor proteins involved inclathrin-mediated endocytosis in both yeast and mammalian cells, where theirrelatively weak binding interactions with one another prevents protein coat as-sembly in solution, but transitions to a rapid assembly on the plasmamembrane.

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Posters

Protein Structure and Conformation I

236-Pos Board B1A Discrete Loop of the SERCA N-Domain Interacts with Phospholambanand Stabilizes a Compact Conformation of the SERCA CytosolicHeadpieceOlga N. Raguimova, Nikolai Smolin, Daniel Blackwell, Elisa Bovo,Aleksey Zima, Seth Robia.Cell and Molecular Physiology, Loyola, Maywood, IL, USA.The cardiac sarco/endoplasmic reticulum (ER) calcium ATPase (SERCA) andits inhibitor phospholamban (PLB) are key regulators of calcium levels in car-diac cells. In previous molecular dynamics (MD) simulations of SERCA spon-taneous headpiece closure, we identified a discrete N-domain beta 5,6-loop thatdirects the closure of SERCA headpiece. Here we use additional MD simula-tions to pinpoint specific negative residues that facilitate SERCA headpiececlosure. In addition, fluorescence resonance energy transfer (FRET) measure-ments showed that mutation of these key residues slows down calcium-dependent SERCA headpiece closure, as predicted from MD calculations.The functional significance of impaired SERCA headpiece closure was demon-strated by a 60% decrease in SERCA ATPase activity with no detectablechange in calcium sensitivity. Additionally, loop mutations reduced transportof calcium as measured by a low affinity calcium sensor (R-CEPIA1er) inthe ER of HEK cells. Interestingly, MD simulations of the SERCA-PLB regu-latory complex model showed that PLB phosphorylation on Serine 16 stabi-lized a compact, ordered SERCA headpiece through an interaction with thebeta 5,6-loop. Fluorescence lifetime imaging microscopy (FLIM) measure-ments also detected a conformational change of the SERCA-PLB regulatorycomplex after phosphomimetic mutation of PLB. Overall, the data suggestthat this discrete loop is important for SERCA transport activity and functionalregulation by PLB. This novel structural element may represent a target formodulation of SERCA activity.

237-Pos Board B2Preparation of a Orexin Precursor Protein by Chemical DigestionNatsumi Mitsuoka, Shigeru Shimamoto, Yuji Hidaka.Kindai University, Higashi-Osaka, Osaka, Japan.Endogenous peptide hormones are often produced in vivo in the form of precur-sor proteins that contain pre- and pro-leader sequences, which are subsequentlyprocessed into the biologically active mature peptide. Therefore, it is importantto study the folding of precursor proteins to understand the mechanisms of post-translational modifications and the formation of the active conformation of themature peptide. However, it is generally difficult to obtain precursor proteinsfrom intact organs because only small amounts are produced in vivo. To over-come this issue, an E. coli expression system is employed to prepare recombi-nant proteins.Pro-orexin consists of orexin A, B, and a C-terminal peptide. To investigate themechanism responsible for the processing and post-translational modificationsof orexin peptides, we tried to express the recombinant pro-orexin in E. colicells. However, recombinant pro-orexin was not expressed in E. coli cells. Inaddition, it was also difficult to obtain pro-orexin, even though Trx-fusedpro-orexin was readily produced in E. coli cells because the digestion ofTrx-fused pro-orexin with an enzyme yielded significant amounts of non-specific digests. Therefore, to solve this issue, we employed a chemical methodto release pro-orexin from fusion proteins.K. Pane et al. recently reported a chemical method to obtain a target proteinfrom an onconase-fused (ONC-fused) protein. They applied a chemical cleav-age of the fusion protein at the Asp-Pro sequence with acetic acid. We em-ployed this chemical method to produce recombinant pro-orexin. ONC-fusedpro-orexin was successfully over-expressed as an inclusion body in E. coliBL21(DE3) cells and treated with acids under several sets of conditions tocleave the Asp-Pro sequence of ONC-fused pro-orexin. The results will be dis-cussed in this paper.

238-Pos Board B3A Simulation Based Analysis of the Oligomeric Plasticity of Sm ProteinAssembliesCharles E. McAnany, Berk Ekmekci, Peter Randolph, Cameron Mura.Chemistry, University of Virginia, Charlottesville, VA, USA.RNA-associated Sm proteins can be found in all three domains of life. In eu-karya, Sm proteins are well-studied in connection with their roles mRNAsplicing. In bacteria, the Sm protein Hfq acts as an RNA chaperone, playingvital roles in mRNA-sRNA annealing and RNA-based regulatory networks.

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In archaea, the functional roles of Sm proteins remains an open question. Smproteins assemble into cyclic oligomers of 5, 6, 7, or 8 subunits, and the assem-blies can be either homo- or heteromeric. Bacterial Sm proteins have only beenfound as homo-hexamers, while eukaryotic Sm proteins typically assemble intohetero-heptamers. Archaeal Sm proteins have been found as homomeric hex-amers, heptamers, and octamers. Despite this variation in quaternary structure,all Sm monomers exhibit nearly identical tertiary structures. How can this be?What is the origin of this oligomeric plasticity, if not encoded in the monomer?We have used a systematic array of molecular dynamics simulations toexamine the interfaces between Sm subunits, and have developed several quan-titative relations that link the results of dimer simulations to the behavior ofcomplete rings. The simulations reveal that Sm oligomers are remarkably flex-ible. Sm dimers can adopt multiple conformations, and Sm rings are distinctlyasymmetric. In particular, the octameric ring of an Sm-like archaeal proteindeforms drastically, and dimers of that protein appear to adopt a non-Sm-liketertiary structure. For a dimer of the E. coli Sm protein, our simulationsshow one monomer twisting nearly fifteen degrees from its position in the crys-tal structure. The surprising flexibility of Sm oligomers may be related to thedynamical effects of RNA binding and we are currently investigating theseeffects in a variety of Sm systems.

239-Pos Board B4An NMR Study of Pin1- Histone H1 InteractionsDinusha S. Jinasena, Hawa Gyamfi, Nicholas C. Fitzkee.Chemistry, Mississippi State University, Starkville, MS, USA.Pin1 is an essential Peptidyl-prolyl isomerase (PPIase) that catalyzes cis-transprolyl isomerization in proteins containing pSer/Thr-Pro motifs. It has an N-ter-minal WW domain and a C-terminal PPIase domain. Pin1 targets pSer/Thr-Promotifs by its WW binding domain and catalyzes isomerization through itsPPIase domain. Recently, Pin1 was shown to modify the conformation of phos-phorylated Histone H1 and to stabilize the chromatin-H1 interaction byincreasing its residence time. The Pin1/Histone H1 interaction plays a keyrole in pathogen response in infection and cell cycle control; therefore, anti-Pin1 therapeutics is an important target for treating infections as well as cancer.The H1 Histones (H1.0-H1.5) each contain several potential Pin1 recognition(pT/pS)-P motifs. To understand this interaction fully, we have investigatedhow both the isolated WW domain and full length Pin1 bind to these H1Histone substrates. NMR spectroscopy has been used to measure the bindingaffinities of Pin1 with select H1 sequences (H1.1, H1.4 & H1.5), and theinter-domain dynamics upon binding theses sequences have been measured.We observe different Kd values depending on the histone binding site, suggest-ing that energetics play a role in guiding the Pin1-Histone interaction. Relaxa-tion data has revealed that bound Pin1 can behave as two independent domainsconnected by a flexible linker, or as a single rigid molecule, depending on theHistone H1 sequence. In the long term, we believe these studies will help toreveal not only the specifics of the Pin1-Histone H1 interaction, but also howPin1 interacts with its substrates in general.

240-Pos Board B5Nanomechanical Properties of Polymorphic Human Islet AmyloidPolypeptide Protofibrils in the Multiple Physiological and MechanicalConditionsMyeongsang Lee.Department of Mechanical Engineering, Korea University, Seoul, Korea,Republic of.Fibrillar and oligomeric amyloids are the origin of neuro-degenerative anddegenerative diseases as type II diabetes, Alzheimer’s diseases, and so forth.The common properties of amyloids are located near functional cells, not easilydegraded, and disrupt their own functions. These amyloids had polymorphiccharacteristics under physiological conditions such as thermal effect, pH, ionicstrength, metal ions and internal fluctuations. Developed from denatured andmisfolded amyloid monomers, polymorphic amyloids are mainly existed asparallel or antiparallel composition along to fibril axis and lateral thicknesscomposition by adding unit amyloid protofilament through the experimentaltechniques. Despite experimental effort of polymorphic structures of amyloidproteins, there are some lacks of knowledge that relationship between structuralcomposition of polymorphic amyloids and their stabilities in detail. The me-chanical characterization as bending, stretching can give insight for under-standing the structure-property relationship of amyloid protofilaments.Considering that amyloids are developed through the repetitive fragmentationand elongation mechanisms, it is crucial to understand the structural stabilitiesof polymorphic amyloid protofibrils. Here, we reported the polymorphic char-acteristics of hIAPP amyloid protofibrils in mechanical insight via equilibriumand steered molecular dynamics (MD and SMD). Through the principalcomponent analysis of polymorphic hIAPP protofibrils after equilibrium MD

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simulations, we found the different deformation mode (i.e. bending, stretchingand torsion) of polymorphic hIAPP protofibrils. Using eigenvalue analysis,antiparallel models had considerable structural stability than parallel modelsdue to the asymmetric hydrogen bond network composition. Furthermore, byapplying the constant force bending and constant velocity tensile SMD simula-tions, we found the dominant material characteristics that homo compositionhad ductile feature, while hetero composition had brittle characteristics. Weprovide the relationship between structural composition and their properties,which are related to growth mechanism of amyloids. Our study also providesthe possibilities for the basic template for functional biomaterials in the future.

241-Pos Board B6Protein Structure Determination by High-Precision FRET and MolecularModelingMykola Dimura1, Thomas Peulen1, Holger Gohlke2, Claus A.M. Seidel1.1Molecular Physical Chemistry, Heinrich-Heine-Universit€at D€usseldorf,D€usseldorf, Germany, 2Pharmazeutische und Medizinische Chemie,Heinrich-Heine-Universit€at D€usseldorf, D€usseldorf, Germany.A comprehensive methodology for FRET based modeling of biomolecules ispresented. Conformations of six multi-state model proteins are determinedbased on synthetic FRET data in the given benchmark. The workflow consistsof five steps:1. Initial conformational ensemble generation with NMSim geometric sim-ulations utilizing available prior knowledge like homology model or crystalstructure of the one of the states.2. Identification of the most informative labeling positions and FRET pairsfor fluorescence measurements based on proposed ensemble generated at firststep.3. State-representative conformer identification, based on their agreementwith experiment, using FRET-screening and Accessible Volume simulations.4. Expansion of the initial conformational ensemble with FRET-guidedNMSim Markov Chain Monte Carlo simulations based on FRET data. As aresult conformations with better FRET-agreement are identified.5. Precision and accuracy assessment and quality control to outline the capa-bilities and limitations of the shown methodology.Using these steps, conformations of the model proteins were determined accu-rately. Confidence levels were determined together with the corresponding con-fidence levels and accuracy measures. Performance capabilities and limitationsof the methodology are rigorously assessed. The software package is presentedon site.

242-Pos Board B7Apo- and Antagonist-Binding Structures of Vitamin D Receptor Ligand-Binding Domain Revealed by a Combination Andlysis of MD Simulationsand SAXS ExperimentsYasuaki Anami1, Nobutaka Shimizu2, Toru Ekimoto3, Daichi Egawa1,Toshimasa Itoh1, Mitsunori Ikeguchi3, Keiko Yamamoto1.1Showa Pharmaceutical University, Tokyo, Japan, 2Photon Factory, Instituteof Material Structure Science, High Energy Accelerator ResearchOrganization, Tsukuba, Japan, 3Yokohama ciry university, Yokohama,Japan.Vitamin D receptor (VDR) is a member of the nuclear receptor (NR) family andregulates the expression of genes related to calcium homeostasis , immunomo-dulation, and cell differentiation and proliferation. The VDR and other NRs areinvolved in human diseases, therefore, understanding of their regulation byligand binding is a pharmaceutical demand for structure-based drug design.According to X-ray crystallographic analysis of NRs and other experimental re-sults, the ligand-binding domain (LBD) undergoes conformational changeupon ligand binding, and the local conformational change around helix 12 iskey to regulating agonism/antagonism.Many crystal structures of agonist/antagonist-binding VDR-LBD have beensolved so far. However, all the crystal structures are almost identical, regardlessof agonist/antagonist binding, and those crystal structures are considered as theagonist form. Because the conformation of helix 12 is key, the crystal structuresdo not provide structural insight into the mechanism of antagonist activity. Inaddition, no crystal structures of apo form has been reported, and the exactconformation of apo form remains unknown.To reveal the apo and antagonist-binding forms of VDR-LBD, we analyze themby a combination approach of small-angle X-ray scattering (SAXS) and molec-ular dynamics (MD). We obtained SAXS profiles of both forms, however, theprofiles were different from the theoretical profiles calculated from crystalstructures. To clarify the solution structures at atomic resolution, we conductedMD and collected each structural ensemble. Comparing the SAXS profiles andthe MD results, we report a reliable structure for each form. In both forms, helix12 is partially unraveled, and does not adopt the active form, preventing co-

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activator binding for transactivation. In apo form, helix 11 bends outward bya kink-centered hinge-bending motion, and the motion creates wide entranceof the ligand-binding pocket. In antagonist-binding form, loop 11-12 remark-ably flexible compared to that in apo form.

243-Pos Board B8Conformational Fluctuations as an Intrinsic Mechanism of Action: TheLipase-Specific Foldase of Pseudomonas Aeruginosa Studied by HybridFluorescence Spectroscopy and MD SimulationsJakub Kubiak1, Neha Verma2, Peter Dollinger3, Filip Kovacic3,Karl-Erich Jaeger3, Holger Gohlke2, Claus A.M. Seidel1.1Institute for Molecular Physical Chemistry, Heinrich-Heine-Universit€atD€usseldorf, D€usseldorf, Germany, 2Institute for Pharmaceutical andMedicinal Chemistry, Heinrich-Heine-Universit€at D€usseldorf, D€usseldorf,Germany, 3Institute of Molecular Enzyme Technology, Heinrich-Heine-Universit€at D€usseldorf, D€usseldorf, Germany.The description of protein function requires structural information, not onlyfrom static structural models but also of flexibility and dynamics. An investiga-tion of highly flexible proteins or IDPs requires techniques sensitive to a varietyof time- and distance-scales. Fluorescence (FRET, FCS) is known to providegood temporal and spatial coverage but it is a label-based technique and lacksatomistic-details. All-atom MD, on the other hand, is challenged by simulationof slow processes and accurate description of all interactions. The lipase-specific foldase (Lif) from Pseudomonas aeruginosa is a highly flexible proteinwith residual secondary and tertiary structure. It is mandatory to produce lipaseA (LipA) in an enzymatically active conformation. Using a combination offluorescence techniques (single molecule FRET, fluorescence lifetime analysisand fFCS) and all-atomMDwe propose a description of structure and dynamicsof Lif in relation to LipA. In the Lif:LipA complex, Lif forms flexible a-helicalscaffold embracing LipA in headphone-like structure. In the unbound form, Lifdoes not stay in the hollow ‘‘headphone’’ conformation but rather exhibitslarge-scale conformational dynamics, where its a-helical structure undergoesreversible collapses and extensions as well as unfolding, on the sub-microsecond to sub-millisecond timescale. This process allows Lif to bindLipA despite the fickleness of structure. We show how the multitude of statesin fast exchange provides a basis for binding. In addition we show how thishybrid approach benefits FRET (insight into the molecular structure) andMD (sampling).

244-Pos Board B9Does Cas9-Catalyzed DNA Cleavage Generate Blunt Ends or StaggeredEnds? Insight from Molecular Dynamic SimulationsZhicheng Z. Zuo, Jin Liu.UNT Health Science Center, Fort Worth, TX, USA.The CRISPR-associated endonuclease Cas9 from Streptococcus pyogenes(spCas9) along with a single guide RNA (sgRNA) has merged as a versatiletoolbox for genome editing. Despite recent advances in the mechanistic studieson spCas9-sgRNA-mediated target double-stranded DNA (dsDNA) recogni-tion and cleavage, it is still unclear how the catalytic Mg2þ ions induce theconformation changes toward the catalytic active state. It also remains contro-versial whether Cas9 generates blunt-ended or staggered-ended breaks withoverhangs in the target DNA. To investigate these issues, here we performedthe first all-atom molecular dynamics simulations of the spCas9-sgRNA-dsDNA system with and without Mg2þ bound. The simulation results showthat binding of two Mg2þ ions at the RuvC domain active site could lead tostructurally and energetically favorable coordination ready for the non-targetDNA strand cleavage. Importantly, we argue that Cas9-catalyzed target DNAcleavage produces 1-bp staggered ends rather than generally assumed bluntends.

245-Pos Board B10Methionine Sulfoxide Formation by Cigarette Smoke is Associated withthe Degradation of Plasma ProteinsAbdullah Qassab, Rohana Liyanage, Wesley Stites.Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR, USA.Background: High level of protein carbonyls have been found in plasma pro-teins in smokers compared to non-smokers. While oxidation may directly inter-fere with activity, the extent to which oxidation affects protein turnover is lessclear.Objectives: To determine levels of oxidized serum proteins cleared in the urineof smokers and non-smokers with focus on methionine sulfoxide (MSO) forma-tion in Human Serum Albumin (HSA) and to determine the effect of methio-nine oxidation on the turnover of HSA.Method: 100 mL of urine were obtained from smoker and non-smokers.proteins were concentrated by reducing the sample size to 1.5% of the originalvolume. 200 mL of the concentrate then were separated by SDS-page gel

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electrophoresis. The band with intact HSA was cut out and the remainder of thegel was cut into four different pieces. Gel sections then were digested withtrypsin. Levels of MSO in the resulting peptides were assessed by LC-MS/MS and data analysis was performed using the Skyline software package.Results: A group comparison between non-smokers (control) and smokersshowed a slight increase in the levels of MSO found in intact HSA of smokersrelative to non-smokers. Regions of gels with proteins of lower mass than intactHSA showed that degraded fragments of HSA in urine of both smokers andnon-smokers have higher levels of MSO than are found in intact HSA.Conclusions: HSA in smokers has statistically significant higher levels of MSOthan HSA in non-smokers. However, the higher levels of oxidation in smokersare concentrated in partially degraded HSA. At the moment it is not possible tosay unequivocally whether oxidized HSA is more likely to be cleaved andcleared, if cleaved protein is more likely to be oxidized before clearance, orboth.

246-Pos Board B11Dynamics and Energetics of Elongation Factor SelB in the TernaryComplex and the RibosomeLars V. Bock1, Niels Fischer2, Holger Stark2, Holger Stark2,Helmut Grubm€uller1.1Theoretical and Computational Biophysics, MPI for biophysical chemistry,Goettingen, Germany, 2Structural Dynamics, MPI for biophysical chemistry,Goettingen, Germany.SelB is an elongation factor specialized to deliver the selenocysteine (Sec)tRNA to the ribosome by recoding the UGA stop codon on the mRNA. Initiallythe tRNA is in complex with selB and GTP forming the ternary complex (TC).High-resolution cryo-EM structures of intermediates of the Sec incorporationpathway uncover large-scale conformational changes of the ribosome and theTC. To complement the structural information with energetics and rapid dy-namics, we performed extensive all-atom molecular dynamics simulations ofthe ribosome with bound TC as well as of the free TC in solution. The simula-tions of the free TC were started after extracting the TC from the ribosome-bound cryo-EM structures. The TC was found to rapidly interconvert betweenthe different conformations allowing us to construct the free-energy landscapeof the involved motions. This free-energy landscape indicates that the intrinsiclarge-scale conformational changes of the tRNA and SelB during the deliveryto the ribosome are not rate-limiting to the process. In simulations of the freeTC started from the GTPase-activated ribosome-bound conformation, the TCrapidly transitions into an inactivated conformation, showing that theGTPase-activated state is strongly stabilized by the ribosome. The simulationsof the full ribosome with bound TC in the intermediate states allow us to iden-tify the motions that are rate-limiting to the process of tRNA delivery and toidentify the molecular mechanism of the domain closure of small ribosomalsubunit upon tRNA decoding.

247-Pos Board B12Polyethylene Glycol Conjugation Enhances Mosquito-Larvicidal Activityof Lysinibacillus Sphaericus BinA ProteinMahima Sharma1,2, Ramesh S. Hire2, Ashok B. Hadapad2,Gagan Deep Gupta2, Vinay Kumar2,3.1Life science, Homi Bhabha National Institute (HBNI), Mumbai, India,2Bhabha Atomic Research Centre, Mumbai, India, 3Homi Bhabha NationalInstitute (HBNI), Mumbai, India.Mosquitoes are known to spread human diseases like West Nile fever, dengue,malaria, zika etc. accounting for millions of deaths worldwide. Lysinibacillussphaericus, a gram positive, spore producing commensal soil bacterium, hasbeen used worldwide for controlling mosquito population like Culex andAnopheles, and is regarded as safe against non-target organisms. Binary toxin,composed of BinA (41.9 kDa) and BinB (51.4 kDa) component proteins, isresponsible for the high larvicidal activity of several L. sphaericus strains.The two proteins exert high toxicity when administered together. BinA alonedisplays larvicidal activity, in the absence of BinB, albeit at reduced levels.But instability, shorter half-lives and rapid proteolytic digestion can limit theiruse as an effective insecticide. We for the first time demonstrate the beneficialeffect of PEGylation (covalent attachment of polyethylene glycol) onmosquito-larvicidal activity of BinA protein. PEG-protein conjugates weresynthesized using PEG-isocyanate polymer. The resulting bio-conjugateswere purified to homogeneity by column chromatography methods. Thesewere characterized by various biophysical methods like MALDI-TOF, DLS,DSF and CD. Two different isoforms of PEG-BinA conjugates are expectedfrom biophysical analysis, which appear to be mono-PEGylated but may differin the site of PEG attachment to BinA protein. The PEGylated proteins dis-played preservation of protein’s native structure and exhibited improved ther-mal stability by about 3-5 oC. The PEGylated proteins were also checked for

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stability against complex proteolytic environment. Regardless of the site ofmodification, the two isoforms showed a remarkable 7-fold improvement inthe larvicidal activity of BinA protein against 3rd instar Culex larvae, comparedto the unmodified protein. The PEGylation of recombinantly produced BinAcan be achieved easily. It promises a judicious approach towards mosquito pop-ulation control.

248-Pos Board B13A Novel Electrostatic Regulatory Mechanism in a Calcium Binding Pro-tein, L-plastinJonn Keenan Fanning, Van A. Ngo, Hiroaki Ishida, Hans Vogel,Sergei Yu. Noskov.Biological Sciences, University of Calgary, Calgary, AB, Canada.Plastins are a group of highly conserved actin binding proteins. L-plastin is a hu-man isoform of this protein found active in hemopoietic cells. This isoform isalso expressed in cancer cells. The N-terminal of this isoform contains aCalcium-binding EF-hand domain that allosterically regulates this protein forbinding to its target. It is, however, very difficult to delineate the effects that un-derlie the allostericmechanism. To investigate the regulatorymechanisms of theactivation of this protein domain, we have used both computational and exper-imental methods. Working with calcium in MD simulations has previouslycaused significant problems as classical force fields are not well equipped todeal with calcium. This is because calcium is a divalent ion, which can inducea strong local electrostatic field, and is capable of charge transfer. Although clas-sical force fields are insufficient, a new polarizable force field now known as aDrude force field, provides a more accurate solution to this problem. Wedescribe results from simulations with both Classical and Drude force fieldsfor the calcium-sensitive regions of L-plastin. These ongoing simulationshave already provided valuable insight an unexpected and previously unseenelectrostatic regulatory mechanism of L-plastin. In addition to computationalsimulations, to further validate this novel mechanism we obtained promisingexperimental validation using a number of biophysical methods includingisothermal calorimetry (ITC) and differential scanning calorimetry (DSC).Although we are looking into this mechanism in L-plastin, the conserved natureof this protein may indicate that this mechanism is present in a wide range ofrelated proteins, and may help provide us with a deeper understanding of themechanisms associated with calcium activation and deactivation.

249-Pos Board B14Structural Destabilization of Tropomyosin Induced by a Cardiomyopathy-Linked MutationThu N. Ly1, Inna Krieger2, Young-Ho Yoon3, Dmitri Tolkatchev1,Fadel A. Samatey3, Alla S. Kostyukova1.1Chemical Engineering and Bioengineering, Washington State University,Pullman, WA, USA, 2Department of Biochemistry and Biophysics, TexasA&M University, College Station, TX, USA, 3Trans-Membrane TraffickingUnit, Okinawa Institute of Science and Technology, Okinawa, Japan.In striated muscle, thin filaments, composed of F-actin, and thick filamentsconstitute the basic contractile units called sarcomeres. Tropomyosin (Tpm),a two-stranded coiled-coil protein, binds along the actin filaments throughhead-to-tail polymerization, protects and stabilizes the thin filaments. TheN-terminus of Tpm orients toward the pointed end of thin filament, where it in-teracts with pointed end-binding proteins such as tropomodulin (Tmod) andleiomodin (Lmod) to maintain the uniform filament length critical for propersarcomeric functions. Recently, a hypertrophic cardiomyopathy-associated mu-tation, R21H, has been identified in striated muscle Tpm (Tpm1.1) with molec-ular mechanism of perturbing muscular function unknown. We designed,expressed, and purified the Tpm chimeric peptide aTM1a1-28Zip. The peptideconsists of 28 N-terminal residues of Tpm1.1 followed by 18 C-terminal resi-dues of the GCN4 leucine zipper domain. The peptide was crystallized and itsstructure was solved. To study how this mutation affects Tpm1.1, we intro-duced the mutation R21H in the peptide. An effect of the mutation was studiedin silico using molecular dynamics simulation (MDS) and in vitro by circulardichroism (CD). Temperature measurements using CD were conducted tocharacterize the effect of the mutation R21H on thermal stability of theaTM1a1-28Zip peptide alone and its complexes with Tmod and Lmod frag-ments containing Tpm-binding sites. CD data showed that the mutationR21H caused a significant decrease in the helical content and structural stabilityof aTM1a1-28Zip. Complexes formed between the aTM1a1-28Zip[R21H] pep-tide and Tmod or Lmod fragments were less stable than those formed withwild-type aTM1a1-28Zip. All CD data were in agreement with MDS resultswhich showed that the mutation R21H significantly altered the coiled-coilstructure of aTM1a1-28Zip. We suggest that the mutation R21H destabilizesTpm structure by disrupting local salt bridges formed between residuesArg21 and Glu26 on opposite strands.

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250-Pos Board B15Probing Conformational and Functional Substates of Calmodulin by High-Pressure FTIRNelli Erwin, Satyajit Patra, Roland Winter.TU Dortmund, Dortmund, Germany.In response to intracellular Ca2þ-concentration changes, the highly dynamicand flexible Ca2þ-sensing protein calmodulin (CaM) interacts with more than300 diverse target proteins that are involved in numerous signaling pathwaysin eukaryotic cells. This unique promiscuous target binding behavior and theunderlying functional versatility of CaM is a result of its structural flexibility.CaM spans multiple conformational substates in solution providing adaptablebinding surfaces for different target proteins. K-Ras4B, known as a key regu-lator in the ERK pathway, was identified as specific binding partner of CaM,causing the dissociation of the lipidated K-Ras4B from the plasma membraneand thus reducing its activity. However, the interaction of K-Ras4B and CaMhas not been elucidated in detail until now. In order to evaluate the conforma-tional space of CaM and shed more light on the mechanism of subsequent targetrecognition and protein function, identification and characterization of func-tionally relevant conformational substates is mandatory. Applying pressure incombination with spectroscopies such as FTIR spectroscopy enables to popu-late and probe otherwise transient low-lying excited conformational substatesof CaM close in energy to ground state, but with a smaller partial volume.Evidence has grown that these states are functionally relevant, for instance inrecognition and ligand binding events. The pressure-induced conformationalchanges of CaM were studied in its Ca2þ-free and Ca2þ-bound state and inthe presence of the C-terminal K-Ras4B peptide. We show that not onlyCa2þ-binding, but also the presence of the target peptide has a drastic effecton the conformational dynamics of the protein.

251-Pos Board B16Obscurin Acts as a Variable Force ResistorNathan T. Wright, Aidan M. Willey.Chemistry and Biochemistry, James Madison University, Harrisonburg,VA, USA.Obscurin, a giant cytoskeletal protein, functions in part to connect distal ele-ments within a large variety of cell types. Due to this cellular role, obscurinlikely changes shape as its molecular targets move throughout the cell. Inthis way, obscurin must intrinsically resist force. Here, we describe the molec-ular etiology of force resistance through the study of multiple tandem obscurinIg domains. Using a combination of experimental and computational tech-niques, we find that obscurin acts as a differential force resistor. Differencesin tandem Ig domain flexibility are dictated primarily by linker sequence andnot linker length. These studies provide insight into the basic mechanisms ofhow cells respond to and resist stretch force.

252-Pos Board B17Structural and Functional Insight into Recombinant Lung SurfactantProtein B (rSP-B)Tadiwos G. Asrat, Valerie Booth.Biochemistry, Memorial University of Newfoundland, St. John’s, NL,Canada.Surfactant associated-protein B (SP-B) is the most essential protein componentof lung surfactant; its absence is fatal. Lung surfactant associated proteins,especially SP-B, assist in the organization and rearrangement of the lipid mono-layers and multilayers that coat the air-water interface of the alveolus, and sohelp to reduce the work of breathing and prevent lung collapse. Due toSP-B’s unique hydrophobic nature however, the overall three dimensionalstructure of the protein is not yet determined, therefore the molecular basisfor its activity is not clearly understood. Recently, our lab has managed to pro-duce the protein in bacteria using recombinant DNA technology. RecombinantSP-B (rSP-B) is shown to retain the expected alpha helical conformation indifferent membrane mimetic conditions, over a range of temperatures asobserved by far UV circular dichroism (CD) spectroscopy. Dynamic LightScattering (DLS) of rSP-B suspended in detergent micelles indicates two pop-ulations, one with a hydrodynamic size of ~3 nm and the other at ~100 nm.Nanoparticle Tracking Analysis (NTA) confirmed the ~100 nm species tobe prominent. Preliminary 1D solution NMR experiments have also been car-ried out.Moreover, rSP-B also shows promising results in in vitromeasures of function-ality when tested with a Langmuir-Blodgett trough. Lipid films in the presenceof rSP-B are able to promote multilayer formation when compressed to a suf-ficiently high surface pressure, comparable to that of clinical lung surfactants.What is more, films in the presence of rSP-B demonstrate superior compress-ibility and film recovery when compared to films compressed without rSP-B.

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253-Pos Board B18Regulation of Folding of de novo Designed Peptides by a-Helix FormationSaya Nishihara, Kosuke Toyama, Shigeru Shimamoto, Yuji Hidaka.Kindai University, Higashi-Osaka, Japan.Uroguanylin folds into its correct conformation with assistance by the propep-tide region. Our previous study of the disulfide-coupled folding of pro-uroguanylin suggested that a mis-bridged disulfide isomer is kineticallystabilized by the a-helical structure at the processing site between the propep-tide region and the mature region at the early stage of the folding reaction. Themis-bridged form is then converted to the native conformation via the forma-tion of a b-sheet structure between the mature and the propeptide region alongwith disulfide exchange reaction.To further investigate and utilize the intra-molecular chaperone function of thepropeptide, we designed a de novo peptide (NDD hybrid peptide) which foldsinto its bioactive conformation (G-type) with difficulty, and also designed ade novo protein (pro-NDD hybrid protein), which is able to fold into only aG-type, via the fusion of the propeptide region of uroguanylin. Our previousresults for their refolding reactions suggested that the G-type conformation isformed via the mis-bridged disulfide isomer (R-type). Considering these resultsand the propeptide-mediated folding of pro-uroguanylin, we proposed that theformation of the a-helical structure at the processing site is important in form-ing the folding intermediate.In this study, to validate this hypothesis, a series of the de novo peptide analogscontaining extended N-terminal sequences was prepared and their folding reac-tions were examined under kinetic or thermodynamic control. The results indi-cated that the formation or stabilization of the a-helical structure regulates theformation of the disulfide isomer as the proper intermediate under kinetic con-trol. The results of our studies will be discussed in this paper.

254-Pos Board B19Characterization of Protein Kinase a Free Energy Landscape by NMR-Restrained MetadynamicsYingjie Wang1,2, Carlo Camilloni3, Jonggul Kim1,2, Michele Vendruscolo3,Jiali Gao1, Gianluigi Veglia1,2.1Department of Chemistry, University of Minnesota, Minneapolis, MN,USA, 2Department of Biochemistry, Molecular Biology and Biophysics,University of Minnesota, Minneapolis, MN, USA, 3Department ofChemistry, University of Cambridge, Cambridge, United Kingdom.The free-energy landscape of a protein underlies the conformational transi-tions that are vital to its biological function. Recent advances in experimentaland computational methods are making it possible to characterize these freeenergy landscapes. In particular, the use of enhanced sampling techniquesin molecular dynamics simulations, including the replica average metadynam-ics (RAM) method, have partly alleviated the sampling bottleneck andbridged the gap between simulations and experiments. Here we appliedRAM to study the free energy landscape of the catalytic subunit of protein ki-nase A in the apo, binary (with ATP), and ternary (with ATP and pseudosub-strate, PKI5-24) forms. We used backbone NMR chemical shift restraints in allthree states to bias the conformational search toward conformations morereflective of experimental observables. Through this rigorous approach, wewere able to characterize the rugged free energy landscape of protein kinaseA and identify its modulation by ligand binding: whereas the apo state ex-hibits heterogeneous conformations, nucleotide binding partly reduces theconformational plasticity, and subsequent inhibitor binding further quenchesthe fluctuations. We conclude that NMR-Restrained Metadynamics is apromising approach to describe the free energy landscapes of complex pro-teins at atomic resolution through the integration of experiments andsimulations.

255-Pos Board B20Thermal Response of Inner and Outer Transmembrane Segments of CoraProtein by a Coarse-Grain Monte Carlo SimulationRas Pandey1, Sunan Kitjaruwankul2, Channarong Khrutto3,Pornthep Sompornpisut3, Barry Farmer4.1University of Southern Mississippi, Hattiesburg, MS, USA, 2KasetsartUniversity, Sriracha Campus, Thailand, 3Chulalongkorn University,Bangkok, Thailand, 4Air Force Research Laboratory, Dayton, OH, USA.Inner (i.corA) and outer (o.corA) transmembrane (TM) components of CorAprotein perform specific functions in transport of Mg2þ across the ion channels.The monomer protein (with 351 residues) consists of its outer (o.corA, residues1-290) and an inner (i.corA, residues 291-351) segments that form the pore. Weinvestigated the structure and dynamics of CorA protein and its inner (i.corA)and outer (o.corA) TM components as a function of temperature by a coarse-grained Monte Carlo simulation in an implicit solvent. We found that the

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thermal response of i.corA differed considerably from that of the outer compo-nent o.corA. Analysis of the radius of gyration revealed that the inner TMcomponent underwent a continuous transition from a globular conformationto a random coil structure on raising the temperature. In contrast, the outertransmembrane component exhibited an abrupt (almost discontinuous) thermalresponse in a narrow range of temperature. Scaling of the structure factorshowed a globular structure of i.corA at low temperature with an effectivedimension D ~ 3 and random coil at high temperature with D ~ 2. The residuedistribution in o.corA is slightly sparser than that of i.corA in a narrow thermos-responsive regime. The difference in thermo-response characteristics of thesecomponents (i.corA, o.corA) may reflect their unique transmembrane func-tions. Attempts are being made to incorporate such realistic features as explicitsolvent and membrane; corresponding results may also be presented as databecome available.

256-Pos Board B21The Competition between Electrostatic-Steering and ConformationalDynamics in the Diffusion-Limited Association of Calcineurin andCalmodulinPeter M. Kekenes-Huskey1, Bin Sun1, Eric C. Cook2, Trevor P. Creamer3.1Chemistry, UK, Lexington, KY, USA, 2Biochemistry, University ofKentucky, Lexington, KY, USA, 3Chemistry, University of Kentucky,Lexington, KY, USA.Calcineurin (CaN) is a serine/threonine phosphatase that regulates a variety ofphysiological and pathophysiological processes in most mammalian tissue. Inits inactive state, the CaN catalytic domain is inhibited by the auto-inhibitorydomain (AID); the active state is obtained upon CaM binding to the CaN reg-ulatory domain (RD). It has been established that the RD is highly disorderedwhen inhibiting CaN, yet it undergoes a disorder-to-order transition uponbinding calmodulin (CaM) to activate the phosphatase. Given that the RDis richly populated with polar and charged amino acids, arguably electrostaticinteractions may influence the rate of CaM association. However, it is likelythat properties of the RD conformational ensemble, such as its ‘effective vol-ume’ and accessibility of its CaM binding motif, influence the CaM/CaNassociation rate. In present study, we investigated via computational modelingthe extent to which electrostatics and structural disorder co-facilitate or hinderCaM/CaN binding kinetics. We examined several peptides containing theCaM binding motif, for which lengths and amino acid charge distributionswere varied, to isolate the contributions of electrostatics versus conforma-tional diversity to predicted, diffusion-limited association rates. These rateswere predicted using Replica Exchange Molecular Dynamics (REMD)and Brownian Dynamics (BD) simulations. Our results indicate that associa-tion rates vary as a function of increasing CaN RD length (beyond therequired CaM recognition sequence), thus indicating that RD conformationalensemble properties influence CaM binding. Second, we found that increasingthe solvent ionic strength generally depressed CaM/CaN association rates,owing to the attenuation of long-range electrostatic interactions that wouldnormally accelerate protein-protein association. Finally, CaN peptides withpositively-charged amino acids substituted at native negatively-charged siteshad complex effects on the predicted association rate, owing to ‘off-target’ in-teractions that in some cases competed with the intending binding site. Ourfindings detail the interplay between conformational diversity andelectrostatically-driven protein-protein association involving CaN, whichare likely to extend to wide-ranging processes regulated by intrinsically-disordered proteins.

257-Pos Board B22Equilibrium Molecular Dynamics of the Monomer and Dimer Units ofStreptococcus Pnuemonae and Corynebacterium Diphtheriae PiliEmmanuel Naziga, Jeff Wereszczynski.Illinios Institute of Technology, Chicago, IL, USA.Pili are elongated protein structures that enhance the adhesive abilities andvirulence of bacteria. As a result, it is important to understand their structureand mechanical properties. Pili are constructed by joining monomeric units (orpilins) into polymeric fibers via intermolecular isopeptide bonds. Interest-ingly, the pilins additionally possess intramolecular isopeptide bonds thatare formed between the sidechains of lysine and asparagine residues in thesame domain of the protein, which are believed to impart mechanical stabilityto the pili. In this work, we use all atom molecular dynamics simulations tostudy the conformational dynamics of monomeric and dimeric forms of thepilins of streptococcus pnuemonae (RrgB) and corynebacterium diphtheriae(spaA) in the presence and absence of intramolecular isopeptide bonds. Theresults reveal how the energetics and dynamics of these subunits are affectedby intramolecular isopeptide bonds, and demonstrate how they affect pilistability.

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258-Pos Board B23Biophysical and Structural Characterization of Antibody Responses toMalaria AntigensStephen Scally1, Alexander Bosch1, Brandon McLeod1, Gianna Triller2,Katharina Imkeller2, Rajagopal Murugan2, Sebastian R€amisch3, Rick King4,William Schief3, Hedda Wardemann2, Jean-Philippe Julien1.1Molecular Structure & Function, The Hospital for Sick Children ResearchInstitute, Toronto, ON, Canada, 2Max Planck Institute for Infection Biology,Heidelberg, Germany, 3The Scripps Research Institute, San Diego, CA, USA,4Malaria Vaccine Inititive, Washington, DC, USA.Malaria is a global health priority: 214 million malaria cases were reported in2015 alone, predominantly in Africa and resulting in 438,000 deaths - 70% ofwhich were in children below five years of age. Reverse vaccinology holdspromise to design effective immunogens for the development of malaria vac-cines. This concept is based on interrogating the B cell repertoire of vaccinatedor infected subjects to identify protective antibodies that will guide immunogendesign. Our efforts have focused on plasmodium falciparum targets associatedwith the development of pre-erythrocytic and transmission-blocking vaccines.We isolated B cells from individuals naturally exposed to plasmodium falcip-arum, and from vaccinated animals and humans having undergone controlledinfection. We performed extensive binding experiments to characterize affin-ities and competition of dozens of antibodies by isothermal titration calorimetryand biolayer interferometry. These studies uncovered several epitope bins,which give biophysical insights into immunodominant and protective B cellresponses. X-ray crystallography, small-angle X-ray scattering and single-particle electron microscopy were used and integrated to define antibody recog-nition at the molecular level. Our structural delineation of protective epitopesprovides the blueprints to engineer optimized antigens that can be formulatedand tested as pre-erythrocytic and transmission-blocking malaria vaccines.

259-Pos Board B24Structural Analysis of the Precursor Protein of Atrial Natriuretic PeptideSumika Futori, Satomi Higashigawa, Shigeru Shimamoto, Yuji Hidaka.Kindai university, Higashi-Osaka, Osaka, Japan.Atrial natriuretic peptide (ANP) is produced in the atrium and functions as avasodilator. ANP is expressed in the form of a precursor, prepro-ANP, and isthen processed into pro-ANP, which consists of the pro-peptide (98 residues)and mature ANP (28 residues, one disulfide bond) regions, in the endoplasmicreticulum. The biological role of ANP has been studied extensively. However,our knowledge of the role of the pro-peptide region of pro-ANP is still unclear.Therefore, to study the processing mechanism of pro-ANP and the role of thepro-peptide region, recombinant pro-ANPwas prepared by anE. coli expressionsystem and its conformation examined by means of circular dichroism (CD).The recombinant pro-ANP, which was readily over-expressed as a soluble formin E. coli cells, was purified by ion exchange chromatography, and identified byMALDI-TOF/MS.Toobtain structural information regardingpro-ANP,CDmeasurementswere car-ried out in 20mM Tris/HCl buffer (pH7.4). However, the results suggested thatpro-ANP does not possess specific secondary structures similar to other intrinsi-cally disordered proteins. Therefore, to further investigate the tertiary structure ofpro-ANP, we employed trifluoroethanol (TFE) as a structural mediator andcollected CD spectra of pro-ANP under several sets of conditions. The resultsindicated that TFE dramatically induced the formation of an a-helical structurein pro-ANP, suggesting that the pro-peptide region of pro-ANP is able to forman a-helical structure under a hydrophobic environment. Therefore, we proposethat the tertiary structure of pro-ANPmaybe stabilized by interactingwith hydro-phobic compounds, such as serum albumin or membrane lipids, in vivo.

260-Pos Board B25Inhibition of Aggregation in B-SheetModel Peptide by PPII Helix CappingHeng Chi1, Min Zhou1, Timothy A. Keiderling2.1pharmacy, Jiangsu Food and Pharmaceutical Science College, Huaian,China, 2Chemistry, University of Illinois at Chicago, Chicago, IL, USA.A 12mer peptide (Beta, SWTVEGNKYTYK-NH2) was designed based on theTrpzip model. Beta can form an aggregate with fibril like morphology imagedby TEM. The secondary structure of the fibril was characterized to be anti-parallel b-sheets by FT-IR spectra. VCD (Vibrational Circular Dichroism)spectrum demonstrated the supramolecular chirality of the fibril and UV-CDspectrum of this peptide confirmed the tertiary contact between aromatic resi-dues in the sequence. Upon mutation with an added PPII helix inducing comboto the C-terminal of this peptide, a new peptide BP (SWTVEGNKYTYKNGAPPPK-NH2) was synthesized. BP did not enhance ThT fluorescence, its sec-ondary structure was characterized to be unordered by UV-CD and FT-IRspectra. Further FRET analysis with a dansylated version of BP shows thatthe end to end distance is consistent with the proline mutations disrupting thearomatic contacts and thus stabilized the unordered conformation. In contrast,

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1:1 ratio mixture of a 9mer PPII conformation peptide and Beta did not changethe aggregation behavior of Beta. This study points out that sequence mutationis crucial in order to change the propensity of aggregation in peptides.

261-Pos Board B26Protein Folding as a Resonance Phenomenon, with Folding Free EnergiesDetermined by Protein-Hydration Shell InteractionsSungchul Ji.Pharmacology and Toxicology, Rutgers University, Kendall Park, NJ, USA.The single-molecule enzyme-turnover-time histogram of cholesterol oxidase[1] resembles the blackbody radiation spectrum at 4000 �K. This observationmotivated the author to generalize the Planck radiation equation (PRE), Sl =(8phc/l5)/(ehc/lkT � 1), by replacing the universal constants and temperatureby free parameters, resulting in the Planckian Distribution Equation (PDE),y = (A/(x þ B)5)/(eC/(x þ B) � 1) [2]. Since the first factor in PRE reflectsthe number of standing waves generated in the blackbody and the second factorthe average energy of the standing waves [3], it was postulated that any mate-rial system that generates data fitting PDE can be interpreted as implicatingstanding waves with associated average energies [2]. PDE has been found tofit the long-tailed histogram of the folding free-energy changes measuredfrom 4,300 proteins isolated from E. coli [4]. One possible interpretation ofthis finding is (i) that proteins (P) and their hydration shells (HS) are organizedsystems of oscillators with unique sets of natural frequencies, (ii) Ps assumetheir conformations whose standing waves are frequency-matched (or resonate)with the standing waves of their HSs, and (iii) the folding free energies aredetermined by the resonance frequencies of the P-HS complexes.[1] Lu, H. P., Xun, L. and Xie, X. S. (1998). Single-Molecule Enzymatic Dy-namics. Science 282, 1877-1882.[2] Ji, S. (2012) Isomorphism between Blackbody Radiation and EnzymeCatalysis. In:Molecular Theory of the Living Cell: Concepts, Molecular Mech-anisms and Biomedical Applications. Springer, New York, pp. 343-368. http://www.conformon.net/wp-content/uploads/2012/11/Isomorphism_blackbody_radiation_enzymic_catalysis_p343_p368.pdf[3] Blackbody radiation. http://hyperphysics.phy-astr.gsu.edu/hbase/mod6.html[4] Dill, K. A., Ghosh, K. and Schmidt, J. D. (2011) Physical limits of cells andproteomes. PNAS 108:17876-82.

262-Pos Board B27Reconstruction of Primordial P-loop NTPase PrecursorsMaria Luisa Romero-Romero.Weizmann Institute of Science, Rehovot, Israel.In collaboration with: Agnes Toth-Petroczy, Alexander Goncearenco and IgorBerezovsky (inference of the ancestral P-loop sequence) and Lin Yu-Ru andDavid Baker (computational protein design).Short functional peptides are likely to have served as crucial intermediates be-tween a primordial RNA world and the extant protein world. Our working hy-pothesis is that relics of these ancestral peptides still exist in the form of keymotifs in active sites of present-day proteins. One such motif, probably themost obvious one, is the P-loop (Walker A motif) that typically binds the trans-ferred phosphate moiety of ATP. The P-loop’s extended motif comprises ab-strand and an a-helix connected by the P-loop – a glycine-rich phosphate-binding loop. The P-loop containing nucleoside triphosphate hydrolase foldincludes this motif, and is one of the most ancient folds and by far the mostabundant (10-18% of all ORFs). In the present work, the sequence of a primor-dial b-Ploop-amotif was inferred. We further showed that this b-Ploop-amotif(~25 amino acids) could have served as a building block for the creation ofancestral globular domains by means of duplication and fusion. Computationaldesigned proteins were generated consisting of 2 repetitions of the ancestralmotif grafted into an ideal b/a- repeat protein. Our results indicate that thesesimple, repetitive proteins avidly bind ssDNA and RNA (possibly a crucialstep in the transition from the RNA world to a protein world). Further, ab-a-b fragment of this protein (< 40 amino acids) was found to self-assemble and thereby confer ssDNA binding. Overall, our results show thatP-loop NTPases could have emerged from a relatively short P-loop containingpeptide and that self-assembly played a key role in endowing biochemical func-tion despite limited size and complexity.

263-Pos Board B28Probing Protein Folding and Interaction in Live Cells using OsmoticPerturbationsShahar Sukenik1, Martin Gruebele2.1Center for Physics of Living Cells and the Department of Chemsitry,University of Illinois, Urbana-Champaign, Urbana, IL, USA, 2Department ofPhysics and Department of Chemistry, University of Illinois, Urbana-Champaign, Urbana, IL, USA.

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Cells rapidly change their volume in both optimal conditions and underduress, causing changes to their internal solute concentrations. Proteinsare known to respond to such fluctuations in solution concentrations evenfaster, well before the cell can initiate regulatory response. We take advan-tage of this response to probe protein conformation and interactions in livecells, under biologically relevant conditions. We induce a cellular volumechange in cells by subjecting them to a jump in media osmotic pressure.Protein dynamics are observed by FRET signal collected on an epifluores-cent microscope from adherent cells expressing mCherry/GFP tagged pro-teins. Our experiments reveal protein conformational changes, as well asbinding of weakly bound protein complexes, depending on the labelingscheme used. We observe that proteins can bind and unbind, and structurescan expand and contract reversibly when the cell volume increases or de-creases. Our results indicate that some proteins structures are more sensi-tive than others to cellular volume change, and that altering the internalconcentrations within the cell can alter protein processes, including foldingand binding.

Protein Structure, Prediction, and Design I

264-Pos Board B29Understanding the Role of Chain Flexibility in Amyloid Protein Aggrega-tion through Rationally Designed Protein SequencesSteven Z. Vance1, Xavier Redmon2, Rachel Hall1, Colman Moore1,Gram Booth2, Christa Hestekin2, Melissa Moss1.1Biomedical Engineering, University of South Carolina, Columbia, SC, USA,2Chemical Engineering, University of Arkansas, Fayetteville, AR, USA.The aggregation of amyloid proteins is associated with a myriad of medicalconditions including Alzheimer’s disease (AD), diabetes, and Parkinson’sdisease. While attributable to different amyloid proteins, these proteinsall share a common feature: a periodic glycine motif (GxxxG). Thisglycine motif, associated with increased backbone flexibility, is extendedin a number of familial mutations that significantly increase severity ofAD. A better understanding of the role that chain flexibility plays in pro-tein aggregation will allow for new and innovative protein engineeringstrategies for nanotech development as well as give insight into therapeuticstrategies.In this study, the glycine motif is targeted via either extension, by introductionof additional periodic glycine, or contraction, by replacement of glycine witha bulky or constrained amino acid. Modifications that extend periodic glycineinclude those that align with familial AD mutations. To examine how thesealterations to the glycine repeat motif impact aggregation kinetics, aggrega-tion was monitored via thioflavin-T fluorescence and fit using a novel kineticequation that accounts for unique features observed at late stages of aggre-gation. Aggregation products were visualized using transmission electron mi-croscopy to examine morphological features. In addition, aggregates werefractionated by size exclusion chromatography for size analysis via light scat-tering and morphology analysis via surface hydrophobicity. Results indicatethat increased chain flexibility correlates with faster nucleation as well as alarger quantity of small, intermediate aggregates that exhibit reduced fibrilmorphology with unchanged surface hydrophobicity. Taken together withobservations that smaller aggregates are more physiologically active, theseresults support the hypothesis that increases in protein chain flexibility,including that associated with familial mutations, may contribute to diseaseprogression.Future work will extend studies to other amyloid proteins, such as amylin andchaplin H.

265-Pos Board B30Probing Interactions between the Curli Accessory Protein CsgE andHuman Islet Amyloid PolypeptideTanya J. Espino, Sharon Patray, Isamar Aranda, Karen Guerrero,Sajith Jayasinghe.Biochemistry, CSUSM, San Marcos, CA, USA.Curli fibers are known to be involved in bacterial adhesion to surfaces, cell ag-gregation, and biofilm formation. Curli belong to a class of fibers known as am-yloids. Curli assembly requires 5 proteins: CsgA, CsgB, CsgE, CsgF, andCsgG. CsgA and CsgB are the major structural components of curli. CsgG isan outer membrane protein responsible for the secretion of CsgA and CsgBto the extracellular surface. CsgE and CsgF are thought to be chaperon proteinswhich are responsible for the transport of CsgA and CsgB in the periplasm. Ithas been shown that CsgE inhibits the aggregation of CsgA, the main compo-nent of curli fibers. However, not much is known about the specific interactionsbetween these two proteins. We employed a series of single cysteine mutants ofCsgE and fluorescence quenching to investigate the interaction of CsgE with

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CsgA and with human islet amyloid polypeptide another amyloidogenic proteinunrelated to curli formation.

266-Pos Board B31Non-Standard Protein Engineering at the Boundary of MolecularMechanics and Quantum Chemistry: Halogen-Based Design of InsulinAnalogsMichael A. Weiss1, Nelson F. Phillips1, Faramarz Ismail-Beigi2,Vijay Pandyarajan1, Yanwu Yang1, Yen-Shan Chen1,Nalinda Wickramasinghe1, Brian Smith3, John G. Menting4,Michael C. Lawrence4, Krystel El-Hage5, Markus Meuwly5.1Biochemistry, Case Western Reserve University, Cleveland, OH, USA,2Medicine, Case Western Reserve University, Cleveland, OH, USA,3Chemistry & Physics, La Trobe University, Melbourne, Australia,4Structural Biology, Walter and Eliza Hall Institute of Medical Research,Parkville, Australia, 5Chemistry, University of Basel, Basel, Switzerland.The broad utility of halogens in medicinal chemistry has motivated applica-tion of hybrid quantum- and molecular-mechanical methods. Extending theseconcepts to a therapeutic protein (insulin), iodination of a conserved tyrosineat position B26 was recently shown to enhance key properties of a rapid-acting clinical analog, including its thermodynamic stability and resistanceto fibrillation. Here, we (i) describe quantitative atomic-level simulations ofthe mono-iodininated insulin to predict its structural features and (ii) test thesepredictions by X-ray crystallography and multidimensional NMR spectros-copy. Using an electrostatic model of the modified aromatic ring based onquantum chemistry, the calculations suggested that the analog—as a dimerand hexamer—exhibits subtle differences in aromatic-aromatic interactionsat the dimer interface. Eight aromatic rings at this interface (residues B16,B24-B26 and their dimer-related mates) must reorganize to enable packingof the hydrophobic iodine atoms within the core of each monomer. Strikingly,these features were observed in the crystal structure of the mono-iodonatedinsulin analog (determined as a zinc hexamer at 2.3 A resolution). Giventhat residues B24-B30 detach from the core on receptor binding, the environ-ment of the 3-iodo-tyrosine at position B26 in a hormone-receptor complexmust differ from that in the free hormone. Based on the recent structure ofa ‘‘micro-receptor’’ complex, we predict that more marked reorientation ofthe modified tyrosine at the receptor interface enables directional halogenbonding and halogen-directed hydrogen bonding: favorable electrostaticinteractions exploiting, respectively, the halogen’s electron-deficient s-holeand electronegative equatorial band. Thus, inspired by quantum chemistryand molecular dynamics, such ‘‘halogen engineering’’ promises to extendprinciples of medicinal chemistry to proteins. Extensions of this approachto fluoro-aromatic, chloro-aromatic systems, and bromo-aromatic systems innon-standard protein design and their potential therapeutic applications willalso be discussed.

267-Pos Board B32Sweeter and Stronger: Structural-Driven Molecular Design to EnhanceSweetness and Stability of the Single Chain Monellin MNEISerena Leone1, Andrea Pica1, Federica Donnarumma1,Alessandro Emendato1, Rocco Di Girolamo1, Roberta Spadaccini2,Piero A. Temussi3, Delia Picone4.1Dept. of Chemical Sciences, University of Naples Federico II, Napoli, Italy,2Department of Science and Technology, University of Sannio, Napoli, Italy,3Department of Basic and Clinical Neurosciences, King’s College London,Napoli, United Kingdom, 4Department of Chemical Sciences, University ofNaples Federico II, Napoli, Italy.Sweet proteins are a family of proteins with no structural homology, able toelicit a sweet sensation by interacting with the dimeric T1R2-T1R3 sweet re-ceptor [Picone, D. and Temussi, P.A., 2012, Plant science, 195, 135-142].Our studies focus on MNEI, a single chain derivative of the natural proteinmonellin, representing one of the sweetest molecules known to date. MNEIstimulates a strong interest for its biological properties, but is also a wellaccepted model for protein folding and aggregation studies. In the frameworkof the ‘‘wedge model’’ [Temussi, P.A., 2009, TIBS, 34, 296-302], we have in-tegrated computational and experimental techniques to enhance sweetness andstability of MNEI against physical and chemical agents [Leone S., et al., 2016,Scientific Reports, 6, 34045]. Based on molecular modeling, NMR [Spadaccini,R., et al., 2016, FEBS Lett, (in press)], and X-ray crystallography, we designedthe sweetest protein ever obtained, which combines a sweetness threshold ofabout 25 nM with a very high and pH-independent thermal stability. Dockingstudies have provided a rationale basis to explain these properties, hinting at apreviously unpredicted role of plasticity in the interaction between this proteinand the sweet receptor. Additional aspects of protein aggregation and unfoldingproperties studied by Molecular Dynamics [Leone S. and Picone, D., 2016,

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PloS one, 11 (6), e0158372], Atomic Force Microscopy and Optic Spectros-copies will be also discussed.

268-Pos Board B33Molecular Determinants of Specificity in the Dpr-DIP InteractionNetworkAiman Sherani1, John M. Jumper2, Engin Ozkan3, Tobin R. Sosnick3,Benoit Roux3.1Biophysical Sciences, University of Chicago, Chicago, IL, USA,2Chemistry, University of Chicago, Chicago, IL, USA, 3Biochemistry andMolecular Biology, University of Chicago, Chicago, IL, USA.Understanding the physical and chemical principles governing specificity inprotein—protein binding is important both for revealing mechanisms of molec-ular recognition and for designing novel biomolecular systems. The proteinfamilies Dprs and DIPs, comprised of 21 and 9 immunoglobulin superfamily(IgSF) proteins, respectively, mediate neural cell adhesions and are criticalfor synapse formation specificity during development in arthropods. Previousresearch has recently characterized the Dpr-DIP interaction network and foundthat each Dpr selectively interacts with specific DIPs; the synaptic connectivitypattern in the fly brain is encoded by this molecular interaction network. Thegoal of this project is to determine the molecular basis of the specificity of theseinteractions. To achieve this goal, we are computing the binding free energiesof each possible Dpr-DIP interaction using both the Molecular Mechanics-Poisson Boltzmann Surface Area (MM-PBSA) approach and potential ofmean force (PMF) based framework, constructed from all atom molecular dy-namics (MD) simulations. We are also employing a new near-atomic level MDprogram, Upside, to rapidly sample possible poses. Due to the lack of high-resolution crystal structures for most of these interactions, homology modelingwas used in order to generate the initial structures. The calculated binding freeenergies will be used to predict possible Dpr-DIP interactions. Ultimately, wehope to use our current approach to predict potential protein—protein interac-tions between other IgSF proteins, which make up the second largest proteinfamily in the human proteome, and are crucial for the development and func-tion of the nervous and immune systems.

269-Pos Board B34Prediction of Protein and RNA Structures by Co-Evolution: Going BeyondAnecdotal Cases towards Large-ScaleGuido Uguzzoni1, Shalini John Lovis2, Francesco Oteri1, Hendrik Szurmant3,Weigt Martin1, Alexander Schug2.1Sorbonne Universites, UPMC, Institut de Biologie Paris-Seine, CNRS,Paris, France, 2SCC, Karlsruhe Institute of Technology, Karlsruhe, Germany,3College of Osteopathic Medicine of the Pacific, Western University ofHealth Sciences, Los Angeles, CA, USA.Structural characterization of many important proteins and protein complexes -typically preceding any detailed mechanistic exploration of their function- re-mains experimentally challenging. Novel statistical tools such as DirectCoupling Analysis (DCA) take advantage of the explosive growth of sequentialdatabases and trace the co-evolution of amino acids to predict secondary andtertiary contacts for proteins [1] and RNAs [2]. These contacts can be exploitedas spatial constraints in structure prediction workflows leading to excellentquality predictions [1,2,3,4]. We demonstrate for two-component signal trans-duction systems (TCS), a ubiquitous signal response system, how different sub-families of TCS can be identified based on genomic data [unpublished data].Going beyond anecdotal cases of a few protein families, we have applied ourmethods to a systematic large-scale study of nearly 2000 PFAM protein fam-ilies of homo-oligomeric proteins [unpublished data]. Also, we can applyDCA to infer mutational landscapes by capturing epistatic couplings betweenresidues and can assess the dependence of mutational effects on the sequencecontext where they appear [5].References[1] Weigt M et al., Proc Nat Acad Sci USA (2009) 106, 67-72; F. Morcos et al.,Proc Nat Acad Sci (2011) 108, E1293-E1301.[2] E. De Leonardis et al., Nucl Acids Res (2015), gkv932.[3] Schug A et al., Proc Nat Acad Sci USA (2009) 106, 22124-22129.[4] Dago A et al., Proc Nat Acad Sci USA (2012), 109: E1733-42.[5] M. Figliuzzi et al., Mol. Bio. Evol. (2016), 33:268-280, msv211.

270-Pos Board B35Protein-Protein Complex Structure Prediction using the Solution Theoryin the Energy RepresentationKazuhiro Takemura1, Akio Kitao1, Nobuyuki Matubayasi2.1IMCB, University of Tokyo, Tokyo, Japan, 2Grad. Sch. Eng. Sci, OsakaUniv., Osaka, Japan.Proteins conduct their functions through interactions with other molecules.Thus, accurate prediction of protein complex is a key to understand functional

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mechanisms. We have developed a method to evaluate binding free energy dif-ferences of complex models generated by docking prediction using the all-atommolecular dynamics simulation and solution theory in the energy representa-tion. By evaluating binding free energy difference with the method, we previ-ously show that ‘‘near-native’’ models similar to crystal structure aresuccessfully selected as the lowest energy structures in two protein-proteincomplexes, bovine trypsin with CMTI-1 squash inhibitor and RNase SA withbarstar [Takemura, J. Chem. Phys. 2012]. The method requires relatively shortMD simulations (2 ns) and can calculate binding free energy difference amongseveral hundreds of complex models. As a protein-protein complex structureprediction method, we combined the evaluation method with CyClus [Omori,Proteins, 2013] that performs fast clustering/reranking using a cylindricalapproximation of interface and improves the results of the rigid-body docking.The initial complex models were prepared using the protein-protein rigid bodydocking program, ZDOCK. The generated complex models were then clusteredand reranked using CyClus. Free energy analysis for top 100 or 300 complexmodels returned by CyClus were conducted using conformational energies, so-lute entropies, and solvation free energies calculated with the solution theory inthe energy representation. Our analysis improved the results of CyClus and thecomplex models similar to the native structure have the lowest binding free en-ergies, suggesting that this procedure is effective in protein-protein complexstructure predictions. We further improved the procedure by placing interfacewaters into protein-protein interface before free energy evaluations.

271-Pos Board B36Refinement of Protein Docking with Atom-Atom Contact Potentials, Back-bone Flexibility and Side-Chain RepackingTaras Dauzhenka, Ivan Anishchenko, Petras J. Kundrotas, Ilya A. Vakser.Center for Computational Biology, University of Kansas, Lawrence,KS, USA.Protein-protein docking is a challenging task for proteins with large conforma-tional changes upon binding (unbound/bound interface RMSD &gt 3 A). Theprotein-protein docking approaches typically involve a refinement stage thatfollows the lower-resolution global rigid-body search. Most refinement proto-cols utilize the same rotational/translational degrees of freedom as at the globalscan stage, with a more thorough sampling and sophisticated scoring. TheRosettaDock protocol also employs side-chain torsional degrees of freedomin the form of rotamers, and simultaneously optimizes the side-chain conforma-tions and the rigid-body position. In our approach, we incorporate rigid-bodyrotational/translational and side-chain and backbone loop torsional degrees offreedom into a single automated pipeline, that utilizes (i) our previously devel-oped semi-empirical atom-atom contact potentials for the rigid-body adjust-ment of the protein positions; (ii) a neighbor-dependent Ramachandranprobability distributions for the backbone loops torsional degrees of freedom;and (iii) our library of protein-protein interface side-chain rotamers. The pro-cedure was benchmarked on the Dockground (http://dockground.compbio.ku.edu) X-ray unbound set 4.0, which contains 396 co-crystallized protein-proteincomplexes and corresponding unbound structures for both proteins. The pro-cedure was evaluated by CAPRI assessment criteria for its ability to refinedocking predictions with ligand RMSD < 10 A, classified as incorrect, to theacceptable or better quality category, using only the atom-atom contact poten-tials and the rotational/translational degrees of freedom.

272-Pos Board B37Protein Folding upon Binding Revealed by Molecular DynamicsSimulationChris Neale1, Regis Pomes2, Rachel Sterne-Marr3, Angel Garcıa1.1Center for Nonlinear Studies, Los Alamos National Laboratory, LosAlamos, NM, USA, 2Biochemistry, University of Toronto, Toronto, ON,Canada, 3Biology, Siena College, Albany, NY, USA.Macromolecules frequently associate in living cells and tissues, where specificbinding interactions based on dynamic conformation regulate molecular local-ization and activity. Despite the rapidly expanding experimental characteriza-tion of the structural proteome, the combinatorial explosion of its interactomeprovides a strong motivation for the development of new tools to predictmacromolecular binding modes. Molecular simulation is one such promisingapproach, where advances in hardware, software, and parameterization aredriving increasingly accurate models of protein folding. Here, we use temper-ature replica-exchange simulations to study folding at a protein-protein inter-face and reveal the disorder-to-order transition that is necessary for bindingbetween a G protein-coupled receptor and either a G protein or a regulatory ki-nase. Specifically, we recapitulate a known G protein binding mode and predictthe experimentally uncharacterized binding mode of G protein coupled receptorkinase 2. The macromolecular signaling assembly that we conformationallydefine has important implications for kinase function and provides new insightinto biased signaling from these receptors.

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273-Pos Board B38Using Molecular Dynamics Simulations to Understand Pattern Formationin PolymersAnna Vernon1, Paul Fenimore1, Charlie Strauss2, Chang-Shung Tung1,Daan Frenkel3, Eugene Terentjev4.1Theoretical Biology and Biophysics, Los Alamos National Laboratory, LosAlamos, NM, USA, 2Bioscience Division, Los Alamos National Laboratory,Los Alamos, NM, USA, 3Department of Chemistry, University ofCambridge, Cambridge, United Kingdom, 4Cavendish Laboratory,University of Cambridge, Cambridge, United Kingdom.We use Molecular Dynamics simulations to study single chain polymer dy-namics and collapse to imitate the early stages of protein folding, as wellas polymer brush collapse, to understand the physics and morphology ofcollapsing polymer brushes. Simulations of this nature can help us understandcomplex phenomena such as glass transition, molecular crowding and com-plex higher order organisation. As an example, we demonstrate a study ofthe physical origin of the polymer glass transition from the point of viewof marginal rigidity, which is achieved above a certain number of intermolec-ular contacts. We find that when the average number of contacts per monomer(covalent and non-covalent) exceeds the critical value z*=4, the system be-comes solid and the dynamics arrested - a state that we declare the glass.We also look at polymer brushes and describe spinodal decomposition-likeprocesses that take place upon brush collapse. It is often impossible tosimulate large systems without a certain level of sophisticated coarse graining.We introduce a possible model for studying higher order organisation ofchromatin, a biological complex of DNA and histone proteins within the nu-cleus of a cell. Chromatin organisation is a fascinating topic that resemblesprotein folding, however, it is much more complex due to multiple levels atwhich chromatin folding and dynamics have to be understood. Using a simpli-fied force field, we also study helical structures formed by peptoids, smallpeptidomimetic molecules that are of importance to the pharmacologicalindustry.

274-Pos Board B39Molecular Interactions of Cannabinoid Receptor Interacting Protein 1 Aand B with Cannabinoid Receptor 1Pratishtha Singh1, Anjali Ganjiwale2, Allyn C. Howlett3, SudhaM. Cowsik4.1School of Life Sciences, Jawaharlal Nehru University, New Delhi, India,2Institute of Bioinformatics and Applied Biotechnology, Bangalore, India,3Department of Physiology and Pharmacology, Wake Forest School ofMedicine, Winston-Salem, NC, USA, 4Jawaharlal Nehru University,New Delhi, India.Cannabinoid receptor interacting protein isoform 1a (CRIP1a) modulatesthe Cannabinoid receptor 1 (CB1R) activity, which binds to D9-tetrahydro-cannabinol (main active ingredient of marijuana).The CB1R has importantphysiological roles in synaptic plasticity, analgesia, appetite, and neuro-protection. Studies indicate that CRIP1a suppresses tonic inhibition ofvoltage-gated Ca2þ channels without altering its agonist dependent activ-ities. There is another isoform of CRIP, known as CRIP1b (128 amino acids)shares same functional domain (residue in 34-110 region) with CRIP1a (164amino acids). Although CRIP1b also interacts with CB1R, but its functionalsignificance is not known. As not experimental structure is not available forboth, here we have attempted to investigate structures and interactions ofCRIP1a and b by in-silico methods. The intense homology searches didnot suggest any PDB structures with significant identity, but Fold DomainRecognition suggested Rho GDP-Dissociation Inhibitor 2(pdb id 1DS6_B)as suitable template for both the targets i.e., CRIP1a and CRIP1b. TheCRIP1a/b sequences were aligned manually using Discovery Studio2.5, fol-lowed by model calculations. The outlier residues were indentified and byloop refinement protocol. The models were optimized by energy minimiza-tion using Gromacs, and showed favorable Ramachandran plot statics withall the residues in favorable regions. Both the models were also supportedby their respective secondary structure data. The CRIP1a and 1b both foldinto beta sandwich fold model having a beta sheets by loops. The obtainedstructure of CRIP1a contains nine beta sheets while CRIP1b contain eightbeta sheets. Both the models were subjected to molecular docking studieswith CB1R-C terminus peptide (S464TDTSAEAL472), which is minimal re-gion involved interactions with CRIP1a/b. Both the complexes were sub-jected to molecular dynamics simulations, followed by interactionanalysis.Both CRIP1a and CRIP1b shows multiple hydrogen bonds withCB1R peptide, revealing the key residues involved in interaction. Sinceno crystal/solution structures are available for CRIP1a and CRIP1b, thebinding sites revealed from this study can be utilized for site directed muta-genesis and can serve as major target for diseases related to central nervoussystem.

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275-Pos Board B40Polarizable Amoeba Force Field Metadynamics with Minimization Pre-dicts Missing Protein LoopsArmin Avdic1, Mallory R. Tollefson2, Nicole Tatro2, Stephen D. LuCore2,Jacob M. Litman3, Timothy D. Fenn4, Michael J. Schnieders2,3.1Carver College of Medicine, The University of Iowa, Iowa City, IA, USA,2Biomedical Engineering Department, The University of Iowa, Iowa City,IA, USA, 3Biochemistry Department, The University of Iowa, Iowa City, IA,USA, 4Boehringer Ingelheim, Ridgefield, CT, USA.Computational methods developed to find the global free energy minimum ofamino acid sequences are increasingly successful, but limitations in both accu-racy and efficiency remain. Optimization algorithms are typically focused onproteins of modest size (i.e. of approximately 100 residues) and utilize potentialenergy functions based on fixed charged force fields, statistical or knowledgebased potentials, and/or potentials incorporating experimental data. Althoughthe aforementioned methods are widely used, known limitations include 1)search protocols that are inefficient or not deterministic due to rough energylandscapes characterized by large energy barriers between multiple minimaand 2) use of a target function whose global minimum does not correspondto the actual free energy minimum. To overcome the first limitation, thiswork describes a global optimization approach based on metadynamics to drivethe search of conformational space toward unexplored regions by adding atime-dependent bias to the objective function. To overcome the second limita-tion, a hybrid objective function is defined as the sum of the polarizableAMOEBA polarizable force field and an experimental X-ray crystallographytarget. As metadynamics drives the search, periodic quenching via local mini-mization is used to access structure quality via evaluation of Rwork. Thus, theoverall method is called AMOEBA Metadynamics with Minimization(AMM), and is suitable for optimization of side-chains, ligand binding poses,protein loops or even protein complexes. Here we focus on characterizingthe ability of AMM to elucidate the structural details of missing protein loops,which are often excluded from experimental X-ray crystallography structuresdue to conformational heterogeneity and/or limitations in the resolution ofthe data. We first show that the correlation between experimental data andAMOEBA structural minima is stronger than that for OPLS-AA/L (i.e. a fixedcharge force field). Next, missing protein loops are optimized using 5 nsec ofsampling for both AMM and simulated annealing with OPLS-AA/L. TheAMM procedure provides more accurate structures in terms of both experi-mental (i.e. lower Rfree values) and structural metrics (i.e. MolProbity). In addi-tion to providing more accurate loop conformations, AMM converged fasterthan the simulated annealing protocol. Overall, this work suggests that AMMis well-suited to refine or predict the coordinates of missing amino acid residuesand/or protein loops due to both the increased accuracy of the target functionrelative to OPLS-AA/L and more rapid convergence of the metadynamicsdriven search compared to simulation annealing.

276-Pos Board B41Improving 3D Structure Prediction of Beta-Barrel Membrane ProteinsWei Tian1, Hammad Naveed2, Jie Liang1.1University of Illinois at Chicago, Chicago, IL, USA, 2Toyota TechnologicalInstitute at Chicago, Chicago, IL, USA.Beta-barrel membrane proteins are found in the outer membrane of gram-negative bacteria, mitochondria, and chloroplasts. They carry out diverse bio-logical functions, including pore formation, membrane anchoring, enzymeactivity, and bacterial virulence. In addition, beta-barrel membrane proteinsincreasingly serve as scaffolds for bacterial surface display and nanopore-based DNA sequencing. Due to difficulties in experimental structure determi-nation, they are sparsely represented in the protein structure databank andcomputational methods are required to understand their biophysical principles.We have developed a novel 3D structural template for the construction of beta-barrel membrane proteins, which captures major geometric properties of theseproteins. With the help of the prediction procedure of strand registers that wedeveloped, we have achieved a high accuracy for the structure prediction ofbeta-barrel proteins. In addition, for the beta-barrel proteins with irregular bar-rel shape, we have further developed an elastic model, which characterizesbending and curvatures of the barrels, to improve the structure prediction ofthese proteins.

277-Pos Board B42De Novo Protein Structure Prediction by Big Data and Deep LearningSheng Wang, Jinbo Xu.Toyota Technological Institute at Chicago, Chicago, IL, USA.Recently ab initio protein folding using predicted contacts as restraints hasmade some progress, but it requires accurate contact prediction, which by ex-isting methods can only be achieved on some large-sized protein families with

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thousands of sequence homologs. To improve contact prediction for small-sized protein families, we employ the emerging deep learning techniquefrom Computer Science, a powerful technique that can learn complex patternsfrom large datasets and has revolutionized object and speech recognition, ma-chine translation and the GO game. Our deep learning model for contact pre-diction is formed by two deep residual neural networks. The first one learnsrelationship between contacts and sequential features (residue conservationand predicted secondary structure) from thousands of protein families, whilethe second one learns the occurring patterns of contacts and their relationshipwith pairwise features such as contact potential, residue co-evolution strengthand the output of the first network. Experimental results suggest that our deeplearning method greatly improves contact prediction and contact-assistedfolding, especially for small-sized protein families. Tested on 579 proteins dis-similar to training proteins, the average top L (L is sequence length) long-rangeprediction accuracy of our method, the representative direct evolutionarycoupling method CCMpred and the CASP11 winner MetaPSICOV is 0.47,0.21 and 0.30, respectively; their average top L/10 long-range accuracy is0.77, 0.47 and 0.59, respectively. Even without using force fields, our predictedcontacts allow us to correctly fold 203 test proteins, while MetaPSICOV andCCMpred contacts can do only 79 and 62 proteins, respectively. In the threeweeks of blind test with the weekly benchmark CAMEO (http://www.cameo3d.org/), our method successfully folded three large hard targets witha new fold and only 1.3L-2.3L sequence homologs while all template-basedmethods failed.

278-Pos Board B43Next Generation Evolutionary Sampling and Energy Function Guidedab initio Protein Structure PredictionAvdesh Mishra, Md Tamjidul Hoque.Computer Science Department, University of New Orleans, New Orleans,LA, USA.The conformation of a protein is vital to understand its function. Homology andab initio modeling are the two major strategies to solve the protein structureprediction from sequence. The homology methods are not applicable in theabsence of homologous sequences. This makes the ab initiomodeling unavoid-able. The development of ab initio method hinges on the effective conforma-tional space sampling and the accurate energy function to guide the searchprocess. In addition, recent studies demonstrates that it is possible to sampleand predict good quality protein structure without using native fragmentsfrom known protein structures. Towards this goal, we developed an ab initiomethod that applies a memory assisted Evolutionary Algorithm (EA) to samplethe energy hyper-surface of the protein folding process, looking for the globalminimum or the native fold of the protein. Sampling of the energy hyper-surface of the protein is achieved by novel mutation and crossover operationsbased on angular rotation and translation capabilities. Furthermore, the cross-over operations in current generation are enhanced by the use of the best parentselected from previous generation. In addition, we developed and employed aknowledge-based novel energy function, called 3DIGARS, which can dif-ferentiate the native structure that corresponds to the most thermodynamicallystable state, compared to the possible decoy structures most effectively. The3DIGARS energy function is an optimized combination of crucial propertiessuch as hydrophobic versus hydrophilic properties, sequence-specific predictedaccessibility and ubiquitous phi-psi angular characterization. The samplesobtained from the sampling of the energy hyper-surface are scored using3DIGARS and passed through the EA operators for natural selection and pro-cessing. The merits of the proposed approach has been rigorously examined andfound to be effective.

279-Pos Board B44A Predicted Structure of the Angiomotin Lipid Binding DomainAnn C. Kimble-Hill, Cameron J. Peck, Piiamaria S. Virtanen.Biochemistry & Molecular Biology, Indiana University School of Medicine,Indianapolis, IN, USA.Amots are a family of adapter proteins that modulate cellular polarity, differ-entiation, proliferation, and migration. Amot family members also have a char-acteristic lipid-binding domain, the coiled coil homology (ACCH) Domain thatselectively targets the protein to membranes, which has been directly linked toits regulatory role in the cell. Therefore, we endeavored to understand thestructure-function relationship of this domain with the desire to find ways tomodulate these signaling pathways. After many failed attempts to crystallizethe ACCH domain of each of the Amot family members for structural analysis,we decided to pursue homologous models that could be refined using smallangle x-ray scattering data. Theoretical models were produced using the Zhangsuite programs I-TASSER and LOMETS and then refined and analyzed usingCoot and PyMol modeling software based on RMSD, C-score, TM-score, and

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template redundancy. Top models were then compared to SAXS data forfurther model selection and refinement. As a result, we present a theoreticalmodel of the domain that is driven by alpha helices and short random coil re-gions. These alpha helical regions form a classic dimer interface followed bytwo wide spread legs that we predict to be the lipid binding interface. Finally,we validate the model presented with several lipid binding assays, which leadsto a suggested mechanism that links ACCH lipid binding, membrane deforma-tion, and vesicle fusion functions.

280-Pos Board B45Self-Association and Conformational Stability of NAMPT ProteinTrivikram R. Molugu1, Udeep Chawla1, Annie Huang1, Radu C. Oita2,Ting Wang2, Michael F. Brown1,3, Joe G.N. Garcia2.1Chemistry and Biochemistry, University of Arizona, Tucson, AZ, USA,2Department of Medicine, University of Arizona, Tucson, AZ, USA,3Department of Physics, University of Arizona, Tucson, AZ, USA.Nicotinamide phosphoribosyltransferase (NAMPT), also known as Visfatinand Pre B-cell colony enhancing factor (PBEF), is a rate-limiting enzyme inthe salvage pathway required for nicotinamide adenine dinucleotide (NAD)biosynthesis. It is a highly conserved 52-kDa protein, found in living speciesfrom bacteria to humans [1]. The protein serves as a cytokine and is involvedin cellular regulation influencing cancer, ischemia, obesity, and type-II diabetes[2, 3]. Despite being involved in many regulatory pathways, there is a paucityof information concerning the function of NAMPT, due to the limitations ofin vivo assays, and lack of expression systems for the protein. Here, we success-fully expressed the NAMPT protein using the pET-SUMO expression vector inE.coli strain SHuffle containing a hexa-His tag for protein purification. Activityassays demonstrated functionality of the protein. Moreover, initial biophysicalcharacterization of the protein using circular dichroism revealed secondarystructural elements consistent with crystallographic data. Dynamic light scat-tering showed the protein exists as large oligomeric units potentially involvedin the NAMPT signal amplification pathway. Hydropathy analysis indicatedpossible hydrophobic patches on the protein surface that explains the nativeoligomeric state. Most striking, we discovered that NAMPT can be solubilizedin n-dodecyl-b-D-maltopyranoside detergent in monomeric form. These find-ings open opportunities for further structural and functional investigations.Presently we are optimizing conditions for NMR experiments on NAMPT pro-tein. These methods [4] are complementary to X-ray crystallography, and pro-vide valuable information on the structure and dynamics, offering an importanttool for understanding biological functioning.[1] T. Wang et al. (2006) Nat. Struct. Mol. Biol 13, 661.[2] S. M. Camp et al. (2015) Sci. Rep. 5, 13135.[3] A. Fukuhara et al. (2005) Science 307, 426.[4] T. R. Molugu et al. (2016) Chem. Rev. (in press).

281-Pos Board B46The Interval Branch-And-Prune Algorithm for the Protein StructureDeterminationTherese E. Malliavin1, Bradley Worley1, Benjamin Bardiaux1,Guillaume Bouvier1, Mohamed Machat1, Andrea Cassioli2, Carlile Lavor3,Leo Liberti2, Michael Nilges1.1Institut Pasteur, Paris, France, 2Ecole Polytechnique, Paris, France,3University of Campinas, Campinas, Brazil.A general trend of structural biology is the switch of a rigid description of pro-tein structures, as in the first X-ray structures in the 50’s, to a flexible descrip-tion, in which each protein populate several distinct conformations or even acontinuum of conformations. This flexibility was shown in numerous casesto have a crucial importance in the function of proteins. Most of the methodsfor bio-molecular structure calculations are, up to now, based on a combinationof sampling and optimization, which does not allow a systematic exploration ofthe conformational space. But, in the case of highly flexible bio-molecules, asystematic (or global) exploration of the conformational space would be verywelcome.On the other hand, the interval branch-and-prune (iBP) approach has beenproposed as a method for allowing a global optimization of molecular struc-ture under distance restraints, the so-called Distance Geometry problem. Arecursive implementation of this algorithm has permitted to apply thisapproach on small structures of proteins in alpha-bundles for which fewlong-range distance restraints were known. Here, we are going to presentthe results obtained on a set of protein structures with sizes from 24 to100 residues, displaying various secondary structures and topology. The dis-tance restraints present on these structures were chosen to contain exclu-sively short-range information.The results obtained with various sets of distance restraints, including exactvalues and interval of values, will be presented, in order to experimentally eval-

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uate the complexity of the algorithm on real-case of protein structure determi-nation. Several procedures of acceleration will be used in order to allow acomplete exploration of the tree describing the molecular Distance Geometryproblem. The efficiency of the exploration will be evaluated using the self-organizing map (SOM) clustering approach, and the quality of obtained confor-mations with respect to Ramachandran plots and steric clashes will beevaluated.References:Cassioli A, Bardiaux B, Bouvier G, Mucherino A, Alves R, Liberti L, Nilges M,Lavor C and Malliavin TE. An algorithm to enumerate all possible protein con-formations verifying a set of distance constraints. BMC Bioinformatics,28;16:23 (2015).Lavor C., Alves R., Figueiredo W., Petraglia A., Maculan N. Clifford Algebraand the Discretizable Molecular Distance Geometry Problem. Adv. Appl. Clif-ford Algebras 25 (2015), 925-942.

Protein Stability

282-Pos Board B47Biological Roles of Protein Hyperstability: Implications for BiotechnologyWilfredo Colon, Ke Xia, Jennifer Church, Jayeeta Sen, Jane Thibeault,Hannah S. Trasatti.Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy,NY, USA.Although most proteins in nature are marginally stable, some proteins are hy-perstable, as indicated by their resistance to degradation, even under relativelyharsh conditions. The hyperstability of such proteins is usually under kineticcontrol due to a high-energy barrier for unfolding that virtually traps them ina specific conformation. Although the protective role of protein kinetic stabilityis well established, relatively little is known about the extent of biological rolesrelated to this biophysical property. We have demonstrated a correlation be-tween kinetic stability and a protein’s resistance to the denaturing detergentSDS, and have developed electrophoresis assays that in combination with pro-teomics analysis allow the identification of kinetically stable proteins incomplex biological samples. We have applied these methods to discover hyper-stable proteins in diverse systems, including mesophilic and thermophilic bac-teria, beans, and human plasma. The results of these studies have revealednovel insight about the biological significance and roles of protein kinetic sta-bility. In addition, the analysis of a growing list of SDS-resistant proteins gener-ated from these studies is enhancing our understanding of the structural basis ofprotein kinetic stability.

283-Pos Board B48Volumetrically Derived Thermodynamic Profile of Interactions of Ureawith a Native ProteinIkbae Son, Tigran Chalikian.University of Toronto, Toronto, ON, Canada.We report the first experimental characterization of the thermodynamic pro-file of urea binding to a native protein. We measured the volumetric param-eters of lysozyme at pH 7.0 as a function of urea within a temperature rangeof 18 to 45 �C. At neutral pH, lysozyme retains its native conformationbetween 0 and 8 M urea over the entire temperature range studied. Conse-quently, our measured volumetric properties solely reflect the interactionsof urea with the native protein and do not involve contributions fromurea-induced conformational transitions. We treated our data within theframework of a statistical thermodynamic analytical model in which urea-protein interactions are viewed as solvent exchange in the vicinity of theprotein. The van’t Hoff analysis of the temperature dependence of the equi-librium constant, k, for the urea-protein binding reaction produced changesin free energy, DG�, enthalpy, DH�, and entropy, DS�, accompanying thebinding. The thermodynamic profile of urea-protein interactions, in conjunc-tion with published MD simulation results, is consistent with the picture inwhich urea molecules, being underhydrated in the bulk, form strong, en-thalpically favorable interactions with the surface protein groups whilepaying a high entropic price. We discuss ramifications of our results forproviding insights into the combined effect of urea, temperature, and pres-sure on the conformational preferences of proteins.

284-Pos Board B49Sub-State Conformations of the Mesophilic and Psychrophilic LactateDehydrogenases Preceding Irreversible Thermal InactivationSergei Khrapunov, Eric Chang, Robert Callender.Biochemistry, Albert Einstein College of Medicine, Bronx, NY, USA.The thermodynamics of oxamate binding and the temperature stability ofthe glycolytic enzyme lactate dehydrogenase from porcine heart, phLDH(mesophilic Sus scrofa) and from mackerel icefish, cgLDH (psychrophilic

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Chamapsocephalus gunnari) have been investigated. A novel fluorescenceassay was utilized that simultaneous monitors changes to the global proteinstructure, structural changes near the active site, and aggregation of the enzymein response to increasing temperature and increasing concentration of the nat-ural osmolyte, trimethyl amine N-oxide (TMAO), a stabilizer of protein struc-ture. Using this assay, the reverse changes of stability and affinity for oxamatewere established for both, phLDH and cgLDH. Importantly, a low-temperature(pre-denaturation) structural transition was found that precedes the high-tem-perature (denaturation) transition for both LDHs and coincides with increasingenzymatic activity. The structural transitions of the global protein structure andthe active site are concerted for the rigid (phLDH) and not concerted for theflexible (cgLDH) LDHs. The profound contribution of entropy to G alongwith the higher structural flexibility increases functional plasticity of the psy-chrophilic cgLDH. TMAO increases stability and shifts all structural transitionsto the higher temperatures for both orthologs and simultaneously reduces theircatalytic activity. The multiple active and inactive along with intermediate sub-state conformations of the enzyme exist in equilibrium at the stage precedingirreversible thermal inactivation. This equilibrium is an essential selective fac-tor for the adaptation of an enzyme to the environmental temperature. It seemsalso possible that thermal adaptation of proteins may be complemented by evo-lution of the cellular milieu.

285-Pos Board B50Denatured State Loop Formation Thermodynamics of a HybridPolypeptideMoses Leavens, Bruce E. Bowler.University of Montana, Missoula, MT, USA.Previous work with the four-helix bundle protein cytochrome c’ in Rhodopseu-domonas palustris using histidine-heme loop formation thermodynamicmethods revealed fold-specific deviations from random coil character in its de-natured state ensemble. To examine the generality of this finding, we extendthis work to a three-helix bundle polypeptide, the human DNA excision repairprotein’s second ubiquitin-associated (UBA) domain, UBA(2). We use yeastiso-1-cytochrome c as a scaffold, fusing the UBA(2) domain to the N-terminusof iso-1-cytochrome c. Using site-directed mutagenesis, we have engineeredhistidine into solvent accessible surface residue positions within the all-alphafold, creating eight single histidine variants. Isothermal equilibration denatur-ation studies reveal that the fusion protein unfolds in a 3-state process,commencing with iso-1-cytochrome c followed by UBA(2). Thermodynamicstability experiments also demonstrate that the histidine residues in theUBA(2) domain strongly destabilize iso-1-cytochrome c. Furthermore, histi-dine-heme loop formation equilibria show lower apparent pKa’s compared tothe pseudo-wild type variant, indicating significant interactions in the dena-tured state. We will compare the degree of deviation of loop stability versusloop size, relative to predictions of the Jacobson-Stockmayer relationshipused in our previous work on cytochrome c’. This comparison will allow eval-uation of sequence-based conformational bias in the denatured state of thisprotein.

286-Pos Board B51Regulation of Protein Folding using Organic Solvents and Ionic LiquidsYuji Hidaka, Ryosuke Nishimura, Shigeru Shimamoto.Kinki University, Higashi-Osaka, Japan.The Escherichia coli expression system is frequently used to prepare variousproteins in a highly efficient manner using information obtained by the HumanGenome Project. However, recombinant proteins are often expressed as inclu-sion bodies which are biologically inactive, and a refolding reaction is abso-lutely required to form the correct tertiary structure. For this purpose, in vitrorefolding reactions of recombinant proteins are generally performed in aqueoussolutions. However, it is still difficult to efficiently fold recombinant proteinsinto their biologically active form in aqueous solutions because aggregatesare formed as the result of the hydrophobic folding intermediates. Ionic liquidshave recently been employed for refolding reaction of proteins in place ofaqueous solutions and successfully folded into their native conformations canbe accomplished, although the folding yield was still low. In addition, organicsolvents, such as trifluoroethanol, are generally used for protein folding toinduce a-helix formation and may reduce the extent of hydrophobic interac-tions. Therefore, to regulate or suppress hydrophobic interactions duringthe refolding reaction, organic solvents and ionic liquids were examined in at-tempts to construct the native conformation or to produce a proper foldingintermediate.Dioxiane and 1-hexyl-3-methylimidazolium chloride (HMIM) were examinedfor the refolding reaction of prouroguanylin, as a model protein, which containsthree disulfide bonds. Prouroguanylin was able to fold into its native conforma-tion at a low concentration of HMIM or dioxane. Surprisingly, prouroguanylin

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was still able to form native disulfide bonds even at 80% HMIM or dioxanealthough prouroguanylin was not able to fold into its native structure underdenaturing condition using urea or guanidine hydrochloride. Therefore, the re-sults indicate that those solvents are suitable candidate solvents for use in pro-tein folding studies. The results will be discussed in this paper.

287-Pos Board B52Glycine Betaine Reverses Osmotic Shock Induced Protein Destabilizationin Living CellsSamantha S. Stadmiller, Gary J. Pielak.Chemistry, University of North Carolina at Chapel Hill, Chapel Hill,NC, USA.Cells utilize several mechanisms for adapting to changes in osmotic pressure.Bacteria, including Escherichia coli, can grow in a wide range of osmolarities.Increasing the external osmolarity (i.e., hyperosmotic shock) causes waterefflux, reduction in cell volume and accumulation of osmolytes such as glycinebetaine. This volume reduction increases the crowded nature of the cytoplasm,which is expected to affect protein stability. In contrast to traditional theory,which predicts that more crowded conditions can only increase stability, recentwork shows that crowding can destabilize proteins through transient attractiveinteractions. Here, we quantify protein stability in living E. coli cells before andafter osmotic shock in the presence and absence of glycine betaine. The 7-kDaN-terminal SH3 domain of Drosophila signal transduction protein drk (SH3) isused as the model protein because it exists in an equilibrium between a foldedstate and an unfolded ensemble. Labeling SH3 with a fluorine on its sole tryp-tophan facilitates NMR-based detection of both states simultaneously, allowingquantification of the free energy of unfolding in vitro and in living E. coli cells.We find that hyperosmotic shock decreases SH3 stability, consistent with theidea that weak interactions are important under physiologically relevantcrowded conditions. Subsequent uptake of glycine betaine returns SH3 to thestability observed without osmotic shock. These results highlight the effectof transient interactions on protein stability in cells and provide a new explana-tion for why stressed cells accumulate osmolytes.

288-Pos Board B53Fluorescence Evidences for Non-Homogeneity and Residual Structure ofDenatured StatesKatherina Hemmen1, Dmitro Rodnin1, Igor Markovic1,Thomas Otavio Peulen1, Suren Felekyan1, Ralf Kuehnemuth1,Hugo Sanabria2, Claus A.M. Seidel1.1Institute of Molecular Physical Chemistry, Heinrich-Heine-University,D€usseldorf, Germany, 2Department of Physics & Astronomy, ClemsonUniversity, Clemson, SC, USA.About 30 % of human proteins do not fold into a stable 3D arrangement of sec-ondary structure elements, but stay predominantly unfolded -similar to proteinsunder highly denaturing conditions. These proteins are involved in many cellsignaling processes. Their characterization poses a great challenge for currentexperimental methods as they consist of an ensemble of rapidly interconvertingconformations. Intense debate exists on the possibility that they show, to certainextent, residual structure, which might facilitate folding or enhance ligandbinding. To study the unfolded state conformational heterogeneity usingForster resonance energy transfer (FRET), we used the lysozyme from thephage T4 (T4L) in denaturing conditions as a model system. We built an elasticnetwork model that spans T4L’s topology in order to evaluate local and globalconformational changes by combining ensemble (ensemble time-resolvedfluorescence lifetime and anisotropy) and single-molecule spectroscopic(multiparameter fluorescence detection, photon distribution analysis, (filtered)fluorescence correlation spectroscopy) methods. Through extensive compari-son of models, we identified regions with apparent residual structure underhighly denaturing conditions, which might serve as folding nuclei; andadditionally we showed that chemically denatured T4L is not a random coilas previously thought. By using obtained distance restraints we determinedthat denatured T4L shows a native-like mean structure, albeit larger in sizecompared to the native state. We demonstrate here the necessity of carefuldata interpretation, but also the potential of a multidimensional approach tocharacterize an ensemble of states, which can be applied generally to unstruc-tured or denatured proteins.

289-Pos Board B54Selection Maintaining Protein Stability at EquilibriumSanzo Miyazawa.Gunma Univ., Kiryu, Japan.Recently it was indicated that fitness costs due to misfolded proteins are a deter-minant of evolutionary rate and selection originating in protein stability is adriving force of protein evolution. Here we examine protein evolution underthe selection maintaining protein stability.

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Protein fitness studied is a generic form of fitness costs due to misfolded pro-teins; s = k exp(DG / kT) ( 1 - exp(DDG / kT)), where s and DDG are selectiveadvantage and stability change of a mutant protein, DG is the folding freeenergy of the wild-type protein, and k is a parameter representing proteinabundance and indispensability. The distribution of DDG is approximated tobe a bi-Gaussian distribution, which represents structurally slightly- orhighly-constrained sites. Also, the mean of the distribution is negatively pro-portional to DG.The evolution of this gene has an equilibrium point DGe, the range of which isconsistent with observed values in the ProTherm database. The probability dis-tribution of Ka/Ks, the ratio of nonsynonymous to synonymous substitution rateper site, over fixed mutants in the vicinity of the equilibrium shows that nearlyneutral selection is predominant only in low-abundant, non-essential proteins ofDGe > �2.5 kcal/mol. In the other proteins, positive selection on stabilizingmutations is significant to maintain protein stability at equilibrium as well asrandom drift on slightly negative mutations, although the average is lessthan 1. Slow evolutionary rates can be caused by high protein abundance/indis-pensability and large effective population size, which produce positive shifts ofDDG through decreasing DGe, and by strong structural constraints, whichdirectly make DDG more positive. Protein abundance/indispensability moreaffect evolutionary rate for less constrained proteins, and structural constraintfor less abundant, less essential proteins.(Reference: J. Theor. Biol., 391, 21-34, 2016.)

290-Pos Board B55Effects of Flanking Disorder on the Behaviour of Ordered DomainsKatie R. Kemplen1, Petur O. Heidarsson2, Lasse Staby1, Charlotte O’Shea1,Karen Skriver1, Birthe B. Kragelund1.1Biocenter, University of Copenhagen, Copenhagen, Denmark, 2Universityof Zurich, Zurich, Switzerland.Both structured proteins and those containing intrinsic disorder have been thesubject of much investigation; especially as disorder relates to protein-proteininteractions. However, there has been little exploration into the influence ofthese elements on each other. Do IDPs regulate the stability and folding mech-anisms of connected structured regions and vice versa do ordered domainsaffect the affinity of IDRs for their ligands e.g. by affecting the association ki-netics? To try and answer these questions we use biophysical methods and pro-gressive deletions to vary the length of disordered regions flanking ordereddomains in two model systems of one and two globular domains, respectively.

291-Pos Board B56The Effect of Polydisperse Crowding on Protein StabilityAlan van Giessen, Anastasia Osti.Chemistry, Mount Holyoke College, South Hadley, MA, USA.The dense, heterogeneous cellular environment is known to affect protein sta-bility through interactions with other biomacromolecules. The effect ofexcluded volume due to these biomolecules, also known as crowding agents,on a protein of interest, or test protein, has long been known to increase the sta-bility of a test protein. The cellular environment is heterogeneous not only interms of its chemical composition, but also in terms of the sizes of thebiomacromolecules, or crowding agents, present. It has been shown experimen-tally that the effect of polydisperse or mixed crowding agents has a non-addi-tive effect, i.e. that there is an optimal mixing ratio where the effect of thecrowding agents is larger than that of monodisperse systems of each crowder.Here we investigate the role of polydisperse crowding on two small test pro-teins: the helical trp-cage and the beta-hairpin GB1m3. For each test protein,a series of simulations using crowding agents of two different sizes in variousratios were conducted. Crowding agents used were either spherical excluded-volume only crowders or proteins with fixed backbones. In particular, we relatethe non-additivity to the excluded volume of the crowding agents. We alsoshow that protein-crowder interactions can play a large role in either enhancingor offsetting the effect of the crowding agent excluded volume.

292-Pos Board B57PAPS-Synthase: Dissecting Folding of a Large and Naturally FragileProtein In Vitro and In CelluloOliver Brylski1, Jonathan Wolf Mueller2, Simon Ebbinghaus1.1Physical Chemistry II, Ruhr-University Bochum, Bochum, Germany,2Institute of Metabolism and Systems Research, University of Birmingham,Birmingham, United Kingdom.PAPS synthases are bifunctional enzymes providing the cell with the sulfatedonor PAPS (3’-phosphoadenosine-50-phosphosulfate), which is further usedby sulfotransferases for modification of several biomolecules (e.g. steroids).Isoform PAPS synthase 2 (PAPSS2) has been shown to be fragile withinin vitro experiments, but is stabilized by binding of its endogenous ligands. Mu-tations affecting its activity lead to disease states like bone and cartilage mal-

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formation as well as metabolic diseases. Compared to in vitro conditions, thecellular milieu is crowded by large biopolymers resulting in intermolecular in-teractions and excluded-volume effects affecting each biopolymer inside thecell.To understand the stability of this large enzyme (70 kDa), we conducted bio-physical studies on the individual domains, APS kinase and ATP sulfurylase,as well as the full length protein. Using Fast Relaxation Imaging we studieddisease-relevant mutants of PAPSS2 directly within the cell. The data providenovel thermodynamic insights into PAPSS2 stability inside the cellular milieuand its influence on the naturally destabilized protein. These results also un-ravel new insights into disease mechanisms of PAPSS2 mutations.

293-Pos Board B58Effects of Salt or Cosolvent Addition on Thermal Stability of a Protein:Relevance to those on Solubility of a Hydrophobic Solute in WaterShota Murakami1, Tomohiko Hayashi2, Masahiro Kinoshita2.1Graduate School of Energy Science, Kyoto University, Uji, Kyoto, Japan,2Institute of Advanced Energy, Kyoto University, Uji, Kyoto, Japan.The thermal stability of a protein is changed upon addition of a salt or cosol-vent. The solubility of a hydrophobic solute (e.g., argon or methane) in wateris also influenced by the addition. Interestingly, the addition which decreasesthe solubility usually enhances the thermal stability. This suggests that the hy-drophobic effect is a principal factor governing the stability change, becausethe decrease and increase in the solubility, respectively, are ascribed toenhancement and reduction of the effect. However, urea decreases the solubi-lity but lowers the stability. Bromide and iodide ions decrease the solubility butlower the stability of a protein with a large, positive net charge. In these cases,the stability change is influenced by the changes in not only the hydrophobiceffect but also other physical factors. We show for hydrophobic solutes thatthe integral equation theory where the solute and solvent particles are modeledas hard spheres with different diameters can reproduce the experimental datafor the following items: salting out by an alkali halide and salting in by tetra-methylammonium bromide, increase in solubility by a monohydric alcohol, anddecrease in solubility by sucrose or urea. The orders of cation or anion speciesin terms of the power of decreasing the solubility can also be reproduced foralkali halides. With our model, the analyses are focused on the roles of entropyoriginating from the translational displacement of solvent particles. As theproducts, we clarify the pivotal physical origin of the hydrophobic effect andpresent a new view on the Hofmeister series. We show how the series is ex-pressed when the hydrophobic effect dominates and how it is modified whenother physical factors are also influential.

294-Pos Board B59Residual Structure in the Denatured State of a Three-Helix Bundle ProteinDustin Becht, Klara Briknarova, Bruce Bowler.University of Montana, Missoula, MT, USA.The denatured state of a protein is canonically described as an ensemble of non-interacting random coil conformations. By studying a helical bundle protein un-der denaturing conditions, we have found that residual structure persists in itsdenatured state. Our work uses the upstream Ubiquitin-Associated domain,UBA(1), of the HHR23A protein as a model system to study residual structurein the denatured state ensemble. Multi-dimensional nuclear magnetic resonance(NMR) experiments 1H-15N HSQC, HNCO, and HNCAwere used to find back-bone atom secondary chemical shifts which correspond to protein secondarystructure with amino acid resolution. By comparing chemical shifts in moder-ate- to highly denaturing conditions of 4M, 5M, and 6M guanidine HCl to areference state of 7M GdnHCl, UBA(1) shows residual helical content amongall three helices. In contrast, the helical propensity is predicted by the programAgadir to be higher in helix 2 and negligible elsewhere, with the assumptionthat helices do not interact. The presence of relatively uniform helical contentamong all three helices may indicate stabilizing tertiary interactions despitehighly denaturing conditions. To further probe tertiary interactions, single-res-idue mutants of UBA(1) will be used to determine if residues in the hydropho-bic core or turn regions are contributing to stability of residual structure in thedenatured state.

295-Pos Board B60Phosphorylation Induced Global Structural Destabilization of a SmallProtein DomainAshleigh Bachman, Radwan Ebna Noor, Dimitra Keramisanou,Ioannis Gelis.Chemistry, University of South Florida, Tampa, FL, USA.Protein phosphorylation at a single or multiple sites is utilized to regulate pro-tein functional outcomes and overall cellular activities through signaling path-ways. At a molecular level, the addition of a phosphoryl group may alter thefunction of a protein through distinct and versatile mechanisms. These include

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allosteric structural and dynamic changes, direct positive or negative modula-tion of affinities, masking or unmasking of cofactor binding sites, autoinhibi-tion and local disorder-to-order or order-to-disorder transitions. Here wepresent evidence from NMR spectroscopy and other biophysical techniquesthat protein phosphorylation may lead to a global domain destabilization.Upon phosphorylation a small protein domain acquires a molten globule state,where at least two stable folding intermediates exist in equilibrium with a het-erogeneous conformational ensemble. This conformational transition modu-lates in turn the affinity for protein partners but also permits rapid domainrefolding upon removal of the phosphoryl group.

296-Pos Board B61Promiscuous Contacts and Heightened Dynamics Increase Thermosta-bility in an Engineered Variant of the Engrailed HomeodomainMichelle E. McCully1,2, Valerie Daggett2.1Biology, Santa Clara University, Santa Clara, CA, USA, 2Bioengineering,University of Washington, Seattle, WA, USA.The Engrailed Homeodomain (EnHD) is a three-helix-bundle transcriptionfactor that is popular within the protein folding community due to its ultra-fast un/folding kinetics. In 2007, the Mayo group used EnHD as a backbonetemplate for their repacking algorithm and designed UVF as a proof ofprinciple. UVF not only folded as designed but was extremely stable; its Tm

is >99�C whereas the Tm of EnHD is 52�C. Here, we present a structuraland dynamical explanation for UVF’s increased thermostability based on1 ms of all-atom, explicit-solvent molecular dynamics simulations of the twoproteins at room temperature (25�C) and high temperature (100�C). UVFhad heightened dynamics at room temperature, relative to EnHD, as measuredby Ca motion and interchangeability of side-chain contacts. UVF maintainedthese dynamics at 100�C whereas EnHD began to unfold. UVF’s binary distri-bution of amino acids, with only hydrophobic residues at buried positionsand only hydrophilic residues on the surface, allowed buried residues tomove independently from the surface, effectively acting as an entropy sink.Hydrogen bonds and salt bridges between buried and surface residues ofEnHD coupled core motion to surface motion, resulting in EnHD denaturingat 100�C. Based on our simulation data, we hypothesize that UVF benefitsfrom more favorable entropy of folding in addition to its rationally designed,more-favorable enthalpy of folding. Future work will investigate this hypoth-esis experimentally and determine how the heightened dynamics effect nativeDNA-binding function.

297-Pos Board B62Rational Design of a Synthetic Peg-Like Polymer for Protein StabilizationChristopher DelRe1, Brian Panganiban1, Tim Li1, Charley Huang1,Monica Olvera de la Cruz2, Patrick Dennis3, Ting Xu1.1University of California, Berkeley, Berkeley, CA, USA, 2NorthwesternUniversity, Evanston, IL, USA, 3Air Force Research Laboratory, Dayton,OH, USA.Organophosphorus hydrolase (OPH) is an enzyme that can deteriorate acutelytoxic chemicals known as organophosphates, which are ubiquitously employedas pesticides and are thus considerable environmental hazards. However, theproclivity of OPH to aggregate in aqueous media severely hinders its biologicalactivity. Inspired by natural chaperones, we have designed and synthesized het-eropolymers to encapsulate and stabilize OPH. When OPH is complexed withthe designed heteropolymer, it exhibits a drastic increase in biological activityin a buffer solution - approximately 15 times greater than the native biologicalactivity of pure OPH in the same environment. We have confirmed via dynamiclight scattering that while pure OPH aggregates in the buffer solution, the het-eropolymer prevents aggregation from occurring.Two model proteins, bovine serum albumin and lysozyme, have also been stud-ied with the heteropolymer to aid our understanding of the protein-polymer in-teractions that occur in our system. The structure and particle size of thedifferent protein-polymer mixtures at several temperatures have been probedvia dynamic light scattering, circular dichroism, and Fourier transform infraredspectroscopy. These characterization techniques have elucidated the mecha-nisms of interactions between the polymer and proteins.

298-Pos Board B63Stability of Hsp60 from Helicobacter Pylori: Effect of GTP BindingKarina Guadalupe, Jose Mendoza.California State University of San Marcos, San Marcos, CA, USA.The heat shock protein Hsp60 is one of the most abundant proteins observed inH. pylori. Since the sequence of Hsp60 is similar to that of the chaperonin fromE. coli, GroEL, it is hypothesized that their functions are similar as well. How-ever, unlike GroEL which binds to ATP, Hsp60 from H. pylori also binds toGTP. It was previously shown that the binding of GTP caused alpha-crystallin,

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a protein that functions as a molecular chaperone, to be less stable. Here, weinvestigated the effect of GTP on the stability of H. pylori Hsp60 by using fluo-rescence. Hsp60 does not contain any tryptophans. Therefore, a mutant ofHsp60 containing a tryptophan replacement at residue 202 (Y202W) wasused for fluorescence studies. Samples of 1 mL were prepared containing50 mM Tris-HCl pH 7.5, 1 uM Y202W, 0.25 mM GTP, and varying urea con-centrations (0-6 M). All aliquots were incubated for 24 h before their fluores-cence was measured between 310 nm and 370 nm. Samples were excited at280 nm. The native state of the Hsp60 mutant displayed a maximum fluores-cence intensity at 331 nm. The Hsp60 mutant appeared to be denatured with4 M urea or higher concentrations. The denatured state of the Hsp60 mutant dis-played a maximum fluorescence intensity at 353 nm. Fluorescence was thenused to determine whether the binding of GTP affected the stability againsturea denaturation. Our results show that Hsp60 has a relatively low stabilityagainst urea denaturation (C1/2 = 2.75 M) and that the binding of GTP had asmall destabilizing effect. A C1/2 of 2.50 M was observed for the Hsp60 mutantin the presence of GTP. This nucleotide did not have any significant effect onthe thermal stability of Hsp60.

Folding Pathways

299-Pos Board B64In Vitro Studies of the Folding and Assembly Mechanism of HemoglobinPremila P. Samuel, William Ou, George N. Phillips, John S. Olson.Biosciences, Rice University, Houston, TX, USA.Deriving the pathway for human hemoglobin assembly is important both fordeveloping treatments for hemoglobinopathies and for designing robust acel-lular hemoglobin oxygen carriers. The adult human hemoglobin (HbA) com-prises two globin heterodimers, (a1b1)(a2b2), with each subunit containing aheme group for coordination of oxygen and having a tertiary structure verysimilar to the monomeric paralog, myoglobin.In order to determine the complete folding mechanism, we have measured andanalyzed guanidinium-induced unfolding curves for both apo- and holo-hemoglobin. This approach expands on previous work done in our lab withmyoglobin and has allowed us to deconvolute the globin unfolding pathwayand the heme-binding effects for the various folding states. When heme is ex-tracted from human Hb, the resulting apohemoglobin dissociates to an a1b1 het-erodimer. Unfolding occurs in two major phases. The first involves melting ofthe heme pockets to generate a dimeric molten globule, followed by dissocia-tion into almost completely unfolded monomers. Structural identification ofthese phases was achieved by comparing the unfolding curves for nativeHbA, recombinant HbF, a N-to-C termini linked a Hb variant, and a seriesof Hb mutants with large, hydrophobic residues replacing the distal histidineand valine in the a and b heme pockets.Previously, we showed that monomeric apoglobin stability, and not hemin af-finity, is the determining factor for myoglobin expression. In the case of hemo-globin, the individual monomers are unstable, but associate strongly to form astable molten globule dimeric state, which binds hemin and facilitates the over-all assembly of the hemoglobin.Supported by the NIH Grant HL110900 and by Grant C-0612 from Robert A.Welch Foundation.

300-Pos Board B65An Evolutionary Trend towards Kinetic Stability in the Folding Trajec-tory of RNases HShion An Lim1,2, Kathryn M. Hart3, Michael J. Harms4,5, Susan Marqusee1,2.1Department of Molecular and Cell Biology, University of CaliforniaBerkeley, Berkeley, CA, USA, 2Institute of Quantitative Biosciences (QB3),University of California Berkeley, Berkeley, CA, USA, 3Department ofChemistry, University of California Berkeley, Berkeley, CA, USA, 4Instituteof Molecular Biology, University of Oregon, Eugene, OR, USA, 5Departmentof Chemistry and Biochemistry, University of Oregon, Eugene, CA, USA.Proper folding of proteins is critical to producing the biological machineryessential for cellular function. Over the course of evolution, the rates and ener-getics of a protein’s folding landscape must be maintained such that the proteinfolds and remains folded over its biological lifetime. Developing a comprehen-sive understanding of how a protein’s folding process is modulated during evo-lution is critical to our understanding and engineering of protein biophysicalproperties. In this study, we characterized the folding trajectories of ancestralproteins of the ribonuclease H (RNase H) family by using ancestral sequencereconstruction to access the evolutionary history between RNases H from mes-ophilic and thermophilic bacteria. We find that the overall folding pathway ofRNase H is preserved over billions of years of evolution. Although thermody-namic stabilities diverge between the mesophilic and thermophilic lineages,

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kinetic stability increases along both, with the last common ancestor foldingand unfolding faster than the modern descendants. The conserved folding inter-mediate permits this paradoxical uncoupling of thermodynamics and kinetics,and allows for the folding landscape to independently respond to different se-lective pressures on global stability and kinetic barriers.

301-Pos Board B66Interpreting Phi-Values using Protein Folding Transition PathsRobert Best1, Gerhard Hummer2.1Laboratory of Chemical Physics, National Institutes of Health, Bethesda,MD, USA, 2Department of Theoretical Physics, Max Planck Institute ofBiophysics, Frankfurt, Germany.It is now possible to watch proteins folding and unfolding spontaneously in mo-lecular dynamics simulations using all-atom force fields. Nonetheless, recentresults have shown that several force fields may be able to fold the same pro-tein, but with very different mechanisms. Distinguishing which of these is cor-rect by comparison to experiment is tricky due to the transient nature of foldingtransitions. Very useful information is furnished by protein folding phi-values,which yield residue-resolved information on the relative effect of mutations onprotein folding rates compared to protein stability. Since computing phi-valuesby brute force is very inaccurate and computationally costly, we have devel-oped a theory for estimating phi-values based on folding transition-paths.Applying the theory to all-atom folding simulations by DE Shaw, we findthat in many cases the agreement with experimental data is reasonable. Weare able to resolve a long-standing controversy in phi-value interpretation,i.e. do fractional phi-values come from parallel pathways or partially formedcontacts? The answer is some of each. We show that the diversity of foldingpathways can be probed by varying the strength of the perturbing mutation ata given residue. Lastly, we have compared the results directly with all-atomfolding simulations using a Go model in order to assess the importance ofnon-native interactions in protein folding pathways.

302-Pos Board B67Reconstructing the Folding of Luciferase to Elucidate the VectorialFolding Pathways of Large, Multidomain ProteinsZackary N. Scholl1, Weitao Yang2, Piotr Marszalek2.1Program in Computational Biology and Bioninformatics, Duke University,Durham, NC, USA, 2Duke University, Durham, NC, USA.Proteins obtain their final functional configuration through incremental foldingwith many intermediate steps in the folding pathway. If known, these interme-diates could be valuable new targets for designing therapeutics and could pro-vide information on the mechanism of chaperones. However, determining theseintermediate steps is hardly an easy feat, and has been elusive for most proteins,especially large, multidomain proteins. Here, we effectively map out the major-ity of a folding pathway for the model large multidomain protein, Luciferase,by combining coarse-grained simulation and single-molecule force-spectros-copy experiments. Simulations indicate that there are several consistent and sta-ble core structures of various sizes nucleating in different regions of Luciferase,each of which has different propensities for propagating to the final foldednative state. We identified, using Monte Carlo simulations of Markov chainsgenerated from simulation, that Luciferase most often folds along a pathwayoriginating from the nucleation of the N-terminal domain, and that this pathwayis the least likely to form non-native structures. We engineered truncated var-iants of Luciferase whose sequences corresponded to the putative nucleatedcores and using atomic force spectroscopy we determined their unfoldingand stability. The experimental results corroborate the structures predictedfrom the folding simulation and strongly suggest that they are intermediatesalong the folding pathway. The simulation also identified non-native structuresoriginating mainly from the C-terminal domain. Taken together, our resultssuggest a pathway for cotranslational folding of Luciferase and also suggesta mechanism that chaperones may exploit to prevent misfolding.

303-Pos Board B68Using Single Molecule Force Spectroscopy to Detect High-Energy Inter-mediates on Protein Folding PathwayHa H. Truong, Emily J. Guinn, Susan Marqusee.California Institute for Quantitative Biosciences, University of California,Berkeley, Berkeley, CA, USA.Single-molecule force spectroscopy allows for molecular insight into thebehavior under force and the folding mechanism of proteins. High-energyon-pathway intermediates do not accumulate significantly under native condi-tions, but may be populated at high force. Here, we use optical tweezers toinvestigate a high-energy intermediate and the force dependent movement ofthe transition state ensemble. We also apply different pulling geometries todetermine the effects of the direction and magnitude of the applied force on un-folding trajectories and compare those results to computational predictions.

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304-Pos Board B69Folding Pathways of Evolutionarily Related Proteins Probed by HydrogenExchange Mass SpectrometryEric Bolin, Shion An, Susan Marqusee.UC Berkeley, Berkeley, CA, USA.The process by which proteins adopt their native structure has been an activefield of study for decades. However, many basic questions about this proteinfolding process remain unanswered. For instance, the role of sequence indetermining the pathway through which a protein folds has been difficult tocharacterize since most techniques to characterize pathways are slow, requirelarge amounts of protein, or only probe folding in a small region of the pro-tein. Hydrogen exchange measured by mass spectrometry (HX/MS) hasproven to be a useful tool that can quickly determine folding pathway of aprotein by measuring the buildup of hydrogen bonding along the protein back-bone. In addition to its relative speed, HX/MS is able to measure to probefolding across the entire protein sequence allowing a pathway to be deter-mined from a single experiment and uses nanomolar quantities of protein.Using HX/MS to determine the folding pathway for the RNases H fromE. coli and T. thermophilus shows that despite folding to the same finalstructure these proteins fold through distinct pathways. Applying this tech-nique to proteins from an ancestral sequence reconstruction of the RNase Hfamily further refined the sequence changes responsible for this shift infolding pathways. This has led to a model that intrinsic helicity of stretchesof the RNase H sequence may determine the earliest steps in the foldingpathway.

305-Pos Board B70How Hydrodynamic Interactions affect the Folding Rate of ProteinsFabio C. Zegarra1,2, Dirar Homouz1,3, Margaret S. Cheung1,2.1Department of Physics, University of Houston, Houston, TX, USA, 2Centerfor Theoretical Biological Physics, Rice University, Houston, TX, USA,3Department of Applied Math and Sciences, Khalifa University, Abu Dhabi,United Arab Emirates.Hydrodynamic interaction (HI) arises by the solvent flow generated by themovement of a particle that affects other particles. This long-range interactionis particularly important in the dynamics of proteins that are immersed in thecrowded milieu of cells. Therefore, there is a need for a computationally andtheoretical quantitative evaluation of its role in protein folding. Usingcoarse-grained molecular simulations and theoretical calculations accordingto the Energy Landscape Theory of protein folding, we assess the impact of hy-drodynamic interaction in the folding of two model proteins, the 64-residueprotein chymotrypsin inhibitor 2 (CI2), and the 57-residue alpha-spectrinSrc-homology 3 (SH3) domain. In our study, we included hydrodynamic inter-action into the equations of motion from Brownian dynamics by computing thediffusion correlation matrices between each residue. We investigated thefolding kinetics as well as the thermodynamics of each protein in the presenceand in the absence of hydrodynamic interaction. Our results suggest that theimpact from hydrodynamic interaction is dependent on the topology of a pro-tein, and temperature.

306-Pos Board B71Folding Mechanisms of Small Proteins GB1 and LB1Qianyi Cheng1, Insuk Joung1, Keehyoung Joo1, Kunihiro Kuwajima2,Jooyoung Lee1.1School of Computational Sciences, Korea Institute for Advanced Study,Seoul, Korea, Republic of, 2Department of Physics, University of Tokyo,Tokyo, Japan.The B1 domains of protein G (GB1) and protein L (LB1) are two small proteinsthat binds to antibody immunoglobulin G (IgG). GB1 and LB1 are similar insize (about 60 residues), and also have an overall similar structure(b-hairpin–a-helix–b-hairpin). However their sequences are very different,possessing only 15% identity in a structure-based alignment. Therefore, thereare interesting similarity and differences in their folding mechanisms. Experi-mental evidence indicated that LB1 folds in a two-state manner; while GB1folds in a more complex way – an early stage intermediate may exist in thefolding path. Till now, the folding mechanisms are still under extensive exper-imental and computational study. Structure-based modeling is one of the lesscostly computational methods. It has a simple formulated potential energyfunction summing over various geometrical restraints from one or more tar-geted structures. Here, we used a new all-atom structure-based method to inves-tigate the folding mechanisms of GB1 and LB1. In this approach, foldedstructures of the two proteins were used to construct the restraints and theyare stabilized by Lorentzian attractive term instead of conventional harmonicterm.3 Our model is able to identify two-state and non-two-state proteins,and gives us more insights of the their folding pathways.

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1. Scalley, M. L., Yi, Q., Gu, H., McCormack, A., Yates, 3rd, J. R., and Baker,D. Biochemistry 36: 3373-82, 1997.2. McCallister, E., Alm, E., and Baker, D. Nat. Struct. Biol. 7: 669-673, 2000.3. Lee, J., Joo, K., Brooks, B., and Lee, J. J. Chem. Theory Comput. 11: 3211-3224, 2015.

307-Pos Board B72A Novel Trp Cage Conformer Revealed by Combining High PressureNMR and MD SimulationsMartin J. Fossat1, Soichiro Kitazawa1, Scott McCallum2, Angel Garcia3,4,Catherine Royer1.1Biology, Rensselaer Polytechnic Institute, Troy, NY, USA, 2Center forBiotechnology and Interdisciplinary Studies, Rensselaer PolytechnicInstitute, Troy, NY, USA, 3Department of Physics, Rensselaer PolytechnicInstitute, Troy, NY, USA, 4Center for NonLinear Studies (CNLS), LosAlamos National Laboratory, Los Alamos, NM, USA.We have used the tryptophan cage variant Tc5b, a widely used model proteinfor benchmarking of force fields and water models, to probe pressure effects onproteins using a combination of high pressure 2D proton NMR TOCSY andmolecular dynamics simulations. Understanding the key factors of the pressurestability of protein is an essential step towards the development of better forcefields and increased predictive power of simulation. We show that results ofMD simulations on Tc5b as a function of pressure using the Amber 99sb forcefield show remarkable agreement with the pressure-dependent NMR data. Pres-sure perturbation in different solvent conditions yielded novel information onthe structural changes in Tc5b induced by pressure. We identified a sub-ensemble in the folded basin that is strongly destabilized by pressure. Hence,most of the pressure effect on Tc5b between 1-2500 bar arises from modulationof the sub-populations of the folded state. The residues observed in the NMRexperiments to be strongly affected by pressure coincided precisely with thoseimplicated in the pressure-dependent change in Tc5b secondary structure re-vealed by the simulations. The relative populations of two folded state con-formers, in which the central 3-10 helix and the following bend are inverted,changed upon pressurization, while the total secondary structural content re-mained the same. These results highlight the existence of significant plasticityin the secondary structure of folded proteins.

308-Pos Board B73Microfluidic Turbulent Mixers, Time Resolved SAXS and Folding Inter-mediates of CheYSagar V. Kathuria1, Osman Bilsel1, Srinivas Chakravarthy2,C Robert Matthews1.1Biochemistry and Molecular Pharmacology, University of MassachusettsMedical School, Worcester, MA, USA, 2BIOCAT, Argonne NationalLaboratories, Argonne, IL, USA.The process of folding a polypeptide chain from an unstructured random coil indenaturant to a functional 3 dimensional form is a rapid process that is perhapsbiased very early on during the initial steps, even as the solvent conditions aretransitioning from favoring denaturation to favoring folding. Access to kineticsin the microseconds to milliseconds time scale by multiple spectroscopicprobes is crucial to interpreting these initial steps of folding. Time resolvedsmall angle X-ray scattering (trSAXS) is a powerful technique for studying 3dimensional shapes of proteins as they transition from unfolded to native struc-tures. By interfacing microfluidic devices with powerful x-ray sources we canaccess timescales in the sub-hundred microseconds range. Structural detailsdetermined using trSAXS of the kinetic refolding process of CheY reveal aburst phase in the sub 50-microsecond time-scale, that is perhaps structuredaround a local-in-sequence cluster of hydrophobic residues.

309-Pos Board B74Thermal and Chemical Unfolding of Cytochrome C in the Presence ofHofmeister IonsEric S. Peterson, Collin A. O’Leary, Sean J. Steinke, Mikayla J. Freese.Chemistry and Biochemistry, University of Northern Iowa, Cedar Falls,IA, USA.The ferric cytochrome c (Cyt c) (un)folding mechanism in the presence of ionsfrom the Hofmeister series is examined. Unfolding was initiated both thermallyand with chemical denaturants. Hofmeister ions were added singly and in pairsto alter the stability of the native folded state, the unfolded state, and twopartially folded intermediates. Protein stability was characterized by eitherthe midpoint of the chemical denaturization curve or by the melting tempera-ture in the thermal studies. UV/VIS absorption spectroscopy and a basis spectrafitting analysis were used to determine the populations of each protein confor-mation along the folding pathway. These species can be differentiated by their

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axial heme ligands. Four species exist in solution: the native HM state (His18/Met80), the partially folded HW (His18/water) and HH (His18/His33) interme-diates, and the 5C (water) unfolded state. The results indicate that the thermaland chemical denaturization pathways are not the same and that both involvesignificant backbone rearrangement. The relative populations of the conforma-tional states depend on how the protein is denatured. For the same concentra-tion, guanidinium causes more unfolding than does urea. The thermal unfoldingpathway appears to involve a more gradual unfolding of the protein and adifferent sequence of change in the heme iron axial ligands. Additionally, itwas found that addition of multiple ions changed the protein’s stability in anadditive manner. These results are discussed in terms of the hydrophobic effect,partitioning of the ions to the protein surface, and an altered water structurearound the protein.

310-Pos Board B75Uncovering Conformational Substates and Kinetic Constants by PressureModulationRoland Winter.TU Dortmund University, Dortmund, Germany.Regulation of protein function is often linked to a conformational switch trig-gered by chemical or physical signals. To evaluate such conformationalchanges and to elucidate the underlying molecular mechanisms of subsequentprotein function, experimental identification of conformational substates andcharacterization of conformational equilibria and reaction constants are manda-tory. We applied pressure modulation in combination with various spectros-copies and scattering experiments to reveal equilibria between substates ofbiomolecular systems, such as of the lipidated signaling protein Ras and its as-sociation with membrane systems. We show that not only nucleotide bindingbut also the presence of the membrane has a drastic effect on the conforma-tional dynamics and selection of conformational substates of the protein, anda new substate appearing upon membrane binding could be uncovered. In a sec-ond example, we explored the effect of pressure perturbation on phospholipaseA2, which catalysis the hydrolysis reaction of sn-2 fatty acids of membranephospholipids, to reveal new mechanistic information about the membraneassociation and subsequent enzymatic reaction. To this end, high-pressureFourier-transform infrared and high-pressure stopped-flow fluorescence spec-troscopies were employed. Finally, we discuss the combined effects of temper-ature, pressure, confinement and osmolytes on the kinetics of enzymaticreactions (e.g., fibril-based enzymes) and polymerization processes (e.g., ofactin) using high-pressure stopped-flow experiments in combination with rapidfluorescence and UV/Vis absorption detection. We show that the compatibleosmolyte trimethylamine-N-oxide (TMAO) is not only able to compensatefor the strongly retarding effect of chaotropic agents such as urea on actin poly-merization, it is also able to largely offset the deteriorating effect of pressure onactin polymerization, thereby allowing biological cells to better cope withextreme environmental conditions. Hence TMAO serves also as efficient coun-teractant of pressure-induced depolymerization reactions, thereby justifying itslabel piezolyte.

311-Pos Board B76Investigating Cotranslational Folding in Membrane Proteins usingFragment-Based Structure PredictionEleanor C. Law1, Saulo H.P. de Oliveira1, Sebastian Kelm2, Jiye Shi2,Charlotte M. Deane1.1Department of Statistics, University of Oxford, Oxford, United Kingdom,2Department of Informatics, UCB Pharma, Slough, United Kingdom.There is experimental evidence that both soluble and membrane proteins startto fold during the process of translation, while still attached to the ribosome. Inthe case of most alpha-helical membrane proteins, their insertion into themembrane is simultaneously carried out by the Sec complex. We have foundevidence to suggest that membrane proteins are also forming tertiary interac-tions cotranslationally. We have tested two structure prediction protocols usingour software SAINT2: one to imitate cotranslational folding which grows thenascent peptide from the N-terminus, and one to imitate in vitro folding whichstarts from an extended chain. The cotranslational method generates more ac-curate models, suggesting that it could be simulating the in vivo folding mech-anism better. We are adapting the SAINT2 score to reflect the membraneenvironment in order to improve the accuracy of models produced and tomore closely imitate in vivo folding. We are also exploring the effect of begin-ning from a segment of the correct native structure and using SAINT2 to com-plete the structure. This is to simulate a folding mechanism where the first partof the protein adopts part of its final fold even before the whole protein issynthesised.

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Protein Dynamics and Allostery I

312-Pos Board B77Molecular Dynamics Simulations for Understanding IR Spectra and theDistribution of Environments Around the Phosphopantetheine Arm ofAcyl Carrier ProteinsMichael R. Jordan, Louise K. Charkoudian, Casey H. Londergan.Chemistry, Haverford College, Haverford, PA, USA.Natural product biosynthesis in polyketide synthases (PKSs) and fattyacid synthases (FASs) is heavily reliant on both specificity and selectivityof protein-protein interactions. Interactions are mediated by the acylcarrier protein (ACP), which utilizes a 40-phosphopantateine (Ppant) armattached to a conserved serine residue to shuttle carbon intermediates be-tween partner enzymes. Recent studies have shown that site-specific vibra-tional spectroscopy can identify the local environment of the arm throughcyanylation of the terminal sulfur of the 40Ppant arm and introduction ofa unique SCN residue. We aim to use molecular dynamics simulations inGROMACS to simulate IR spectra from atom-level physical interactionsand better elucidate the dynamics and structural distribution of the Ppantarm. A strong understanding of the behavior of the ACP’s Ppant arm willprovide both a reliable and generalizable method for confirming experi-mental IR data, and inform bioengineering efforts by further characterizingACP’s interactions with PKSs and FASs that give rise to complex naturalproducts.

313-Pos Board B78Caught in the Act: Trapping an Acyl Carrier Protein Interacting with aKetosynthaseGrace A. Thiele.Chemistry, Haverford College, Haverford, PA, USA.Polyketide synthases (PKSs) and fatty acid synthases (FASs) in microorgan-isms rely on selective and specific protein-protein interactions to build struc-turally complex molecules. These synthases utilize acyl carrier proteins(ACPs) to carry reactive intermediates and substrates to different stops onthe enzyme assembly line. ACPs collaborate with their partner enzymesby tethering substrates and intermediates onto its highly conserved 40-phos-phopantetheine (40-Ppant) arm. Previously, we showed that the installationof a site-specific thiocyanate vibrational spectroscopy probe onto the 40-Ppantarm allows us to visualize and evaluate the solvation environment of the40-Ppant arm using infrared spectroscopy. Herein, we extend these studiesto show that the thiocyanate probe can be used to probe ACP-protein interac-tions. We characterized a potential ACP-enzyme interaction using a rangeof biochemical and biophysical techniques including FTIR spectroscopy,SDS PAGE, exclusion chromatography, and tandem proteolysis mass spec-trometry. The ability to induce a covalent link between an ACP and itscatalytic partner could represent a powerful tool for exploring the possibilityof synthesizing ‘‘unnatural’’ natural products through the bioengineering ofPKSs and FASs.

314-Pos Board B79Allosteric Modulation of a Serine Protease by Conformationally SelectiveNanobodiesTobias Kromann-Hansen.Department of Chemistry and Biochemistry, UC San Diego, San Diego,CA, USA.Trypsin-like serine proteases regulate many important physiological pro-cesses including digestion, blood coagulation, tissue remodelling, comple-ment activation and fibrinolysis. It is well known that ligand binding toallosteric sites modulates the function of the active site region throughconformational and/or dynamic changes leading to a change in protease ac-tivity and altered substrate specificity. Furthermore, increasing biophysicalevidence supports the existence of multiple active and inactive conforma-tional states. However, molecular details about sparsely populated proteaseconformations, and how ligands modulate the activity of a given protease re-mains largely elusive. Here we report the X-ray crystal structure of an unap-preciated inactive conformational state of the serine protease urokinase-typeplasminogen activator (uPA). Next, two distinct uPA conformations werestabilized by different single domain camelid antibodies – an active confor-mation captured by an active site binding (orthosteric) nanobody (Nb22) andan inactive conformation captured by an allosteric nanobody (Nb7). Bycombining X-ray crystallography and hydrogen deuterium-exchange massspectrometry, we identify the mechanism of allostery, which explains atthe molecular level how ligand binding regulates uPA activity by changingthe dynamics of several surface-exposed loops surrounding the active site re-gion. Collectively, our data supports a model which assumes equilibrium

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amongst at least three conformational states and shows that ligands modulatethe biological activity of the protease by differentially stabilizing a specificprotease conformation.

315-Pos Board B80Allosteric Transmission Along a Loosely Structured Backbone Allows aCardiac Troponin C Mutant to Function with only One Ca2D ionMayra A. Marques1, Jose R. Pinto2, Adolfo H. Moraes3, Anwar Iqbal1,Mariana T.Q. de Magalhaes1, Jamila Monteiro1, Murilo M. Pedrote1,Martha M. Sorenson1, Jerson L. Silva1, Guilherme A.P. de Oliveira1.1Medical Biochemistry Institute, Rio de Janeiro, Brazil, 2Department ofBiomedical Sciences, College of Medicine, Florida, FL, USA, 3Departmentof Chemistry, Institute of Exact Sciences, Minas Gerais, Brazil.Hypertrophic cardiomyopathy (HCM) is one of the most common cardiomyop-athies, and a major cause of sudden death in young athletes. The Ca2þ sensor ofthe sarcomere, cardiac troponin C (cTnC), plays an important role in regulatingmuscle contraction. Although several cardiomyopathy-causing mutations havebeen identified in cTnC, limited information about their structural defects hasbeen mapped to the HCM phenotype. Here, we use high-resolution electron-spray ionization mass spectrometry (ESI-MS), Carr-Purcell-Meiboom-Gillrelaxation dispersion (CPMG-RD) and affinity measurements of cTnC forthe thin filament in reconstituted papillary muscles to provide evidence of anallosteric mechanism in mutant cTnC that may play a role to the HCM pheno-type. We show that the D145E mutation leads to altered dynamics on a ms-mstimescale, deactivates both divalent-cation binding sites of the cTnC C-domainand modifies cTnC binding to the troponin I inhibitory peptide (cTnI128-147).CPMG-RD captured a low-populated conformation triggered by the Glu145replacement of Asp. Paradoxically, although D145E C-domain is unable tobind Ca2þ or Mg2þ, these changes along its backbone allowed it to attachmore firmly to thin filaments than the wild-type isoform, providing evidencefor an allosteric response of the Ca2þ -binding site II in the D145E N-domain.Our findings explain how the effects of an HCM mutation in the C-domainreflect up into the N-domain to cause an increase of Ca2þ affinity in site II,thus opening up new insights into the HCM phenotype.

316-Pos Board B81Dynamics and Assembly of ASB-Containing E3 Ubiquitin LigasesRyan Lumpkin1, Alla Ahmad2, Melinda Chan1, Elizabeth Komives1.1Chemistry and Biochemistry, University of California San Diego, La Jolla,CA, USA, 2Chemistry and Biochemistry, University of California Irvine,Irvine, CA, USA.E3 Ubiquitin Ligases (UBLs) facilitate the highly-specific covalent attachmentof activated ubiquitin to bound substrate proteins through an isopeptide bond onan exposed lysine residue. Ubiquitin signaling regulates cellular protein degra-dation which is essential for proper cell functioning. The multi-subunit Cullin-RING ligase (CRL) represents the most prominent form of E3 Ligases, and it isresponsible for a substantial percentage of ubiquitin-directed protein degrada-tion through the K48 ubiquitin linkage. CRLs share a common structure,composed (in order of arrangement) of a substrate receptor with a suppressorof cytokine signaling (SOCS) domain, one or two adapter proteins, a Cullinprotein, and a RING-box (RBX) protein that recruits E2 enzymes chargedwith activated ubiquitin. Among the substrate receptors belonging to this classof UBLs, all 18 proteins in the Ankyrin Repeat and SOCS-box (ASB) family ofproteins associate with Cullin 5 and RBX2 through the Elongin B and C (EB/C)adapter proteins. ASB9 has previously been shown to tightly and specificallybind to Creatine Kinase (CK). I am building the entire seven-protein ASB9-containing E3 ligase complex in order to characterize its dynamics. I haveexpressed and purified all components of the ASB9 UBL, along with the neces-sary Ubiquitin and Nedd8 conjugating enzymes. I have obtained crystals ofCK-ASB9-EB/C for structural determination, and I performed hydrogen-deuterium exchange (HDX) on the same complex to characterize how proteincomplex formation influences the dynamics of each subunit in the complex. CKbinding to ASB9 reduced exchange across both of the first two Ankyrin repeats,while the removal of the SOCS box increased deuterium exchange in the finalAnkyrin repeats in ASB9. This demonstrates the complex relationship betweenthe components of the E3 ligase and the sophisticated substrate recognition byASB proteins.

317-Pos Board B82NFkB and IkBa Engage in an ‘I Fold You, You Fold Me’ InteractionExcluding DNAKristen M. Ramsey, Holly E. Dembinski, Yi Chen, Elizabeth A. Komives.Chemistry & Biochemistry, University of California San Diego, San Diego,CA, USA.Binding of IkBa to NFkB robustly causes cessation of NFkB signalling. Thisphenomenon is facilitated by the sequestering of p50/p65 NFkB dimers in the

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cytosol via IkBa’s masking of the p65 NLS. Interestingly, IkBa is one of thegenes upregulated by NFkB binding to its promoter upon translocation intothe nucleus; IkBa then facilitates dissociation of NFkB from DNA, creatinga negative feedback loop to turn off the NFkB response. NFkB dimers consistof two domains, a dimerization domain and an N-terminal domain (NTD),which are responsible for dimerization/IkBa binding and DNA binding respec-tively. There are no direct contacts between IkBa and the NTDs and constructslacking the NTDs bind IkBa with comparable KD values. I performedhydrogen-deuterium exchange mass spectrometry to analyze changes in amideexchange in NFkB when in complex with IkBa and DNA. As expected, the re-sults show decreases in amide exchange at the binding interface for both part-ners. Surprisingly, we observe significant folding and stabilization in the NFkBNTDs when IkBa is bound, particularly in the regions containing the DNA con-tacting residues in p65. Previous studies have shown that IkBa’s ankyrin re-peats (AR) 5 and 6 fold upon binding to NFkB; when NFkB binds a IkBamutant that is prefolded in ARs 5 and 6 we observe no folding in the NFkBNTDs, further supporting a synergistic ‘I fold you, you fold me’ interactionwherein folding in the C-terminus of IkBa facilitates folding and stabilizationof the NFkB NTDs which locks NFkB into a conformation that is unable tobind DNA.

318-Pos Board B83HDXMS Reveals Dynamic Changes in the Anticoagulant W215A Mutantof ThrombinRiley Peacock, Jessie Davis, Sofia Zaragosa, Elizabeth Komives.University of California San Diego, San Diego, CA, USA.The serine protease, thrombin, regulates the balance between the anticoagula-tion and coagulation in the blood clotting pathway. Thrombin ordinarilycleaves fibrinogen generating fibrin, which forms the blood clot. Binding ofthrombomodulin to thrombin switches its catalytic activity away from fibrin-ogen and towards activation of protein C, which initiates the anticoagulationpathway. Recently Trp 215 was shown to be a key residue in determining sub-strate specificity. The mutant W215A was reported to lose significant activitytowards fibrinogen without significant loss of activity towards protein C- shift-ing thrombin’s activity toward anticoagulation, even in the absence of thromo-bomodulin. Our lab has obtained experimental and computational resultssuggesting that enzyme dynamics are important for connecting the main allo-steric site on thrombin, where thrombomodulin binds, to the active site. In orderto understand how mutation of Trp215, which is not on the pathway betweenthe allosteric site and the active site may alter thrombin specificity, I carriedout hydrogen-deuterium exchange experiments (HDXMS) to compare theenzyme dynamics of the W215A mutant with those of wild type thrombin.HDXMS results indicate that amide exchange throughout much of the thrombinmolecule remains unchanged in the W215A mutant. However, amide exchangein the 220s loop was strongly increased in the W215A mutant as compared towild-type, and amide exchange in the beta-sheet leading up to position 215 (res.209-214) was markedly decreased. We hypothesize that these changes aredirectly due to changes in solvent accessibility of the active site if thrombin,which appears to be remodeled in the W215A mutant. It is likely that the mu-tation directly alters substrate specificity rather than allosteric pathways withinthrombin.

319-Pos Board B84Insights into Dynamics of Thrombin W215 Mutants using Synapt G2Siwith Ion Mobility SeparationJessie R. Davis, Riley Peacock, Elizabeth Komives.Chemistry/Biochemistry, University of California San Diego, San Diego,CA, USA.Thrombin is a serine protease central to regulation of the blood clottingcascade. Active wild-type thrombin acts as a procoagulant and cleaves thezymogen fibrinogen to produce fibrin, which forms the scaffold structure ofa blood clot. Thrombin contains many intrinsically disordered loops nearit’s active site and exosites that are believed to facilitate specificity ofthrombin towards it’s substrates through small time scale entropic movementswhen cofactors are bound. To study the dynamic properties of these mutants,we produced W215I and W215A mutants of human thrombin and carriedout hydrogen-deuterium exchange (HDXMS) experiments using the SynaptG2Si instrument with ion mobility separation. This new instrument gavemuch higher coverage of the thrombin sequence and we were able todiscover where the dynamics of the thrombin had changed with the mutations.Amide exchange in the 220s loop was strongly increased in the W215Amutant as compared to wild-type, and amide exchange in the beta-sheet lead-ing up to position 215 (res. 209-214) was markedly decreased. These resultshelp to explain why mutants at Trp215 have markedly different substratespecificity.

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320-Pos Board B85The Effects of Protein Dynamics on Immune System Signaling PathwaysHelen T. Hobbs1, Susan Marqusee2, John Kuriyan2.1Chemistry, University of California, Berkeley, CA, USA, 2Molecular andCell Biology, University of California, Berkeley, CA, USA.Activation of signaling pathways in immune cells can be initiated by binding ofmembrane proteins, known as B-cell receptors (BCRs) or T-cell receptors(TCRs), to fragments of an antigen. The proteins spleen tyrosine kinase(Syk) and zeta-chain associated protein kinase 70 (ZAP-70) are among the firstproteins to be activated upon engagement of the BCR or TCR. Recruitment ofthese proteins to phosphorylated immunoreceptor tyrosine activation motifs(ITAMs) associated with the TCR or BCR activates the kinase whereupon itinitiates downstream signaling pathways, eventually leading to an immuneresponse. Syk and ZAP-70 are paralogs, with Syk being present in B-cellsand ZAP-70 in T-cells. Additionally, ZAP-70 and Syk share the domain archi-tecture of tandem SH2 domains (tSH2) linked to a kinase domain. The tSH2 isresponsible for binding the phosphorylated ITAMs. Despite similarities in thestructure and function of these two kinases, there are key differences in theirintrinsic activity and specificity. Using the technique of hydrogen exchangeby mass spectrometry (HX/MS) we have identified the Inter-SH2 linker as be-ing significantly different in ZAP-70 compared to Syk. HX/MS experimentsindicate that the Inter-SH2 linker of the Syk tSH2 is very flexible and poten-tially disordered unless bound to a phosphorylated ITAM. This is not thecase for the ZAP-70 tSH2, which shows protection even in the apo state. Thesefindings suggest that the intrinsic flexibility of these proteins may be the causeof some of the observed differences in auto-inhibition, activity, and specificity.

321-Pos Board B86Imatinib Binding to Human c-Src is Coupled to Inter-Domain Allosteryand Suggest a Novel Kinase Inhibition StrategyYuko Tsutsui1, Daniel Deredge2, Patrick L. Wintrode2, Franklin A. Hays1.1Department of Biochemistry and Molecular Biology, University ofOklahoma Health Sciences Center, Oklahoma City, OK, USA, 2Departmentof Pharmaceutical Sciences, University of Maryland School of Pharmacy,Baltimore, MD, USA.One of non-receptor tyrosine kinases (nRTKs), c-Src, is a multi-modular pro-tein kinase. Because c-Src is involved in numerous oncogenic signaling cas-cades, it is an important drug target to treat myeloid leukemia andgastrointestinal stromal tumors. Imatinib is a prototype nRTK inhibitor andbinds to c-Src as well as another nRTK, c-Abl, with different affinities despitehigh sequence and structural conservations in their ATP/drug binding pockets.Structural dynamics of c-Src has been extensively investigated to elucidate un-derlying causes of the affinity difference between c-Src and c-Abl. One of suchstudies suggests the presence of a conformer that plays an important role in se-lective imatinib binding. However, because such a conformer accumulatesslower than the timescale of conventional biophysical and computational ap-proaches, its structural identity cannot be studied using those techniques. Weused hydrogen-deuterium exchange and mass spectrometry (HDX-MS) tostudy structural dynamics of such a key c-Src conformer in the presence of im-atinib. While most previous biochemical/biophysical studies used isolatedc-Src kinase domain, we used purified human c-Src spanning residue 83-536containing SH3, SH2, and kinase domains. This allowed us to visualize changesnot only in the structural dynamics of the drug binding pocket in kinase domainbut also of functional regulatory sites in distant SH3 and SH2 domain upon im-atinib binding, suggesting an allosteric communication among those sites. Ourresults also indicate similarities in structural changes between imatinib bindingand c-Src activation steps. Such changes include loosening of the interface be-tween N- and C-lobes of kinase domain, loss of kinase domain anchoring toSH2-kinase connecting loop, and disassembly of the hydrophobic spine. Basedon our findings, we propose a new cancer therapeutic strategy to improve bind-ing of currently available nRTK inhibitors.

322-Pos Board B87A Phospho-Induced Theft of a Salt Bridge in RKIP Links Map Kinase andG Protein-Mediated SignalingTobin R. Sosnick1, John J. Skinner1, Sheng Wang2, Jiyoung Lee3,Ruth Sommese4, Sivaraj Sivaramakrishnan4, Wolfgang Kolmel5,Maria Hirschbeck5, Hermann Schindelin5, Caroline Kisker5,Kristina Lorenz6, Marsha R. Rosner3.1Department of Biochemistry and Molecular Biology, University of Chicago,Chicago, IL, USA, 2Toyota Institute of Technology at Chicago, Chicago, IL,USA, 3Ben May Department for Cancer Research, University of Chicago,Chicago, IL, USA, 4Department of Genetics, Cell Biology and Development,University of Minnesota, Twin Cities, MN, USA, 5Rudolf Virchow Centerfor Experimental Biomedicine, University of W€urzburg, W€urzburg,

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Germany, 6Institute of Pharmacology and Toxicology, University ofW€urzburg, W€urzburg, Germany.Phosphorylation is a ubiquitous post-translational modification that has beenimplicated in a myriad of biological functions but the underlying mechanismof action can be unclear. Here we study the phosphorylation-induced partialunfolding reaction in Raf Kinase Inhibitory Protein (RKIP), a dual functionprotein that regulates key pathophysiological states including heart diseaseand cancer. RKIP transitions between inhibition of Raf/MAPK to activationof Protein Kinase A via phosphorylation of a serine on RKIP. We showby NMR and X-ray crystallography that switching is due to a ’theft’ bythe phosphoserine of a lysine involved in a salt bridge with a pair of car-boxylic acids. The helical region containing the phosphorylation site re-mains intact whereas the region with the acidic groups unfolds, therebyswitching RKIP’s preferred binding partner. A database search finds candi-dates that have the same structural motif underlying the theft mechanism.Three of them, Bax (1), troponin I & C (2), and Early endosome antigen1 (3), had been more extensively characterized by mutations, and the resultscan be explained by a salt-bridge theft. These findings identify a facile andevolutionarily accessible mechanism for reorganizing a salt bridge networkwith only a single mutation to trigger a functional switch. We anticipate thatthe salt-bridge theft mechanism can be identified in other proteins andcomplexes.1. Arokium, H., et al., (2007) J Biol Chem 282, 35104. 2. Kooij, V et al., (2013)PLoS One. 8, e74847. 3. Ramanathan, H. N., Zhang, G., and Ye, Y. (2013) CellBiosci 3, 24.

323-Pos Board B88Characterization of Disease Causing Mutations Associated with FGFReceptor Tyrosine Kinases using NMR SpectroscopyWilliam M. Marsiglia1, Huaibin Chen2, Min-kyu Cho1,Moosa Mohammadi2, Nathaniel J. Traaseth1.1New York University, New York, NY, USA, 2New York University Schoolof Medicine, New York, NY, USA.The kinase domain of receptor tyrosine kinases (RTKs) is a key factor inthe initiation of intracellular signaling cascades involved in developmentand metabolism. Pathogenic mutations in this domain are generally locatedwithin an allosteric network of residues involved in kinase activation, andhijack this pathway to enhance the kinase domain’s intrinsic activity. Thisleads to overstimulation of resulting signal cascades to cause many types ofcancers and growth disorders. Although many crystal structures of kinasedomains harboring pathogenic mutations have been solved, little is knownabout how these mutations affect the dynamics and stability of the kinasedomain. Here we assessed the dynamics and stability of pathogenic muta-tions within the kinase domain from the Fibroblast Growth Factor receptorfamily using the Carr-Purcell-Meiboom-Gill (CPMG) experiment and differ-ential scanning calorimetry, respectively. Results from the CPMG experi-ments show millisecond-to-microsecond motions for residues involved inthe kinase allosteric activation network, and provide a metric to quantifythe population of the active conformation imparted by the pathogenic muta-tions. Differential scanning calorimetry experiments provide evidence thatpathogenic mutations can lower kinase stability by increasing inter-lobal flex-ibility, and become stabilized in the presence of the ATP mimic AMP-PCP.Together, these data provide detailed insight into the mechanisms of howpathogenic mutations alter the FGFR kinase domain to overstimulate signalcascade processes, and provide a direction for the development of futuredrugs.

324-Pos Board B89Conformational Flexibility of HIV Vif in Complex with Host ProteinsK. Aurelia Ball1, John D. Gross2, Matt P. Jacobson2.1Chemistry, Skidmore College, Saratoga Springs, NY, USA, 2PharmaceuticalChemistry, University of California, San Francisco, San Francisco, CA, USA.Like many viruses, HIV hijacks the host cell’s apparatus for normal proteinubiquitination and degradation, using it to eliminate antiviral proteins. Under-standing how a virus recruits and targets the ubqiuitination complex is criticalfor developing therapeutics to prevent it. One HIV protein responsible for thishijacking is Virion infectivity factor (Vif). Vif is intrinsically disordered butloses flexibility as it binds more host proteins, a process that may be crucialfor function. We are investigating the complex formed with Vif and the hostproteins EloB, EloC, and CBF-beta. Using molecular dynamics simulationsand NMR spectroscopy we have found that this Vif-host protein complex ex-hibits global dynamics and occupies alternate conformational states. Wehave also characterized how these motions are affected by the binding of an

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additional host protein, Cul5. Understanding the dynamics and conformationalensemble of the Vif-host protein complex could reveal future directions fortherapeutics.

325-Pos Board B90Binding-Interface Dynamics between Calmodulin and its Targets Revealedusing Nonperturbative Infrared Probe GroupsCasey H. Londergan, Kristen L. Kelly.Chemistry, Haverford College, Haverford, PA, USA.Calmodulin (CaM) binds to a wide array of natural targets, and most of thesebinding interactions have not been characterized structurally. The artificialSCN functional group was placed at chosen sites on both a target peptide ofCaM and on CaM itself to characterize site-specific details of the bindingbetween these two species. The infrared CN stretching band of the probegroup reports mainly on solvent exposure, and reveals a dynamic distributionof environments at each chosen site that report directly on the bound conforma-tional distribution of CaM and its partner. Dramatic site-specific changes areseen when CaM is free of calcium ions, saturated with calcium ions, or boundto the target peptide. Isothermal titration calorimetry experiments indicatesurprisingly that the SCN probe group does not substantially perturb bindingthermodynamics, even when placed at ‘‘key’’ hydrophobic sites. New insightsinto CaM-target binding will be discussed, as will the prospects for applica-tion of this versatile and non-perturbative probe methodology to otherwiseuncharacterized CaM-target interactions and other protein-protein bindinginterfaces.

Enzyme Function, Cofactors, and Post-translational Modifications

326-Pos Board B91Nedd4-2 is a Functional Oligomer Exhibiting Cooperative AllostericKineticsDustin R. Todaro, Allison C. Augustus-Wallace, Jennifer M. Klein,Arthur L. Haas.Biochemistry and Molecular Biology, LSU Health Sciences Center, NewOrleans, New Orleans, LA, USA.The Hect ligase Standard Model posits binding of the E2~ubiquitin co-substrate to a single site within a monomeric Hect domain followed by trans-thiolation to form a Hect~ubiquitin thioester intermediate. Transfer of theHect-bound ubiquitin to lysyl residues on target proteins is then catalyzedby the ligase, processesively assembling polyubiquitin chains by distal addi-tion. Recently, we demonstrated that the Hect ligase Nedd4-2 catalyzes pol-yubiquitin chain assembly by cooperative allosteric kinetics ([S]1/2=93520nM; kcat=0.06750.008 s�1; nH=1.950.6) and exhibits substrate inhibitionabove 0.5 mM Ubc5B~125I-ubiquitin, requiring the enzyme to function asan oligomer with two ordered E2~ubiquitin binding sites of different affin-ities. These data support an alternative model for Hect ligase conjugationand are in agreement with our previous observations for E6AP. The currentstudies employ kinetic and biophysical methods to further explore the mech-anism of Nedd4-2. Gel filtration chromatography and dynamic light scat-tering are consistent with trimerization. Truncation of the N-terminusyielding GST-Nedd4-2HECT (residues 597-955) reveals cooperative ki-netics for polyubiquitin chain assembly (nH=2.651) with a ca. 104-foldreduction in kcat (1.950.2 x 10�6 s�1), demonstrating allostery intrinsic tothe Hect domain and N-terminal contributions supporting oligomerization.Processing of GST yields monomeric Nedd4-2HECT resulting in monoubi-quitination with hyperbolic kinetics (KM=3957 nM; kcat=6.050.2 x 10�6

s�1) and loss of polyubiquitin chain assembly and substrate inhibition. Addi-tion of Nedd4-2DHECT (residues 1-596) to wild type Nedd4-2 quantita-tively inhibits activity; however, addition of the dominant negative Nedd4-2C922A mutant inhibits Nedd4-2 to 30% limiting activity, requiring thetwo sites to function in trans. These data support a model requiring trimeri-zation and intersubunit interactions for polyubiquitin chain assembly consis-tent with our model of Proximal Indexation. [Supported by GM34009 toA.L.H.]

327-Pos Board B92Ubiquitination of Substrates by E6Ap/UBE3A LigaseVirginia Ronchi, Arthur Haas.Biochemistry and Molecular Bilogy, Louisiana State University, NewOrleans, LA, USA.Only a narrow range of E6AP ubiquitin ligase activity is allowed for normalneural development. Enzyme activation by Ube3A gene duplication is linked

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with familial autism spectrum disorder while loss of function results in theAngelman syndrome neurological disorder. BIOGRID recognizes 174 E6AP-interactors but only a few have been validated as substrates for ubiquitination.Recently, phosphorylation on E6APT485, an autism related residue, affectedRPN10/S5A substrate ubiquitination in cells. The current aim included kineticanalysis of E6AP-catalyzed conjugation of target proteins to provide new in-sights into the mechanism of substrate ubiquitination. E6AP-dependentLys48-polyubiquitin chain assembly in the absence of substrate requires twofunctionally distinct UbcH7~ubiquitin binding sites on the ligase surface andoligomerization. Here, the E6AP ubiquitin-function was analyzed in the pres-ence of RNP10/S5A (regulatory subunit of proteasome 26S) and PRDX1 (anti-oxidant enzyme). Rates of substrate ubiquitin adduct formation were analyzedunder E6AP rate-limiting conditions. Adducts of PRDX1-Ub1 or RPN10/S5A-Ub1 showed Km values of 851 mM and 0.250.06 mM, respectively; while thekcat values were 0.3 s

�1, comparable to 0.5 s�1 observed for polyubiquitin chainassembly without substrate and indicating that the ligase cannot distinguish thelysine nucleophile in Lys48-ubiquitin and Lys-target protein ubiquitination.Analysis of pH-dependent E6AP ligase function inferred a pKa of ~8.4, eitherin the absence or presence of PRDX1. Removal of the first 250 N-terminal res-idues reduced ubiquitination of both substrates supporting the presence of pre-viously unrecognized substrate binding domains in this region. A T485Dmutation mimicking E6AP phosphorylation, or a D212A mutation, an Angel-man syndrome mutation, abrogated substrate ubiquitination, although they re-tained the polyubiquitin chain assembly function. The results provide newinsights of the E6AP ubiquitination mechanism in the presence of target pro-teins that might explain the deleterious effect of some mutations associatedwith Angelman syndrome.

328-Pos Board B93Mechanistic Insights into Ubc13-Catalyzed UbiquitinationIsaiah Sumner1, R. Hunter Wilson1, Walker M. Jones1, Aaron G. Davis1,Serban Zamfir2.1Chemistry & Biochemistry, James Madison University, Harrisonburg,VA, USA, 2Chemistry, Virginia Commonwealth University, Richmond,VA, USA.Ubc13 is an E2 enzyme that catalyzes lysine ubiquitination, a type of proteinpost-translation modification. Ubiquitinating a protein can signal for itsdegradation and affect its activity. Ubiquitination also plays a role in DNArepair and inflammatory response. Defects in this process are linked todifferent disorders including cancer, Parkinson’s and Alzheimer’s diseases.The accepted mechanism for Ubc13-catalyzed ubiquitination is a stepwisepathway that proceeds through an oxyanion intermediate. This intermediateis hypothesized to be stabilized by a nearby asparagine residue, which isknown as the ‘‘oxyanion hole.’’ However, recent experimental results onmutated Ubc13 have suggested an alternate role for the asparagine. In ourstudy, we use a combination of simulation techniques on the wild-type andmutated Ubc13 to examine its catalytic mechanism. Our calculations indicatethat several different intermediates are possible, that water may stabilize theintermediate, and that the asparagine serves to stabilize a random coil nearthe active site.

329-Pos Board B94Characterization of the Essential Residues of Cyclooxygenase-1 and �2Responsible for their Inter-Subunit Communications Upon their Bindingto the Corresponding Substrates and InhibitorsInseok Song.University of Seoul, Seoul, Korea, Republic of.Various conformational changes of a protein upon interaction with its endog-enous partner molecules or artificial synthetic compounds imply often anessential role structurally or functionally. Induction triggered by its bindingto small ligands can contribute a long-range communication between intra-or inter-subunits, which has been exemplified in several model studies.Among these, cyclooxygenases (COX-1 and �2), also known as prostaglandinendoperoxide synthases, display a differential binding pattern between twosequentially identical monomers. COXs catalyze the first committed stepin the conversion of arachidonic acid into prostaglandins and thromboxanes.Potential drug-like compounds against COXs have been enormously devel-oped and characterized up to date, which are exploited in this study withan aim to find core regions or residues responsible for their inter-subunitand domain-domain communications. In addition to a careful examinationof the COX crystallographic data, extensive analysis of docking experi-ments with categorized NSAIDs and prediction of hot-spot(s) for protein-protein interaction were performed, which suggest the theoretical basisfor the functionally heterodimeric nature and half-of-the-sites behavior ofCOXs.

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330-Pos Board B95A Computational Investigation into the Mechanism of the Histone Acetyl-transferase, Gcn5R. Hunter Wilson, Isaiah Sumner.Department of Chemistry and Biochemistry, James Madison University,Harrisonburg, VA, USA.Post-translational modifications (PTMs) can have a profound effect on proteinstructure and function. One such PTM is the acetylation of histone (a proteininvolved in DNA binding). In this reaction, an enzyme catalyzes the transferof the acetyl group from acetyl CoA to a free lysine on the histone. This transferneutralizes the positively charged lysine, which ultimately allows the DNA tobe exposed for transcription. In our study, we focus on the acetyltransferase,Gcn5. Details regarding the reaction mechanism used by Gcn5 remainobscured. However, current mechanistic hypotheses suggest that the reactionoccurs through a tetrahedral oxyanion intermediate, which is stabilized by ahydrogen bond to a nearby residue, i.e. an oxyanion hole. We utilize moleculardynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) cal-culations in order to further probe possible mechanistic schemes of thisreaction.

331-Pos Board B96Molecular Simulations of Bacterial Lipoprotein BiogenesisPhillip J. Stansfeld.Biochemistry, University of Oxford, Oxford, United Kingdom.Lipoproteins perform critical roles in bacterial physiology, pathogenicity, andantibiotic resistance. Their roles include modulation of the cell envelope struc-ture, signal transduction and transport. Lipoproteins are processed by a pathwayof membrane proteins - Sec, Lgt, LspA, Lnt and Lol - which insert, cleave andtransport the protein substrate, while affixing lipid moieties to an absolutelyconserved cysteine and therefore permitting their tethering to the cell envelope.The recent determination of the three-dimensional protein structures of Lgtand LspA have enlivened lipoprotein research. Furthermore, the latest struc-tures of the Sec translocon with associated signal peptide provide a meansto study the initiation point of this pathway and mechanism by which the lipo-proteins are inserted into the cell membrane. Structural studies of mature lipo-proteins have also recently come to the fore, including the BAM complex, ofwhich four of the five proteins are lipoproteins, and LptDE, an outer membranebarrel with a lipoprotein plug. Both complexes are essential to gram-negativebacteria; respectively, folding the protein-conduits that enable nutrient trans-port through the outer membrane and assembling the outermost bacterialfortifications.Here we have used a range of molecular simulation, modelling and bioinfor-matics methods to study this pathway. With initial focus on the first two en-zymes of the pathway, Lgt and LspA, our studies have elucidated the modeof binding of signal peptides to both enzymes and interpreted from this themolecular mechanisms involved in the enzymatic reactions. These studieshighlight key roles for the most highly conserved residues, whilst alsoproviding a means to inhibit the enzymes, as illustrated by the antibiotic, glo-bomycin, bound to LspA.We have also developed molecular parameters for the cysteine lipid-moeities,and applied these post-translational modifications to simulate the dynamics ofboth individual lipoproteins and the lipoprotein complexes of BAM and LptDEwithin the bacterial outer membrane.

332-Pos Board B97Heme Trafficking by the Cytochrome C Biogenesis PathwaysMolly C. Sutherland1, Joel A. Rankin2, Robert G. Kranz1.1Washington University in St. Louis, St. Louis, MO, USA, 2Michigan StateUniversity, East Lansing, MI, USA.Cytochromes function in electron transport chains to perform critical cellularfunctions, such as respiration and photosynthesis. Cytochromes c are uniquedue to their requirement for the covalent attachment of heme via two thioetherbonds at a conserved CXXCH motif. Three pathways have been identified forcytochrome c maturation: System I (prokaryotes), System II (prokaryotes) andSystem III (eukaryotes). System I consists of 8 integral membrane proteins(CcmABCDEFGH), System II is comprised of 2 membrane proteins (CcsBA)and these pathways will be the focus of this presentation. Trafficking of hemefrom the site of its synthesis (cytoplasm) to the site of attachment to apocyto-chrome c (periplasm) is critical for cytochrome c biogenesis, yet little directevidence of heme trafficking exists. The study of heme trafficking has provedelusive in this and most other systems due to tight cellular regulation, as well asthe cytotoxic and amphipathic nature of heme. Here, we use key putative hemetransporters in the prokaryotic pathways as model systems to develop a noveltechnique to covalently ‘trap’ heme during the trafficking process. First, theconserved WWD domain, which is predicted to interact with heme in the

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periplasmic space and is found in both System I and System II pathways, wasused to develop the heme trapping technique. Heme was covalently trapped inthe WWD domains of CcmC (System I) and CcsA (System II), providing thefirst direct evidence of how heme is trafficked to the periplasm in cytochromec biogenesis. Currently, this trapping approach is being used on other integralmembrane proteins in the pathway to capture heme intermediates and delineatethe exact paths for trafficking in vivo. We envision that this approach will beapplicable to other heme transporters and trafficking pathways from prokary-otes through eukaryotes.

333-Pos Board B98Designed Enzymes and the Driving Forces Behind Interdomain ElectronTransferMia C. Brown, Kelly Greenland, Lei Zhang, Ronald L. Koder.City College of New York, New York, NY, USA.Synthetic biology and biodesign approaches to redox active enzymes willrequire the construction of artificial electron transport chains, particularlychains which can interconvert between one- and two-electron chemistry. Toboth demonstrate this capability and explore the engineering parameters neces-sary for rapid and efficient electron transport through artificial electron trans-port chains, we have constructed a natural protein-designed protein chimerain which the diflavin domain of P-450 BM3 is connected to a de novo designed,heme binding four helix bundle. This single chain protein contains one FMN,one FAD, and two heme cofactors. This chimera reacts with NADPH, taking inits two electrons at the FAD cofactor, breaking them into single electrons at theFMN cofactor, and then transferring them into the artificial heme domain. Wehave tested three different heme analogues with varying mid-point potentials toexamine the effect of driving forces on interdomain electron transfer rates.Finally, as our heme-binding domain is capable of binding oxygen in thereduced state, I will present some results using this construct as an artificialnitric oxide dioxygenase, which can perform NADPH-driven catalysis.

334-Pos Board B99Factors Governing Autooxidation of Human HemoglobinAndres S. Benitez Cardenas1, John S. Olson2.1Biosciences, Rice University, Houston, TX, USA, 2Biosciences, RiceUniversity, HOUSTON, TX, USA.Determining mechanisms for the autooxidation of hemoglobin is required forunderstanding and treating unstable hemoglobinopathies and for developingmore stable hemoglobin based O2 carriers. Previous studies suggested signifi-cant differences in autooxidation rates of a and b subunits. We used an azidereaction assay to measure the concentrations of ferric a and b chains at differenttime points during autooxidation. Our results showed no differences betweenthe subunits. To obtain more accurate time courses for autooxidation, we de-convoluted observed spectra into the decay of HbO2, metHb appearance, hemi-chrome generation, and increases in turbidity due to hemin loss and apoproteinprecipitation. The time courses for HbO2 decay at high concentrations(R 100mM heme) accelerate implying cooperative autooxidation, where asat low concentrations (% 10uM) the time courses are biphasic. These resultssuggest that the biphasic time courses at low hemoglobin are due to differencesbetween tetramers and dimers. We have also measured autooxidation rates for arecombinant hemoglobin, rHb0.1, that contains a genetically crosslinked di-asubunit. This hemoglobin shows a monophasic time course for autooxidationat both high and low protein concentrations, and the azide binding assayshowed equal amounts of ferric a and b subunits. We have also examined re-combinant mutant hemoglobins to examine the structural factors that governautooxidation. Increased rates of autooxidation were found for rHb Providence,rHb Bethesda, rHb Presbyterian, and rHb Kirklareli. We have also confirmedthat the rate of autooxidation shows a bell-shaped dependence on oxygen con-centration and increases markedly as the pH is decreased.Supported by NIH Grant P01 HL110900 and by Grant C-0612 from the RobertA. Welch Foundation.

335-Pos Board B100Assessing the Spectroscopic Properties and Enzyme Activity of Fluores-cent Caspase SubstratesGena Lenti, Nicholas Tassone, Srirajkumar Ranganathan, Caitlin Karver,Cathrine A. Southern.Chemistry, DePaul University, Chicago, IL, USA.Inflammatory caspases (caspase-1, �4 and �5 in humans and caspase-11 inmice) are cysteine-dependent, aspartate-specific proteases implicated in inflam-matory, autoimmune and autoinflammatory disorders. To date, assays seekingto test the activity of caspases-1,�5 and�11 have all used Ac-WEHD-AMC astheir fluorogenic substrate. To explore the possibility that alternative fluoro-genic peptides may exhibit enhanced assay properties, we have designed,synthesized, and characterized several novel fluorogenic peptides containing

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coumarin derivatives. The coumarin derivatives were incorporated into pep-tides with various amino acid residues: WEHDA, WEHD, LEVD, LEHD aseither a side chain of a non-natural amino acid, or at the C-terminus. The fluo-rescence quantum yields of these peptides were obtained, allowing the viabilityof these substrates to enhance the signal to noise ratio in caspase enzyme assaysto be assessed. Biochemical assays were then carried out to determine if thesignal to noise ratio indicated by the fluorescence quantum yield results corre-lated with caspase activity and could be applied to inhibitor screening assays.

336-Pos Board B101Single Molecule Enzymology with Outer Membrane Protein GBach G. Pham.University of Massachusetts - Amherst, Amherst, MA, USA.We have developed a nanopore sensor based on Outer membrane protein G(OmpG) to study enzyme kinetics at the single molecule level. OmpG is ab -barrel porin with seven flexible loops that we had previously exploited inprotein sensing. A recognition peptide sequence was inserted into one ofOmpG’s loops allowing it to undergo enzymatic reaction. Caspase 7(casp-7), a protease implicated in apoptosis, was chosen as the target enzymeto interact and chemically alter the OmpG sensor. Currently, we can unambig-uously detect the enzyme-substrate complex of casp-7 and OmpG, as well asthe OmpG cleavage product in real time. We found that the key to the robustefficiency of casp-7 activity (and many other caspases) is that substrate isalways cleaved once bound to casp-7. Casp-7 cleavage is essentially an irre-versible reaction (k-1 = 0; k2 = 8.3 s�1). In addition, we manipulated the pHand observe the effects on k2 that support the catalytic mechanism of cysteineproteases. Our results allow us to probe more closely the catalytic mechanismof an enzyme which cannot be probed using conventional ensemble assays.Thus, we can use our single molecule OmpG enzymology platform to a varietyof other enzyme targets in a similar fashion.

337-Pos Board B102Protein Semi-Synthesis to Characterize Phospho-Regulation of HumanUNG2Brian P. Weiser, James T. Stivers, Philip A. Cole.Pharmacology & Molecular Sciences, Johns Hopkins University, Baltimore,MD, USA.The human nuclear Uracil DNA Glycosylase (hUNG2) initiates the base exci-sion repair pathway that removes uracil from genomic DNA. The catalyticdomain of hUNG2 is preceded by a disordered N-terminus that containsnumerous sites for post-translational modifications. We hypothesized thatcertain modifications might affect hUNG2 activity and/or its interactionswith its protein binding partner, Proliferating Cell Nuclear Antigen (PCNA),which is thought to bind hUNG2 residues 4-11. Using protein semi-synthesis, we prepared full-length hUNG2 with a phosphorylation at eitherThr6 or Tyr8, and these constructs were purified to >95%. The uracil excisionactivity of phospho-hUNG2 proteins was comparable to unmodified hUNG2when using a 19mer duplex DNA substrate. However, fluorescence anisotropymeasurements showed that the phospho-hUNG2s had >10-fold weaker affin-ities for free PCNA. The PCNA affinity of a synthetic peptide that correspondsto UNG2 residues 1-19 was similar to that of full-length hUNG2. Our data in-dicates that the N-terminal 11 residues of hUNG2 are necessary and sufficientfor high-affinity binding to PCNA, and that phosphorylation within this motifdisrupts binding. How phosphorylation affects other aspects of hUNG2 activityis now being explored.

338-Pos Board B103Voltage Dependent Phosphatase Activity is Enhanced by IntracellularAcidificationAngeliki Mavrantoni, Kirstin Hobiger, Dominik Oliver,Christian R. Halaszovich.Neurophysiology, University Marburg, Marburg, Germany.Voltage sensitive phosphatases (VSPs) are PI(4,5)P2/PI(3,4,5)P3-5- and PI(3,4)P2/PI(3,4,5)P3-3-phosphatases. For non-mammalian VSPs, this activity is regu-lated by membrane voltage via a voltage sensor domain (VSD). For mamma-lian VSPs the VSD seems insensitive to voltage changes yet still essentialfor control of the phosphatase activity. Under physiological conditions, thenon-mammalian VSPs strongly deplete PI(4,5)P2 in a voltage dependentmanner. The physiological regulator of mammalian VSP activity remainselusive. VSPs are suggested to play a role in fertilization and development,where changes in intracellular pH are known to occur. They are found to be ex-pressed in tissues like kidney, stomach, sperm, and ovary which are known toundergo such pH changes. Therefore, we speculated that intracellular pH mightmodulate VSP activity.To test this hypothesis we performed whole-cell patch-clamp experiments inCHO cells expressing diverse VSPs and fluorescent PI(4,5)P2 reported

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domains, the latter allowing for monitoring VSP activity by means of total in-ternal reflection microscopy (TIRF-M). The whole-cell patch-clamp configura-tion allowed for control not only over membrane voltage but also intracellularpH by dialysing the cell with solutions with the desired pH.We find that acidification of the cytoplasm results in increased PI(4,5)P2 deple-tion, accompanied by a shift of the apparent voltage dependence towards morenegative potentials. An increase in intracellular pH has the opposite effect. Thevoltage dependence of sensing currents was unaffected by the pH changes, sug-gesting that alterations of the VSD are not causal for the observed changes involtage dependent activity. Similar effects were observed in all tested VSPsWeconclude that the overall activity of the phosphatase is enhanced under acidicand diminished under alkaline conditions. Kinetic modeling predicts a shift inapparent voltage dependence under these circumstances that is in agreementwith the observed shift.In conclusion, we suggest that intracellular pH can play a role in the regulationof the activity of VSPs.This work was supported by a research grant of the University Medical CenterGiessen and Marburg (UKGM32/2011MR) to C.R.H and by Deutsche For-schungsgemeinschaft (SFB593 TP A12) to D.O.

339-Pos Board B104Electric Field Effects in the Active Site of a Thermophilic Enzyme asObserved by FTIR and 2D IR SpectroscopyTayler D. Hill, Hannah H. Lepird, David A. Price, Sean D. Moran.Chemistry and Biochemistry, Southern Illinois University Carbondale,Carbondale, IL, USA.Our research aims to understand how changes in ultrafast dynamics compareand correlate to thermophilic enzyme activity. We observe fluctuating electricfield effects in a promiscuous, hyperthermophilic ene-reductase from Pyrococ-cus horikoshii (PhENR) to address this. This enzyme catalyzes the reduction ofactivated alkenes/alkynes to their respective alkanes/alkenes via proton and hy-dride transfers from a flavin cofactor in the active site. We exploit the promis-cuity of PhENR in order to incorporate a variety of substrates and substrateanalogs into the active site for these studies. We have synthesized a set ofcovalently-attached substituted N-phenylmaleimide infrared labels, whichmimic the structures of the enzyme’s substrates, and contain unique vibrationalchromophores to probe the enzyme’s active site dynamics. Current studiesfocus on the vibrational frequencies and lineshapes of nitrile labels such asthose of 4-cyano-N-phenylmaleimide, which sits proximal to the catalyticflavin and can be attached in multiple orientations within the active site.When compared to the label in solution, the covalently attached label un-dergoes significant inhomogeneous broadening in its FTIR spectrum reflectingthe distribution of active site microenvironments. Additionally, protein-basednon-natural amino acid labels such as methionine to azidohomoalanine substi-tutions are also being incorporated into the distal side of the flavin cofactor forsimilar studies in different location within the enzyme’s active site. Using 2DIR spectroscopy, we are examining the contributions of femtosecond to pico-second active site dynamics to the lineshapes of both the covalently attachedprobes as well as the incorporated non-natural amino acid labels. Futureresearch aims to break the thermophilicity of the enzyme via specific mutationsin order to compare the active site dynamics to a corresponding mesophilicversion of the protein.

340-Pos Board B105Infrared Structural Biology: How to Detect Protonation States of HistidineSide Chains in ProteinsAihua Xie, Charle Liu, Matthew Cavener.Physics, Oklahoma State University, Stillwater, OK, USA.The imidazole group of histidine residues are found functionally important ina vast number of catalytic proteins. The remarkable catalytic power of histi-dine side chains originates from its ionizable imidazole ring armed with apair of different tertiary amines and the ability of adopting three protonationstates near physiological pH environment. Knowledge on the protonationstates of key histidine side chains in enzymes at rest and during catalytic ac-tions is indispensable to elucidation of the structure-function relationship un-derlying enzymatic catalysis. We report a rigorous method on how to detectthe three protonation states of functionally important histidine imidazolerings in the static and dynamic states of enzymes using infrared structuralbiology. First principle computational methods based on density functionaltheory were employed to develop two vibrational structural markers(VSM) of the imidazole group: VSMq for the charged states of the imidazolegroup, while VSMt for distinguishing the D and E tautomers of chargeneutral histidine. The accuracy of the VSMs is assessed by comparison ofcalculated VSMs with experimental FT-IR data of the 4-ethyl-imidazolemodel compound. We will discuss how these VSMs may be employed in

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structure-function studies on functionally important histidine residues inenzymes.

341-Pos Board B106Origin of Chain Length Specificites of Starch Branching EnzymeHadi Nayebi Gavgani, Remie Fawaz, Zahra Assar, Alireza Ghanbarpour,David Walls, Sarah McGovern, James H. Geiger.Chemistry, Michigan State University, East Lansing, MI, USA.Starch Branching enzyme (BE) is one of the three enzymes involved in starchbiosynthesis. It is responsible for synthesizing the alpha-1,6-glucan branches,remodeling the linear alpha-1,4-glucan polymer to produce amylopectin. Thereare at least two isoforms of the enzyme in most plants, with each having distinctreactivities and product chain-length specificities. Though the chemistry of theactive site of branching enzyme is relatively well studied, the origin of the chainlength specificity is yet to be understood. Using protein crystallography andbiochemical studies on rice branching enzyme, we aim to understand the fac-tors controlling the chain-length specificity of branching enzymes. The mech-anism of branching enzyme involves two steps: (1) An oligosaccharide (donorchain) binds to the enzyme and is cleaved by the action of a nucleophilic aspar-tate residue to form a covalently-linked enzyme-glucan intermediate. (2) A sec-ond oligosaccharide (acceptor chain) then reacts, by nucleophilic attack of oneof its alpha-1,6-hydroxyl groups, to form a new alpha-1,6-branch. We havefocused on discovering the surface glucan binding sites in branching enzymebecause they are likely essential to understanding the specificity of the enzyme.To this end we have obtained a crystal structure of an oligosaccharide (M12)-bound rice branching enzyme, which reveals oligosaccharide binding from theouter surface of the enzyme almost to the active site. Mutations of the residuesinteracting with the M12 can substantially compromise the enzyme’s activity,though none have affected the branch chain specificity. On the other hand,comparison of an isoamylase maltoheptaose (M7)-bound structure with ricebranching enzyme suggested another potential glucan surface binding site.Interestingly, mutations in this new site did effect the chain-length specificityof the enzyme. These results suggest that the bound M12 is part of the acceptorchain and the newly identified binding site hosts the donor chain. Together, thedata allow us to, for the first time propose a detailed mechanism for the enzyme,explaining how disparate surface glucan binding sites far from the active sitecreate the enzyme’s activity and specificity against its polymeric substrate.

342-Pos Board B107Modifications of Alpha and Beta Carboxy-Terminal Tails Regulate Micro-tubule Severing by KataninMadison Tyler1, Corey Reed1, Dan Sackett2, Jennifer Ross1.1Physics, University of Massachusetts, Amherst, Amherst, MA, USA,2National Institutes of Health, Bethesda, MD, USA.Microtubules are part of a dynamic cytoskeletal network that is constantly be-ing reorganized to control cell processes such as neuronal development andmaintenance, cell division, and cargo transport. Many stabilizing and destabi-lizing enzymes function to reorganize these networks for the specific needs ofthe cell in a spatiotemporal manner. Katanin p60 is a microtubule destabilizingenzyme from the ATPases Associated with various Activities (AAAþ) family.It recognizes the tubulin carboxy-terminal tails (CTTs) to sever microtubules.Our lab has previously shown free tubulin dimers and CTTs alone can inhibitkatanin severing. We seek to determine the manner that tubulin CTTs sequencecan regulate katanin activity using polypeptide sequences of CTTs of differenttubulin isoforms. We find that the sequence’s ionic, hydrophobic, and stericfeatures play a role in determining katanin’s activity.

Ribosomes and Translation

343-Pos Board B108Protein Synthesis Times Scale with Gene Length because the Determinantsof Translation Speed are Randomly Distributed Across GenesEdward P. O’Brien, Ajeet Sharma.Department of Chemistry, Penn State University, University Park, PA, USA.Many of the molecular factors influencing codon translation speed have beenidentified, and their relative contributions estimated. These factors includetRNA concentration, the presence of charged nascent-chain residues in theribosome exit tunnel, mRNA secondary structure, proline residues at the Aor P sites of the ribosome and steric interactions between ribosomes translatinga transcript. Here, we combine this information with genomic information fromE. coli, yeast and humans in a simulation model of translation to estimate thesynthesis time of cytopolasmic proteins. We find that regardless of the organ-ism, the synthesis time of a protein scales linearly with the length of mRNA’scoding sequence even though there is a large variation in the translation speedof individual codons. We demonstrate that this scaling arises because the

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molecular determinants of translation speed are distributed randomly acrossthe mRNA transcripts and that this distribution is generated from a Poissonpoint process. As a consequence, the Law of Large Numbers is followedand for any given transcript fast-translating segments are canceled out by asimilar number of slow-translating segments resulting a constant average codontranslation rate between different transcripts. This means that a protein’saverage synthesis time can be accurately predicted based solely on the corre-sponding gene length, provided the average codon translation speed is known.Thus, although evolution has biased codon usage between different genes inan organism, there is still a large degree of randomness associated with howindividual codon translation speeds have been distributed. These results alsoprovide an explanation for the observation from ribosome profiling thatdifferent transcripts have the same average codon translation speed inmouse-stem cells.

344-Pos Board B109The Role of L11 Stalk Fluctuations in aa-tRNA AccommodationHuan Yang, Paul Charles Whitford.Physics, Northeastern University, Boston, MA, USA.The ribosome is a large ribonucleoprotein complex that is responsible fortranslation of proteins in all living organisms. Accommodation is a keyconformational change during transfer RNA (tRNA) selection that allowsfor each molecule to fully associate with the ribosome. During tRNA accom-modation, there are large-scale fluctuations in the L11 stalk and the associ-ated protein. Experimental and theoretical work has shown that the L11protein and RNA exhibit dynamic behavior, where their precise positioncan significantly affect the dynamics of elongation. To explore the impactof L11 stalk mobility on the kinetics of aa-tRNA accommodation, we usedmolecular dynamics simulations with a simplified model to evaluate thefree-energy as a function of aa-tRNA position. We find that as the flexibilityof L11 is decreased, the position of the free-energy minima is robust. Incontrast, the magnitude of the free-energy barrier for accommodation de-pends on the precise scale of L11 fluctuations. We find that increasing therigidity of the L11 stalk can destabilize the A/T ensemble. In addition tothe close relationship between L11 mobility and the free-energy landscapeof accommodation, the direction of L11 stalk movement is also correlatedwith aa-tRNA displacements. To probe the collective motion of the L11stalk, we performed Principal Component Analysis on the simulated trajec-tories. When aa-tRNA is first delivered to the ribosome, the stalk movesaway from the tRNA and adopts extended configurations. It then relaxes dur-ing the accommodation process. Together, these calculations reveal a corre-lation between fluctuations in the L11 stalk and the dynamics of aa-tRNAduring accommodation. This provides a quantitative foundation for interpret-ing experimental measures of accommodation and suggests how the dy-namics of the L11 stalk may contribute to aa-tRNA proof-reading andaccommodation.

345-Pos Board B110Simulating Movement of the Ribosome Head during TranslocationKarissa Y. Sanbonmatsu1,2, Wataru Nishima2.1Los Alamos National Laboratory, Los Alamos, NM, USA, 2New MexicoConsortium, Los Alamos, NM, USA.Intrasubunit head movement has been identified as an essential motionrequired for translocation of mRNA through the ribosome. Recent single mole-cule FRET data has suggested that exaggerated motions of the head, beyondwhat are observed in structural studies, are required for the rate-limitingstep of translocation. We use molecular simulations of the 70S ribosome trans-location complexes to explore these exaggerated motions and identify regionsof the ribosome that place constraints on the maximal displacements of thehead.

Nucleic Acid Structure and Dynamics I

346-Pos Board B111Effects of 5-Hydroxymethylcytosine Epigenetic Modifications within theVEGF Promoter Region on G-Quadruplex and I-Motif DNA Structureand StabilityMichael M. Molnar1, Rhianna K. Morgan2, Tracy A. Brooks2,Randy M. Wadkins1.1Chemistry & Biochemistry, University of Mississippi, Oxford, MS, USA,2Pharmacology, University of Mississippi, Oxford, MS, USA.Epigenetic modifications to DNA base sequences may regulate gene expres-sion. CpG islands can contain methylated (5mC) or hydroxymethylated(5hmC) cytosine. Most CpG islands are found primarily in promoter regionsthat may also contain a high number of repeated cytosines and/or guanines.G-quadraplexes (G4) and i-motifs (iM) are two unique DNA secondary struc-

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tures that can form in repeating sequences of either guanine or cytosine, respec-tively. Both G4 and iM sequences may contain CpG sequences that can bemethylated or hydroxymethylated. The effects of CpG islands on DNA second-ary structures were determined by incorporating a single 5hmC at varying po-sitions in the Vascular Endothelial Growth Factor (VEGF) G4 and iMsequences. An Olis DSM-20 spectropolarimeter and a Cary 100 UV-visiblespectrometer were used to monitor the effect of 5hmC on G4 and iM thermalstability. Two of the three 5hmC-containing loops showed a notable decreasein stability for G4’s and increased intermolecular structure formation. Contrast-ingly, the iM stability increased when 5hmC was incorporated into itssequence. Additionally, there was little change in the iM pka. In summary,our results suggest the 5hmC has little effect on iM structures, but can destabi-lize the G4’s.

347-Pos Board B112Temperature Dependence of L-Proline RNA Duplex DestabilizationJeffrey J. Schwinefus, Kalpit Modi, Nadia Baka.Chemistry, St. Olaf College, Northfield, MN, USA.L-Proline, an osmoprotectant and non-essential amino acid, destabilizes thesecondary and tertiary structures of nucleic acids. L-proline interactions withthe surface area exposed in unfolding RNA duplexes were quantified usingm-values for ten RNA dodecamer duplexes that varied in GC content from17% to 83%. The RNA dodecamer unfolding transitions were facilitated bythermal denaturation and isothermal titration with proline, both monitored us-ing ultraviolet spectroscopy. The m-values from isothermal titration at 22 �Cwere independent of GC content. However, m-values from thermal denatur-ation were greater in magnitude and more negative for higher GC contentRNA indicating more favorable proline interactions with the accessible surfacearea exposed in unfolding high GC content duplexes. Since RNA duplex tran-sition temperatures increase with GC content, the more favorable L-proline in-teractions with the high GC content duplex surface area result from thetemperature dependence of L-proline interactions rather than the RNA surfacechemical composition. The enthalpy contribution to the m-value is positive andsmall (indicating a slight increase in duplex unfolding enthalpy with L-proline)while the entropic contribution to the m-value is positive and increases withtemperature. Our results help facilitate using L-proline as a probe of solventaccessible surface area changes during biochemical reactions at different reac-tion temperatures.

348-Pos Board B113Engineering DNA Looping in E. ColiNicole A. Becker, Tanya L. Schwab, Karl J. Clark, L. James Maher III.Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA.DNA looping plays an important role in many cellular functions includingreplication, transcription, DNA packaging, and recombination. DNA repres-sion loop formation in prokaryotes acts to regulate transcription in responseto a changing environment. One example of gene control by DNA loop for-mation occurs in the well-studied lac operon. E. coli lac loop formation oc-curs when LacI, a tetrameric protein, simultaneously binds two DNAoperators within close proximity, resulting in a DNA loop. This structure re-presses transcription of downstream operon genes. We are curious if repres-sion loop formation can be targeted in vivo. Our goal is an engineered proteinfusion with both sequence specific DNA binding function and chemicallyinduced dimerization (CID) function. The chemically dimerized proteinwould have the ability to bind the DNA in a bidentate manner, analogousto LacI, resulting in DNA looping. Targeted DNA loop formation couldact in gene repression and might be tunable by the concentration of CIDagent. Using designed transcription activator-like effector (TALE) proteinsallows creation of DNA binding proteins with arbitrary DNA sequence spec-ificity. TALEs have been used for in vivo genome-engineering applications.FK506 binding protein (FKBP) provides a well-studied protein domainwhose ability to undergo CID is exploited in our designs. We utilize our es-tablished lac promoter looping system to assess TALE-FKBP protein fusionstargeted to lac operators.

349-Pos Board B114Investigating the Stability of DNADuplex Hairpins using Optical TweezersLeah Furman1, Micah McCauley2, Catherine A. Dietrich3,Mark C. Williams2, Megan E. Nunez1.1Department of Chemistry, Wellesley College, Wellesley, MA, USA,2Department of Physics, Northeastern University, Boston, MA, USA,3Department of Chemistry, Mount Holyoke College, South Hadley,MA, USA.Covalent modifications and replication errors lead to DNA damage and muta-tion. Such errors occur frequently throughout the genome, disrupting the stabil-ity of the double helix. The 8-oxoguanine (8-oxoG) lesion, one of the most

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widespread forms of DNA damage, mimics guanine with respect to hydrogenbonding, but perturbs base stacking, destabilizing the double helix. Other com-mon forms of damage include mismatches, such at G-T and C-T base pairs, thatare unable to form canonical hydrogen bonds. We have investigated the desta-bilizing effects of three abnormal base pairs (8-oxoG-C, G-T, and C-T) on thethermodynamic stability of DNA hairpins at the single molecule level. Eachdamaged hairpin shows a distinct pattern of unfolding that allows us to quantifyhairpin destabilization. These data are combined with an mFold-based analysisto create energy landscapes of the unfolding pathway for perfectly matched,damaged, and mismatched hairpin duplexes.

350-Pos Board B115Untwisting of Double-Stranded DNA and RNA Investigated by MolecularDynamics SimulationsKorbinian Liebl.Technical University Munich, Garching, Germany.The response of dsDNA and dsRNA to torsional stress influences many oftheir biological functions, including binding by proteins, transcription initia-tion and genome packaging. So far, the twist flexibility of DNA and RNAhas been studied comparatively with single-molecule experiments. However,while these studies capture the global deformability of duplexes with a lengthof several thousand base pairs (bp), detailed insight into conformationalchanges on the base pair level remains elusive. We performed all-atom Mo-lecular Dynamics (MD) simulations on 15 bp long DNA and RNA duplexesat femtosecond resolution. Employing an advanced sampling method basedon a torsion-like restraining potential, we were able to untwist the moleculesto induce localized melting. The simulations allowed us to determine therelative free energy and structural changes as a function of the mean twist.Suppression of local bending has a strong influence on the onset of DNAmelting. Significant differences in the response were also observed forDNA and RNA. The results can have important implications for understand-ing the mechanism of replication and transcription of DNA the function ofRNA molecules.

351-Pos Board B116Translocation of Structurally Defined Branched DNA through NanoporesPhilipp Karau, Kyle Briggs, Vincent Tabard-Cossa.University of Ottawa, Ottawa, ON, Canada.We use solid-state nanopores to study the translocation characteristics ofdifferent structurally defined DNA topologies. Site-specific modificationswith non-natural nucleotides along the backbone of DNA fragments allowgrafting of side branches at specific locations by ‘‘click’’ chemistry. We pro-duce T- and pi-shaped DNA molecules with a 50bp double-stranded DNAbackbone and either 25nt single-stranded or 25bp double stranded DNAbranches. Nanopores ranging in size from 3 to 10nm are fabricated bycontrolled breakdown (CBD) in ultra thin 10-nm SiN membranes and usedto electrophoretically translocate these short branched DNA molecules. Wecan distinguish the topologies of these DNA molecules through analysis ofthe ionic current blockages. Such structurally defined branched DNA mole-cules can be used for the development of multiplexed nanopore-basedassays, and as position-controlled building blocks of much larger DNApolymers, to further our understanding of the fundamentals of moleculartransport through nanopores by precisely measuring intra molecular velocityfluctuations.

352-Pos Board B117Thermodynamic Linkage Analysis of pH-Induced Folding and UnfoldingTransitions of I-MotifsByul Kim, Tigran Chalikian.Pharmaceutical Sciences, University of Toronto, Toronto, ON, Canada.We describe the pH-induced folding/unfolding transitions of i-motifs by a link-age thermodynamics-based formalism in terms of three pKa’s of cytosines,namely, an apparent pKa in the unfolded conformation, pKau, and two apparentpKa’s in the folded state, pKaf1 and pKaf2. For the 50-TTACCCACCCTACCCACCCTCA-3’ sequence from the human c-MYC oncogene promoter region,the values of pKau, pKaf1, and pKaf2 are 4.8, 6.0, and 3.6, respectively. Withthese pKa’s, we calculate the differential number of protons bound to the foldedand unfolded states as a function of pH. Analysis along these lines offers analternative interpretation to the experimentally observed shift in the pH-induced unfolded-to-i-motif transitions to neutral pH in the presence of cosol-vents and crowders. Our simulations reveal that a significant increase in thetransition midpoint pH can be achieved by an increase in the equilibrium con-stant between the folded and unfolded DNA conformations due to the excludedvolume effect.

BPJ 7720_7

353-Pos Board B118Live Cell Imaging of Genomic Loci using Fluorescent RNA AptamersAdam Cawte1, Sunny Jeng2, Peter Unrau2, David Rueda1.1MRC Clinical Science Centre, Imperial College London, London, UnitedKingdom, 2Department of Molecular Biology and Biochemistry, SimonFraser University, Burnaby, BC, Canada.In recent years, there has been an explosion of SELEX-evolved fluorescentRNA aptamers, such as Spinach and Mango, with enhanced folding, fluores-cence and a high affinity for their dyes. These aptamers have a drasticallyimproved fluorescence contrast relative to EGFP and hold great promise forvisualising vital cellular processes involving RNA molecules. However, theuse of aptamers in live-cell imaging has experienced limited resolution andapplicability due to the dynamic nature of RNA. We have recently developeda new method that combines CRISPR/Cas9-based nuclear localization withfluorescent RNA aptamer-based imaging. A major difference with existing ap-proaches is that this system contains an engineered fluorescent RNA aptamerwithin the sgRNA scaffold in lieu of a fluorescent dCas9-EGFP fusion. Thismethod enables the direct visualisation of genomic loci and their diffusion dy-namics within live cells. We anticipate the development of this technology toimprove our ability to target specific regions of the genome, as well as todevelop multi-colour imaging using different fluorescent sgRNA constructs.

354-Pos Board B119Elucidating the Role of Transcription in Shaping the 3D Structure of theBacterial GenomeHugo Brandao1, Xindan Wang2, David Rudner2, Leonid Mirny3.1Harvard University, Cambridge, MA, USA, 2Harvard Medical School,Boston, MA, USA, 3Massachusetts Institute of Technology, Cambridge,MA, USA.Active transcription has been linked to several genome conformation changesin bacteria, including the recruitment of chromosomal DNA to the cell mem-brane and formation of nucleoid clusters. Using genomic and imaging dataas input into mathematical models and polymer simulations, we sought toexplore the extent to which bacterial 3D genome structure could be explainedby 1D transcription tracks. Using B. subtilis as a model organism, we investi-gated via polymer simulations the role of loop extrusion and DNA super-coiling on the formation of interaction domains and other fine-scale featuresthat are visible in chromosome conformation capture (Hi-C) data. We thenexplored the role of the condensin structural maintenance of chromosome com-plex on the alignment of chromosomal arms. A parameter-free transcriptiontraffic model demonstrated that mean chromosomal arm alignment can bequantitatively explained, and the effects on arm alignment in genomically rear-ranged strains of B. subtilis were accurately predicted.

355-Pos Board B120Investigation of the Melting Thermodynamics of a DNA 4-Way Junction:One Base at a TimeRachel E. Savage1, Wujie Wang2, Francis W. Starr2, Ishita Mukerji1.1Molecular Biology and Biochemistry Department, Wesleyan University,Middletown, CT, USA, 2Physics Department, Wesleyan University,Middletown, CT, USA.DNA four-way junctions are branched structures that form between two homol-ogous chromosomes. These junctions play key roles during several cellular pro-cesses, including meiosis and DNA double-stranded break repair, however theirmechanism of formation and separation are not well understood, particularlywith respect to branch migration. We have investigated the thermodynamic sta-bility of the DNA four-way junction J3 using the fluorescent pteridine nucleo-side analogues, 6-MAP and 6-MI, which provide site-specific information onthe melting process. We have incorporated these probes at different locationsthroughout the junction to determine the influence of position on junction sta-bility. Preliminary fluorescence data suggest that the central region of the four-way junction, a region under much torsional strain, is in fact more stable thanpreviously hypothesized. We have also investigated the relative stability of thedifferent arms, by incorporating probes on each arm approximately the samedistance from the junction center. These results are compared with those pre-dicted from coarse-grained simulations of the junction using the 3 sites pernucleotide (3SPN.2) model. Already, we have demonstrated the ability ofthis model to reproduce many experimentally determined aspects of DNA junc-tion structure and stability, including the temperature dependence of melting onsalt concentration, the bias between open and stacked conformations, the rela-tive populations of conformers at high salt concentration, and the inter-duplexangle between arms. We are now using a replica-exchange molecular dynamicsapproach to evaluate the fraction of bonded bases along each arm of the junc-tion over the temperature range associated with melting. This approach allowsus to determine base-by-base the local melting temperature along the arm. We

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specifically compare the patterns of melting observed experimentally withthose obtained from the coarse-grained simulations.

356-Pos Board B121Role of Watson-Crick-Like Mismatches in DNA Replication FidelityEric S. Szymanski1, Isaac J. Kimsey2, Hashim M. Al-Hashimi1.1Dept. of Biochemistry, Duke University, Durham, NC, USA, 2Nymirium,Ann Arbor, MI, USA.DNA replication, transcription, and translation rely on the strict Watson-Crickbase pairing rules to ensure faithful transmission of genetic information. TheWatson-Crick pairing rules are determined by the predominant neutral tauto-meric forms of the nucleic acid bases. Incorrect base pairing during replication,if left unrepaired, leads to transition or transversion point mutations. Sponta-neous mutagenesis from replication errors is believed to be a prominent sourceof base substitution errors in tumor suppressor genes in multiple forms ofcancer. Rare tautomeric and ionized nucleotide bases can form mismatchesthat conform to the Watson-Crick like geometry, subverting proof readingmechanisms. These tautomeric and anionic mismatches have long been sus-pected to contribute to spontaneous replication errors; they have proveddifficult to visualize as the conformational changes are subtle and involvethe rearmament of protons. Nuclear magnetic resonance relaxation dispersiontechniques have allowed for the characterization of a highly sequence-dependent kinetic network connecting the wobble dG$dT mismatch to multipleWatson-Crick-like tautomeric and anionic dG$dT mismatch ‘excited states’.We have obtained evidence in support of a kinetic model for misincorporationwhich introduces a rate-limiting on-pathway tautomerization or ionizationstep that leads to Watson-Crick-like mismatches prior to incorporationthrough the canonical synthesis pathway. This kinetic model can account fori) the three orders of magnitude difference seen in vitro between rates of correctand incorrect nucleotide incorporations, ii) nucleotide selectivity fidelity as lowas 10�6, and iii) the poorly understood sequence dependence of polymerizationerrors.

357-Pos Board B122Direct Observation of Single Biopolymer Folding and Unfolding Processby Solid-State NanoporeXin Shi, Rui Gao, Shao-Chuang Liu, Qiao Li, Yi-Tao Long.East China University of Science and Technology, Shanghai, China.Biomolecular conformation and their transition play a crucial role in variousin vivo or in vitro system. The most of the practical techniques for resolvingthe secondary structures of biomolecules could provide quite precise structuralinformation for their solid-state or steady state, even at atomic resolution. Forexample, Cryo-EM determines high-resolution structures for the frozen-hydrated specimens of biomolecules. polymers, but it is still challenging toresolve the dynamic process of multiple functional conformational states forbiomolecules at single-molecular scale. Here, we direct observed DNA foldingand unfolding process in real-time by using sub-5 nm solid-state nanopores. Inour experiments, a single-stranded DNA adhered to single monovalent strepta-vidin could be reversibly trapped in a solid-state nanopore. Then, the fluctua-tions of the blockade current could be recorded, which reveals the dynamicstructural transitions among DNA secondary structures. For example, aftertrapping the cytosine-rich DNA strains in slightly alkaline solution, the forma-tion of multiple unstable and semi-folded i-motif structures could be observed.More important, well time-resolved transitions between these structures couldbe obtained. When using slightly acidic solution, the stable structures withstable blockade current could be found. With this new approach, we candirectly observe the dynamic conformational change of biomolecules atsingle-molecular scale, which would be of great help for resolving single mole-cule interactions, designing single-molecule machine and understanding theworking process of biomolecular in biological system.

358-Pos Board B123Tuning Up Tethered Particle MotionDaniel T. Kovari, Eric Weeks, David Dunlap, Laura Finzi.Physics, Emory University, Atlanta, GA, USA.Tethered Particle Motions assays are a simple but powerful tools for monitoringthe effective length of individual DNA strands and other linear bio-polymers inreal-time. The technique has been employed in various capacities includingcharacterization of DNA topology, transcription factor - DNA interactions,and monitoring progress of enzymes that translocate along DNA. At its corethe technique is relatively simple to implement, only requiring a research-grade microscope and a video camera; however, it is important to note that op-tical resolution, frame rate, exposure time, particle size, and solution viscosityall affect the ability to discriminate different tether lengths and detect changes.

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Through a combination of simulations and control experiments we demonstratethese limits, and present a collection of best-practices.

359-Pos Board B124Small Molecule Aptamers for BiosensingGregory Wiedman, Yunan Zhao, David Perlin.Public Health Research Institute, Rutgers New Jersey Medical School,Newark, NJ, USA.In this work, we used modified Synthetic Evolution of Ligands through Expo-nential Enrichment (SELEX) to discover a DNA aptamer recognizing azoleclass antifungal drugs. This aptamer undergoes a secondary structural changeupon binding its target molecule as shown through fluorescence anisotropybased binding measurements. Using circular dichroism spectroscopy, we founda unique double G-quadruplex structure that was essential for binding to thetarget: azole antifungal drugs. This type of aptamer has the potential to beused as a small molecule captor component of a device for therapeutic drugmonitoring.

360-Pos Board B125Improved Sampling in Molecular Dynamics Studies of DNA and the B ToZ[WC] To Z-DNA TransitionLam T. Nguyen, Ashutosh Rai, Micaela E. Bush, Alma Gracic,Ahsan A. Khoja, Jinhee Kim, Sunil Pun, Alexander K. Seewald,Benjamin L. Yee, Michael G. Lerner.Physics and Astronomy, Earlham College, Richmond, IN, USA.Although DNA is most commonly found in the right-handed B-DNA structure,it is known that biologically active systems also contain left-handed ZII-DNA. We used both steered and targeted molecular dynamics in combinationwith umbrella sampling to produce potentials of mean force for the B to ZIItransition along both the direct B-ZII pathway as well as the B-Z[WC]-ZIIpathway. Full pathway sampling is not feasible on smaller computer clusters,so we used Hamiltonian Replica Exchange to relax individual portions of thepathway. This technique is generalizable to larger systems and larger computerclusters.

361-Pos Board B126Energetic Contributions of Plectoneme Tips and TailsAndrew Dittmore, Keir C. Neuman.NIH, Bethesda, MD, USA.Global DNA topology is sensed locally by enzymes that act on plectonemes insupercoiled DNA. Here we report that the formation and diffusion of plecto-nemes are determined by the energetic contributions of their tips and tails. First,to systematically vary the geometry and formation energy of plectoneme end-loops, we introduced base-pair defect regions of variable size (1-16 bp) using acassette based single-strand nicking template generated by PCR. Direct manip-ulation measurements with magnetic tweezers revealed that even a singlemismatch or abasic site is sufficient to nucleate formation of a plectoneme. Pre-sentation of the defect precisely at an extruded plectoneme tip potentiallyserves as a damage-sensing mechanism and may facilitate the search processof repair enzymes. Second, our measurements unexpectedly revealed that aftertwisted DNA abruptly buckles into an initial plectoneme loop, further plecto-neme extrusion occurs through a cascade of additional buckling steps in whichthe torque changes by roughly half of the initial overshoot value. These discretesteps do not match any obvious scale of the system but are consistent withdiscontinuous feed-in of curving plectoneme tails. In light of these results, theo-retical models of plectonemes should include their overall structure, includingthe often neglected tips and tails.

362-Pos Board B127Multi-Scale Structure and Conformational Dynamics of Scaffolded DNAOrigami NanoparticlesWilliam Bricker, Keyao Pan, Mark Bathe.Massachusetts Institute of Technology, Cambridge, MA, USA.Synthetic DNA can be programmed into self-assembled 3D nanoparticles us-ing a DX design motif and the principle of scaffolded DNA origami. A top-down design procedure (DAEDALUS) (Veneziano, Ratanalert, et al., Science,2016) facilitates the design of arbitrary DNA nanoparticle geometries on the 5to 100 nanometer scale, which we investigate in detail here using multi-scalestructural modeling. While coarse-grained modeling is useful for generatingequilibrium structures of DNA nanoparticles (Pan et al., Nat. Comm.,2014), only all-atom models reveal fine structural details and mechanicalproperties that contribute to overall structure and conformational dynamics.Here, we first use all-atom molecular dynamics (MD) to simulate two 0.5 -1.0 MDa DNA polyhedral nanoparticles: a tetrahedron with 63 base pair

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(bp) edge lengths, and an octahedron with 52 bp edge lengths. Using 150 nstrajectories, we are able to elucidate subtle structural features seen in exper-imental cryo-EM maps, including right-handed twisting of the vertices inthe octahedron and outward bowing of the edges in the tetrahedron. Next,these results are compared with all-atom MD simulations of unconstrainedvertex building blocks including the tetrahedron (3-arm vertex) and octahe-dron (4-arm vertex). In these simulations, a notable feature is the significantout-of-plane bending angle away from the minor groove at the vertex dueto the chirality of dsDNA. Finally, equilibrium solution structures of 45DX-based DNA origami nanoparticles are predicted by implementing an up-dated bulge stiffness parameter for our coarse-grained FE model CanDo (Kimet al., Nucleic Acids Res., 2012). These multi-scale structural results show theinterplay between coarse-grained and all-atom models in the ab initio predic-tion and elucidation of complex features of DNA nanoparticles seen in exper-imental cryo-EM maps.

RNA Binding

363-Pos Board B128Shedding Light on Cas9 Target SearchViktorija Globyte1, Seung Hwan Lee1, Luuk Loeff1, Jin Soo Kim2,Chirlmin Joo1.1TU Delft, Delft, Netherlands, 2Seoul National University, Seoul, Korea,Republic of.The CRISPR/Cas adaptive immune system provides prokaryotes with a defen-sive mechanism against invading viruses and plasmids. The invading viralDNA fragments are incorporated into the CRISPR (Clustered Regularly Inter-spaced Short Palindromic Repeats) locus in the bacterial genome and are laterused to recognize and destroy the invader when it returns. In the last stage ofCRISPR immunity, called the interference stage, Cas (CRISPR-associated)proteins assemble with short guide RNA molecules which are transcribedfrom the CRISPR locus. These guide RNA molecules can be programmed torecognize any DNA sequence. In recent years the CRISPR/Cas adaptive im-mune system has seen an immense growth in interest with the type IICRISPR-Cas9 system being in the center of attention. In this system, theDNA of the invading virus is recognized and cleaved by a single proteinCas9 which is guided by an RNA duplex. Due to its simplicity, CRISPR-Cas9 system is a promising tool in gene engineering as its guide RNA canbe programmed to recognize virtually any sequence in the genome. TheCRISPR-Cas9 has been demonstrated to work in a variety of organisms, how-ever, despite the large interest in this system, the precise mechanism by whichCas9 finds and cleaves its target remains ambiguous. We utilize biophysicalsingle-molecule techniques, namely total internal reflection fluorescence mi-croscopy (TIRFM) together with Forster resonance energy transfer (FRET)to investigate the mechanics of Cas9 target search with nanometer sensitivity.We are probing one-dimensional diffusion of the protein along the DNA strandand investigating the effects different DNA sequences have on the mechanicsof Cas9 target search.

364-Pos Board B129To Cleave or not to Cleave: Predicting the Target Specificity of CRISPR-Cas Systems through Theoretical ModelingMisha Klein, Martin Depken.Kavli institute of Nanoscience, Departement of BioNanoScience, TU Delft,Delft, Netherlands.Many prokaryotes employ the CRISPR-Cas (Clustered Regularly InterspacedShort Palindromic Repeat – CRISPR associated) system to fend off attacksby hostile genetic elements. This adaptive immune system recognizes in-vaders based on their level of sequence complementarity with RNA tran-scribed from a library of past invasions stored at the CRISPR locus. Toavoid infection, the CRISPR interference complex must be able to singleout a short target sequence (20 – 40 nt) among a total of 105-106 nt in thecell, while at the same time recognizing targets that have evolved awayfrom the record stored at the CRISPR locus. Despite the tremendous interestthe CRISPR-Cas9 system has gained as a novel genome editing tool, thesequence preference of the interference complex remains poorly understood.Experiments have shown that it is not just the amount of mutations, but alsotheir placement along the guide/target, that determine the level ofinterference.Through kinetic modeling we provide simple rules to assess sequence speci-ficity based on mismatch patterns, and quantitatively explain the experimen-tally observed seed region over which no mismatches are permitted. Movingbeyond sequence complementarity, we also quantify how changes in theconformation of the interference complex can serve to improve specificity dur-ing target recognition. Finally, by fitting our model to published experimental

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data, we give estimates for the base-pairing energies within the interferencecomplex for various CRISPR systems.

365-Pos Board B130Conformational Dynamics of Cas9 during DNA BindingYavuz S. Dagdas1, Janice S. Chen2, Samuel H. Sternberg3,Jennifer A. Doudna2,3, Ahmet Yildiz2,4.1Biophysics Graduate Group, University of California, Berkeley, Berkeley,CA, USA, 2Department of Molecular and Cell Biology, University ofCalifornia, Berkeley, Berkeley, CA, USA, 3Department of Chemistry,University of California, Berkeley, Berkeley, CA, USA, 4Department ofPhysics, University of California, Berkeley, Berkeley, CA, USA.Cas9 is an RNA-guided endonuclease that cleaves double-stranded DNAusing two conserved nuclease domains, RuvC and HNH, as part ofCRISPR-Cas bacterial adaptive immune systems. Together with single-guide RNAs, Cas9 is also widely utilized as a programmable genome editingtool. DNA cleavage activity is controlled directly by the conformational stateof the HNH nuclease domain, but Cas9 conformational dynamics during DNAbinding remain poorly characterized. Using single-molecule FRET assays, weidentified a long-lived intermediate state of S. pyogenes Cas9. Upon DNAbinding, the HNH nuclease reversibly transitions between RNA-bound,DNA-bound and docked conformations, before it docks irreversibly into itscatalytically active conformation for DNA cleavage. Docking of HNH to itsactive state requires divalent cation, and HNH remains in the docked state af-ter cleavage of a complementary target. Increasing the number of mutations inthe target region distal to the protospacer adjacent motif (PAM) prevents tran-sitions from intermediate to the docked conformation. The results provide astructural explanation for the lack of DNA cleavage activity when Cas9 bindsto off-target sites.

366-Pos Board B131Repetitive Loop Formation by the CRISPR-Cas3 HelicaseLuuk Loeff1, Stan Brouns1,2, Chirlmin Joo1.1Technical University Delft, Delft, Netherlands, 2Wageningen University,Wageningen, Netherlands.E. coli maintain CRISPR-Cas adaptive immune systems to protect the cellagainst invading genetic elements. Immunity relies on the RNA guided surveil-lance complex Cascade (CRISPR-associated complex for antiviral defense) andthe trans-acting Cas3 protein with helicase and nuclease activities. We recentlyshowed that Cas3 generates degradation products ranging from 30 to 150 nt thatact as pre-cursors for primed spacer acquisition. However, it remains unclearwhich mechanism drives the generation of these fragments with a specificsize. Here we employed single-molecule FRET to probe the molecular dy-namics of Cas3 in real-time. Our data shows that Cas3 repeatedly generatesDNA loops in the target strand whilst remaining in tight contact with Cascade.DNA loops are generated by breaking open the dsDNA helix in distinctive stepsof 3 bp, arising from the RecA like folds of the helicase domain. Repetitiveunwinding achieved by slipping of the helicase domain, which limits theaverage translocation distance to ~90 nt. Taken together, our data suggestthat the inherent helicase properties of Cas3 drive the generation precursorsof adequate size for primed spacer integration.

367-Pos Board B132The Impact of DNA Topology on Target Selection by a Cytosine-SpecificCas9Tsz Kin Martin Tsui, Travis H. Hand, Hong Li.Institute of Molecular Biophysics, Florida State University, Tallahassee,FL, USA.Cas9 is an RNA-guided DNA cleavage enzyme being actively developed forgenome editing and gene regulation. To be cleaved by Cas9, a double strandedDNA, or the protospacer, must be 1) complementary to the Cas9-bound guideRNA and a short Cas9-specific sequence adjacent to protospacer, called Proto-spacer Adjacent Motif (PAM). Understanding the correct juxtaposition in timeand space of the protospacer- and PAM-interaction with Cas9 will enabledevelopment of versatile and safe Cas9-based technology. We report identifica-tion and biochemical characterization of Cas9 from Acidothermus cellulolyti-cus (AceCas9). AceCas9 depends strictly on a 50-NNNCC-30 PAM and ismore efficient in cleaving negative supercoils than relaxed DNA. We furthershowed that mismatches to the guide RNA on a supercoiled protospacer aretolerated by AceCas9, whereas the same mismatches on its relaxed formwere not. The cytosine-specific and DNA topology-sensitive properties of theAceCas9 maybe explored for chromosome domain specific genome editing ap-plications. Finally, our preliminary data showed that AceCas9 can disrupttarget DNA in in vivo assays, demonstrating its utility as a genome editingenzyme.

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368-Pos Board B133CRISPR-Cas9:Computational InsightsToward ImprovedGenomeEditingGiulia Palermo1, Yinglong Miao1, Ross C. Walker1, Martin Jinek2,J. Andrew McCammon1.1Department of Pharmacology, University of California at San Diego/Howard Huges Medical Institute, La Jolla, CA, USA, 2Department ofBiochemistry, University of Zurich, Zurich, Switzerland.Life sciences are undergoing a transformative phase due to an emerginggenome-editing technology based on the RNA-programmable CRISPR-Cas9(clustered regularly interspaced short palindromic repeats-CRISPR associatedprotein 9) system. In this system, the endonuclease Cas9 associates with a guideRNA to match and cleave complementary sequences in double stranded DNA,forming an RNA:DNA hybrid and a displaced non-target DNA strand.Although extensive structural studies are ongoing, the conformational dy-namics of Cas9 and its interplay with the nucleic acids during associationand DNA cleavage are largely unclear. This missing aspect hampers the precisestructure-based design of CRISPR-Cas9 genome-editing tools with improvedspecificity. Here, we report the first biophysical study – based on extensivemulti-microseconds molecular simulations integrated with structural data –revealing the conformational plasticity of Cas9 and identifying the key deter-minants that allow its large-scale conformational changes during nucleic acidbinding and processing. We identify a remarkable conformational plasticityas an intrinsic property of the nuclease HNH domain, being a necessary factorallowing for the HNH domain repositioning during catalysis. More impor-tantly, we disclose a key role of the non-target DNA during the process of acti-vation of the HNH domain, showing how the non-target DNA positioningtriggers local conformational changes that favor the formation of a catalyticallycompetent Cas9. Our outcomes further suggest new and precise protein-engineering modifications, which are of fundamental importance for therational design of more effective genome-editing tools. Overall, these novelfindings constitute a reference for future experimental studies aimed at a fullcharacterization of the dynamic features and at the improvement of biologicalapplications of the CRISPR-Cas9 system.

369-Pos Board B134Structural Insights into G-tract Recognition by the hnRNP H-RNA Recog-nition MotifSrinivasa R. Penumutchu.Chemistry, Case Western reserve University, Cleveland, OH, USA.The heterogeneous nuclear ribonucleoprotein H (hnRNP H) family of proteinsare involved in RNA splicing of cellular and viral mRNAs. These proteinsfunction as both splicing activators and repressors. The hnRNP H family pro-teins have previously been found to interact with poly-G sequences (G-tracts)of cellular and viral mRNAs using quasi RNA recognition motifs (qRRMs)including human immunodeficiency virus (HIV). hnRNP H proteins arecomposed of three qRRMS, which are separated by linkers and two Glycinerich domains at C-terminal. The qRRM1 and qRRM2 domains are located atthe N-terminus and separated by 10-residue linker, whereas qRRM3 is locatedtowards the C-terminus. To gain structural insights into hnRNP H protein, herewe solved the solution structure of the HRRM12 domain of hnRNP H usingNMR spectroscopy. We used paramagnetic relaxation enhancement (PRE)and Residual dipolar couplings (RDC) to obtain distance restraints and orien-tational restraints between the HRRM1 and HRRM2 domains. T1, T2 andNOE experimental data is consistent with calculated structure of HRRM12domain and reveals that HRRM12 adopt the compact (closed) structure. To bet-ter understand principals of hnRNP H-RNA recognition, we systematicallyscreened G-tracts of RNA oligos against HRRM12 by using isothermal titrationcalorimetry (ITC) and NMR spectroscopy. We examined the minimal G-tractRNA (–GGG–) sequence requirements for HRRM12 binding. The HRRM12domain of hnRNP H exhibited no substantial differences in binding affinitiesfor G-tracts of RNA (AGGGX) and HRRM12 residues involved in bindingG-tracts of RNA were detected by NMR chemical shift perturbation experi-ments and a data-driven model of the complex was determined usingHADDOCK. Taken together, this study provides the molecular insights for bet-ter understanding the role of the qRRM domains of hnRNP H in RNA splicingof cellular and viral mRNAs.

370-Pos Board B135Blind Predictions of RNA/Protein Relative Binding AffinitiesKalli Kappel, Inga Jarmoskaite, Pavan P. Vaidyanathan,William J. Greenleaf, Daniel Herschlag, Rhiju Das.Stanford University, Stanford, CA, USA.Interactions between RNA and proteins are pervasive in biology, shaping pro-cesses such as mRNA translation, localization, and alternative splicing. Devel-oping a predictive understanding of the energetics of these systems would allow

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us to model biologically relevant mutations of these interactions and ultimatelydesign novel interactions. Despite recent advances in high throughput experi-mental technologies that measure the energetics of these systems, quantitativecomputational prediction of relative RNA/protein binding affinities has re-mained a challenge. This is partly due to the observation that computationalbinding affinity prediction methods typically break down when the moleculesare highly flexible or undergo significant conformational changes, situationsthat often arise in RNA/protein binding. Here, we present a novel frameworkwithin Rosetta for predicting RNA/protein relative binding affinities that beginsto address this issue. Specifically, we show that the nearest neighbor energies,which are typically used for RNA secondary structure prediction, can be used toapproximate the unbound free energy of the RNA, thus eliminating the need toexplicitly account for the flexibility of the unbound RNA or conformationalchanges of the RNA upon binding. Using this method of calculating the un-bound RNA free energy significantly improves the prediction accuracy overa more typical 3D structure-based approach. We optimized this method usinga subset of published MS2 coat protein affinities and ultimately made predic-tions for the system with 1.11-1.28 kcal/mol root mean square (RMS) error.Additionally, we show that this method is able to predict relative binding affin-ities for four diverse RNA/protein systems with 1.48 kcal/mol RMS error.Finally, to more rigorously assess this method, we independently measuredand made blind predictions for PUF3 and PUM2 binding affinities with RMSerrors of 1-2 kcal/mol, which is comparable to the accuracy achieved by predic-tion methods for other types of systems.

371-Pos Board B136Biophysical Studies of Liposome Encapsulated Pokeweed Antiviral Proteinand its use as a HIV TherapeuticO’Jay Stewart1, Artem Domashevskiy2.1CUNY - John Jay College, Manhattan, NY, USA, 2Department of Sciences,CUNY - John Jay College, Manhattan, NY, USA.Human Immunodeficiency Virus (HIV) is a virus that attacks the human im-mune system, compromising its effect in regards to disease prevention. HIV re-sults in the destruction of CD4 cells, which are vital in the defense againsthuman immune responses. HIV can severely damage the immune system andlead to Acquired Immunodeficiency Syndrome (AIDS). There is presently nocure or effective HIV vaccine and as a result it is important to seek alternativemeasures in the defense against HIV/AIDS. Pokeweed Antiviral Protein (PAP),a protein isolated from pokeweed plant, Phytolacca americana, provides a newand profoundly promising direction in the field of HIV/AIDS research. PAPplays a vital role in the immune system of pokeweed and is a ribosome inacti-vating protein (RIP), inhibiting viral protein production. PAP possesses anti-viral properties, and reduces the infectivity of many plant and animalviruses, including HIV-1. My project focuses on mechanisms using liposomalencapsulated PAP, targeted to infected cells as a therapeutic for HIV infection.Steady state fluorescence was used to identify the thermodynamic parametersof interaction between PAP and HIV RNA. PAP binds to the m7 GTP capand the 50 UTR region of HIV RNA. Various isoforms of PAP were testedand the optimal affinity was identified as a Km of 20.09 nM. Fluorescenceobserved V.S [m7 GTP] was plotted through nonlinear regression using thePrism 6.0 program. Varying liposomes were prepared in order to identify theoptimal characteristics for PAP encapsulation. Liposomes were lyophilizedthrough rotary evaporation and later extruded to ensure homogeneity. Fluores-cent titration enables the testing of liposomal PAP encapsulation efficiency. Itwas concluded that a 1/1 v% mixture of liposomes DOPE-DOTAP is most effi-cient for PAP encapsulation. These accomplishments will enable further modi-fication of the liposomes by covalently conjugating HIV-specific monoclonalantibodies (anti-pg120 and/or anti-pg41), thus facilitating the selectivity ofthe liposomal PAP to the target tissues (HIV-infected CD4þ lymphocytes).With the aforementioned completed the basis for PAP-liposome interactionscan be established and also the modulation of these interactions in order totarget HIV-infected cells.

372-Pos Board B137A Dead-Box Protein Acts through RNA to Promote HIV-1 Rev-RREAssemblyRajan Lamichhane, John A. Hammond, Raymond F. Pauszek,Ingemar Pedron, Edwin van der Schans, James R. Williamson,David P. Millar.Integrative Structural and Computational Biology, The Scripps ResearchInstitute, La Jolla, CA, USA.The HIV-1 Rev (Regulator of Expression of Virion) protein activates nuclearexport of unspliced and partially spliced viral mRNAs, which encode the viralgenome and the genes encoding viral structural proteins. Rev interacts with ahighly conserved region, the Rev Response Element (RRE), located within

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the viral mRNA. In order to activate nuclear export, multiple Rev proteins mustassemble on the RRE. The host DEAD box protein 1 (DDX1) enhances theRNA export activity of Rev through an unknown mechanism. We used asingle-molecule assembly assay utilizing immobilized full length RRE andfluorophore-labeled Rev to monitor each step of Rev-RRE assembly, in thepresence or absence of DDX1. More Rev monomers were observed to bindto the immobilized RRE in the presence of DDX1, indicating that DDX1 pro-motes oligomeric Rev-RRE assembly. Further experiments using specificDDX1 mutants that are defective in either Rev binding or RNA binding indi-cate that DDX1 must be capable of associating with RNA in order to promoteassembly of the Rev-RRE complex. Single-molecule Forster resonance energytransfer (smFRET) experiments show that DDX1 transiently interacts with theRRE and that both DDX1 and Rev can occupy the same RRE molecule. Takentogether, these results suggest that DDX1 acts as an RNA chaperone, foldingthe RRE into a conformation that is pre-organized to bind the first Rev mono-mer, thereby promoting the overall Rev-RRE assembly process. Supported byNIH grant GM082545.

373-Pos Board B138Deciphering the ActionMechanism of DDX3: An RNAHelicase Implicatedin Cancer Propagation and Pathogenic Viral InfectionAnthony F. Moore, Aliana Lopez, de Victoria, Eda Koculi.Chemistry, University of Central Florida, Orlando, FL, USA.DDX3 is a human DEAD-box RNA helicase implicated in crucial cellularprocesses including translation initiation, ribosome assembly, RNA transport,and microRNA processing. Consequently, DDX3 is implicated in many viralinfections and cancer cell metabolism. Our goal is to obtain a detailed under-standing of DDX3’s mechanism of action and employ this understanding todiscover DDX3 inhibitors that would serve as lead compounds for drugsthat halt viral infections and cancer cell metabolism. While DDX3 is requiredfor many viral infections and cancer cell propagations, it is not essential forhealthy cell metabolism, making DDX3 an ideal anticancer and antiviraldrug target. Like all the members of the DEAD-box family of enzymes,DDX3 uses ATP binding and hydrolysis to unwind short double-strandedRNA helices. Our data show that different from many members of DEAD-box family of enzymes, monomeric DDX3 is unable to perform RNA unwind-ing, and a multimeric DDX3 complex is required to support DDX3’s helicaseactivity. Furthermore, our data suggests that the single-stranded-double-stranded RNA junction promotes the formation of the DDX3 multimer. Weare in the process of performing mutagenesis studies combined with cross-linking and mass spectrometry to determine the DDX3 amino acids implicatedin mulitmer formation and the amino acids that come in direct contact withRNA during the DDX3 catalytic cycle. These experiments would produce in-formation both on DDX3’s mechanism of action, and elucidate why someDEAD-box proteins have evolved to act as mulitmers. Lastly, we have foundfour natural compounds that are specific inhibitors of DDX3 ATPase activity.These compounds will be used as probes to decipher DDX3’s action mecha-nism and could have translational potential as drugs that stop various viral in-fections and cancer progression.

374-Pos Board B139Interaction of PKR with Single Stranded RNAChristopher B. Mayo, James L. Cole.Molecular and Cell Biology, University of Connecticut, Storrs, CT, USA.Although the antiviral kinase PKR was originally believed to interact withonly duplex RNAs, evidence has accumulated that the enzyme can be acti-vated by a variety of structured RNAs. A potent PKR activating motif consistsof a short stem loop containing single stranded tails (ss-dsRNA). The ssRNAtails contribute to binding and activation. However, PKR does not contain acanonical ssRNA binding domain. Here, we demonstrate that isolated ssRNAsinteract with PKR. Both homopolymeric (rU)30 and heteropolymeric 30 ntssRNAs bind with micromolar dissociation constants. Addition of a 50-triphos-phate slightly enhances binding affinity. A homopolymeric (dT)30 ssDNAbinds more weakly than (rU)30, indicating a contribution of the 20OH moiety.PKR contains a conserved region N-terminal to the kinase that is enriched inbasic residues. ssRNA binds to a construct containing the basic region and ki-nase and also binds to the isolated dsRNA binding domain. Both full lengthPKR and the basic region/kinase domain construct are weakly activated byssRNA. However, the isolated kinase is not activated and does not bindssRNA. Photocrosslinking measurements were performed using a ss-dsRNAcontaining 4-thiouridine and PKR constructs with a TEV protease cleavagesite at different positions between the kinase and dsRNA binding domain.Analysis of the products following crosslinking and TEV cleavage demon-strates that that the basic region interacts with ss-dsRNA in the context offull length PKR. Our results support a model where PKR activation by

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RNAs is regulated in vivo by interaction with both duplex and single strandedregions.

Membrane Dynamics I

375-Pos Board B140Surfactant Micelle Self-Assembly with Coarse-Grained Martini StandardWater and Polarizable WaterEric Sefah, Blake Mertz.Chemistry, West Virginia University, Morgantown, WV, USA.Molecular dynamics (MD) simulations of proteomicelle complexes are usefulfor the investigation of membrane protein dynamics and function. However,modeling assembly and dynamics of complex oligomeric systems requirelength- and timescales that are traditionally inaccessible to all atom MD simu-lations. Coarse grained (CG) force fields such as MARTINI decrease spatialresolution of a system, making biologically relevant time scales for phenomenasuch as membrane protein oligomeric assembly accessible [1]. In order to applyCG force fields to the study of large proteomicelle complexes it is important totest their ability to reproduce experimentally observed properties. In this studywe have characterized the effects of the nonpolarizable and polarizable watermodels of the MARTINI force field [2] on assembly of surfactant micelles.The two surfactant systems studied were a zwitterionic detergent (n-dodecyl-phosphocholine (DPC)) and nonionic detergent (n-dodecyl-b-D-maltoside(DDM)). From a system of 50, 100, 150 and 200 random detergent molecules,stable micelles formed with variable sizes for both models, in general agree-ment with experimental aggregation numbers. However, the polarizable watermodel formed larger micelles than the normal water model in a few cases. Inaddition, the polarizable water model formed stable compact micelles in shortertimes.[1] Marrink SJ, et al. J. Phys. Chem. B. 2007;111:7812.[2] Yesylevskyy SO, et al.PLoS Comp. Biol, 2010;6:e1000810.

376-Pos Board B141Simulations of Glycerol and its Effect on the Phase and Behaviour of DPPCMonolayersJemma L. Trick1, Wachirun Terakosolphan2, Ben Forbes2,Christian D. Lorenz1.1Physics Department, Kings College London, London, United Kingdom,2Institute of Pharmaceutical Science, Kings College London, London, UnitedKingdom.Lung Surfactant (LS), a monolayer coating the alveolar surface undergoeschanges during breathing. Glycerol, a known cryo-protectant is known toinduce folding in LS, stiffening in model LS monolayers, modulate area perlipid, and the transition temperature of such systems experimentally. Usingatomistic molecular dynamics (MD) simulations, we model LS as a pure dipal-mitoylphosphatidylcholine (DPPC) monolayer, in which the concentration ofglycerol and simulation temperature is varied to investigate the molecular basisof such variations in behaviour. Our Simulations suggest a possible dehydrationstage of DPPC head-groups under high concentrations of glycerol, which resultin a change in DPPC transition temperature (6). This change could influence theuse of glycerol in possible aerosol devices and the permeability of the modu-lated monolayer.

377-Pos Board B142Protocol and Validation of CHARMM-GUI Hex Phase BuilderAndrew H. Beaven1, Alexander J. Sodt2, Richard W. Pastor3, Wonpil Im4.1Chemistry, The University of Kansas, Lawrence, KS, USA, 2Unit onMembrane Chemical Physics, National Institutes of Health, Bethesda, MD,USA, 3Laboratory of Computational Biology, National Institutes of Health,Bethesda, MD, USA, 4Department of Biological Sciences andBioengineering Program, Lehigh University, Bethlehem, PA, USA.It is becoming increasingly apparent that protein dynamics, conformation, andtherefore, function are dependent on the stresses within the bilayer – one ofwhich being bending frustration. The amount of bending frustration is depen-dent on the bending modulus (kc) and spontaneous radius of curvature (R0

–1)for a given lipid composition. These quantities are difficult to obtain computa-tionally, particularly by all-atom molecular dynamics (MD) simulation. Formany years, experimentalists (and much more recently, simulators) haveused the lipid hexagonal phase to obtain these quantities. The inverse lipid hex-agonal phase exists in high temperature and low water domains of the certainlipid’s phase diagram. In this phase, lipids aggregate with their hydrophilicheads oriented along hexagonally packed water pores. Although this phasespontaneously assembles, the equilibration time necessary for this type of pro-cess is unfeasible for typical all-atom MD simulations. Here, a simple andreproducible methodology, Hex Phase Builder, is described and validated as

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an integrated part of the CHARMM-GUI framework (http://www.charmm-gui.org/input/hexphase). By calculating the pressure within the water pore, kc andR0

–1 can be obtained for any lipid composition. This sensitive technique pro-vides energetic insight into lipid systems and allows for further validation ofthe oft-used CHARMM C36 lipid force field.

378-Pos Board B143Reproduction of a Three-Component (DPPC/DOPC/Cholesterol) PhaseDiagram using Coarse Grained Molecular DynamicsCameron Montour1, Timothy S. Carpenter2, Felice C. Lightstone2.1Dept. of Biochemistry & Molecular & Cellular Biology, GeorgetownUniversity, Washington, DC, DC, USA, 2BBTD, Lawrence LivermoreNational Laboratory, Livermore, CA, USA.The plasma membrane consists primarily of a mixture of lipids and proteins,responsible for creating a homeostatic environment inside the cell. The plasmamembrane is also responsible for mediating cell-cell communication; specif-ically, membrane proteins send and receive signals across the plasma mem-brane. However, in some cases the membrane proteins require lipids tocreate a locally optimized environment to create functional groupings. Lipidsmay facilitate protein localization by forming phases of differing lateral orga-nization, which proteins are likely to segregate into based on overall lipid dy-namics. These phases occur mainly as liquid-disordered (Ld) or liquid-ordered(Lo); the Ld phase is composed of mainly unsaturated lipids, while the Lophase is composed of mainly saturated lipids and cholesterol. Membranescan exist as a single fluid phase or coexistence of phases under the right com-positions and conditions. It is established that phase separation can occur incertain models but the ordering progression of the single fluid phase andwhether or not a threshold event occurs between the Ld and Lo phases is stilllargely unidentified. In this study we performed coarse-grained MARTINI mo-lecular dynamic simulations on a ternary system, consisting of a saturated lipid,dipalmitoylphosphatidylcholine (DPPC), an unsaturated lipid, dioleoylphos-phatidylcholine (DOPC), and cholesterol (CHOL). A primary goal of the studywas to observe how well the MARTINI forcefield could reproduce theseternary phase diagrams. As such, no changes were made to the MARTINIforcefield, and no biases were imposed on the system. The MARTINI forcefieldperformed remarkably well, replicating all of the single fluid phases and mostof the phase separated systems. This work were performed under the auspicesof the U.S. Department of Energy by Lawrence Livermore National Laboratoryunder Contract DE-AC52-07NA27344, Release LLNL-ABS-704312.

379-Pos Board B144Molecular Dynamics Simulations of 38 Types of Ganglioside in Homoegne-ous Membrane BilayersSteve Kim1, Wonpil Im2.1University of Kansas, Lawrence, KS, USA, 2Lehigh University, Bethlehem,PA, USA.Ganglioside is a membrane lipid in which the hydrophilic head group is acomplex oligosaccharide containing one or more sialic acids. The oligosac-charides of the ganglioside extend out from the cell surface, interactingwith various signaling molecules. Some of their functions include regulationof protein activities, recognition of specific molecules, and communication be-tween cells. Gangliosides are especially abundant in the brain, with threetimes as much in grey as in white matter. It is believed that gangliosidesare involved in pathological states such as cancer, Tay-Sachs disease, Hun-tington’s disease, Alzheimer’s disease, et certera. To explore and understandthe orientation, structure, and dynamics of gangliosides, molecular dynamicssimulations on 38 different types of gangliosides in homogenous membranebilayer (POPC) were performed. CHARMM-GUI was utilized to build themembrane bilayers, and a ganglioside was inserted in both the upper leafletand the bottom leaflet, with three simulations for each ganglioside. Each sys-tem was ran for up to 50 ns and multiple analyses were ran. Through compar-ative analysis, the difference in properties and dynamics due to differentglycan sequence will be studied. Future work includes running the simulationlonger up to 200~300 ns and assembling the information gathered throughmultiple analyses for greater understanding of gangliosides, which will aidin glycome studies.

380-Pos Board B145Vitamin E does not Preferentially Bind to Polyunsaturated Lipids asRevealed by Umbrella Sampling MD SimulationsXiaoling Leng1, Andres Cavazos1, Bruce Ray1, Mikel Ghelfi2,Jeffrey Atkinson2, Fangqiang Zhu1, Stephen Wassall1.1physics, IUPUI, Indianapolis, IN, USA, 2chemistry, Brock University,St. Catharines, ON, Canada.Alpha-tocopherol (atoc) is the active form of vitamin E that is retained in thehuman body. It is a lipid-soluble antioxidant, which protects polyunsaturated

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fatty acids (PUFA) by terminating a chain of chemical reactions thatfollow free radical attack. Due to the low global concentration of atoc in theplasma membrane, we have proposed that atoc preferentially interacts withthe PUFA-containing lipids to optimize its local concentration. Here we testedthis hypothesis by calculating the binding energy between atoc and twodifferent phospholipids. 1-stearoyl-2-docosahexaenoylphosphatidylcholine(SDPC, 18:0-22:6PC) was used to represent a PUFA-containing lipid, while1-stearoyl-2-oleoylphosphatidylcholine (SOPC, 18:0-18:1PC) was used as amonounsaturated control. Umbrella sampling molecular dynamics (USMD)simulations of atoc in bilayers composed of these lipids were performedwith 45 windows separated by 1 A and arranged from the center of bilayerall the way to the bulk water to calculate the free energy as a function of atoc’slocation. The results show slightly less binding energy between atoc andSDPC, which we ascribe to a more loosely packed arrangement of lipids inthe PUFA-containing membrane. NMR experiments measuring the affinityof atoc for SDPC and SOPC are currently ongoing in support of thesimulations.

381-Pos Board B146RBL-2H3 Proliferation is Modulated by Treatments that Shift TransitionTemperatures in Isolated Plasma Membrane VesiclesRohan P. Desai, Sarah Veatch.Biophysics, University of Michigan, Ann Arbor, MI, USA.Giant plasma membrane vesicles (GPMVs) isolated from a variety of cell typesappear homogeneous at physiological temperatures but separate into distinctliquid-ordered and liquid-disordered phases below their transition temperature(Tmix). Previous work in the lab has shown that Tmix is higher in GPMVs iso-lated from sparsely plated cells than those plated at a higher density (Gray et al.PLoS One 10(9):e0137741 (2015)). Since these cells are also contact inhibited,we hypothesize that differences in Tmix are related to their different growthrates. This previous study also found that Tmix is elevated in GPMVs isolatedfrom cells preparing to undergo cell division compared to cells in other phasesof the cell cycle. Based on these past results, we hypothesized that compoundswhich shift GPMV Tmix also affect the rate of cell growth when incubatedwith intact cells. To test this hypothesis, we incubated intact RBL-2H3 cellsin n-alcohols and combinations of n-alcohols which we have previouslyshown to alter Tmix when added to isolated GPMVs (Machta et al. Biophys.J. 111(3):537-45 (2016)). We find that these treatments also alter cell growthas measured by counting cells present after 24h of incubation with compoundsin complete media at 37�C. Specifically, fewer cells are counted in dishes con-taining n-alcohols that lower Tmix in GPMVs than that of a control, whereas aneven greater number of cells are counted in dishes containing an n-alcohol thatraises Tmix in GPMVs. In addition, we find that cells adapt their membranecomposition to compensate for the effect of n-alcohols on GPMV Tmix whenincubated with n-alcohols for 24h. Ongoing experiments are being conductedto illuminate the biochemical pathways underlying the observed changes incell proliferation.

382-Pos Board B147Lipid Lateral Ordering Defined by High-Field EPRZahra Hayati, Pavanjeet Kaur, Likai Song.National High Magnetic Field Laboratory, Florida State University,tallahassee, FL, USA.EPR spectra at 94 GHz or higher are sensitive to lipid lateral ordering, whichprovides key information on the structure and dynamics of biologicalmembranes as well as protein–lipid interactions. High-frequency high-fieldEPR improves spectral resolution through increased g-factor resolution,enabling the determination of the motionally averaged gxx–gyy anisotropy,which reflects lipid lateral ordering. Lipid rafts are domains of plasma mem-branes enriched with cholesterol and sphingolipids and are essential forcellular functions. Here, we investigated the lateral ordering of lipid raftsusing high-field EPR. The analyses reveal that lateral ordering is moresensitive to raft-domain formation than the conventional order parameter.Lateral ordering is a sensitive parameter for both lipid phase transitionand cholesterol/sphingomyelin content. Our data also show that raft andnonraft domains exhibit distinct lateral order profiles across the lipid bilayer.In addition, we characterized the membrane interaction and peptide-induced lipid-lateral-ordering changes of an antibacterial g-AApeptide(AA1). The results illustrate that AA1 interacts strongly with bacterial mem-branes, induces significant lipid lateral ordering and membrane thinning, andsubsequently disrupts bacterial membranes. Our findings suggest that AA1 in-teracts with and disrupts bacterial membranes through a carpet-like mecha-nism. Taken together, these results highlight the sensitivity of high-fieldEPR for elucidating membrane order and dynamics as well as protein–lipidinteractions.

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383-Pos Board B148Azobenzene-Cholesterol as a Photoactivator in Biomimetic Membranes: 1.Lipid DynamicsChen Shen1, Jacques Ollivier2, Judith Peters3, Jorg Pieper4, Beate Klosgen1.1Department of Physics, Chemistry and Pharmacy, University of SouthernDenmark, Odense, Denmark, 2Institut-Laue-Langevin, Grenoble, France,3Universite Grenoble Alpes, Grenoble, France, 4Institute of Physics,University of Tartu, Tartu, Estonia.Properties of biomembranes are modified in presence of additives such ascholesterol (chol) or proteins. Azobenzene-cholesterol (azo-chol) is a photoac-tive variety of chol since its azo-headgroup exhibits a reversible change of theconformation upon illumination (365nm: trans to cis; 455nm: cis to trans).Quasielastic neutron scattering (QENS) was applied to resolve lipid dynamicswithin biomimetic membranes consisting of a 1-palmitoyl-2-oleoyl-sn-phos-phatidyl-choline (POPC) matrix with embedded azo-chol in either of the twoconformational states. A pure POPC membrane and also a POPC membranewith inactive chol were used as control systems. The results report on thefast intramolecular motion of the protons in the POPC chains, a slow hopdiffusion and an even slower mode both of the whole POPC molecule. The in-tramolecular motion and the hop diffusion are modified by the presence of azo-chol in the host membrane as compared with the control systems, but do notdepend on the isomerization state. The slowest mode is though sensitive tothe isomerization. The findings show the capacity of azo-chol for tuning the dy-namics and most probably also the structures of the host lipid membrane thoughphotoactivation.

384-Pos Board B149Anomalous Behavior in Lipid Bilayer MembranesMatthew R. Cheetham1, Helena L.E. Coker2, Mark I. Wallace1.1Department of Chemistry, King’s College London, London, UnitedKingdom, 2Physical and Theoretical Chemistry, University of Oxford,Oxford, United Kingdom.In contrast to the diffusion of membrane components in artificial lipid bilayers,diffusion in cells is both slower and non-ergodic. The physical origins ofsuch anomalous diffusion are poorly understood, though cytoskeletal-membrane interactions and nanoscopic heterogeneity in the membrane haveboth been suggested to play an important role. There remains however alack of controlled model systems on which to test these potential causes.Here we present a model system for reproducing this anomalous diffusionthat utilizes supported lipid bilayers (SLBs) with varying degrees of excludedarea fraction. By varying the concentration of PEG-lipids, we observe a dra-matic and controllable switch in anomalous diffusion as we cross the estimatedpercolation threshold. This provides a simple model system for investigatingthis phenomenon in lipid membranes. The behavior is well described byboth theory and Monte-Carlo simulation of the anomalous crossover as a func-tion of obstacle density.

385-Pos Board B150All-Atom Molecular Dynamics Simulation of Stealth LiposomesSeyed Hamid Tabari, Jeevapani Hettige, Mahmoud Moradi.University Of Arkansas, Fayetteville, AR, USA.Pegylation of liposomes has been widely used in the field of drug delivery toincrease the bloodstream circulation time of liposomes; however, simulationstudies have focused on simple model of liposomes, including only one ortwo different kinds of lipids simulated by simplistic coarse-grained modelsthat lack the atomic/chemical details. Implementing all-atom molecular dy-namics (MD) simulations, stable structure of solvated stealth liposomes areachieved. The liposomes are composed of different DOPC (dioleoylphosphati-dylcholine)/cholesterol/PEG2000-DSPE (polyethylene glycol 2000-distearoyl-phosphatidylethanolamine) molar ratio. Here we used coarse-graining/backmapping method to speed up mixing of the stealth liposome molecules.The MD simulations revealed that not only are geometric structure of lipo-somes dominated by the cholesterol molecules, but also PEGylated lipidsreduce leakage of contents from liposomes.

386-Pos Board B151Lipid Diffusion in Membrane Junctions Measured by Single-MoleculeTrackingVivek Ramakrishna, Mark I. Wallace.King’s College London, London, United Kingdom.The interface between lipid membranes is an important site for biochemical ac-tivity. Cellular signalling processes are often mediated by the diffusion of lipidsand other components in the membrane. We present a system comprising a sup-ported lipid bilayer and a giant unilamellar vesicle as a model for membrane

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interfaces. Using single-molecule tracking, we study the diffusion of membranecomponents in the interfacial region and the effect of lipid composition andenvironmental conditions on membrane behaviour.

387-Pos Board B152Influenza Binding Avidity Governed by Sterol-Dependent GangliosideDynamicsIsabel Goronzy1, Robert Rawle2, Peter Kasson2, Steven Boxer1.1Department of Chemistry, Stanford University, Stanford, CA, USA,2Department of Molecular Physiology and Biological Physics, University ofVirginia, Charlottesville, VA, USA.Influenza virions attach to host membranes by binding cell-surface glycanscontaining sialic acid, initiating the viral life cycle and infecting cells. Whilethe primary determinant of viral binding is thought to be the glycan chemicalstructure, single interactions between a viral hemagglutinin binding site and asialic acid are weak, so binding specificity is presumed to occur via multiva-lent interactions. Here, we show that target membrane composition can alterinfluenza binding while total receptor content is held constant. We hypothe-size that this effect occurs by changing the spatial distribution of target recep-tors. Viral binding was quantitated by monitoring individual viruses bindingto synthetic supported lipid bilayers using fluorescence microscopy. Addingcholesterol to target bilayers increased binding in a dose-dependent manner.Furthermore, membranes containing 5-cholesten-3-one, an oxidized variantof cholesterol, displayed significantly higher binding avidity compared tomembranes supplemented with an equal amount of cholesterol. When sterolmole fractions were held constant, viral target receptor concentration andbinding exhibited a sigmoidal relationship, confirming the multivalent natureof virus receptor binding. The cooperativity required for binding establishedthe potential for local receptor concentration to regulate membrane bindingaffinity. To develop a physical model for sterol-induced changes in the lateralorganization of GD1a receptors, we performed coarse grained molecular dy-namics simulations of target lipid bilayers. Irrespective of sterol content,GD1a receptors showed strong tendencies to self-associate. The inclusion ofcholesterol in membranes increased pairwise receptor contacts, suggestingthat sterol addition stabilized target receptor clustering. This observationsupports the hypothesis that sterols can modulate membrane nanoscale orga-nization and thus alter viral binding avidity, illuminating a previously under-appreciated mechanism by which target membrane context can influenceinfluenza infectivity.

388-Pos Board B153Secrets of the Enigmatic Lipid II Revealed by Molecular Dynamics Simu-lationsSyma Khalid1, Firdaus Samsudin1, Timothy S. Carpenter2, Sarah Witzke1.1School of Chemistry, University of Southampton, UK, Southampton, UnitedKingdom, 2Lawrence Livermore National Laboratory, Livermore, CA, USA.Lipid II is critical for the biosynthesis of peptidoglycan; the main component ofthe bacterial cell wall. Lipid II is targeted by antibiotics such as the lantibiotics,which achieve their function by disrupting the biosynthesis of the cell wall.Currently there is an the urgent need for development of novel antibiotics tocounter the growing threat of pathogenic bacteria becoming resistant tocurrently used antibiotics. To achieve this, it is imperative we gain a detailedunderstanding of the molecules targeted by antibiotics.Relatively little is known about the conformational dynamics of Lipid II, inparticular about the unusually long tail. To this end, we present a molecular dy-namics simulation study of the conformational dynamics of Lipid II within adetailed model of the Staphylococcus aureus cell membrane. We show thatLipid II is able to adopt a range of conformations even within the packed lipidicenvironment of the membrane. Furthermore we present energetic analysis thatreveals the free energy associated with removing Lipid II from the S. aureusmembranes compared to other lipids. Thus, we provide unprecedented insightsinto the conformational dynamics of Lipid II within a Gram-positive bacterialmembrane.

389-Pos Board B154Evaluating Bilayer Mechanical Properties in Protein Reconstituted GUVsNestor Lopez Mora, Heather Findlay, Paula Booth.Chemistry, King’s College London, London, United Kingdom.Biological membranes are complex systems where membrane proteins aresurrounded by a bilayer composed of different types of lipids, with mem-branes from different organisms and organelles varying greatly in theircomposition and therefore physical properties. The mix of lipids in the mem-brane has additionally been shown to influence the activity, stability andconformation of many integral membrane proteins. In order to investigate

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the effect of incorporating protein on the mechanical properties of lipidbilayers we have reconstituted the Escherichia coli transporter lactosepermease (LacY), a transmembrane protein representative of the major facil-itator superfamily, into synthetic lipid vesicles. We observed directly the fluc-tuation of the lipid bilayer in these protein reconstituted GUVs, with sizes inthe order of micrometers, by phase contrast microscopy. This allowed thedetermination of the bending rigidity, which characterizes the ability of mem-branes to bend under low stress, by fluctuation analysis. Changes in thebending rigidity parameter allowed us to get better insights into the effectof lipids and protein on the mechanical properties of GUVs in a quantitativefashion.

390-Pos Board B155Shear Stress Stimulated MSC Activities: Direct Changes of MembraneTension or Cytoskeletal Stress?Mohammad Mehdi Maneshi1, Frederick Sachs2, Susan Zonglu Hua1,2.1Mechanical and Aerospace Engineering, University at Buffalo, Buffalo, NY,USA, 2Physiology & Biophysics, University at Buffalo, Buffalo, NY, USA.Fluid shear stress induced membrane transport such as Ca2þ influx and theactivation of ion channels has been widely reported. The shear stress canbe mediated by a direct change in bilipid membrane tension and/or by achange in cytoskeletal stress via binding proteins that link channels to actin,but how the shear stress is coupled to ion channels is unclear. Using narrowshear pulse stimuli generated by a pressure servo in a microfluidic chamber,we measured the changes of membrane tension and cytoskeletal protein stresssimultaneously in astrocytes. The membrane tension was reported using mo-lecular motor probes (2-carboxy-2-cyanovinyl)-julolidine farnesyl ester(FCVJ) and cytoskeletal tension reported by genetically encoded force probeactinin-cpst-FRET. Our results show that the changes of membrane tensionare highly localized and the gradient is relevant to the flow directions. A shearstress pulse (23 dyn/cm2, 400 ms duration) caused a rapid increase in mem-brane tension at the front edge of the cell with respect to the flow and adecrease in tension (compression) at the distal edge. The rise time was lessthan 30 ms, and the tension dropped to the initial state within ~30 ms poststimulus, showing a typical elastic behavior. In contrast, the same shear pulsegenerated profound and long-lasting tension in cytoskeletal cross-linking pro-teins a-actinin at the front edge of the cell, and the tension persisted for theentire experimental duration of 60 s. In situ Ca2þ imaging showed that theinitial Ca2þ influx was strongly correlated with the region having high cyto-skeletal tension, but weakly linked to the bilayer tension. The results suggestthe cytoskeletal tension plays primary role in shear stress activated Ca2þ

influx. This work was funded by NINDS.

391-Pos Board B156Stress Propagation through Biological Lipid-Bilayers Revealed by Atom-istic and Coarse-Grained SimulationsCamilo Aponte-Santamaria1,2, Frauke Gr€ater1,2.1Molecular Biomechanics, Heidelberg Institute for Theoretical Studies,Heidelberg, Germany, 2Interdisciplinary Center for Scientific Computing,Heidelberg University, Heidelberg, Germany.Membrane tension has been shown to play various critical roles in cellsignaling. We here asked if and how pulses of local stress dynamically propa-gate through membranes, and propose this mechanism as a novel way ofquickly propagating signals along the membrane.In both atomistic and coarse-grained MARTINI molecular dynamics simula-tions of biological lipid-bilayers, we observed short stress pulses to very effi-ciently propagate laterally at a velocity of the order of km/s, in closeagreement with the expected speed of sound. The temperature dependence ofpulse propagation shows tendencies very comparable to analogous experiments[1,2,3], with insightful differences between the atomistic and coarse-grainedsimulations. Remarkably, the propagation of the lateral stress was damped atlength scales in the ~100 nm range. Our data supports the notion of lateral stresspropagation through membranes as a potential ultrafast way of short-rangesignal propagation in biology [4].[1] W. Schrader, et al. J. Phys. Chem. B. 106:6581-6586 (2002)[2] S. Shrivastava S and MF Schneider. J. R. Soc. Interface 11:20140098(2014).[3] J. Kappler and R. Netz. EPL. 112: 19002 (2015)[4] C. Aponte-Santamarıa and F. Gr€ater. In preparation

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392-Pos Board B157Interdependence between Collective Thermal Fluctuations and Elastic andViscous Properties in Model Lipid BilayersMichihiro Nagao1,2, Elizabeth G. Kelley1, Rana Ashkar3,Robert Bradbury1,2, Paul D. Butler1,4.1NIST Center for Neutron Research, National Institute of Standards andTechnology, Gaithersburg, MD, USA, 2Center for Exploration of Energy andMatter, Indiana University, Bloomington, IN, USA, 3Biology and Soft MatterDivision, Oak Ridge National Laboratory, Oak Ridge, TN, USA,4Department of Chemical and Bimolecular Engineering, University ofDelaware, Newark, DE, USA.Lipid membranes undergo an array of conformational and dynamic transitions,ranging from individual lipid motions to undulations of micron-sized patches ofthe membrane. However, the dynamics at intermediate length scales are largelyunexplored due to experimental challenges in accessing the appropriate lengthand time scales. Over the past several years our group has used neutron spinecho spectroscopy (NSE) to provide unique insights into these elusive dy-namics in model lipid bilayers, measuring collective bending and thicknessfluctuations. These thermally induced collective membrane fluctuations arecontrolled by elastic and viscous properties of the membranes. It has longbeen known that the bending fluctuations are characterized by the bendingmodulus, k, of the membranes and the motion is damped by the viscosity ofsolvent, h. By contrast, according to a recent theory proposed by Bingham,Smye and Olmsted, the collective thickness fluctuations are characterized bythe bilayer area compressibility modulus, KA, which is damped by the mem-brane and solvent viscosities, m and h, respectively. Therefore, by measuringthese two collective membrane fluctuations the membrane’s elastic and viscousparameters can be evaluated. Here we use this novel method to determine thesecharacteristic parameters of lipid bilayers from neutron scattering data for acouple of simple saturated phosphatidylcholine bilayers. The estimated valuesare k ~ 10�19 J, KA ~ 0.3 to 0.4 N/m, and m ~ 10 nPa s m, which are all consis-tent with literature values.

393-Pos Board B158Hydration-Mediated Elastic Deformations in Biological MembranesTrivikram R. Molugu1, Soohyun K. Lee1, Xiaolin Xu2, Rami Musharrafieh1,K.J. Mallikarjunaiah1, Constantin Job1, Michael Brown1,2.1Department of Chemistry and Biochemistry, University of Arizona, Tucson,AZ, USA, 2Department of Physics, University of Arizona, Tucson, AZ, USA.Lipid membranes are excellent examples of biological soft matter [1]. Manyfunctions of biomembranes involve collective phenomena with motional time-scales spanning several decades (10�12 s to s). For liquid-crystalline mem-branes atomistic interactions often explain bulk material properties inrelation to key biological functions. Solid-state 2H NMR spectroscopy providessuch information by simultaneously probing structure and dynamics [2]. Herewe examine the effect of hydration on the liquid-crystalline properties of mem-branes using NMR relaxation methods. We performed 2H NMR longitudinal(R1Z) and transverse quadrupolar-echo decay (R2

QE) experiments on DMPC-d54 bilayers, to study membrane-lipid dynamics. Plots of the R1Z rates versussquared segmental order parameters (SCD

2) follow an empirical square-lawshowing the emergence of collective lipid dynamics [3]. Such a functionalbehavior characterizes 3-D order-director fluctuations due to the onset ofmembrane elasticity over mesoscopic dimensions [3]. The R2

QE rates alsoshowed similar results. At high hydration there is an R2

QE enhancement ofthe functional square-law for the segments deeper in the bilayer. Additionalcontributions from slower dynamics involving water-mediated membranedeformation are evident over mesoscopic length scales on the order of bilayerthickness. Such membrane deformations are also evident from bilayer struc-tural parameters calculated using a statistical mean-torque model [4]. In addi-tion, the square-law confinement must be due to water penetration into thehydrophobic interior of the bilayer. The slow dynamics at high hydrationmust be a consequence of modulation of membrane elastic properties. TheQCPMG frequency dispersions provide quantitative viscoelastic properties ofthe liquid-crystalline membranes. Such studies on model membranes give in-sights into lipid rafts and membrane compositions relevant for biomembranefunctions. [1] A. Leftin et al. (2014) BJ 107, 2274[2] K.J. Mallikarjuniah et al. (2011) BJ 100, 98.[3] T.R. Molugu et al. Chem. Phys.Lipids. (2016)[4] A. Leftin et al. (2014) eMagRes 4, 199.

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Membrane Fusion and Non-Bilayer Structures

394-Pos Board B159Interactions of Carbon Nanotubes Stabilized by Selected GeminiSurfactants with Model BiomembranesMichalina Skupin1, Justyna I _zykowska1, Weronika Andrzejewska1,Maria Dobies1,2, Stefan Jurga1,2, Maciej Kozak1,3.1Department of Macromolecular Physics, Adam Mickiewicz University,Pozna�n, Poland, 2NanoBioMedical Center, Adam Mickiewicz University,Pozna�n, Poland, 3Joint Laboratory for SAXS studies, Adam MickiewiczUniversity, Pozna�n, Poland.Thanks to the extraordinary mechanical strength and high electrical conductiv-ity multiwalled carbon nanotubes are currently used in electronics, medicine (asbiomedical sensors, transporters or drugs) as well as in the production of light-weight and durable construction. The aim of this study was to determine thepossibility to use different cationic gemini surfactants with different spacerlengths or alkyl chain lengths in more efficient systems for dispersing nano-structures in aqueous solutions. The most important advantages of these sys-tems are their non-immunogenic, biocompatible properties and generally lowtoxicity . Therefore nanotubes, surrounded by surfactants, have the potentialto interact with biological membranes. For this purpose we studied the influ-ence of dispersed CN solution on the phase behavior of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) - a phospholipid most often present inmembranes of nerve cells. The microstructure of the stable suspension ofcarbon nanotubes was investigated using high-resolution Transmission Elec-tron Microscopy and Atomic Force Microscopy. Fourier transform infrared(FTIR) spectroscopy and differential scanning calorimetry (DSC) were usedto analyze the influence of surfactants studied, used for CN dispersion, onthe phase behavior of DMPC bilayers. A series of measurements of toxicityof these systems were performed in HeLa and fibroblast cell cultures. Thiswork was supported by the Ministry of Science and Higher Education, withinthe project ‘‘Najlepsi z najlepszych!’’ dec. DIR.5210.352016/1.

395-Pos Board B160The Bilat: A Free Standing Lipid Bilayer Microarray Platform forMembrane FusionSathish K. Ramakrishnan1,2, Andrea Gohlke2, Paul Heo2, James Rothman1,Frederic Pincet1,2.1Department of Cell Biology, Yale University, Stamford, CT, USA,2Laboratoire de Physique Statistique, Ecole normale superieure, Paris,France.Planar lipid bilayer membranes are crucial for studying many physiologicalprocess such as membrane fusion, transport, interactions and ion channels.Despite the continuing technological progress in planar lipid membranes, thereis a lack of system with high membrane stability, protein mobility and free fromsupport. Most in vitro membrane fusion studies are still carried out either bybulk fusion assays or on supported membranes, where protein mobility is hin-dered. Here, we developed a planar bilayer microarray platform with integratedtransmembrane proteins that mimics in vivo like environment by having supe-rior protein mobility and free from support. We will present our system andshow the significance of freely diffusing transmembrane proteins in artificialmembranes. Both sides of the lipid bilayers can be modified and controlledseparately allowing this setup to be adopted for a wide range of research. Wedemonstrate the ability of the system by mimicking synaptic vesicle fusion dy-namics and regulation at a single vesicle level in a millisecond timescale. Eachmembrane fusion states such as hemifusion and full fusion were monitoredthrough lipid mixing and content release assays. In addition, we report a proto-type of an automated data analysis software, vesicle fusion analyzer, for fastermembrane fusion data analysis. We suggest that the newly developed freestanding bilayer system associated with this software can be used as a generalplatform to study behavior of membrane-related molecules.

396-Pos Board B161Phase Specific Membrane Fusion with SNARE MimeticsBastian Kubsch1, Tom Robinson1, Torben-Tobias Kliesch2,Andreas Janshoff2, Reinhard Lipowsky1, Rumiana Dimova1.1Theory and Bio-Systems, Max Planck Institute of Colloids and Interfaces,Potsdam, Germany, 2Gottingen University, Gottingen, Germany.Biological membrane fusion is involved in a number of essential processes suchas neurotransmission, exocytosis, and viral infection. It is crucial for cells tospatially confine this process to specific organelles or sites in the plasma mem-brane. In nature, the SNARE complex induces fusion in eukaryotic cells. Itsdifferent constituents integrated into opposing membranes approximate thetwo bilayers by interacting in a zipper-like fashion. Different studies examinedmembrane fusion in artificial systems using SNARE mimetics. Fusion between

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pairs of small unilamellar vesicles have been shown using lipidated DNA[Chan et al., Biointerphases, 3:FA17, 2008] and peptide [P€ahler et al., Biophys-ical Journal, 103:2295, 2012] ligand-receptor pairs. Here, we show strong ev-idence of the fusion of large unilamellar vesicles (LUVs) to a spatially confinedregion within liquid-liquid phase-separated giant unilamellar vesicles using theabovementioned (i) hybridised DNA and (ii) coiled-coil peptide pairs asSNARE mimetics. We quantified the (hemi-)fusion process using two differentFRET-based approaches which yielded similar results. Moreover, our systempotentially offers to determine LUV docking and fusion efficiencies. Comple-mentary experiments are currently performed using reconstituted SNARE pro-teins (SNARE proteins were kindly provided by Claudia Steinem, Departmentof Biomolecular Chemistry at Gottingen University). This work is part of theMaxSynBio consortium which is jointly funded by the Federal Ministry ofEducation and Research of Germany and the Max Planck Society.

397-Pos Board B162The Influence of Nanoparticles on SNARE-Mediated Membrane FusionMichael J. Crowe, Jiajie Diao.Cancer Biology Department, University of Cincinnati, Cincinnati, OH, USA.Due to their unique electrical, optical, and thermal properties, nanoparticleshave attracted significant research interest for biomedical applications [1-3].A recent study showed that nanoparticles can affect synaptic transmission bydisrupting lipid composition and Ca2þ homeostasis [4]. To further elucidatethe effects of nanoparticles on the molecular mechanism of synaptic transmis-sion, we utilized in vitro fusion assays based on proteoliposomes reconstitutedwith SNAREs. The v- and t-SNARE proteins are reconstituted in two indepen-dent populations of liposomes that are labeled with acceptor (DiD) and donor(DiI) fluorophores. Upon fusion, lipid molecule mixing results in FRET be-tween donor and acceptor molecules [5].1. Diao, J.J. and Chen, G.D. J. Phys. D: Appl. Phys. 34, L79 (2001).2. Diao, J.J. and Chen, H. J. Chem. Phys. 124, 116103 (2006).3. Chen, Y. et al. J. Phys. Conf. Ser. 59, 548 (2007).4. Bramini, M. et al. ACS Nano 10, 7154 (2016).5. Brunger, A.T. et al. Crit. Rev. Biochem. Mol. Biol. 50, 231 (2015)

398-Pos Board B163Calcium Sensitive Ring-Like Oligomers of Synaptotagmin 1: Implicationsfor Neurotransmitter ReleaseShyam Krishnakumar.Dept of Cell Biology, Yale University, West Haven, CT, USA.The synaptic vesicle protein, Synaptotagmin-1 (Syt1) is required to coupleCa2þ influx to the membrane fusion machinery. However, the structural mech-anism underlying this process is unclear. Using negative stain electron micro-scopy, we find that the Syt1 assembles into ring-like oligomeric structures onlipid monolayers under physiological ionic strength and lipid composition inthe absence of free Ca2þ. These rings oligomers vary in diameter between19 - 42 nm (with an average size of 30 5 5 nm), corresponding to 12- 25 mol-ecules of Syt1. The ring-like oligomers are sensitive to Ca2þ and are disruptedrapidly by the physiological concentrations of free Ca2þ. Analogous ring-likeoligomers assemble from the C2AB domains of other Syt isoforms (Syt2,Syt7, Syt9) as well as related C2 domain containing protein, Doc2B andextended Synaptotagmins (E-Syts). Evidently, the circular oligomerization isa general and conserved structural aspect of many C2 domain proteins,including Synaptotagmins. Further, we find that both Syt1 ring formation andits disruption by Ca2þ principally involve well-established functional surfacesof the C2B domain which are important for neurotransmission. This includesthe Ca2þ -independent binding of the polybasic motif of C2B domain withphosphatidylinositol 4,5-bisphosphate (PIP2) as a prerequisite for rings toassemble. This suggests that ring formation may be triggered at a very earlystep in synaptic vesicle docking, as C2B-PIP2 interaction is required for dock-ing in vivo. Ca2þ binding to the C2B domain and re-orientation/insertion intothe membrane, both required for triggering synaptic transmission, disrupts thering oligomers. We advance a simple and novel mechanism wherein Syt1 ringoligomer act as reversible washer/spacer to synchronize neurotransmitterrelease to Ca2þ influx. Supporting this hypothesis, we find mutations in Syt1that specifically disrupt the ring oligomer formation results in dysregulationneuroexocytosis in PC12 cells.

399-Pos Board B164Towards Understanding the Molecular Mechanism of SynchronousNeurotransmitter ReleaseQiangjun Zhou, Thomas Christian S€udhof, Axel Thomas Brunger.Howard Hughes Medical Institute, Molecular and Cellular Physiology,Stanford University, Stanford, CA, USA.Most communication between neurons is achieved at synapses by theprocess of neurotransmitter release. Neurotransmitter release is initiated

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by depolarization of a neuron, which in turn activates voltage-gated Ca2þ

channels. The resulting Ca2þ influx then triggers the fusion of the synapticvesicles with the plasma membrane. Synaptic vesicle fusion is mediated bya core fusion machinery SNARE complex, a small regulatory factor com-plexin (Cpx), and Ca2þ sensor synaptotagmin (Syt). However, it was un-known how they cooperate to trigger synaptic vesicle fusion. CombiningX-ray crystallography and electrophysiological recording techniques, wedetermined two atomic resolution crystal structures of the synaptic vesiclefusion machinery at different states, revealing a large, specific, Ca2þ-inde-pendent interface which is essential for synchronous neurotransmitterrelease in mouse neuronal synapses. We propose a working model andfurther reveal the molecular mechanism of synchronous neurotransmitterrelease.

400-Pos Board B165a-Synuclein: A Functional Role as a Regulator of SNARE-MediatedFusionSiobhan Toal, Elizabeth Rhoades.Chemistry, University of Pennsylvania, Philadelphia, PA, USA.Fusion of vesicular and plasma membranes is mediated by SNAREproteins. In a vesicular fusion event, the t- and v-SNAREs assemble intoa four-helix bundle pulling the two membranes together to cause fusion.A decrease in neurotransmitter release upon overexpression of theneuronal protein a-Synuclein (aS) has been observed in animal models, sug-gesting that aS may act as a regulator of neurotransmission, alteringSNARE driven fusion of synaptic vesicles. Recent work in our lab hasshown that aS is able to inhibit SNARE-mediated fusion in vitro, althoughthe mechanism appears to be through binding to the lipid bilayer, notthrough direct interactions with SNARE proteins. Here, we investigate thepossibility that aS may also modulate fusion through interactionswith SNARE regulatory proteins, synaptotagmin and complexin, using anin vitro fusion assay. We find that in the presence of synaptotagmin andcomplexin, aS differentially alters SNARE-mediated vesicle fusion in aconcentration dependent manner. At low aS concentrations, fusion is signif-icantly enhanced in a concentration-dependent manner (i.e. increasingfusion with increasing aS). However, once a threshold concentration isexceeded, fusion is again inhibited, again in a concentration dependentmanner (i.e. decreasing fusion with increasing aS). Direct evidence ofprotein-protein interactions was monitored using fluorescence correlationspectroscopy to measure the diffusion times of the protein components.SNARE complex formation can be observed as a function of timethrough an increase in the diffusion time of labeled t-SNARE protein.While aS does not appear to impact the rate of complex formation alone,substantial increases in the diffusion time of the SNARE complex in thepresence of aS and complexin were observed, suggesting an interaction be-tween the two. Taken together, our results suggest that aS may have a dualrole in SNARE-mediated membrane fusion, as a chaperone of SNARE reg-ulatory components as well as, at high enough concentrations, a fusioninhibitor.

401-Pos Board B166Mitochondrial Fusion Proteins: A Tale of Two MembranesAndrew D. Kehr, Marisa A. Rubio, Jenny Hinshaw.NIDDK, National Institutes of Health, Bethesda, MD, USA.Dynamins are a class of GTPase enzymes responsible for the fusion,fission, and vesiculation of cellular lipid membranes throughout thecell. The dynamin-like proteins Optic Atrophy 1 (Opa1) and Mitofusin(Mfn) 1 and 2 are responsible for the fusion of the mitochondrialinner and outer membranes, respectively. Mutations in any of theseproteins can lead to neuropathies including blindness and Charcot-Marie-Tooth, a disease characterized by progressive loss of distal muscletissue. Currently, little is known structurally or biochemically about anyof these proteins. We have developed a protocol for expressing andpurifying biologically relevant and biochemically active shortenedisoforms (OpaGG and Mfn1GG) in sufficient quantity to begin crystallo-graphic studies. Both have comparable GTPase activity compared to full-length, unstimulated Dynamin 1 when assayed at room temperature andinterestingly OpaGG exists as a tetramer when assayed by size exclusionchromatography. In addition, the long, membrane-bound isoforms of Opa1and Mfn1 have been expressed and purified in large quantities. To date,we have shown full length Mfn1 can be incorporated into proteoliposomesand in the presence of GTP forms dense tethers as seen by cryo-EM.This tethering is reversible as shown by confocal microscopy. Currentlywe are developing tethering assays for Opa1 and fusion assays for bothOpa1 and Mfn1.

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402-Pos Board B167Structure of the Ebola Virus Envelope Protein MPER/TM Domain and itsInteraction with the Fusion Loop Explains their Fusion ActivityJinwoo Lee1,2, David A. Nyenhuis1,3, Elizabeth A. Nelson1,4,David S. Cafiso1,3, Judith M. White1,4, Lukas K. Tamm1,2.1Center for Membrane and Cell Physiology, University of Virginia,Charlottesville, VA, USA, 2Departments of Molecular Physiology andBiological Physics, University of Virginia, Charlottesville, VA, USA,3Department of Chemistry, University of Virginia, Charlottesville, VA, USA,4Department of Cell Biology, University of Virginia, Charlottesville,VA, USA.Ebolavirus (EBOV), an enveloped filamentous RNA virus causing severe hem-orrhagic fever, enters cells by macropinocytosis and releases its genetic mate-rial into the cytoplasm after membrane fusion in a late endosomalcompartment. Membrane fusion is governed by the EBOV surface envelopeglycoprotein (GP), which consists of subunits GP1 and GP2. GP1 binds tocellular receptors including Niemann-Pick C1 (NPC1) protein and GP2 isresponsible for membrane fusion at low pH. GP1 undergoes multiple steps ofproteolytic cleavage and binds to NPC1 at endosomal pH. GP2 is rearrangedin a fashion that exposes the hydrophobic fusion loop (FL) of GP2, which isthen inserted into the cellular target membrane, ultimately forming a six-helix bundle structure and resulting in the formation of the fusion pore.Although major portions of the GP2 structure that have been solved in pre-and post-fusion states and the current model places the transmembrane (TM)and FL domains of GP2 in close proximity to each other at critical steps ofmembrane fusion, their structures in membrane environments and especiallyinteractions between TM and FL have not yet been characterized. Here we pre-sent the structure of the membrane proximal external region (MPER) connectedto the TM domain, i.e. the missing parts of the EBOV GP2 structure. Thestructure, solved by solution NMR and EPR spectroscopy in membrane-mimetic environments, consists of a helix-turn-helix architecture that is inde-pendent of pH. Moreover, the MPER region, not TM region, is shown tointeract in the membrane interface with the previously determined structureof the EBOV FL through several critical aromatic residues. Mutation ofaromatic and neighboring residues in both binding partners decreases fusionand viral entry highlighting the functional importance of the MPER/TM - FLinteraction in EBOV entry and fusion.

403-Pos Board B168Leakage Induced by the Influenza Virus Haemagglutinin Depends onTarget Membrane Spontaneous CurvatureSourav Haldar, Elena Mekhedov, Jane Farrington, Petr Chlanda,Paul S. Blank, Joshua Zimmerberg.Section on Integrative Biophysics, NICHD/NIH, Bethesda, MD, USA.A recent cryo-electron microscopy investigation(Chlanda et al. (2016) Nat.Microbiol. 1:16050) of hemifusion structures mediated by the influenza vi-rus haemagglutinin posited that there exist two pathways for hemi-fusion:hemifusion-stalk and rupture-insertion. Depending on target membranematerial properties, such as spontaneous curvature, one pathway will befavored over the other. A prediction of this hypothesis is that leakage of sol-uble content will be greater through the rupture-insertion pathway. To testthis prediction, we have developed a giant unilamellar vesicle (GUV)-baseddye influx assay that provides a direct measure of leakage. Our results showthat leakage (influx of soluble dye in GUV) induced by influenza viruschanges from ~ 80 % to ~ 40 % as the spontaneous curvature is changedfrom �0.02 nm�1 to �0.30 nm�1, supporting the hypothesis that leakageis modulated by membrane spontaneous curvature. Surprisingly, withsome lipid compositions, leakage was sub-maximal, i.e. there was a variabledegree of GUV filling. This result raised the possibility of a transient targetmembrane damage induced by the influenza virus. It was also possiblethat the complete fusion of a leaky virus to a GUV was responsible forthe filling of the GUV. To control for this possibility, we compared leakageinduced by commercially prepared virus (containing significant damagedviral membrane, as evidenced by entry of a cell impermeant nucleotide-binding dye) with lab-grown virus (with apparently minimal damaged viralmembranes).

404-Pos Board B169Viral Fusion Efficacy of Influenza Virus H3N2 Reassortment Combinationto the Suppoered Lipid LayerHunglun Hsu1, Jean Millet2, Deirdre Costello1, Gary Whittaker2,Susan Daniel1.1Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY,USA, 2Department of Microbiology and Immunology, Cornell University,Ithaca, NY, USA.

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Virus pseudotyping is a useful and safe technique for studying entry ofemerging strains of influenza virus. However, few studies have compareddifferent reassortant combinations in pseudoparticle systems, or comparedentry kinetics of native viruses and their pseudotyped analogs. Here, vesic-ular stomatitis virus (VSV)-based pseudovirions displaying distinct influ-enza virus envelope proteins were tested for fusion activity. We producedVSV pseudotypes containing the prototypical X-31 (H3) HA, either aloneor with strain-matched or mismatched N2 NAs. We performed single-particle fusion assays using total internal reflection fluorescence microscopyto compare hemifusion kinetics among these pairings. Results illustrate thatmatching pseudoparticles behaved very similarly to native virus. Pseudopar-ticles harboring mismatched HA-NA pairings fuse at significantly slowerrates than native virus, and NA-lacking pseudoparticles exhibiting theslowest fusion rates. Relative viral membrane HA density of matchingpseudoparticles was higher than in mismatching or NA-lacking pseudopar-ticles. An equivalent trend of HA expression level on cell membranes ofHA/NA co-transfected cells was observed and intracellular trafficking ofHA was affected by NA co-expression. Overall, we show that specificinfluenza HA-NA combinations can profoundly affect the critical roleplayed by HA during entry, which may factor into viral fitness and the emer-gence of new pandemic influenza viruses.

405-Pos Board B170SERINC5 Inhibits HIV Fusion through Inactivation of Env Glycoproteinsand Interference with Productive Refolding of EnvChetan Sood1, Mariana Marin1, Ajit Chande2, Alexa L. Mattheyses3,Khalid Salaita4, Massimo Pizzato5, Gregory Melikian1.1Pediatrics, Emory University, Atlanta, GA, USA, 2University of Trento,Trento, Italy, 3Cell biology, Emory University, Atlanta, GA, USA,4Chemistry, Emory University, Atlanta, GA, USA, 5University, Trento, Italy.The multispan membrane proteins, SERINC3 and SERINC5, have beenrecently shown to incorporate into HIV-1 particles and compromise theirability to fuse with target cells – an effect that is antagonized by the viralaccessary protein Nef. Env glycoproteins from different HIV-1 strainsexhibit variable levels of sensitivity to SERINC-mediated restriction. Themechanism by which SERINCs interfere with HIV-1 fusion remains un-clear. Here, we show by real-time single particle imaging that incorporationof SERINC5 into virions in the absence of Nef inhibits the formation ofsmall fusion pores between viruses and cells. This effect was notrelated to the SERINC5’s ability to oligomerize in the membrane or targetthe virus to degradation in lysosomes. Strikingly, we found that SERINC5promotes spontaneous inactivation of sensitive, but not resistant Env glyco-proteins, and enhances the exposure of the conserved gp41 domains by de-laying the HIV-1 fusion reaction. Super-resolution imaging revealed thatSERINC5 also interferes with the formation of Env clusters on mature vi-rions, a step that is thought to be required for efficient HIV-1 fusion. Theseresults show that SERINC5 restricts HIV-1 fusion at a step prior to smallpore formation by selectively inactivating sensitive Env glycoproteins andinterfering with the function of the remaining active Env, likely by prevent-ing the formation of large Env clusters and slowing down Env refolding.This work was partially supported by the NIH R01 grant GM054787 toG.B.M.

406-Pos Board B171SERINC Inhibits HIV-1 Env Induced Membrane Fusion and Slows FusionPore EnlargementRuben M. Markosyan1, Shan-Lu Liu2, Fred S. Cohen1.1Rush University Medical Center, Chicago, IL, USA, 2Department ofMolecular Microbiology and Immunology, University of Missouri School ofMedicine, Columbia, MO, USA.The SERINC family of proteins are integral membrane proteins that regulatethe incorporation of serine into phospholipids, to create PS, and into sphingo-lipids. It has recently been shown that two members of the family, SERINC3and SERINC5, inhibit HIV infectivity. Using a cell-cell fusion system todetermine the extent to which inhibition of infectivity is due to reducedfusion, we found that the presence of SERINC3 or SERINC5 in either effectoror target cells slows the kinetics and reduces the extent of fusion induced byHIV-1 Env. These two incorporators of serine greatly retard fusion poreenlargement, as determined by the rate of aqueous dye transfer once a poreforms. Nef is an auxiliary protein of HIV that is well-known to enhanceHIV infectivity. The presence of SERINC5 and Nef in effector cells leadsto the same extent of fusion induced by expression of Env alone, showingthat Nef eliminates the reduction of fusion caused by SERINC. (R01 GM101 539).

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407-Pos Board B172Probing Induced Structural Changes in Biomimetic Bacterial CellMembrane Interactions with Divalent CationsAllison Whited, Alexander Johs, John Katsaras, Robert Standaert,Aaron Jubb.Oak Ridge National Lab, Knxoville, TN, USA.Biological membranes, formed primarily by the self-assembly of complexmixtures of phospholipids, provide a structured scaffold for compartmentali-zation and structural processes in living cells. The specific physical propertiesof phospholipid species present in a given membrane play a key role in medi-ating these processes. Phosphatidylethanolamine (PE), a zwitterionic lipidpresent in bacterial, yeast, and mammalian cell membranes, is exceptional.In addition to undergoing the standard lipid polymorphic transition betweenthe gel and liquid-crystalline phase, it can also assume an unusual polymor-phic state, the inverse hexagonal phase (HII). Divalent cations are amongthe factors that drive the formation of the HII phase, wherein the lipid mole-cules form stacked tubular structures by burying the hydrophilic head groupsand exposing the hydrophobic tails to the bulk solvent. Most biological mem-branes contain a lipid species capable of forming the HII state suggesting thatsuch lipid polymorphic structural states play an important role in structuralbiological processes such as membrane fusion. In this study, the interactionsbetween Mg2þ and biomimetic bacterial cell membranes composed of PE andphosphatidylglycerol (PG) were probed using differential scanning calorim-etry (DSC), small-angle x-ray scattering (SAXS), and fluorescence spectros-copy. The lipid phase transitions were examined at varying ratios of PE toPG and upon exposure to physiologically relevant concentrations of Mg2þ.An understanding of these basic interactions enhances our understanding ofmembrane dynamics and how membrane-mediated structural changes mayoccur in vivo.

408-Pos Board B173Role of trans to cis Transition in Viral Fusion Pore DilationBrett E. Alcott1, Zhenyong Wu2,3, Josie Bircher4, Erdem Karatekin2,3,Ben O’ Shaughnessy5.1Biochemistry and Molecular Biophysics, Columbia University, New York,NY, USA, 2Cellular andMolecular Physiology, Yale University, New Haven,CT, USA, 3Nanobiology Institute, Yale University, West Haven, CT, USA,4Molecular Biophysics and Biochemistry, Yale University, New Haven, CT,USA, 5Chemical Engineering, Columbia University, New York, NY, USA.Fusion of viral and host membranes is a key step during infection bymembrane-enclosed viruses. The fusion pore plays a critical role, and mustdilate to release the viral genome. Previous studies of fusion mediated by influ-enza A hemagglutinin (HA) revealed ~2-5 nm pores that flickered beforedilating to >10 nm. The mechanisms are unknown.Here we studied HA-mediated fusion pore dynamics using a novel single-poreassay, combined with computational simulations accessing extraordinarily longms-s timescales. We measured pores between HA-expressing fibroblasts andbilayer nanodiscs. From pore currents we infer pore size with millisecondtime resolution. Unlike previous in vitro studies, use of nanodiscs limited themembrane contact areas and maximum pore sizes, better mimicking the initialphases of virus-endosome fusion. With wild type (WT) HA, fusion poresflickered about a mean pore size ~1 nm. By contrast, fusion pores formed byGPI-anchored HA nucleated at half the WT rate and were significantly larger.We developed radically coarse-grained, explicit lipid molecular dynamics sim-ulations of the fusion pore reconstituted with post-fusion, trans HA hairpins.With WT HA, fusion pores were small, similar to experiment. Over time hair-pins gradually converted from trans to cis, but contrary to a common view, cishairpins accumulated on the ‘‘viral’’ membrane, not the pore waist, due to thelow mobility HA transmembrane domains. With GPI-HA the anchoring lipidswere far more mobile and the trans-cis transition much accelerated. Once mosthairpins had converted to cis, because apposing membranes were released thefusion pore dilated significantly.Our results suggest pore dilation requires the trans-cis transition. We hypothe-size that this transition is accelerated in GPI-HA by the more mobile lipid an-chor, explaining the larger observed pores.

409-Pos Board B174The Influence of Membrane Composition on the Kinetics of InfluenzaVirus Fusion Measured using a Single Particle ApproachGuus van der Borg1, Scarlett Braddock1, Jelle S. Blijleven1,Antoine M. van Ooien2, Wouter H. Roos1.1Molecular Biophysics, Zernike Institute, Groningen, Netherlands, 2Schoolof Chemistry, Wollongong, Australia.

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In order to infect a host cell the influenza virus fuses its envelope with the hostcell membrane. This fusion of the viral and cell membranes is mediated by theviral surface protein hemagglutinin (HA) which both docks the virus and fusesthe membranes. Using Total Internal Reflection Fluorescence (TIRF) micro-scopy and a planar, fluid bilayer it is possible to study the kinetics ofhemifusion in single virus particles. We have used this technique to studythe yield and kinetics of the fusion of influenza with multiple different bilayercompositions. This allows us to better understand the physics behind HAmediated membrane fusion, and thus to better understand the process of influ-enza infection.

410-Pos Board B175Revisit the Correlation between the Elastic Mechanics and Fusion of LipidMembranesZih-An Fan, Kuan-Yu Tsang, Si-Han Chen, Yi-Fan Chen.Department of Chemical and Materials Engineering, National CentralUniversity, Taoyuan County, Taiwan.Membrane fusion is a vital process in key cellular events. The fusion capabilityof a membrane depends on its elastic properties and varies with its lipid compo-sition. It is believed that as the composition varies, the consequent change in C0

(monolayer spontaneous curvature) is the major factor dictating fusion, owingto the associated variation in GEs (elastic energies) of the fusion intermediates(e.g. stalk). By exploring the correlations among fusion, C0 and Kcp (monolayerbending modulus), we revisit this long-held belief and re-examine the fuso-genic contributions of some relevant factors. We observe that not only C0

but also Kcp variations affect fusion, with depression in Kcp leading to suppres-sion in fusion. Variations in GE and inter-membrane interactions cannotaccount for the Kcp-fusion correlation; fusion is suppressed even as the GEsdecrease with Kcp, indicating the presence of factor(s) with fusogenic impor-tance overtaking that of GE. Furthermore, analyses find that the C0 influenceon fusion is effected via modulating GE of the pre-fusion planar membrane,rather than stalk. The results support a recent proposition calling for a paradigmshift from the conventional view of fusion and may reshape our understandingto the roles of fusogenic proteins in regulating cellular fusion machineries.

411-Pos Board B176Hemagglutinin Palmitoylation Contributes to Membrane Curvature inInfluenza a Virus Assembly and Membrane FusionPetr Chlanda1, Elena Mekhedov1, Hang Waters1, Alexander Sodt2,Paul S. Blank1, Joshua Zimmerberg1.1SIB DBTB NICHD, NIH, Bethesda, MD, USA, 2UMCP DBTB NICHD,NIH, Bethesda, MD, USA.Three cysteine residues in the cytoplasmic tail of influenza virus glycoproteinhemagglutinin (HA) are covalently modified by three fatty acids and highlyconserved among HA subtypes. The importance of these S-acylation post-translational modifications is highlighted by a strain-dependence to their role invirus replication either in assembly or in fusion, but the mechanisms by whichthe modifications exerts any effect are unknown. We studied the effects of HAacylation on influenza virus-like particle (VLP) morphology, glycoproteinspacing, protein incorporation, HA induced curvature, and membrane fusion us-ing cryo-electron tomography (cET), VLP-cell and cell-cell fusion assays, andmolecular dynamics. Acylation has a significant effect on VLP envelope curva-ture but is not a determinant of either VLP morphology or HA lateral spacing.De-acylated mutant HA is correlated with a flatter envelope curvature of thereleased particles in the absence of the M1 layer compared to wild type HA.The de-acylated mutant HA failed to incorporate an M1 layer within sphericalVLP consistentwith alteredHA-M1 interactions. In cell-cell fusionassays, fusionpore enlargement was not observed, regardless of which strain of influenza wasde-acylated (H2 (A/Japan/305/57), H3 (A/Aichi/2/68), H3 (A/Udorn/72)), sug-gesting that the role of acylation in membrane fusion is viral strain independent.Fusion without pore enlargement could be partially rescued by the expression ofM1 and M2 proteins. The spontaneous curvature of palmitate was calculated bymolecular dynamic simulations, and was found to be comparable to curvaturevalues derived from VLP size distributions. Our studies indicate that HA acyla-tion is important for both influenza virus assembly and membrane fusion by con-trolling membrane curvature and modifying HA’s interactions with M1.

412-Pos Board B177HIV Entry: Receptors Cooperate with Membrane Domain Boundaries toform Entry Sites in Host CellsSung-Tae Yang, Volker Kiessling, Lukas K. Tamm.University of Virginia, Charlottesville, VA, USA.It has been proposed that lipid rafts of host cell membranes play pivotal rolesfor cell entry of many enveloped viruses including HIV. However, it remains

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largely unknown why virions would prefer nanoscopic ordered lipid domainsover uniformly fluid membrane regions. Here, we show that HIV does notenter cells from within ordered membrane regions, but rather at the bound-aries between raft-like and non-raft-like regions of the plasma membrane.Using cell-derived giant plasma membrane vesicles (GPMVs), which arephase-separated into large-scale liquid-ordered (Lo) and liquid-disordered(Ld) membrane domains, we demonstrate that the HIV receptor CD4 is sub-stantially sequestered into Lo domains while the coreceptor CCR5 localizespreferentially at Lo/Ld domain boundaries. Lo/Ld phase coexistence is notrequired for HIV attachment, but the recognition of Lo/Ld boundaries is aprerequisite for successful fusion of the viral envelope with the cell mem-brane. We propose that virions localized to membrane domain boundariesutilize their interfacial energy as an additional driving force for fusion andcell entry. This study provides surprising answers to the long-standing ques-tion about the roles of lipid rafts in cell entry of HIV and perhaps other en-veloped viruses.

413-Pos Board B178Single-Virus Observation of pH-Triggered Zika Fusion in the Absence of aCellular ReceptorRobert J. Rawle1, Elizabeth Webster2, Isabel Goronzy2, Steven Boxer2,Peter Kasson1.1Molecular Physiology and Biological Physics, University of Virginia,Charlottesville, VA, USA, 2Chemistry, Stanford University, Stanford,CA, USA.Zika virus is a membrane-enveloped flavivirus which has garnered interna-tional attention as an emerging pathogen with causal links to birth defectsand neurological sequelae following infection. Because Zika virus has onlyrecently been the subject of intense scientific study, little is known aboutthe entry of Zika virus into host cells. From limited studies and by drawingparallels to closely-related flaviviruses, Zika virus is presumed to first bind toa (as yet unknown) receptor on the cell surface, and then become internalizedby endocytosis. At some point during the endocytic pathway, the viral E pro-tein is triggered by an unknown factor or factors, initiating membrane fusionwith the endosomal membrane. To identify the factor(s) which trigger Zikavirus fusion, as well as to study the subsequent fusion kinetics, we used syn-thetic DNA-lipid conjugates to tether Zika virus to target model lipid mem-branes in the absence of receptor, a strategy demonstrated earlier forinfluenza virus (Rawle et al., 2016, Biophysical Journal). This enabled usto screen triggering conditions for Zika virus fusion and to monitor the result-ing single virus fusion kinetics by quantitative fluorescence microscopy.We demonstrate that low pH, mimicking that inside the endosome, is suffi-cient to trigger Zika virus fusion (lipid mixing). We also present the pH-dependence of the Zika virus fusion kinetics, as well as implications for afusion mechanism.

414-Pos Board B179Molecular Atlas Imaging and Osteoclast Formation: Multiscale Study ofCell-Cell Fusion MechanismsJesse L. Silverberg1, Pei Ying Ng2, Roland Baron2, Peng Yin1.1Wyss Institute, Harvard University, Boston, MA, USA, 2Harvard School ofDental Medicine, Harvard University, Boston, MA, USA.Advanced strategies for synthetic biomaterial design is a critical applicationof fundamental research in tissue-scale biomechanics. Top-down experi-ments seek to understand emergent material properties from the organizationof individual constituents. While illuminating, bottom-up approaches are anecessary compliment since they provide information hidden by the com-plex interactions in many-component multi-scale tissues. Thus, reducingcomplexity and studying minimal interactions allows us to better anticipateand predict phenomenology in biomaterials engineered with living cells.As a concrete example, fracture resistance in bone arises from mineralturnover driven by osteoclast and osteoblast cells. Osteoclasts are particu-larly interesting since these large multi-nuclear cells resorb bone matrix.While it is widely accepted that efficient bone resorption requires osteo-clasts to become multi-nucleated, the exact biological mechanisms initiatingand driving cell-to-cell fusion remains poorly understood. In this work,we are using Molecular Atlas Platform imaging technology to study theformation of osteoclast cells from the fusion of progenitor bone marrowmacrophages. These experiments utilize multiscale imaging to study thecytoskeleton of progenitor cells fusing to create multi-nuclear osteoclasts.At the smallest scales, super-resolution imaging enabled by DNA-PAINTallows us to observe the mechanisms when two progenitor cells comeinto physical contact and their membranes fuse. At the largest scales, westudy osteoclast development over fields of view spanning several mm. By

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imaging across 5 orders of magnitude in length, we are able to both findthese rare fusion events, and study a heterogeneous population of osteoclastsfor a contextualized understanding of this important cell type. The insightsgained in this work explore fundamental features of cell fusion requiredfor development of successful next generation biomaterials that cater tobasic osteoclast biology requirements and ensure better replication of thein vivo environment.

415-Pos Board B180Optimizing Excitation Polarization to Probe Fusion Pore Dynamics usingTIRF MicroscopyKasey Hancock1,2, Joerg Nikolaus3, Erdem Karatekin3, David Baddeley4.1Yale University, New Haven, CT, USA, 2Truman State University,Kirksville, MO, USA, 3School of Medicine, Cellular and MolecularPhysiology, Molecular Biophysics and Biochemistry, Yale University, WestHaven, CT, USA, 4Department of Cell Biology, School of Medicine, YaleUniversity, New Haven, CT, USA.During hormone or neurotransmitter release via exocytosis, fusion pores mayopen and close (flicker) repeatedly before resealing (‘‘kiss & run’’ fusion) ordilating (full fusion) irreversibly. Pore dynamics regulate the amount and ki-netics of cargo release, and determine the mode of recycling, but mechanismsthat govern pore dynamics are not understood. This is in large part due to alack of reconstituted assays with single-pore sensitivity and millisecondtime resolution. We recently described a polarized total internal reflection(pTIRF) microscopy assay to monitor fusion of proteoliposomes to planarlipid bilayers supported on a soft polymer cushion with single molecule sensi-tivity and ~15 ms temporal resolution. Fluorescently labeled small unilamellarvesicles, reconstituted with exocytotic/neuronal v-SNAREs (v-SUVs), fusewith a supported bilayer containing the cognate t-SNAREs (t-SBL). Eachfusion event is accompanied by changes in the total fluorescence intensity sur-rounding the fusion site, as the lipid-linked labels diffuse from the liposomeinto the supported bilayer through the fusion pore. Analysis of the intensitychanges, combined with a mathematical model, provides information onpore dynamics (Stratton et al. Biophys. J. 2016). In principle, three factorscan contribute to intensity changes upon fusion: 1) dequenching of fluoro-phores, 2) evanescent field decay and 3) a change in the average orientationof the fluorophore dipole moments with respect to the excitation polarizationfield, as the fluorophores move from the round liposome into the flat bilayer.Here, we systematically varied the polarization of the excitation field andquantified its contribution to intensity changes for different lipid-linked fluo-rophores. Large increases facilitate detection of fusion events and quantifica-tion of lipid release kinetics.

Membrane Structure I

416-Pos Board B181Analysis of Lipid Domains in Bilayer Simulations using Observables forLipid PackingSoohyung Park, Wonpil Im.Biological Sciences, Lehigh University, Bethlehem, PA, USA.Lipid rafts are micro- or nano-sized dynamic domains of membranes en-riched in cholesterols, glycosphingolipids (and other saturated lipids), andspecific membrane proteins, which are involved in many important biolog-ical processes such as membrane trafficking, signaling, protein sequestra-tion, and so on. Though the existence of lipid rafts has been widelyaccepted, the study of their dynamics (size and lifetime) is still challengingdue to the limitations in experimental techniques. In this context, computa-tional studies can be a useful approach that complements experiments andprovides insight into the properties and dynamics of lipid rafts by moni-toring and analyzing membrane structures at the atomic resolution. So far,the analysis of lipid domains in computational studies has been mostly basedon the order parameter and neighbor composition of lipids, which could becomplicated in the analysis of multicomponent bilayers. To address thisissue, we propose a simple yet robust method for the analysis of lipid do-mains in bilayer simulations using physically transparent observables forlipid packing, area and thickness. In our method, the ordered states of lipidsare inferred by a hidden Markov model analysis. Then, the ordered statemap onto the Voronoi tessellation of lipids is analyzed using the Getis-Ord local spatial autocorrelation statistics to obtain clusters of ordered lipids(lipid domains). The method is general and can be applied to various situa-tions in that 1) it does not require information other than the mechanicalproperties of lipids to assign their ordered states, and 2) the spatial autocor-relation statistics allows a robust assignment of the clusters of ordered and

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disordered regions. The usefulness of the method is illustrated by analyzingthe concentration effects on the membrane order for the bilayers consistingof 1) ganglioside GM1 and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocho-line (POPC), and of 2) cholesterol, 1,2-dimyristoyl-sn-glycero-3-phospho-choline (DMPC), POPC, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamin(DMPE), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine(POPE).

417-Pos Board B182Software for Direct Comparison of Membrane Scattering ExperimentsData to Molecular Dynamics SimulationsYevhen Cherniavskyi1, Svetlana Baoukina1, Bryan W. Holland1,2,Norbert Ku�cerka3,4, Peter Tieleman1.1Biological Sciences, University of Calgary, Calgary, AB, Canada,2Simulations Plus, Inc., Lancaster, CA, USA, 3Frank Laboratory of NeutronPhysics, Joint Institute for Nuclear Research, Dubna, Russian Federation,4Department of Physical Chemistry of Drugs, Comenius University,Bratislava, Slovakia.Detailed information on lipid bilayers structure is very important for under-standing the processes in cellular membranes. Nowadays X-ray and neutronscattering experiments are widely used as a source of lipid bilayers structuralparameters (area per lipid, thickness, etc.), but in contrast to scattering pat-terns from crystal structures, lipid membranes produce very broad peaks inscattering intensity profiles due to their fluid nature. This makes the processof retrieving structural details indirect and introduces approximations toconvert experimental structure factors to electron or neutron scattering pro-files. On the other hand, molecular dynamics simulations are able to providedirect sub-atomic level information on lipid bilayers structure, but can pro-duce unreliable data because of inaccuracy of the force fields. Simulation toExperiment (SIMtoEXP) software is designed to facilitate direct, model free,comparison of MD simulations to experimentally measured structure factorsfor lipid bilayers. We present SIMtoEXP version 2.0 - an updated version ofthe original software. The new version of this program is written in Cþþusing the Qt GUI library, instead of the original C/Tcl implementation.New features, including a MD trajectory reading module, were added tosimplify the workflow for users. In future releases SIMtoEXP softwarewill support structure factor calculations from simulations with the coarse-grained Martini force field. The Martini force field provides significantcomputational speedup while retaining significant chemical detail of thelipids. This allows simulating large lipid-protein aggregates on longer time-scales and investigate the structure and lateral organization of cell mem-branes. Incorporation of Martini support in SIMtoEXP will allow directcomparison of coarse-grained simulations to experiments, which will benefitboth Martini force-field parametrization and the interpretation of experi-mental results.

418-Pos Board B183Modeling Ethers with Molecular Dynamics: Updated CHARMM ForceField Parameters for Ethers in Model Compounds and Lipid MembranesAlison M. Leonard.UMCP, Crofton, MD, USA.Small ether molecules such as polyethylene glycol (PEG) have extensiveindustrial and medical applications. Additionally, phospholipids containingether linkages make up 30% of the glycerophospholipids in the humanbrain. Preliminary research suggests that the CHARMM All-Atom AdditiveForce Field does not accurately recreate experimental results for bilayerscomposed of ether lipids such as 1,2-di-O-hexadecyl-sn-glycero-3-phospho-choline (DHPC). 100-ns simulations of pure DHPC bilayers do not repro-duce essential target data such as surface area per lipid and electrondensity profile, which describe the structural properties of the membrane.Additionally, the initial parameterization of ethers tested a limited popula-tion of linear ethers and did not accurately reproduce potential energy scansabout the O-C-C-O dihedral (J. Chem. Theory Comput., 3(3):1120 - 1133).We have used the MP2 density and Dunning diffuse aug-cc-pVQZ basissets to compute the charge distributions of model ethers, including variouslengths of PEG. We have also used Hybrid Methods for Interaction Energies(HM-IE) to compute potential energy scans about the O-C-C-O angle effec-tively at the CCSD(T)/aug-cc-pVQZ level (J. Phys. Chem. A, 108: 107 -112). We found that the charge on ether oxygens is substantially morenegative than the current CHARMM force field suggests. Adjusting thischarge allows more water to penetrate a DHPC bilayer, increasing the sur-face area per lipid and improving the bilayer’s structural representation.Additionally, our new dihedral parameters for the central O-C-C-O anglemore accurately reproduce the quantum potential energy scan and bring

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the free energies of solvation of model compounds closer to experimentalvalues. Further research will involve evaluating the radius of gyration ofPEG in salt solutions to improve the interaction of ether oxygens withions in simulation (Colloid Jour., 72(2): 279 - 281).

419-Pos Board B184Simulation of Linoleoyl-Containing Pure Lipid Bilayer and SoybeanPlasma MembranesXiaohong Zhuang, Anna Ou, Jeffery B. Klauda.Chemical and Biomolecular Engineering, University of Maryland, CollegePark, MD, USA.Molecular dynamics (MD) simulations have been extensively used to studylipid membranes in addition to experimental studies as they help better un-derstand membranes in the atomic level. Computational models of bacterial(E. coli) membranes have been developed and applied to study the antimi-crobial peptide proteins. Plant membranes are less frequently studiedcompared to the bacterial membrane. In this work, we will present the soy-bean plasma membranes models. The compositions of cell plasma mem-branes of soybean vary depending on the species, stage of development,and the part of the plant. The two parts of the plant that we study are thehypocotyl and the root. Each model consists of 100 lipids per leaflet, withthe composition based on the weighted and averaged values from past exper-imental studies. Specifically, the hypocotyl membrane contains 7 types ofunsaturated phospholipids and two types of sterols, while the root membranecontains 8 types of phospholipids and two types of sterols. All types of phos-pholipids in soybean contains the 18:2 (cis D9, 12) linoleoyl tail which wasnot well studied before, therefore, the simulations on the pure 18:0/18:2 and18:2/18:2 phosphocholine (PC) lipid bilayers are also performed. The struc-tural properties such as surface area per lipid, bilayer thicknesses, order pa-rameters, and spin-lattice relaxation time are analyzed for all membranes.Moreover, the analyses of the sterols tilt angle distributions, hydrogenbonding, and clustering are also conducted for the soybean membranes.The structural properties of pure bilayers agree well with NMR experimentaldata validate the accuracy of 18:2 linoleoyl-containing lipids, based onwhich the soybean membrane models also result in reasonable structuralproperties. These results imply that the two soybean membrane models arerealistic, and can facilitate the further study of soybean and other plantmembranes.

420-Pos Board B185Lateral Heterogeneity of Cholesterol on Binary Lipid Mixtures ofPOPC/Chol Imaged with AFMArturo Galvan-Hernandez1, Fernando Favela-Rosales2,Jorge Hernandez-Cobos1, Ivan Ortega-Blake1.1Universidad Nacional Autonoma deMexico, Cuernavaca, Mexico, 2InstitutoTecnologico Superior Zacatecas Occidente, Sombrerete, Mexico.Phase diagrams on ternary and quaternary lipid mixtures showing theexistence of liquid-ordered (lo), liquid-disordered (ld) and a mixed phase(loþld) are now well established. However the existence of such a phasediagram on binary mixtures is still in debate despite reports of a phasediagram on the mixtures POPC/Chol and POPC/Erg and nystatin activityco-related to this purported phase diagram; with a maximal activity ap-pearing in the mixed phase. Recent Molecular Dynamics simulations ofPOPC/Chol at different temperatures and cholesterol concentrations alongthis phase diagram, suggest the existence of cholesterol enriched nano-sites in this mixed phase, indicating the presence of cholesterol lateralheterogeneity. Here we present atomic force microscopy images of sup-ported lipid bilayers made up of POPC and different cholesterol concentra-tions along the phase diagram. We observe structures that are consistentwith the Molecular Dynamics predictions of order parameters and mem-brane thickness, supporting the idea of cholesterol lateral heterogeneity inbinary mixtures, which can enrich the discussion on the existence oflipid rafts and domains in biological membranes. Funding: DGAPA-PAPIIT-RG100416.

421-Pos Board B186Orientational Properties of DOPC/SM/Cholesterol Mixtures: APM-IRRAS StudySabina M. Mate1,2, Romina Vazquez1, Felippe J. Pavinatto3,M. Antonieta Daza-Millone4, Vanesa Herlax1, Laura Bakas5,Osvaldo N. Oliveira Jr3, Marıa E. Vela4.1INIBIOLP-CONICET-UNLP, La Plata, Argentina, 2Comision deInvestigaciones Cientıficas de la Pcia. de Buenos Aires, La Plata, Argentina,3Instituto de Fısica de Sao Carlos, Sao Paulo, Sao Carlos, SP, Brazil., Brazil,

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4INIFTA (CONICET-UNLP), La Plata, Argentina, 5CIPROVE(CONICET-UNLP), La Plata, Argentina.Sphingomyelins (SM) and phosphatidylcholines (PC) are major lipid classesin the external plasma membrane leaflet of mammalian cells. A preferentialinteraction between SM and cholesterol (Cho) in both cell and model mem-branes has been proposed as central for the formation of Cho- and SM-richdomains in membranes. In this context, the relevance of the SM hydro-phobic moiety on its interaction with Cho for domain stabilization hasbeen investigated by our group (1-2). We report here on the effects ofsphingomyelin structure on the orientational and conformational propertiesof monolayers of pure lipids and of two ternary lipid mixtures (DOPC/16:0SM/Cho and DOPC/24:1SM/Cho), which are relevant as mammaliancell membrane models. We investigated interchain interactions, hydrogenbonding, conformational and structural properties using in situ polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS). Ourresults indicate that the particular properties conferred on sphingolipidsby unsaturation have profound implications on membrane organization.Finally, we also explored the orientational and conformational changes inlipid monolayers of DOPC/16:0SM/Cho 2:1:1 after the adsorption/insertionof the active toxin HlyA and its unacylated nonhemolytic precursor ProHlyA,so as to complement our knowledge on the action mechanism of bothproteins.(1) Sabina Mate, Jon V. Busto, Aritz B. Garcıa-Arribas, Jesus Sot, RominaVazquez, Vanesa Herlax, Claude Wolf, Laura Bakas and Felix M. Goni(2014). ‘‘N-nervonoylsphingomyelin (C24:1) prevents lateral heterogeneityin cholesterol-containing membranes’’. Biophys J. Jun 17;106(12):2606-16.(2) Sabina M. Mate, Romina F. Vazquez, Vanesa S. Herlax, Marıa A. DazaMillone, Marıa L. Fanani, Bruno Maggio, Marıa E. Vela, Laura S. Bakas(2014). Boundary region between coexisting lipid phases as initial binding sitesfor Escherichia coli alpha-hemolysin: A real-time study. Biochimica et Bio-physica Acta 1838 (2014) 1832-1841.

422-Pos Board B187Investigating Lipid Domain Formation in Asymmetric Large UnilamellarVesicles using Forster Resonance Energy Transfer (FRET)Johnna R. St Clair, Qing Wang, Erwin London.Biochemistry and Cell Biology, Stony Brook University, Stony Brook,NY, USA.Many living cell membranes display lipid asymmetry, with a distinct dif-ference between the phospholipid and sphingolipid compositions of theinner and outer leaflets of the lipid bilayer. We recently developed improvedmethods to prepare asymmetric lipid vesicles using using alpha-cyclodextrins. These hexasaccharide rings can promote the exchange oflipids between vesicles, but do not transport sterols. This allows the efficientand selective replacement of the outer leaflet lipids of lipid vesicles withoutperturbing sterol content. Here we show, using several types of asymmetricvesicles, it is possible to investigate ordered domain formation in both theinner and outer leaflets of large unilamellar vesicles using FRET. To dothis, large unilamellar vesicles are prepared with FRET acceptor probes ineither only the inner or only the outer leaflet. FRET was used to studycholesterol-containing vesicles in which sphingomyelin was the predominantlipid in either the inner leaflet or the outer leaflet, and the opposite leafletwas composed of unsaturated phosphatidylethanolamine and phosphatidyl-serine, or of unsaturated phosphatidylcholine. Preliminary results showthat in at least some of the lipid compositions studied ordered lipid domainsin one leaflet can induce ordered lipid domains in leaflets composed of lipidswhich do not normally form ordered domains by themselves. These resultssupport the findings of our previous studies of domain formation and inter-leaflet coupling using microscopy, and show that the FRET approach canbe extended to study interleaflet coupling in membranes containing sub-microscopic domains.

423-Pos Board B188Lipid Mixing in Model MembranesRuo-Xu Gu, Svetlana Baoukina, D. Peter Tieleman.Centre for Molecular Simulation and Department of Biological Sciences,University of Calgary, Calgary, AB, Canada.The plasma membrane constitutes the boundary between the cell and itsenvironment. It is believed to have a highly organized lateral structure(the raft hypothesis). Due to the complex composition of plasma membranes,model lipid bilayers of simple composition are often used in experimentsand simulations to study the lateral organization. Here we conducted all-atom (AA) and coarse-grained (CG) simulations of bilayers contain-ing POPC, POPC:cholesterol, DPPC:DOPC, DPPC:DOPC:cholesterol and

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POPE-POPG at different temperatures to investigate bilayer properties andmixing behaviour. We compare the AA and CG results. The POPC:choles-terol bilayer formed a random mixture, with small ordered cholesterolsub-structures. DPPC:DOPC formed a homogeneous liquid phase at310 K, and showed notable lipid segregation at 290 K and 280 K. Choles-terol enhanced lipid segregation in the DPPC:DOPC:cholesterol bilayersby displacing DOPC lipids from the local environment of DPPC. Thecholesterol-cholesterol and cholesterol-phospholipid interactions in AAand CG models were found to differ significantly. Lipid segregation inthe CG simulations showed weak temperature dependence, due to partialsubstitution of entropic components by enthalpic components intrinsic incoarse-graining. In the bilayers containing charged lipids, repulsion be-tween POPG lipids in the CG simulations was stronger than in AA simula-tions, and also differed between the standard and polarizable Martini watermodels.

424-Pos Board B189Glycolipid Crosslinking is Required for Cholera Toxin to Partition intoand Stabilize Ordered DomainsKrishnan Raghunathan1, Tiffany Wong1, Daniel J. Chinnapen2,Wayne I. Lencer3, Michael G. Jobling4, Anne K. Kenworthy1.1Dept of Molecular Physiology and Biophysics, Vanderbilt University,Nashville, TN, USA, 2Harvard Medical School and the Harvard DigestiveDiseases Center, Cambridge, MA, USA, 3Harvard Medical School and theHarvard Digestive Diseases Center, Cambridge, MA, USA, 4University ofColorado Anschutz Medical Campus, Aurora, CO, USA.Current models of lipid rafts propose that lipid domains normally existas nanoscale compositional fluctuations at steady state in cells, but canbe stabilized to form functional entities. However, the mechanismbehind how stabilized rafts assemble and function remains unclear.Here, we test the role of glycolipid crosslinking as a raft targeting andordering mechanism using the well-studied raft marker cholera toxinB pentamer (CTxB) as a model. We show that when bound to cell-derived Giant Plasma Membrane Vesicles, a variant of CTxB containingonly a single functional GM1 binding site exhibits significantly reducedpartitioning in the ordered phase compared to wild type CTxB with fivebinding sites. Moreover, monovalent CTxB does not increase membraneheterogeneity, unlike wild type CTxB. These results support the long-heldhypothesis that CTxB stabilizes raft domains via a crosslinking mechanismand uncover an unexpected role for crosslinking in the targeting of CTxB toordered domains.

425-Pos Board B190Model for Lipid Droplets within Endoplasmic ReticulumGonen Golani1, Michael M. Kozlov2.1School Physics and Astronomy, Tel Aviv University, Tel Aviv, Israel,2Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.Endoplasmic Reticulum (ER) is a continuous network of membrane tubulesand sheets that plays a central role in the biogenesis of Lipid Droplets (LD).The bulk of a LD is filled by hydrophobic molecules while its surface iscovered by a phospholipid monolayer. It has been suggested that LDsform between two leaflets of an ER membrane as a result of accumulationof enzymatically synthesized triacylglycerols and sterol esters. Our workaims at analyzing the shape and stability of a tubular ER membrane contain-ing LDs and determination of conditions of LD detachment from themembrane. We consider the ER membrane as a thin elastic sheet, whosebending energy is determined by Helfrich model. The tubular membraneis assumed to be subjected to a vanishing or small lateral tension, vanishingtrans-membrane pressure difference and periodic boundary conditions. Theshape of an ER tubule is determined by a homogeneous spontaneous curva-ture of the membrane. The LD is modeled as a liquid drop with relativelyhigh surface tension. The transition area between the LD and the ERmembranes is modeled as a line with constant line tension. We usedthe finite element numerical simulations (using Surface Evolver program)to compute the minimal energy configurations of the system. We foundthe conditions under which the LD embedding into the membraneis energetically more favorable than LD release in the cytosol dependingon the LD size, the lateral tension of the tubular membrane, the line tensionof the tubule-LD contact line, the membrane bending and saddle splaymoduli. We showed that the membrane lateral tension and negative saddlesplay modulus favor, whereas the line tension disfavors the LD embeddinginto the membrane. We found that LD growth results in the droplet detach-ment from the membrane.

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426-Pos Board B191Functional and Structural Characterization of Pulmonary SurfactantFractions Obtained from Bronchoalveolar LavagesJose Carlos Castillo-Sanchez, Alejandro Cerrada, Mikel Conde,Jesus Perez-Gil, Antonio Cruz.Dept. of Biochemistry, Faculty of Biology, and Research Institute Hospital 12de Octubre, Universidad Complutense, Madrid, Spain.The alveolar surface of the lung is protected by a lipophilic material, the pulmo-nary surfactant, mainly composed by lipids and a few specific proteins: SP-A,SP-B, SP-C and SP-D. The main function of pulmonary surfactant is to reducethe surface tension of the thin aqueous layer covering the alveolar epithelium,avoiding the collapse of the smaller alveoli during the expiration. To achievethis function, surfactant lipids form an interfacial film at the air-liquid interfaceof this aqueous lining layer. Nevertheless, pulmonary surfactant is synthetized,packed and secreted by type II alveolar pneumocytes as tightly packed mem-branes assembled in specialized organelles, the lamellar bodies. Currently, themolecularmechanisms that let pulmonary surfactant be transformed from the bi-layers of lamellar bodies into the functional interfacial film remain unclear. Thisis in part because most of the previous studies were made using surfactants ob-tained from bronchoalveolar lavages, a complex mixture that likely combinesfreshly secreted surfactant and surfactant that has been already exposed to respi-ratory dynamics and can be at least partially deactivated. In this work, we havecharacterized different fractions obtained by density gradient ultracentrifugationof pulmonary surfactant purified from bronchoalveolar lavages of porcine lungs.The analysis of these fractions by transmission electron microscopy showed aheterogeneous mixture of membranous structures. With regard to composition,each fraction showed important differences in cholesterol and the content ofsurfactant proteins. All fractions presented a relatively good surface activitywhen analyzed in the captive bubble surfactometer, except for some minorityfraction. These results indicate that surfactant from bronchoalveolar lavage iscomposed by a heterogeneous mixture of membranes differing in compositionand structure. We propose that some of these structures could be associatedwith different stages of surfactant dynamics, including freshly secretedmaterial,interfacial structures and recycled membranes.

427-Pos Board B192Simulations Provide Insight into Improving the Tolerance of the E. coliMembranePouyan Khakbaz1, Jeffery Klauda2.1Chemical and Biomolecular engineering, University of Maryland CollegePark, College Park, MD, USA, 2Chemical and Biomolecular engineering,University of maryland college park, College Park, MD, USA.Damage of the cell membrane is identified as the principal mechanism fortoxicity. Partitioning of chemicals like ethanol in the membrane could changemembrane properties and eventually destabilize lipid bilayers. Lipid composi-tion, specifically head group populations, affect membrane properties such asintegrity, electrochemical potential, and intracellular pH. Here, a set of molecu-lar dynamics (MD) simulations were carried out to investigate the effect ofethanol on native and PssA mutant models for the inner membrane of E. coli.Properties such as surface area per lipid, order parameters, rigidity, and thick-nesses were calculated. The main differences between PssA and native modelswere the ratio of phosphoethanolamine (PE)/phosphatidylglycerol(PG) headgroups and the concentration of the cyclic moiety in fatty acid chains. ThePssAmodel had more PE head groups and more unsaturated lipids, while nativemodel had more cyclic moiety in lipid tails. Equilibrium bulk molar concentra-tions of ethanolwith the PssAmodelwere 0%, 0.62%, 1.84%, and 4.56% relativeto water in the bulk. The molar concentration of ethanol in native system was4.64%. For the pure water simulations, the PssA model resulted in a thickermembrane, which may reduce ethanol permeation to cytoplasm of E. coli. Forsimulations with ethanol, higher surface area and less order parameters wereobserved as ethanol concentration increased in PssA and native models. MD re-sults also provedhigher ethanol concentrationwill result in a reduction in rigidityof membranes with the native being less rigid at higher ethanol concentrations.Less hydrophobic core thickness of native model compared to the PssA modelindicated likely increased permeation of ethanol into the cytoplasm. In overall,ourMD simulation results support the hypothesis that PssA is more protective ofthe cell with increased thickness and rigidity.

428-Pos Board B193Intermembrane Crosstalk in E. coliPatrice Rassam, Colin Kleanthous.Biochemistry, University of Oxford, Oxford, United Kingdom.We have previously described how promiscuous protein-protein interactionsbetween outer membrane proteins (OMPs) cause them to self-associate into

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large platforms, which we call OMP islands, and that these supramolecularassemblies are the basis by which OMPs are turned over in bacterial popu-lations (Rassam et al., Nature, 2015; Kleanthous et al., Current Opinion inStructural Biology, 2015). Combining advanced microscopy methods withnew analytical tools, we have discovered that the organisation inherent inthe bacterial outer membrane is reflected in the inner membrane when thetwo become connected by a protein bridge. Periplasmic protein bridgesserve many important functions in Gram-negative bacteria, includingsecreting lipopolysaccharides and virulence factors to the cell surface andimporting iron siderophores across the outer membrane. Current work isaimed at determing the biological consequences of this outer-inner mem-brane cross-talk.

429-Pos Board B194Low-Enthalpy Phase Transitions Yield Entropy-Driven Lateral Reorgani-zation and Phase Separation in Synthetic and Natural Multi-ComponentDIB MembranesGraham Taylor1, Frederick A. Heberle2, Jason Seinfeld3, John Katsaras2,C. Patrick Collier4, Stephen A. Sarles3.1Joint Institute of Biological Sciences (JIBS), UTK-ORNL, Knoxville, TN,USA, 2Shull Wollan Center, Oak Ridge National Laboratory, Knoxville, TN,USA, 3Mechanical, Aerospace, and Biomedical Engineering, UTK,Knoxville, TN, USA, 4Center for Nanophase Materials Sciences, Oak RidgeNational Laboratory, Knoxville, TN, USA.Temperature is an important parameter affecting biomembrane behavior,however, model membrane studies combining electrophysiology and activetemperature control are relatively scarce. Here, we report on the recentlydiscovered ability to use controlled heating to form droplet interface bila-yers (DIBs) composed of lipids that exhibit phase transitions above ambienttemperatures. Using heating-assisted monolayer formation, we formed DIBsfrom single lipids or multi-component lipid mixtures, as well as complexnatural total lipid extracts (TLE) from E. coli and porcine brain tissues.DIB capacitance measurements detected thermotropic phase transitions inTLEs and some phase-separating ternary systems that were not observedwith differential scanning calorimetry, pressure-perturbation calorimetry,or densitometry measurements. We also performed electrical measurementsof membrane protein insertion using the model pore-forming peptideAlamethicin, to directly show how bilayer phase transitions are coupled todramatic transitions in membrane protein behavior. Our results provide anew picture of low-enthalpy phase transitions in complex membranes wherethe ensemble average lipid area, length, and volume do not undergo abruptchanges, but where lateral reorganization of lipids is driven largely byentropic forces. We discuss how a DIB, which is sensitive to lipid orderand packing as well as membrane tension, is especially well-suited to thestudy of such low-enthalpy transitions in complex synthetic and naturalmodel membranes.

430-Pos Board B195Utilizing Asymmetric GUVs to Inspect Plasma Membrane Phase Behaviorand Binding of Polybasic ProteinsJosephine Gonzales1, Milka Doktorova2, Gerald Feigenson1.1Molecular Cellular Biology and Genetics, Cornell University, Ithaca, NY,USA, 2Physiology, Biophysics and Systems Biology, Weill Cornell MedicalCollege and Field of Biophysics, New York City, NY, USA.The lipid distribution is asymmetric across the plasma membrane (PM)with cytosolic leaflet enriched in anionic lipids, and the outer leafletenriched in sphingomyelin and phosphatidylcholine and perhapscontaining ordered, functional domains, or lipid rafts. Our lab has previ-ously analyzed cellular events by using symmetric giant unilamellar vesicles(GUVs) to model peripheral membrane protein binding to thecytosolic leaflet of the PM. As a way to inspect the dynamics anddomain behavior of an asymmetric PM, we make GUVs with asymmetricmembranes. The goal of this project is to prepare asymmetric GUVs as amodel system to study plasma membrane phase behavior and binding ofpolybasic proteins. For convenience in study of protein binding the GUVouter leaflet will have plasma membrane cytosolic composition of PE/PS/cholesterol; the GUV inner leaflet will have plasma membrane exoplas-mic composition SM/PC/cholesterol. We are using a-cyclodextrin to ex-change the initially symmetric GUV of SM/PC/cholesterol withmultilammeller (MLVs) donor vesicles. Initial confocal microscopy imagingresults show good exchange of the GUV outer leaflet, and almost completeremoval of MLV donor vesicles. Currently, leaflet exchange isestimated from the LR-DOPE fluorescence intensity remaining in each

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GUV, along with Bodipy-PC fluorescence intensity added to the GUVsfrom the MLV donors.

431-Pos Board B196The L-Gamma Phase of Pulmonary SurfactantKamlesh Kumar1, Mariya Chavarha1, Ryan W. Loney1, Maayan P. Dagan1,Thomas M. Weiss2, Shankar B. Rananavare3, Stephen B. Hall1.1Oregon Health & Science University, Portland, OR, USA, 2SLAC/SSRL,Menlo Park, CA, USA, 3Portland State University, Portland, OR, USA.When compressed, alveolar films of pulmonary surfactant resist collapse fromthe air/water interface. This physiologically essential characteristic remainsunexplained. Recent studies show that self-assembled films of pulmonarysurfactant formed by adsorption are more resistant to collapse than spreadmonolayers containing the same constituents. One candidate for the self-assembled stable structure is the Lg phase. The unit cell of this structurecontains two lamellar bilayers. Half of the monolayers are ordered, and halfare disordered. Because ordered monolayers resist collapse, formation of theLg phase with the correct orientation at the air/water interface could explainthe stability of the adsorbed film. Preparations related to pulmonary surfactantform the Lg phase over a poorly defined range of hydrations. The studiesreported here used small angle and wide angle X-ray scattering to determinefactors that affect formation of the Lg phase. Variables considered include tem-perature, hydration, the influence of an interface, and important constituents ofpulmonary surfactant. The complete set of surfactant phospholipids formed theLg phase on solid supports at relative humidities from 70-99%. The electrondensity profiles confirmed prior evidence suggesting that the two bilayers inthe Lg phase are equal and asymmetric, with each containing one orderedand one disordered leaflet. The anionic phospholipids were essential for thesestructures, which otherwise formed standard lamellar structures. Physiologicallevels of cholesterol had minimal effect. In dispersed samples, Lg structureswere evident up to a water-content of ~35%. The structures continued to swellwith additional water. Because the lattice-constants for single bilayers at thesehydrations depends on their electrostatic composition, whether our samplesrepresented the Lg phase was uncertain. These results show that pulmonarysurfactant can form the Lg phase approaching physiological conditions,particularly at the solid/liquid interface. Whether this behavior extends to theair/water interface remains unknown.

General Protein-Lipid Interactions I

432-Pos Board B197Lipid-Protein Interactions are Unique Fingerprints for MembraneProteinsValentina Corradi1, Eduardo Mendez-Villuendas1, Helgi Ingolfsson2,Siewert-Jan Marrink2, D. Peter Tieleman1.1Biological Sciences, University of Calgary, Calgary, AB, Canada,2University of Groningen, Groningen, Netherlands.Cell membranes function as physical barriers for the cell and control the ex-change of ions, peptides, and small molecules between the interior and the exte-rior of the cell. The main constituents of cell membranes are lipid molecules,whose hydrocarbon tails provide the barrier-like properties, and membrane pro-teins, which carry out specific functions. Complex lipid-protein interactionstake place in the membrane, where proteins and lipids affect each other, strictlyregulating a wide range of cellular tasks. Here, we use coarse grained (CG) mo-lecular dynamics (MD) simulations to characterize the lipid environment of tenmembrane proteins, which include examples of receptors, transporters, chan-nels, and enzymes. To provide a realistic lipid environment, the proteins areembedded in a model plasma membrane, where more than 60 lipid speciesare represented, asymmetrically distributed between upper and lower leaflet(JACS, 2014, 136, 14554-59). The simulations show in detail how each proteinmodulates its local lipid environment in a unique way through local lipidcomposition, thickness, curvature, lipid dynamics and other properties.

433-Pos Board B198Lipid-Dependence of the Membrane Interactions of the Tim23 ChannelSubunit of the Mitochondrial Protein Import MachineryMelissa K. Skoryk, Kevin J. Boyd, Eric R. May, Nathan N. Alder.Molecular and Cell Biology, University of Connecticut, Storrs, CT, USA.The TIM23 complex of the mitochondrial inner membrane mediates the trans-location and integration of most nuclear-encoded polypeptides that residewithin the mitochondrion. The central subunit of this complex is Tim23,a voltage-gated channel-forming protein that constitutes part of thepolypeptide-conducting channel. Tim23 has a bipartite domain organizationwith a carboxy-terminal integral membrane domain and an amino-terminal

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intrinsically disordered region that resides in the intermembrane space (IMS).Work from our group and others has shown that cardiolipin, the signature phos-pholipid of the mitochondrion, is required for the activity and subunit interac-tions of the TIM23 complex, as well as for specific membrane interactions ofthe Tim23 subunit. In this work we further address the role of cardiolipin inmodulating membrane interactions of Tim23 from Saccharomyces cerevisiae.Using a site-specific fluorescence mapping approach, we show that in organ-ello, key sites in the Tim23 IMS domain partition into a nonpolar environment,consistent with past NMR experiments. Using a reductionist approach withmodel membranes, we measure the cardiolipin-dependence of this interactionas well as the depth of membrane penetration and how variations in cardiolipinphysiochemical properties affect Tim23 bilayer interactions. These results areverified and extended using computational approaches. Based on molecular dy-namics simulations, membrane binding is initiated by interaction of the Tim23amino terminus with the bilayer that is stabilized by electrostatic interactionsbetween lipid phosphate groups and the protein amino group. The presenceof cardiolipin promotes the insertion of key hydrophobic residues into thenonpolar core, stabilizing the membrane bound state of Tim23. We attributethis phenomenon to packing defects in cardiolipin-containing bilayers thatexpose acyl chains to the solvent. These results are discussed in light of heri-table disorders in cardiolipin biogenesis known to disrupt protein-lipidinteractions.

434-Pos Board B199Deciphering Membrane Protein Energetics using Deep Sequencing;Towards Robust Design and Structure Prediction of Membrane ProteinsAssaf Elazar, Jonathan Weinstein, Sarel Fleishman.Biomolecular Sciences, Weizmann Institute of Science, Rehovot, Israel.The energetics of membrane-protein interactions dictates protein topologyand structure, which in turn determines the function and expression levelsof all plasma membrane proteins. However, systematic and reliable quanti-fication of membrane-protein energetics has been challenging. I recentlydeveloped a deep mutational scanning method, dsTbL(Elazar et al.,2016a) (deep-sequencing TOXCAT-b-lactamase), to monitor the effects ofhundreds of point mutations on insertion and association within the bacterialinner membrane. The assay quantifies insertion-energy profiles for eachamino acid residue across the membrane, revealing that the hydrophobicityof biological membranes is significantly higher than appreciated. In addi-tion, a key feature of the dsTbL profiles is that they show asymmetriesfor Arg, Lys, and His, in agreement with the ‘positive-inside’ rule. The threeprofiles, however, are not identical: whereas Lys and Arg are favored by 2kcal/mol near the cytoplasm compared to near the periplasm, the titratableamino acid His shows a more modest asymmetry of 1 kcal/mol;moreover, only Arg stabilizes the segment near the cytosol, whereas Lysand His are nearly neutral at the cytosol- membrane interface. Based onthese findings, together with Jonathan Weinstein, we developed a novelgraphical topology-prediction algorithm named TopGraph(Elazar et al.,2016b), based on a sequence search for minimum energy of insertion usingthe dsTbL experimental insertion scale rather than statistics derived fromknown structures. Unlike many existing predictors, TopGraph exhibitshigh accuracy even on large transporters with no structural homologues.Furthermore, results suggest that the ‘positive-inside’ rule, which is knownto orient segments with respect to the membrane, can also drive insertion ofmarginally hydrophobic segments in large membrane domains. These in-sights may aid structure prediction, engineering, and design of membraneproteins.

435-Pos Board B200Governing Mechanism of Phospholipids Peroxidation via 15-Lipoxyge-nase, A Key Player in Ferroptosis Cell Death PathwayDariush Mohammadyani1, Judith Klein-Seetharaman2, Valerian E. Kagan3.1Biophysics, Johns Hopkins University, Baltimore, MD, USA, 2University ofWarwick, Coventry, United Kingdom, 3University of Pittsburgh, Pittsburgh,PA, USA.Ferroptosis is a newly discovered cell death pathway that is characterizedby the production of lipid hydroperoxides. Although the exact lipid peroxi-dation pathways have not been identified, recent findings indicate theinvolvement of lipoxygenases (LOXs), particularly, 15LOX actingdirectly —independently from phospholipases— on the membrane. Here,a combination of computational approaches was used to study interactionsof human 15LOX-2 with a membrane, and to identify preferred phospho-lipid substrates for this enzyme. Using molecular docking modeling, thebinding poses and the binding affinities of various esterified lipids andarachidonic acid —a known substrate of LOXs—were compared. It hasbeen shown that ferroptosis-triggering events occur predominantly in the

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endoplasmic reticulum. Therefore, coarse-grained molecular dynamics(CGMD) simulations were applied to study the interactions of 15LOX-2with an endoplasmic reticulum membrane mimic. Our modeling data re-vealed that phosphatidylethanolamine and phosphatidylinositol are themost likely membrane substrates for 15LOX-2. CGMD simulations (2 msruns) confirmed that the amino-terminal b-barrel domain (called PLAT) of15LOX-2 is responsible for membrane binding. This domain entered deeplyinto the membrane. To establish the positioning of the catalytic site’sopening and its orientation with respect to the membrane surface, wereconstructed an atomistic model based on the CGMD data. The openingof the J-shaped catalytic site invariably oriented itself toward themembrane surface facilitating phospholipid hydroperoxidation. Accord-ingly, we propose a model indicating that the PLAT domain stronglyentangles 15-LOX in the membrane providing accessibility of the activesite to esterified lipids embedded in the membrane. These datahave been supported by grants P01HL114453, U19AI068021 and HFSP-RGP0013/2014.

436-Pos Board B201Investigation of acyl Protein Thioesterase Activity at the MembraneKathrin Estel, Patricia Stege, Ingrid Vetter.Department of Mechanistic Cell Biology, Max Planck Institute of MolecularPhyisiology, Dortmund, Germany, Dortmund, Germany.As a member of the family of GTPases, Ras proteins officiate asmolecular switches (i.e. cycling between a GDP-bound OFF and aGTP-bound ON state) in many signal transduction cascades. Oncogenic mu-tations in Ras genes can lead to constitutively active proteins, which arefound in 20-30% of all human tumors. Interfering with Ras localization atthe plasma membrane would disturb the oncogenic proliferation signal,making Ras a potential anti cancer target. There are three human Rasisoforms (H/N/K), which are all posttranslationally prenylated. H- andN-Ras are additionally S-palmitoylated. Both modifications increase thehydrophobicity and therefore the membrane affinity of Ras. The reversibilityof palmitoylation allows the spatial regulation of Ras via a reaction-diffusion mechanism called ‘‘acylation cycle’’. This is achieved by an ubiq-uitous depalmitoylation of Ras followed by a rapid diffusion to the golgiapparatus where it gets palmitoylated and transferred back to the plasmamembrane via a vesicular transport. This cycle ensures the enrichment ofRas on the plasma membrane and prevents its mislocalization on othercellular membranes.The depalmitoylation of Ras is catalyzed by the Acyl Protein Thioesterase(APT). Its function is to prevent mislocalization of H- and N-Ras to the endo-membranes and it allows the acylation cycle to reestablish the physiologicalplasma membrane localization. Inhibiton of APT inhibits this cycle and Rasremains distributed on the endomembranes. The absence of Ras from theplasma membrane leads to a weaker Ras mediated proliferation signal. APTitself undergoes a dynamic palmitoylation for steady state membranelocalization.The effect of membranes on APT and their targets remains still unclear.In the presented work, several biochemical methods were used toinvestigate how the physiological membrane environment influences theAPT activity.

437-Pos Board B202MG56, A Membrane Bound O-Acyltransferase Protein, Regulates LipidComposition and Membrane Vesicle Size in Skeletal MuscleMatthew Sermersheim1, Arpad Somogyi2, Jordi Torrelles3, Miyuki Nishi4,Hiroshi Takeshima4, Pei-Hui Lin1, Jianjie Ma1.1Surgery, The Ohio State University, Columbus, OH, USA, 2The Ohio StateUniversity, Columbus, OH, USA, 3Microbial Infection and Immunology, TheOhio State University, Columbus, OH, USA, 4Kyoto University, Kyoto,Japan.The human genome encodes 11 genes belonging to the membrane boundO-acyltransferase (MBOAT) family of proteins. MBOAT family proteinsare known for containing a large number of transmembrane domains andexhibiting acyltransferase enzymatic activity. While collectively theseproteins cover a broad-spectrum of acylation, substrates ranging fromphospholipids to peptides, each member possesses specificity with regardsto the target of acylation and the lipid species utilized. Mitsugumin 56(MG56), also known as hedgehog acyltransferase-like protein, is anMBOAT protein predominantly expressed in cardiac and skeletal muscle.MG56 resides along the terminal cisternea of the Sarcoplasmic Reticulum(SR) in striated muscle. Ablation of MG56 in mice results in seeminglynormal pups at birth, however severe growth retardation is observed aroundday 7, shortly followed by death under starvation conditions. Skeletal

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muscle from knockout mice displays swollen SR and dilated vacuoles withinsarcomeres, however this phenomenon is not seen in cardiac tissue. Lipidanalysis of skeletal muscle via matrix-assisted laser desorption/ionizationfourier transform ion cyclotron resonance mass spectrometry (MALDIFT-ICR) shows a larger abundance of phospholipids in knockout musclecompared to wild type. Interestingly, knockout muscle also shows anincreased abundance of high molecular mass phospholipids, indicative oftri-acyl phospholipids. Tri-acylated phospholipids, such as N-acyl phospha-tidylethanolamine (NAPE), have an additional acyl chain attached to thephosphate group, and have been shown to alter membrane size and serveas precursors to N-acyl ethanolamine (NAE), a hormone shown to be anappetite suppressant in rodents. It is our hypothesis that MG56 functionsto negatively regulate NAPE production, thereby fostering normal muscledevelopment. We are working to identify the high molecular mass lipidsseen in knockout skeletal muscle in hopes of determining the substrate ofMG56.

438-Pos Board B203The Role of Packing Defects in the Stability and Function of the Intramem-brane Protease GlpGRuiqiong Guo1, Zixuan Cang2, Deans Erin3, Guowei Wei2,3,Heedeok Hong1,3.1Department of Chemistry, Michigan State University, East Lansing, MI,USA, 2Department of Mathematics, Michigan State University, East Lansing,MI, USA, 3Department of Biochemistry and Molecular Biology, MichiganState University, East Lansing, MI, USA.Although proteins are efficiently packed, structural analysis has revealedthat packing defects (i.e., voids, tunnels and pockets) are prevalent insideproteins despite their unfavorable contribution to protein stability. It hasbeen speculated that those packing defects may be necessary for ligandbinding, transport or conformational changes required for function. Despitethe potential importance, the role of packing defects in the stability andfunction of proteins is not clearly understood especially for membrane pro-teins. Here, using the rhomboid intramembrane protease GlpG as a model,we test the hypotheses that 1) improving the packing through cavity-filling substitution can be a general way to stabilize a membrane protein,and 2) if packing defects are critical for function, it would be possible tolock the protein conformation into either inactive or constitutively activestate through engineering of the cavities. We first identified packing defectsin several rhomboid proteases from different origins by homology modeling,and mapped conserved cavities onto the structure of GlpG. Then we de-signed cavity-filling substitutions, and measured the stability and activityof those variants. Among the 18 ‘‘cavity-filling’’ substitutions tested, threewere significantly destabilizing (DDGo

U > 1 kcal/mol), three were moder-ately stabilizing (DDGo

U =0.6~1.0 kcal/mol), and the rest retained the sta-bility to the level similar to wild-type. Among the three stabilizingsubstitutions, one was fully active while the other two were inactive. Thosetwo inactivating substitutions did not involve the active-site residues buttargeted the sites that belonged to the previously identified more flexible re-gions in GlpG. This result suggests that the packing defects may be requiredfor the movement of structural elements of proteins, and implies that proteinpacking may have evolved for function through the delicate balance be-tween stability and flexibility.

439-Pos Board B204Dissecting the Side Chain Interaction Energies of the Active Site HydrogenBond Network in a Rhomboid Protease GlpGKristen A. Gaffney1, Jeff Cho1, Heedeok Hong2.1Biochemistry & Molecular Biology, Michigan State University, EastLansing, MI, USA, 2Chemistry and Biochemistry & Molecular Biology,Michigan State University, East Lansing, MI, USA.The intramembrane rhomboid proteases are of particular interest due to theirability to hydrolyze a substrate peptide bond at the active site which is buriedwithin the membrane. The high-resolution structure of the E. coli rhomboidprotease GlpG revealed the catalytic dyad Ser201/His254, which is surroundedby a protein matrix and connected to bulk water via a narrow water channel.Although multiple crystal structures have been solved for GlpG, its catalyticmechanism is not clearly understood. Because it is a serine protease, hydrogenbonding interactions among active site residues are thought to play a criticalrole in the catalytic cycle. In this study we dissected the interaction energiesamong the active site residues His254, Ser201 and Asn154, that form ahydrogen bonding network in the active site of GlpG. To do this, we utilizedthe double-mutant cycle analysis combined with stability measurement bysteric trapping. Steric trapping, which couples the unfolding of a doubly-

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biotinylated protein to the binding of monovalent streptavidin, is advantageousdue to its ability to measure protein stability directly under native conditionswithout using denaturants. Interestingly, in mild detergents, the direct hydrogenbond between His254 and Ser201 was moderately unfavorable, with theinteraction energy DDGo

Interzþ1.0 kcal/mol, while the water-mediatedhydrogen bond between Ser201 and Asn154 was moderately strong(DDGo

Interz�1.5 kcal/mol). His254 and Asn154, which are distantly located,were weakly coupled (DDGo

Interz0 kcal/mol). Our result suggests that thestrength of polar interactions within the membrane are strongly context-dependent and their burial in the membrane have been stringently selectedfor function, not stability, during evolution.

440-Pos Board B205Reconstitution of FtsH-Mediated Membrane Protein Degradation inBicellesYiqing Yang1, Miyeon Kim1, Ruiqiong Guo1, Kristen Gaffney2,Heedeok Hong1.1Chemistry, Michigan State University, East Lansing, MI, USA,2Biochemistry, Michigan State University, East Lansing, MI, USA.Controlled degradation of misassembled and dispensable proteins is a crucialcellular process for maintaining the quality control of proteomes. While a ma-jority of studies have focused on degradation of water soluble proteins, it ispoorly understood how membrane proteins are degraded in cells. This discrep-ancy is largely due to the difficulties in reconstituting the degradation process invitro, which is necessary for biochemical and biophysical analysis of the mech-anism. In this study, we successfully reconstituted ATP-dependent proteolysisof membrane proteins mediated by the membrane-integrated AAAþ proteaseFtsH of E. coli.To achieve this, we established robust overexpression and purification proto-cols of E. coli FtsH, and reconstituted its ATPase and protease activities inTriton X-100 micelles and DMPC/CHAPS bicelles. We successfully trans-formed a stable E. coli membrane protein, the intramembrane proteaseGlpG, into an efficiently degraded substrate of FtsH by fusing the N-terminalor C-terminal degradation markers. This construct was specifically recog-nized by FtsH both in vivo and in vitro. We also developed a fluorescence-based high-throughput assay to monitor FtsH-mediated degradation ofmembrane proteins in real time. These newly developed tools enabled theMichaelis-Menten analysis of membrane protein turnover by FtsH. Ourexperimental system may serve as a versatile platform to study the molecularmechanisms of FtsH-mediated membrane protein degradation and elucidatethe quantitative relationship between folding and degradation of membraneproteins.

441-Pos Board B206Impact of Plasma Protein Binding on Cargo Release by ThermosensitiveLiposomes Studied by Fluorescence Correlation SpectroscopyJudith J. Mittag1, Barbara Kneidl2,3, Tobias Preiß1, Martin Hossann2,Gerhard Winter3, Stefan Wuttke4, Hanna Engelke4, Joachim O. R€adler1.1Department of Physics and Center for Nanoscience, Ludwig MaximilianUniversity of Munich, Munich, Germany, 2Department of Internal MedicineIII, University Hospital Munich, Ludwig Maximilian University of Munich,Munich, Germany, 3Department of Pharmacy, Pharmaceutical Technologyand Biopharmaceutics, Ludwig Maximilian University of Munich, Munich,Germany, 4Department of Chemistry and Center for NanoScience, LudwigMaximilian University of Munich, Munich, Germany.Thermosensitive liposomes (TSLs) whose phase-transition temperature (Tm)lies slightly above body temperature are promising candidates for induceddrug release via local hyperthermia. Recent studies, however, have revealeddisruptive shifts in the transition temperature in mouse plasma, which areattributed to unknown interactions with blood proteins. Here, we study theeffects of four major plasma proteins – serum albumin, transferrin, apolipo-protein A1 and fibrinogen – on the temperature-dependent release of fluores-cein di-b-D-galactopyranoside (FDG) from TSLs. The amount of fluoresceinreleased was quantified by fluorescence correlation spectroscopy (FCS) afterhydrolysis of FDG with b-galactosidase. This approach is more sensitiveand thus superior to established release assays, as it is non-sensitive tothe confounding effects of Triton on conventional fluorescence measure-ments. The assay determines the molar release ratio, i.e. the number ofmolecules released per TSL. We show that shifts in the Tm of release donot correlate with protein affinities for the liposomes derived from adsorp-tion isotherms. A remarkable shift in induced release towards lower temper-atures in the presence of mouse plasma is confirmed. In contrast, exposure torat or human plasma, orfetal bovine serum (FBS), has no effect on therelease profile.

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442-Pos Board B207A Mass-Spectrometry Based Methodology to Unravel the MolecularMechanisms of Sugar TransportChloe Martens, Antoni Borysik, Paula Booth, Argyris Politis.Chemistry, King’s College London, London, United Kingdom.Our work aims at providing a molecular description of the transport mech-anism(s) of sugar transporters from the Major Facilitator Superfamily (MFS)using a novel combination of mass-spectrometry based methods and model-ling. According to the widely acknowledged alternating-access model, MFStransporters have to adopt at least two conformations open to opposite sidesof the membrane to allow vectorial translocation of their substrate. How-ever, a molecular description of the mechanism underlying such transitionis not well established and the actual models fail to integrate the role ofthe lipid environment. By combining reconstitution in different lipid envi-ronments, mutagenesis and modelling, we plan to derive a detailed confor-mational landscape of the transporters XylE and LacY in lipid bilayers. Weuse native mass spectrometry to identify with high-resolution the binding oflipid species, ion-mobility mass spectrometry to assess the global conforma-tional changes and the variation in stability of the protein, and hydrogen-deuterium exchange mass spectrometry to follow the structural dynamics.Preliminary results show that the transporters have a signature pattern ofbound lipids and that this pattern is affected by changes in the conforma-tional equilibrium

443-Pos Board B208Vectorial Cholesterol Transport by STARD4 is Mediated by Specific PIP2

Membrane CompositionDerek M. Shore1, David B. Iaea2, Radda Rusinova1, George Khelashvili1,Michel A. Cuendet1, Olaf S. Andersen1, Frederick R. Maxfield2,Harel Weinstein1.1Department of Physiology and Biophysics, Weill Cornell Medicine, NewYork, NY, USA, 2Department of Biochemistry, Weill Cornell Medicine, NewYork, NY, USA.STARD4 (steroidogenic acute regulatory protein-related lipid-transferdomain containing 4) is known to transport cholesterol from the plasmamembrane (PM) to intracellular organelles, such as the endoplasmic reticu-lum and endocytic recycling compartment, but it has not been known if thetransport is selective. Identification of a surface-exposed basic region en-compassing the C-terminal helix in the crystal structure of STARD4 sug-gested the possibility that anionic lipids confer membrane selectivity. Totest this hypothesis we carried out combined biochemical and computationalstudies. Results of fluorescence-based sterol transport assays show thatphosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P2) selectively increasedthe rate of STARD4-mediated sterol removal from PM-like membranes,whereas phosphatidylinositol-(3,5)-bisphosphate (PI(3,5)P2) increased therate of cholesterol release into membranes. From the analysis of extensiveunbiased atomistic molecular dynamic simulations (20 ms), we identifieddistinct STARD4/membrane binding modes regulated by associations withdifferent PIP2 lipids, and identified primary and secondary binding sites ofSTARD4 to the membranes. Evaluation of STARD4/membrane interactiontrajectories with a Molecular Mechanics-Generalized Born surface areaapproach revealed differences in the binding geometry of STARD4 to mem-branes containing PI(4,5)P2 compared to membranes containing PI(3,5)P2.Thus, the C-terminal helix adopts a smaller angle with the membranenormal in binding to membranes containing PI(4,5)P2, reflecting a differen-tial presentation of the exposed basic region to the two membrane types.Both PI(4,5)P2 and PI(3,5)P2 strongly bind a region of basic residues onthe b1/b2 sheets that serves as a primary PIP2 anchoring site, but PI(3,5)P2 lipids also interact strongly with the distal end of the C-terminal helix.Altogether, the results have identified the basis of selectivity in the compo-sition of the two types of membranes, and the computational analysis hasrevealed a specific structural context for STARD4’s experimentallymeasured functional selectivity at membranes containing different PIP2subtypes.

444-Pos Board B209Chimera of Apolipophorin III and C-terminal Domain of Apolipoprotein Eto Study Apolipoprotein Structure FunctionPaul M. Weers, Leesa M. Kakutani, James V. Horn,Vasanthy Narayanaswami.Chemistry and Biochemistry, California State University Long Beach, LongBeach, CA, USA.Human apolipoprotein E (apoE) is a two-domain anti-atherogenic lipidtransport protein that mediates plasma cholesterol homeostasis by servingas a ligand for the low density lipoprotein (LDL) receptor. Of its three

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isoforms E3, E2, E4, the latter two have been associated with increasedrisk of cardiovascular and Alzheimer’s disease respectively, yet allthree contain an identical C-terminal (CT) domain. The CT domain (resi-dues 201-299) is responsible for tetramerization and has been found toinitiate lipid binding that influences critical functional features of apoE.The N-terminal (NT) domain of apoE has four a-helices arranged in abundle similar to apolipophorin III (apoLp-III), a model insect apolipopro-tein containing five a-helices. To better understand the role apolipoproteindomains play in structure and function, a novel chimeric apolipoproteinwas designed by attaching apoE-CT to apoLp-III. Recombinant apoLp-III/apoE-CT, apoE3, apoE-CT, and apoLp-III were expressed in bacterialcells, purified by affinity chromatography, and purity verified bySDS-PAGE. Western blot analysis using monoclonal apoE-CT specific anti-body confirmed the presence of apoE-CT in the chimera. Crosslinkingstudies using dimethylsuberimidate revealed that the apoLp-III/apoE-CTchimera formed oligomers similar to apoE, while apoLp-III was monomeric.The chimera was highly a-helical, and incubation with 1-anilinona-phthalene-8-sulfonic acid showed increased fluorescence intensity whenapoE-CT was added to apoLp-III, consistent with the presence of water-shielded dye binding sites upon addition of a structured apoE-CT segment.Our results suggest that the chimeric protein has structural features similarto the parent proteins, and that self-association properties of apoE can betransferred to apoLp-III. Future studies will include measuring the abilityof the chimera to solubilize phospholipid bilayers and binding to low densitylipoproteins. The chimeric approach offers the potential to obtain insightinto domain interactions and the structure-function relationships inapolipoproteins.

Membrane Receptors and Signal Transduction I

445-Pos Board B210Computational Model of Integrin Clustering in Response to Actin Turn-overTamara C. Bidone1, Aravind R. Rammohan2, Matt McKenzie2,Gregory A. Voth1.1University of Chicago, Chicago, IL, USA, 2Corning Inc, Elmira, NY, USA.Cell adhesion is a physical process that requires coordinated changes in cellmorphology and interactions with the extracellular matrix (ECM). Couplingbetween reorganization in cell morphology and contacts with the ECM isprovided by dynamic actin filaments and integrin-mediated adhesions. How-ever, owing to the complexity and potential regulators of both actin fila-ments and adhesions, the interplay between actin dynamics and adhesionsassembly is not fully understood. Here, we developed a coarse-grainedBrownian Dynamics computational model, which includes explicit semiflex-ible actin filaments, coupled to particles diffusing on a quasi-2Dsurface, representing integrins on the ventral surface of cells. In the model,integrins can establish interactions with actin filaments and regulate theirassembly and disassembly dynamics. Using this model, we determinedthe effect of tuning ligand affinity, patterns of actin (de)polymerization,and actin flow on integrin clustering and ligand binding. Our results provideinsights into the interplay between these factors in regulating adhesion initi-ation as well as coupled changes in the architecture of a dynamic actinnetwork.

446-Pos Board B211The M2 Muscarinic Receptor Signaling Complex Resolved by SingleMolecule Tracking in Live CellsYuchong Li1, Rabindra V. Shivnaraine2, Huiqiao Ji3, Fei Huang3,Kevin Braeckmans4, James W. Wells3, Claudiu C. Gradinaru1.1Department of Chemical and Physical Sciences, University of TorontoMississauga, Mississauga, ON, Canada, 2Department of Molecular andCellular Physiology and Medicine, Stanford University, Stanford, CA,USA, 3Department of Pharmaceutical Sciences, University of Toronto,Toronto, ON, Canada, 4Department of Pharmacy, Ghent University, Ghent,Belgium.Many aspects of the cellular signaling pathways via G protein-coupled recep-tors (GPCRs) are not completely understood. In particular, three questionshave been the focus of much attention and debate: the oligomeric status ofthe receptor, the coupling strength between the receptor and the G protein,and the regulation of the receptors in response to external stimuli. Here weexamine those questions by 2D tracking the M2 muscarinic receptors with To-tal Internal Reflection Fluorescence Microscopy (TIRFM) in the membrane oflive cells.M2 Receptors and G proteins were genetically fused with fluorescent pro-teins (GFP and mCherry) and expressed in Chinese Hamster Ovary

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(CHO) cells. The distributions of oligomeric sizes and diffusion coefficientsfor both the receptors and the G proteins were extracted from single-particletracking data obtained with TIRFM. Both of the receptors and G proteinswere found to be localized in membrane micro-domains, showing multi-step photobleaching and exhibited a wide range of diffusion behaviors ona slower time scale than single protein controls. Corroborated with dual-color fluorescence correlation spectroscopy on the same samples, we pro-pose that multiple oligomeric receptors and oligomeric G proteins sizesco-exist in close proximity inside a spatially confined signaling scaffoldin the membrane of living cells. In addition, two-color simultaneoustracking of co-expressed receptors and G proteins shows preliminary evi-dence for receptor-G protein decoupling and receptor internalization uponinduction with excessive agonist.

447-Pos Board B212Quantitative Fluorescence Microscopy Reveals Higher Order Oligomeri-zation of FGFR5Romario Regeenes1,2, Pamuditha Silva1,2, Dawn M. Kilkenny1,Jonathan V. Rocheleau1,2.1IBBME, University of Toronto, Toronto, ON, Canada, 2AdvancedDiagnostics, Toronto General Research Institute, UHN, Toronto, ON,Canada.The fibroblast growth factor receptor (FGFR) family of tyrosine kinase re-ceptors transactivate in response to ligand binding (e.g. FGF2) to initiateclassical intracellular signaling cascades. Despite not having an intracellularkinase domain like canonical FGFR1, FGFR5 retains the ability to activatethe MAPK signaling pathway, and particularly in the absence of ligand-receptor binding. Both receptors exhibit high affinity for FGF2, and aretherefore likely to compete for ligand binding. To examine the extent ofcompetition between FGFR1 and FGFR5 for ligand in living cells, we aimedto use the complementary techniques of fluorescence-anisotropy basedhomo-Forster resonance energy transfer (homoFRET) and Number andBrightness analysis (N&B). In this study, we expressed varied combinationsof dark, Venus-tagged, and Cerulean-tagged FGFR5 and/or FGFR1. Homo-FRET imaging using the smaller Forster distance of Cerulean-tagged recep-tor revealed that FGFR5 existed in pre-formed homodimers. However, thelarger Forster distance of Venus-tagged FGFR5 revealed anisotropy lowerthan the tandem Venus-Dimer control, suggesting it more loosely formedhigher order oligomers. Furthermore, N&B imaging revealed that removalof the receptor’s intracellular domain (R5DC) resulted in higher order ag-gregation, suggesting FGFR5 associates through an interaction in eitherthe transmembrane or extracellular domains. Co-expression of Cerulean-tagged FGFR5 and R5DC confirmed this potential alternate interactiondomain. Both techniques suggest FGFR5 forms pre-formed heterodimerswith canonical FGFR1. Interestingly, the addition of FGF2 ligand resultedin no change in FGFR5 aggregation state. These data suggest that FGFR5natively exists as a higher order oligomer with the ability to complexwith FGFR1. Collectively, these findings shed new light on the signalingmechanisms of FGFRs that can be used to develop novel therapeutics totreat a number of diseases.

448-Pos Board B213Investigating the Hetero-Interations of Receptor Tyrosine Kinases in LiveCellsMichael Paul, Fozia Ahmed, Kalina Hristova.Johns Hopkins University, Baltimore, MD, USA.Receptor tyrosine kinases (RTKs) are the second largest class of membrane re-ceptors, and are one of the main players in transducing signals across the mem-brane. They have an important role in development and regulating cell growthand migration, and mutations of these receptors are associated with many can-cers. Although in vitro studies have suggested that receptors from different sub-families of RTKs (Ephs, VEGFRs, FGFRs, etc.) might interact with each other,little is known about whether this actually occurs in cells. We use a quantitativeFRET spectroscopy method known as fully quantified spectral imaging (FSI) toidentify association partners between subfamilies in live cells.

449-Pos Board B214Decoding the Signaling through Homomeric and Heteromeric Cannabi-noid CB1 ReceptorsGuoqing Xiang1, Takeharu Kawano2, Apostolia Baki2,Diomedes Logothetis2.1Physiology and Biophysics, Virginia Commonwealth University,Richmond, VA, USA, 2Pharmaceutical Sciences, Northeastern University,Boston, MA, USA.

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Cannabinoid CB1 receptors are one of the most abundantly expressed G-pro-tein coupled receptors (GPCRs) in the central nervous system and play criticalroles in various neurophysiological processes such as reward, emotion and mo-tor activity. Growing evidence supports that GPCRs form heteromers that causesignificant changes in their signaling. Utilizing electron microscope immuno-cytochemistry, CB1 receptors were found to co-localize with dopamine D2 re-ceptors in the somata and dendrites of GABAergic neurons. Furthermore,fluorescence resonance energy transfer and co-immunoprecipitation studieshave provided evidence for heteromerization of CB1 and D2 receptors in het-erologous expression systems. CB1 receptors primarily signal through Pertussistoxin (PTX) sensitive Gi/o, leading to a decrease in cAMP levels. Interestingly,stimulating the CB1 receptor in a cell line co-expressing CB1 and D2 receptorswas shown to increase cAMP levels, suggesting a possible Gs signaling switchthrough the CB1-D2 heteromer. Our preliminary data using GIRK channels asreporters of GPCR signaling in the Xenopus oocyte heterologous expressionsystem corroborated the Gs switching behavior of CB1 receptors upon hetero-merization with D2 receptors. To increase the signal to noise ratio of the CB1receptor Gs signaling switch, we have turned to a calcium mobilizationassay. Naturally, the promiscuous G alpha 16 belongs to the Gq superfamilyand its activation leads to phospholipase C activation and calcium release.G alpha 16 chimeras in which 11 or 30 amino acids from the C-terminuswere replaced by the corresponding sequence of Go and Gs were constructed.Calcium mobilization was monitored by GCaMP 6s in a FlexStation. We foundthat activation of Gi-coupled D2 receptor causes increases in intracellularcalcium levels with G16/o chimeras but not with G16wt. Gs-coupled Glucagonreceptor signal to both G16/s chimeras and G16wt but G16/s chimeras signif-icantly left-shift the dose-response curve of Glucagon, indicating G16/schimeras as good Gs signaling reporter. We are in the process of testingsignaling of homomeric and heteromeric CB1 receptors through G16 chimerasin this system.

450-Pos Board B215Examining the Effects of Neurotrophin 3 and Nerve Growth Factor on theInteraction of Tropomyosin Receptor Kinase A and CFozia Ahmed, Kalina Hristova.Materials Science and Engineering, Johns Hopkins University, Baltimore,MD, USA.Tropomyosin receptor kinases (Trks) are known to initiate signaling cascadesin response to their ligands, the neurotrophins. Trks are involved in multipleimportant key events responsible for the development of the mammalian ner-vous system. They are known to control synaptic strength, plasticity, neuronalsurvival, proliferation, migration, axonal growth/guidance and patterning,injury protection, and neuronal apoptosis. Trks belong to the second largestclass of membrane proteins, known as receptor tyrosine kinases (RTKs). Thereare three known members of the Trk family: Tropomyosin receptor kinase A, Band C. The signaling mechanism of Trks in response to neurotrophins is com-plex and the exact mechanism of their activation is still under study. We studythe mechanism of activation of the Trk receptors in the presence and absence ofdifferent neurotrophins, and the effects of different neurotrophins on the dimer-ization energetics of each Trk receptor, using quantitative Forster ResonanceEnergy Transfer and Western Blotting techniques.

451-Pos Board B216High Content Analysis of Intracellular Heterogeneity to Study GPCROligomerizationArtu’ Breuer, Samuel Mcewen Walsh, Anna Mantsiou, Dimitrios Stamou.University of Copenhagen, Copenhagen, Denmark.Heterogeneities are a fundamental feature of biology that enable organismresilience through flexible responses. We quantified diffraction limited intra-cellular heterogeneities of recombinant receptors in an in vitro cell cultureusing conventional fluorescence microscopy. We exploited intracellularheterogeneities to quantitate the oligomerization of two prototypical G-pro-tein coupled receptors (GPCRs): b1 adrenergic receptor (b1AR) and the Y2receptor (Y2R). Our results suggest that intracellular heterogeneities canseverely skew single cell, and many cell, average properties.

452-Pos Board B217Interactions between G Proteins and Non-Stimulated GPCRs Revealed byTwo-Photon Polarization MicroscopyAlexey Bondar1, Josef Lazar2.1Center for Nanobiology and Structural Biology, Institute of Microbiology,Nove Hrady, Czech Republic, 2Institute of Organic Chemistry andBiochemistry, Prague, Czech Republic.

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G protein-coupled receptors (GPCRs) and G proteins are key players ofcellular signal transduction. They transduce signals from a multitude ofextracellular stimuli into the cells. Despite a number of studies, many as-pects of molecular mechanisms and spatiotemporal dynamics of G proteininteractions with GPCRs remain unclear. In particular, it is uncertainwhether G proteins can form complexes with inactive GPCRs, and whatis the stability and functional significance of such complexes.In order to address these issues, we have investigated the interactionsbetween the G proteins and various GPCRs using the technique of two-photon polarization microscopy (2PPM). 2PPM, developed in our labora-tory, allows sensitive monitoring of protein conformational changes andprotein-protein interactions, in living cells, in real time, using a single fluo-rescent protein tag. Our results demonstrate that 2PPM indeed allowsobserving interactions between G proteins and representative GPCRs, usinga single fluorescent protein tag. Our experiments reveal an important role ofreceptor basal activity for GPCR - G protein interactions, yielding insightsinto receptor - G protein precoupling and other aspects of GPCR - G proteinsignal transduction.

453-Pos Board B218Investigating MUC1 Transmembrane Dimer Structure using ReplicaExchange Molecular DynamicsChristina M. Freeman, Alexander J. Sodt.NICHD, National Institutes of Health, Bethesda, MD, USA.The epithelial single-pass transmembrane glycoprotein Mucin 1 (MUC1) is amain constituent of mucus and has roles in cell signaling and differentiation.In addition, overexpression and subsequent homodimerization of the C-termi-nal subunit of MUC1 has been implicated in the progression of many cancers,particularly breast cancer.Recent experiments have shown that strong dimerization is dependent on theformation of disulfide bonds between two cysteine residues in the juxta-membrane region, but weak dimers can also be formed without them.Certain mutations in the TMD can also partially disrupt the strength ofthe dimer. This suggests that protein-protein or protein-lipid interactionsare also mediating dimer formation. However, the transmembrane domainof MUC1-C doesn’t appear to contain common oligomerization motifssuch as the Sm-X3-Sm sequence found in glycophorin A and many receptortyrosine kinases.Understanding the physical mechanisms by which MUC1-C dimerizes withinthe surrounding plasma membrane environment will help in creating drugtargets, as well as generating a more comprehensive model of protein-lipid in-teractions. To investigate this, atomistic replica exchange molecular dynamicssimulations are being used to find stable conformations of wild-type and mutantMUC1 TMDs.

454-Pos Board B219Electrostatics Facilitates the Trimer-of-Dimers Formation of the Chemo-receptor Signaling DomainMarharyta Petukh1, Davi Ortega2, Igor B. Zhulin1.1UTK/ORNL, Oak Ridge, TN, USA, 2The California Institute of Technology,Pasadena, CA, USA.Chemoreceptors are crucial components of the bacterial sensory systemthat modulates cellular motility. They detect changes in the environmentand transmit information to CheA histidine kinase, which ultimatelycontrols cellular flagellar motors. The prototypical Tsr chemoreceptor inE. coli is a homodimer containing two principle functional modules: (i) aperiplasmic ligand-binding domain and (ii) a cytoplasmic signaling domaincomprising an antiparallel, four-helix coiled-coil bundle. Receptor dimersare arranged into a trimer-of-dimers, which is a minimal physical unitessential for enhancing the CheA activity several hundredfold. Recentadvances in cryo-electron tomography showed that trimers-of-dimers arearranged into highly ordered hexagon arrays at the cell pole; however, themechanism underlying the trimer-of-dimer and higher order arrayformation remains unknown. Current evidence from structural and bio-chemical studies suggest that trimers-of-dimers are maintained exclusivelyby contacts at the chemoreceptor cytoplasmic tip. Here, using all-atom,microsecond-range MD simulation of the Tsr trimer-of-dimers crystal struc-ture, we show that dimers within the trimer may interact throughout theentire length of the signaling domain. While inter-dimer contacts at thechemoreceptor tip occur via hydrophobic interactions, completedimer ‘‘zipping’’ is facilitated by electrostatics, especially by the polarsolvation component. We also show that many of the residues involved inestablishing hydrogen bonds and salt bridges between dimers are evolu-tionary conserved.

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455-Pos Board B220Investigating Initial Events of IgE Receptor Signaling with Super-Resolution Microscopy and Monte Carlo SimulationsEshan Mitra1, James P. Sethna2, David Holowka1, Barbara Baird1.1Chemistry and Chemical Biology, Cornell University, Ithaca, NY, USA,2Physics, Cornell University, Ithaca, NY, USA.The high affinity IgE receptor (FcεRI) in mast cells plays a central role in initi-ating allergic responses, and also serves as a model system for immune receptorsignaling. FcεRI binds IgE antibody, which confers specificity for antigen. Thecross-linking of IgE-FcεRI by antigen stimulates a transmembrane signal thatleads to downstream events including Ca2þ mobilization and degranulation.Our work seeks to understand the molecular basis of these initial signalingevents.To examine signal initiation experimentally, we performed PALM/STORMsuper-resolution imaging. We employed structurally defined ligands to gainmolecular-level control over the structure of the IgE cluster formed. Two triva-lent ligands studied, dsDNA based Y16 and Y46, differ in the spacing of recep-tor binding sites. With one-color STORM on live cells, we imaged the dynamicclustering of receptors upon stimulation. We found that Y16 and Y46 differ inthe density of receptors in clusters formed, as quantified by autocorrelationfunctions. With two-color PALM/STORM, we quantified the extent to whichthe differing structures of these IgE clusters affect their capacity to recruit othermembrane components, including signaling partner Lyn kinase and markers forliquid ordered (Lo) membrane phase.We used theoretical modeling to further address the potential role of membranelipids in promoting the recruitment of a kinase to a cluster of receptors. Wemodeled the membrane with a 2D Ising model, and calculated the change infree energy associated with recruiting a Lo-preferring kinase into a Lo-preferring receptor cluster. Using this framework, we asked what structural fea-tures of a receptor cluster are most important for effective lipid-mediatedsignaling. Ongoing work uses an extension of the Ising model in order toaddress other models of membrane phase behavior, including microemulsions.

456-Pos Board B221Fluorescence Fluctuation Spectroscopy of Dopaminergic Signaling inPancreatic Beta CellsDaniel J.P. Foust, Alessandro Ustione, David W. Piston.Cell Biology and Physiology, Washington University School of Medicine,St. Louis, MO, USA.Dopamine inhibits insulin secretion from pancreatic beta cells via D2-likedopamine receptors and we seek to understand the molecular details of this in-hibition using the tools of fluorescence fluctuation spectroscopy. Normal insu-lin secretion is critical to maintaining blood glucose homeostasis and theprevention of type II diabetes. Although glucose stimulated insulin secretionis well understood, there are hundreds of receptors expressed in beta cells,including G-protein coupled dopamine receptors, which may provide addi-tional mechanisms for regulating insulin secretion. Previously, we have shownthat D2-like (D2, D3, D4) dopamine receptors confer beta cell sensitivity todopamine. Furthermore, we demonstrated that the D3 homolog is the receptorprimarily responsible for dopamine sensitivity, although the other receptorsmay compensate in a D3-knockout scenario. Electrophysiology has revealedan inwardly-rectified current in dopamine-stimulated beta cells, implicatingion channels as possible downstream signaling targets. G-protein coupledinwardly rectifying Kþ (GIRK) channels are known effectors of D2-like recep-tors, although transactivation of GIRK channels by dopamine receptors has notbeen demonstrated explicitly in beta cells. GIRK channels are activated bytetrameric binding of Gbg subunits dissociated from the trimeric G-proteincomplex. To study dopamine receptor interactions with downstream effectorswe express fluorescently labeled proteins (receptors, G-proteins, channels) inMIN6 cells. Cells are imaged with single- or two-photon excitation and theseimages are analyzed by considering the intensity distribution (photon countinghistogram) and correlation (image correlation spectroscopy). Using these ana-lyses we show that dopamine receptors co-cluster with G-proteins at discretesites on the plasma membrane. At these cluster sites, the apparent brightnessof labeled Gbg subunits increases with dopamine stimulus. Brightness in-creases indicate oligomerization, therefore these results support our hypothesisthat dopamine receptor activation promotes the opening of GIRK channels viatetrameric binding of dissociated Gbg subunits to channels.

457-Pos Board B222Dynamics of Various Phospholipase C-B ComplexesAshima Singla, Suzanne Scarlata.Chemistry and Biochemistry, Worcester Polytechnic Institute, Worcester,MA, USA.

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Phospholipase C-b (PLCb) is activated by G protein signals from different hor-mones and neurotransmitters on the plasma membrane, to mediate increases inintracellular calcium. Surprisingly, we find that PLCb also localizes to the cyto-plasm where it plays an important role in RNA-induced gene silencing by in-teracting with the component 3 promoter of the RNA-induced silencingcomplex (C3PO) as well as one of the key components of the RNA-inducedsilencing machinery, Argonaute 2 (Ago2). We also find that PLCb distributesbetween its two binding partners, Gaq and C3PO/Ago2, to promote calciumsignaling or RNA-induced silencing. For example, Using bimolecular fluores-cence complementation, FLIM and FCS we find that the interaction of PLCbwith Ago2 and C3PO change with neuronal differentiation. We propose thatthe ability of PLCb to shuttle between its two binding partners to carry out thesetwo different functions plays a key role in the normal physiological processesof cells.

458-Pos Board B223Optical Control of cGMP SignalingUlrike Scheib1, Katja Stehfest1, Christine E. Gee2, Heinz G. Korschen3,Shatanik Mukherjee1, Thomas G. Oertner2, Peter Hegemann1.1Experimental Biophysics, Humboldt-Universitaet zu Berlin, Berlin,Germany, 2Institute for Synaptic Physiology, University Medical CenterHamburg-Eppendorf, Hamburg, Germany, 3Molecular Sensory Systems,Center of Advanced European Studies and Research (caesar) Bonn, Bonn,Germany.Zoospores of the aquatic fungi Blastocladiella emersonii are phototactic and arhodopsin-guanylyl cyclase (RhGC) was identified as the putative phototaxisreceptor. Due to the direct linkage between the rhodopsin and the cyclasedomain, RhGC represents the first member of a novel class of enzyme linkedrhodopsins. In this work, we characterized RhGC biophysically. We showthat green light maximally activates RhGC and cyclic GMP is produced. Aftera short green laser flash, RhGC (D525) converts in 8 ms into a blue-shiftedsignaling state P380 and recovers within 100 ms. RhGC expresses well inXenopus oocytes, yeast, CHO cells, mammalian neurons and cyclic GMPproduction was light dose-dependent, rapid and reproducible. Thus, RhGC isa versatile tool for optogenetic analysis of cGMP-dependent signalingprocesses in cell biology and the neurosciences.

459-Pos Board B224Photo Regulation of Small G-Protein Ras using Photochromic PeptideMasahiro Kuboyama, Kaori Masuhara, Shinsaku Maruta, Kazunori Kondo,Kazuo Fujiwara.Soka University, Hachioji, Japan.The small guanine nucleotide binding protein (G-protein), which are known asa molecular switch is central regulator of cellular signaling pathway. Theregulation mechanism of the protein is well studied at molecular level. Rasis one of the G-protein which have essential role in signaling cascade reactionin cell is regulated by guanine nucleotide exchange factor (GEF) and GTPaseactivating protein (GAP). GTP bout Ras induced by GEF is active state andGDP bound Ras induced by GAP is inactive state. The conformational changeinduced by GTP binding enable Ras to transduce a signal into downstreamthrough direct interaction with its effectors. Also, mutant Ras known as con-stant active state cause excess signaling transduction to downstream effectors,which is pathogenesis of canceration, In this study, we focused on the inter-action between Ras and its GEF, Son of sevenless (SOS) in order to controlRas function using photochromic compounds. Previously it is shown thatthe peptide mimicking the aH-helix, Ras binding region of SOS effectivelyinhibits Ras function competing with native SOS. Therefore, it is expectedthat photo-induced structural change of the peptide conjugate with photo-switch enable us to control Ras activity. We designed and synthesized SOSaH-helix peptide which contains two cysteine residues in order to cross-link with bi-functional photochromic molecule of azobenzene derivative,ABDM which change its structure reversibly by UV-VIS irradiation. The pep-tide modified with ABDM exhibited secondary structural change reversiblyupon UV and visible light irradiation. Binding of ABDM-peptide to Rasand SOS dependent nucleotide exchange reaction were also altered photo-reversibly. We also tried to examine the photo-reversible effect of ABDM-peptide for HeLa cell.

Exocytosis and Endocytosis I

460-Pos Board B225Dynamin Independent Endocytic Pathway Operates in a Negative Feed-back Loop to Sense and Regulate the Resting Membrane TensionJoseph J. Thottacherry.

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Cellular Organization and Signalling, National Centre for BiologicalSciences, Bengaluru, India.Cells regulate their membrane area and tension by finely tuned mechanisms.Exo-endocytic processes, by addition or removal of membrane respectively,play a major role in controlling physical properties of the membrane.However, the cellular processes and molecular mechanisms behindthis regulation are poorly understood. Here we have explored the role ofmultiple endocytic pathways in the maintenance of membrane homeostasis.We have used either a custom made cell stretching device, osmoticshocks or deadhering to modulate membrane tension and optical ormagnetic tweezers to measure membrane tension. We find that a specific en-docytic pathway, the dynamin-independent CLIC/GEEC (CG) endocyticpathway is transiently up regulated during change of membrane tensionby stretch-induced relaxation or by multiple other means. By contrast, theextensively studied clathrin-mediated or caveolar endocytic pathways arenot modulated. The CG pathway is a high capacity pathway that rapidlyendocytoses and recycles a major fraction of endocytosed contents withinminutes of internalization. Modulating the CG pathway via perturbationof key regulatory genes correspondingly modifies membrane tensionand membrane retrieval after strain-induced relaxation. Using knock outcell lines we further find that focal adhesion related molecules help insensing and transducing membrane tension to regulate CG pathway. Thesefeatures indicate that the CG pathway could be a vital component of thecellular machinery that senses and responds to regulate plasma membranetension.

461-Pos Board B226Munc13 And Munc18 Cooperate to Properly Assemble SNAREs for FastNeurotransmitter ReleaseYing Lai.Molecular and Cellular Physiology, Stanford University, palo alto, CA, USA.Neuronal SNAREs (synaptobrevin, syntaxin, SNAP-25), synaptotagmin, com-plexin, Munc13, and Munc18 are essential for fast Ca2þ-triggered synapticvesicle fusion. Syntaxin forms a closed complex with Munc18 that preventsSNARE complex formation, and Munc13 catalyzes the opening and transitof syntaxin into the SNARE complex. However, Munc13 knockout in micecannot be fully rescued with a constitutively open mutant of syntaxin. Wereport a new function of Munc13, independent of Munc18: it promotes theproper syntaxin/synaptobrevin subconfiguration during assembly of the ternarySNARE complex. In cooperation with Munc18, Munc13 additionally ensuresthe proper syntaxin/SNAP-25 subconfiguration. In a reconstituted fusion assaywith SNAREs, complexin, and synaptotagmin, inclusion of both Munc13 andMunc18 quadruples the Ca2þ-triggered amplitude, and improves the Ca2þ-sensitivity by an order of magnitude, reaching physiological sensitivity. IfMunc13 or Munc18 is deleted, improperly assembled SNARE complexesmay occur, explaining the observed abrogation of neurotransmitter release inneurons.

462-Pos Board B227Effects of Opsonin Density and type on the Phagocytosis of BeadsSebastian Hendrickx-Rodriguez1, Michael R. Falvo2,E. Timothy O’ Brien III,2, Richard Superfine2.1Physics, New Mexico Institute of Mining and Technology, Socorro, NM,USA, 2Physics, UNC - Chapel Hill, Chapel Hill, NC, USA.When a pathogen enters the body, the immune system releases small proteinsthat opsonize the foreign entity. This serves as a signal to macrophages toengulf the pathogen, a process known as phagocytosis. Depending on thetype of opsonin covering the invasive species, phagocytosis can occur instructurally different ways. My research aimed to examine how differentopsonin types and densities affect phagocytosis both qualitatively and quan-titatively. Microspheres were used to replicate pathogens, and IgG antibodyalongside iC3b complement were the opsonins studied. First, a Dose-Response curve was made to show it was possible to cover the bead withdifferent amounts of opsonin, i.e. change the density of antibody or comple-ment on the bead surface. Then, the distinctly opsonized beads were fedto cells and the effectiveness of phagocytosis was analyzed using twodifferent techniques: time-lapse movies and 3D image reconstruction.Through the former method, it was quantitatively found that more denselyopsonized beads were more effectively phagocytosed, and that cells transi-tioned through phagocytosis quicker with IgG covered beads. This may sug-gest that the actin dynamics and cytoskeleton remodeling occur fasterthrough the IgG signaling pathway than through that of iC3b. Progresswas made through the 3D image reconstruction to better observe the phago-cytic cup structure formed by the cell and to more efficiently note a

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difference between iC3b and IgG consumption in a qualitative sense. Phago-cytic studies can supply useful information for drug treatments involvingpassage of the drug through the cell membrane to reach its activation site.My research showed that opsonin density and type affect phagocytosistremendously.

463-Pos Board B228A Supported Tubulated Bilayer System Shows Effects of Synaptotagmin-7on Membrane CurvaturePeter Dahl1, Joseph Vasquez2, Hai Tran2, Jeff Knight2, Arun Anantharam1.1University of Michigan, Ann Arbor, MI, USA, 2University of Colorado,Denver, CO, USA.Fusion and fission of cellular membranes involve dramatic, preciselyregulated changes in membrane curvature mediated by a numberof proteins. The mechanisms by which proteins influence membrane curva-ture are not well understood, and current methods for investigating curvaturechanges using well-controlled systems are limited. We have developed asystem based on supported lipid bilayers in which lipid tubules spontane-ously form in a manner that can be tuned by varying the ionic characterand strength used during bilayer deposition and imaging. Using thissupported tubulated bilayer system, which we term ‘‘STuBS,’’ we haveinvestigated membrane-targeting C2 domains from synaptotagmin-7, amember of the synaptotagmin protein family that triggers exocytosis inneurons and neuroendocrine cells. We find that addition ofpurified synaptotagmin-7 C2AB domains, but not synaptotagmin-1 C2A,leads to a Ca2þ-dependent disappearance of tubules with concomitantformation of vesicles. These studies demonstrate that synaptotagmin-7 canalter membrane morphology by driving changes in membrane cur-vature. STuBS is a novel experimental system useful for monitoring solute-and protein-mediated effects on membrane topology in aqueous media andin real time.

464-Pos Board B229Exosomes Fractionation by Biophysical PropertiesSoheyl Tadjiki1, Robert Reed1, Samer Al-Hakami2, Mikhail Skliar3.1Postnova Analytics, Salt Lake City, UT, USA, 2University of Utal, Salt LakeCity, UT, USA, 3University of Utah, Salt Lake City, UT, USA.Exosomes are small extracellular vesicles containing nucleic acid andprotein, which have shown a great potential for cancer diagnostics andtherapeutic applications. Characterization of exosomes is challenging dueto their inherent heterogeneity and complexity. A fractionation step isnecessary to provide narrow-sized fractions to enable a more accurate sub-sequent size analysis. Asymmetrical Flow Field-Flow Fractionation(AsFlFFF) is a high resolution elution technique for fractionation of macro-molecules and biological nanoparticles based on their hydrodynamic sizes.In this study the AsFlFFF system was interfaced with Multi Angle StaticLight Scattering (MALS) detector to characterize the MCF-7 tumor exo-some sample. The analysis revealed a size distribution between 30-120nm in diameter. Several narrow fractions were collected along the size dis-tribution and were analyzed by PCR. The study showed that the populationof tumor exosomes in circulation are heterogeneous in their cancerbiomarker miR21.

465-Pos Board B230Membrane Recruitment Enables Weak Binding Endocytic Proteins toForm Stable ComplexesOsman Yogurtcu, Margaret E. Johnson.Biophysics, Johns Hopkins University, Baltimore, MD, USA.Membrane targeting and assembly of proteins is required for vesicle traf-ficking and receptor mediated signaling, but it is not known to what extentthe proteins recruited to these events may have evolved to exploit the 2Dsurface for assembly, versus pre-assembling in solution. We show that thephospholipid targeting proteins of clathrin-mediated endocytosis dramati-cally enhance their effective binding strength and subsequent complex for-mation to one another after surface recruitment in yeast and metazoans. Forproteins such as clathrin that do not directly bind lipids, the enhancement isstill achieved by using three distinct binding sites to stabilize the clathrin toperipheral membrane proteins on the surface. We derive simple formulasthat quantify the degree of binding enhancement as a function of the proteinand lipid concentrations, binding constants, and critically, the ratio of vol-ume to membrane surface area. Our results thus apply to any cell type orgeometries, including in vitro systems and the targeting of internal organ-elles from the cytoplasm. With a sufficient concentration of lipid recruiters,

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such as PIP2, we show that the effective binding strength is enhanced by or-ders of magnitude and becomes, surprisingly, independent of the protein-protein binding strength. We quantify how this effect varies for proteinsinvolved in later stages of vesicle trafficking and cell division in yeast.Coupled with detailed spatially and structurally resolved simulations, wehave further measured the effect of membrane recruitment on controllingthe speed of assembly, and influences of crowding and diffusion on thisprocess.

466-Pos Board B231Mechanoregulation of Clathrin-Mediated Endocytosis in Isolated Cellsand Developing TissuesComert Kural.The Ohio State University, Columbus, OH, USA.Clathrin-coated assemblies bear the largest fraction of the endocytic loadfrom the plasma membrane of eukaryotic cells. However, dynamics ofclathrin-mediated endocytosis (CME) have not been established withintissues of multicellular organisms due to experimental and analyticalbottlenecks in determining the lifespan of clathrin-coated structures. Wefound that clathrin coat growth rates obtained from fluorescence microscopyacquisitions can be utilized as reporters of CME dynamics. Growth ratescan be assembled within time windows shorter than the average clathrincoat lifetime and, thereby, allow probing the changes in CMEdynamics in real time. Furthermore, this novel approach is applicable totissues as it is not prone to particle detection and tracking errors, whichresult in underestimation of the clathrin coat lifetimes. Exploiting these ad-vantages, we detected spatial and temporal changes in CME dynamicswithin Drosophila amnioserosa tissues at different stages of embryo devel-opment. We also found that increased membrane tension impedes CMEthrough inhibition of formation and dissolution of clathrin-coated structures.Therefore, the parameters defining clathrin coat dynamics (i.e., lifetime,formation density and growth rates) can be utilized to monitor the spatio-temporal gradients of the plasma membrane tension during cell migrationand spreading.

467-Pos Board B232Regulation of Lysosomal Exocytosis by Oxidative Stress and Calcium IonsSreeram Ravi, Andrew P. VanDemark, Kirill Kiselyov.University of Pittsburgh, Pittsburgh, PA, USA.Lysosomal exocytosis has emerged as an important mechanism of cellularrepair and clearance. Cellular reactions supported by lysosomal exocytosisinclude membrane repair and expulsion of various toxins. Lysosomal exocy-tosis involves delivery of lysosomes to the plasma membrane followed bySNARE-dependent fusion. The processes of lysosomal delivery and fusionrequire calcium ions. How these processes are regulated is not completelyunderstood, especially how they are modulated by pathological conditions.Our data show that lysosomal exocytosis is biphasically regulated byreactive oxygen species. Low levels of reactive oxygen species increaselysosomal exocytosis, while high levels of oxidative stress suppresses it.Activation of lysosomal exocytosis by reactive oxygen species requiresboth the lysosomal ion channels TRPML1 and calcium entry acrossthe plasma membrane. High levels of oxidative stress suppressedlysosomal acidification and significantly the ability of calcium to stimulatelysosomal exocytosis. We propose that stimulation of lysosomal exocytosisby oxidative stress is a cytoprotective mechanism that limits lysosomalpermeabilization aos cells death under oxidative stress condition.Suppression of lysosomal exocytosis by oxidative stress is a pathologicmechanism probably driven by inhibition of lysosomal delivery to theplasma membrane.

468-Pos Board B233Local Turgor Pressure Reduction via Channel ClusteringJonah K. Scher-Zagier.Physics, Washington University in St. Louis, St. Louis, MO, USA.The primary drivers of yeast endocytosis are actin polymerization andcurvature-generating proteins, such as clathrin and BAR domain proteins.Previous work has indicated that these factors may not be capable of gener-ating the forces necessary to overcome turgor pressure. Thus local reductionof the turgor pressure, via localized accumulation or activation of solutechannels, might facilitate endocytosis. The possible reduction in turgor pres-sure is calculated numerically, by solving the diffusion equation through aLegendre polynomial expansion. We find that for a region of increasedpermeability having radius 45 nm, as few as 60 channels with a spacing

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of ten nanometers could locally decrease the turgor pressure by 50%. Weidentify a key dimensionless parameter, P1 a/D , where P1 is the increasedpermeability, a is the radius of the permeable region, and D is thesolute diffusion coefficient. When p > 0.44, the turgor pressure is locallyreduced by more than 50%. An approximate analytic theory is used togenerate explicit formulas for the turgor pressure reduction in terms ofkey parameters. These findings may also be relevant to plants, where themechanisms that allow endocytosis to proceed despite high turgor pressureare largely unknown.

469-Pos Board B234Effects of Sterol Substitution in Plasma Membrane of Host Cell uponInternalization of Yersinia PseudotuberculosisJiHyun Kim1, Hana S. Fukuto2, James B. Bliska2, Erwin London1.1Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY,USA, 2Microbiology, Stony Brook University, Stony Brook, NY, USA.Cholesterol is a major component of mammalian plasma membrane, andcan participate in the formation of ordered membrane domains (lipid rafts).To investigate its functional role, it can be substituted with varioussterols having different chemical structures and varying abilities to supportor inhibit ordered domains formation by incubating cells with sterol-loadedmethyl b cyclodextrins. We recently used this technique to investigatethe role of sterol properties upon endocytosis of proteins. Several sterolscould support endocytosis. It was found that a sterol having both raft-promoting abilities and a 3b-OH group in its structure is necessary and suf-ficient for that sterol to support endocytosis. These studies have beenextended by investigating the effect of sterol substitution upon entry of abacterial pathogen into host cells. Entry of Yersinia pseudotuberculosisinto the human breast cancer cell line MDA-MB-231 was measured. Thir-teen different sterols/steroids were substituted for cholesterol. In all cases,a similar level of Y. pseudotuberculosis attachment to cells was obtained.However, other than cholesterol, only desmosterol and 7-dehydrocholes-terol could support Y. pseudotuberculosis internalization. The sterols arethe immediate precursors of cholesterol, and thus are very similar tocholesterol in structure. Thus, it appears that Y. pseudotuberculosis inter-nalization into host cells needs more specific property of the sterol thanthe endocytosis. Whether this sterol specificity reflects a requirement foractive bacterial invasion or passive endocytosis by the host cells is beingstudied with Y. pseudotuberculosis mutants lacking one or more surfaceadhesins.

470-Pos Board B235Recovery of ESCRT-III Filaments Subjected to Force: An ‘Invasive Mode’HS-AFM StudyNebojsa Jukic1,2, Lorena Redondo-Morata1,2, Aurelien Roux3,Simon Scheuring1,2.1Weill Cornell Medicine, New York, NY, USA, 2U1006, INSERM / Aix-Marseille Universite, Marseille, France, 3Department of Biochemistry,University of Geneva, Geneva, Switzerland.Endosomal sorting complex required for transport III (ESCRT-III) proteinsare crucial to membrane sculpting processes, including cytokinesis andbiogenesis of multivesicular bodies. How ESCRT-III polymerization gener-ates membrane curvature remains debated. Using High-Speed Atomic ForceMicroscopy (HS-AFM), a versatile technique with unprecedented spatialand temporal resolution, we acquired insights into how Snf7 assemblies,the major component of the ESCRT-III system, changed architecture inthe presence of divalent cations and how they recover after being dissectedby applying increased forces to well-defined delimited areas of the samplesurface. The dissected assemblies present free ends of broken filamentsonto which new monomers from the imaging solution can polymerize. Afterinitial perturbation, the recovering assemblies show a tendency towardmaximization of interfilament contacts, manifesting as nascent filamentsor elongation of broken filaments along pre-existing filaments that act asscaffolds, as well as reparation of broken filaments. Based on these results,we hypothesize about a novel mechanism by which lateral interactions be-tween ESCRT-III filaments drive constriction of the assemblies in orderto induce membrane deformation.

471-Pos Board B236Structural Dynamics of Endocytosis by High-Speed Atomic ForceMicroscopyGrigory Tagiltsev1, Frederic Eghiaian2, Simon Scheuring1.1Weill Cornell Medicine, New York, NY, USA, 2JPK Instruments, Berlin,Germany.

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Clathrin-mediated endocytosis is one of the major endocytosis pathways.Formation of clathrin coated vesicles (CCV) begins from relatively flatplasma membrane regions. Clathrin molecules bind to the CCV buddingsites and assemble into a lattice covering growing vesicle. Initial flat clathrinlattice displays a hexagonal honeycomb architecture whereas the final CCVdisplays a mixture of hexagons and pentagons. The relation between clathrinlattice conversion and curvature generation in that process remains un-known. No dynamic structural analysis of the detailed mechanisms duringCCV budding was achieved so far. High-speed atomic force microscopy(HS-AFM) permits observation of structural dynamics in biologicalprocesses because it allows performing imaging with nanometer spatialand subsecond temporal resolution. To study the CCV transformation byHS-AFM, we use a cell-free endocytosis assay based on plasma membranepatches. Combination of HS-AFM together with cell-free assay shouldpermit direct, real-time observation of clathrin lattice rearrangement duringCCV budding.

472-Pos Board B237Dilation of Fusion Pores by SNARE Protein CrowdingZhenyong Wu1, Oscar D. Bello2, Sathish Thiyagarajan3, Sarah M. Auclair2,Wensi Vennekate1, Shyam S. Krishnakumar2, Ben O’shaughnessy4,Erdem Karatekin1.1Department of Cellular and Molecular Physiology, Yale University, NewHaven, CT, USA, 2Department of Cell Biology, Yale University, NewHaven, CT, USA, 3Department of Physics, Columbia University, New York,NY, USA, 4Department of Chemical Engineering, Columbia University,New York, NY, USA.Hormones and neurotransmitters are released through exocytotic fusionpores that can fluctuate in size and flicker open and shut multiple times.The kinetics and the amount of cargo released, and the mode of vesiclerecycling depend on the fate of the pore, which may reseal or dilate irrevers-ibly. Pore nucleation requires zippering between vesicle-associated v- andtarget membrane t-SNAREs, but the mechanisms governing thesubsequent pore dilation are not known. Past approaches either monitoredsingle exocytotic pores in live cells with unknown biochemistry, or usedreconstitutions that lacked single pore sensitivity. Here, we probed dilationof single fusion pores using v-SNARE-reconstituted ~23 nm diameterdiscoidal nanolipoprotein particles (vNLPs) as fusion partners with cellsectopically expressing cognate, ‘‘flipped’’ t-SNAREs. A flipped t-SNAREcell is patch-clamped in the cell-attached configuration with the vNLPsincluded in the pipette solution. Fusion of a vNLP with the cell surfaceproduces a pore connecting the cytosol with the pipette solution, throughwhich direct-currents are measured under voltage clamp. The magnitudeof the current reports pore size with sub-millisecond time resolution(Wu, Z. et al. Sci. Rep. 2016). We found that pore nucleation required aminimum of 2, and reached a maximum above ~4 copies of v-SNAREsper NLP face. In contrast, the mean conductance of single poresincreased as copy number was increased and was far from saturating at15 copies, the NLP capacity. Thus, very different numbers of SNAREcomplexes cooperate at the distinct stages of fusion pore nucleationand pore dilation. We calculated pore size distributions and free energyprofiles versus pore size. Combined with a mathematical model, theseresults suggest crowding of SNARE complexes at the pore waist drivepore expansion.

473-Pos Board B238Imaging Regulatory Lipids and Protein Kinase C at Sites of ExocytosisAdam J. Trexler, Justin W. Taraska.National Heart Lung and Blood Institute, National Institutes of Health,Bethesda, MD, USA.The control of exocytosis is physiologically essential. In vitro SNARE pro-teins are sufficient to drive model membrane fusion, but in cells there areover twenty additional proteins and lipids that work together to drive effi-cient, fast, and timely release of vesicular cargo. Many of these importantaccessory proteins are controlled via phosphorylation, and protein kinaseC (PKC) has previously been linked to the modulation of exocytosis. Weuse total internal reflection fluorescence microscopy to observe the spatio-temporal dynamics of dozens of proteins and lipids relative to single sitesof exocytosis in living endocrine INS-1 cells. INS-1 cells are a modelsystem for insulin secretion. We use a vesicle cargo marker NPY-GFP toidentify where exocytosis occurs and tag proteins-of-interest with a redfluorescent protein. Lipid species are visualized using lipid sensors: fluores-cent proteins fused to protein domains with known specificity for singlelipids. After stimulating exocytosis using ionomycin, we observe a transient

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accumulation of PIP2- and DAG-sensors at exocytic sites centered on thetime of membrane fusion and only lasting a few seconds. Intriguingly, weobserve a concomitant recruitment of conventional PKC isoforms withkinetics broadly similar to PIP2 and DAG. Novel and atypical PKCs donot appear to be recruited. PIP2, DAG, and PKC recruitment only occurat sites of exocytosis and not at control docked-vesicles nearby to sites ofexocytosis. PKC is known to target several critical exocytic proteins,including munc18 and dynamin-1, and we are currently investigatingwhether these targets are phosphorylated in response to the recruitment ofPKC that we observe. These data suggest that a regulatory lipid cascademay recruit and activate PKC to sites of exocytosis to regulate membranefusion or cargo release.

474-Pos Board B239A CMOS based Sensor Array Platform for Analysis of Exocytosis EventsMeng Huang1,2, John C. Ruelas3, Shailendra S. Rathore2,Joannalyn B. Delacruz4, Manfred Lindau2.1Materials Science and Engineering, Cornell University, Ithaca, NY, USA,2Applied Engineering Physics, Cornell University, Ithaca, NY, USA,3Electrical and Computer Engineering, Cornell University, Ithaca, NY, USA,4Field of Pharmacology, Cornell University, Ithaca, NY, USA.The release of neurotransmitters into the extracellular space is an importantdrug target and crucial for molecular manipulation. Amperometric measure-ments provide details about the amount and kinetics of transmitter release insingle quantal events. However, amperometric spikes vary from cell to celleven under the same condition; therefore, a large number of release eventsmust be analyzed to achieve the statistical significance. We present CMOSchips with 100 or 1,024 active electrodes with surface modifications foramperometry measurements. To enable amperometry measurements,polarizable electrodes such as platinum are patterned on the originalaluminum metal contact on the chip. To prevent potential current leakage,a new shifted electrode design was used to enhance the reliability ofpost-fabrication. Platinum electrode material is deposited on the aluminumcontact extending onto the periphery area and further insulated to redefinethe openings of the active electrodes by lithography and sputtering. Theinsulation of the surface is performed by lithography using photoresist.The 100 sensor array was validated by simultaneous recording from allelectrodes at 1MS/s using dopamine injection and recordings from livechromaffin cells cultured on-chip. A new chip with 1024 working electrodescapable of bi-directional current recordings was developed for fast scancyclic voltammetry. Positive as well as negative currents are measured inthe same working manner, and a current mirror and a biased cascode tran-sistor ensure a gain error <1%. A sign detection function is implemented toreport the polarity of the current input. Simulations of the circuit revealedreliable capacitive current recording and the linearity of the gain over awide range of input with the correct sign-bit output. We anticipate thatthe chip will serve as a universal platform for characterizing various phys-iological events.

475-Pos Board B240Miz-2, A New Catecholamine-Selective Fluorescent Sensor for VisualizingNorepinephrineXin A. Liu1, Le Zhang2, Kevin D. Gillis3,4, Timothy E. Glass2.1Dalton Cardiovascular Research Center, University of Missouri-Columbia,Columbia, MO, USA, 2Department of Chemistry, University ofMissouri-Columbia, Columbia, MO, USA, 3Dalton Cardiovascular ResearchCenter; Department of Medical Pharmacology & Physiology, University ofMissouri-Columbia, Columbia, MO, USA, 4Department of Bioengineering,University of Missouri-Columbia, Columbia, MO, USA.

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We have developed a fluorescence-based turn-on molecular sensor (Neuro-Sensor 521) based on a coumarin aldehyde scaffold that allows for the se-lective recognition and sensing of norepinephrine in live and fixedchromaffin cells. Based on NeuroSensor 521, we have now developedMiz-2: a promising member of a new series of catecholamine-selective sen-sors, which has both a functional aldehyde group to bind to primary amines,and other recognition elements to improve selectivity for binding catechol-amine over other amines. The excitation maximum of Miz-2 is near 488 nm.Fluorescence is quenched when the sensor interacts with epinephrine anddopamine whereas the fluorescence increases upon binding with norepineph-rine. We are using adrenal chromaffin cells as a model cell to test fluores-cent catecholamine sensors and sub-populations of cells enriched inepinephrine versus norepinephrine can be isolated upon gradient centrifuga-tion. Norepinephrine-enriched chromaffin cells exhibit distinct fluorescencepunctae by total internal reflection fluorescence (TIRF) microscopy whenloaded with Miz-2 that are consistent with labeling of granules. Upon stim-ulation with a high-Kþ solution, punctae abruptly disappear during TIRFimaging, consistent with granule fusion and loss of dye via exocytosis.Using underlying transparent electrodes, we recorded amperometric spikesconsistent with quantal exocytosis of catecholamine in cells loaded withthe sensor. Experiments are in progress to combine TIRF imaging withamperometric measurements in underlying transparent electrodes to resolvefeatures of norepinephrine release from individual granule. Supported byNIH R01EB020415.

476-Pos Board B241SNARE Protein Structure Altered in Response to pHKara L. Woodbury, Sam K. Zenger, Peter Weitzel, Curtis D. Nelson,Sterling M. Jones, Trey S. Winter, Wade J. Whitt, Ani C. Henriksen,Dixon J. Woodbury.Brigham Young University, Provo, UT, USA.The SNARE proteins SNAP-25, syntaxin, and VAMP play critical roles inneuronal exocytosis by providing the four helical regions (SNARE domains)that form a coiled-coil complex required for neurotransmitter release. Thiscomplex is continually formed and unwound as exocytotic vesicles fuse andare recycled. Using multiple techniques, we observe clear structural changesof these proteins in response to changes in pH, ionic strength, and oxidationstate. Here we focus on pH changes.Using Circular Dichroism to measure the secondary structure of SNAP-25Bin low ionic strength solutions, we observe an increase in alpha-helical struc-ture when pH is lowered from 7 to 4.5, similar to the helical shift observedwhen SNAP-25 forms a complex with syntaxin and VAMP. This shift isnot observed in 150 mM NaF solutions. This conformation change correlateswith our observation that SNAP-25 precipitates when the pH is dropped from7 to 4.5.Using the fluorescent signal from the single tryptophan in SNAP-25B, weobserve a reversible shift in emission wavelength as the pH is increased from7 to 10. The shift has a pK of ~8.8, similar to the expected pKa for the sulfhy-dryl of cysteine. Replacing all four cysteines with serines prevents this shift.The conformational changes observed above may lead to a change in accessi-bility of the cysteines in these SNARE proteins. Consistent with this hypothe-sis, we observe that decreasing the pH from 7 to 5.5 of solutions containingsyntaxin and SNAP-25B varies the number of cysteines accessible for labelingwith a Biotin-maleimide tag technique developed in our lab (Woodbury et al.,2011. Anal. Biochem. 417:165-173). This correlates with the pKa of Histidine,present in both proteins.These data support the hypothesis that the structures of SNAP-25B and syn-taxin are sensitive to changes in pH, which may contribute to modulation ofexocytosis in vivo.

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Calcium Signaling I

477-Pos Board B242Optogenetic Dissection of STIM1 Conformational Switch and Oligomeri-zationGuolin Ma, Lian He, Qian Zhang, Yubin Zhou.Center for Translational Cancer Research, Institute of Biosciences &Technology, Texas A&M University, Houston, TX, USA.Store-operated calcium release-activated calcium (CRAC) channel constitutesa major route for calcium influx in many cell types. To study the impact ofcalcium signals on specific cellular functions, several approaches based onpharmacological tools or photoactivatable ‘caged’ compounds have beendeveloped to manipulate intracellular calcium concentrations, but they oftenlack rapid reversibility and spatial precision. The recently developed optoge-netic tools provide exciting opportunities to remotely and reversibly modulatecell signaling at superior temporal and spatial resolution. Here we report twomajor optogenetic engineering strategies to confer light sensitivities to CRACchannel (termed ‘‘Opto-CRAC’’) to achieve light-operated calcium entry inmammalian cells: i) fusion of light-inducible dimerization/oligomerizationdomain with the cytosolic domain of STIM1 or ii) fusion of a photoswitchLOV2 with SOAR/CAD to mimic CC1-SOAR/CAD intramolecular trapping.In the first strategy, light induced dimerization or oligomerization overcomesthe autoinhibition of STIM1, much like calcium-depletion induced oligomer-ization of the STIM1 luminal domain, thereby exposing the SOAR/CADdomain to gate ORAI1 channels on the plasma membrane. In the secondapproach, STIM1 cytosolic fragments containing SOAR/CAD domain werefused with LOV2. In the dark, LOV2 sterically masks function of the effectordomain. Upon blue light illumination, LOV2 underwent conformationalchanges to unleash the minimal ORAI-activating domain SOAR/CAD toinduce calcium flux through ORA1 channels. The Opto-CRAC system canbe conveniently used to generated user-defined calcium signals to controlcalcium-dependent activities in cells of the immune system. The similar opto-genetic engineering approach can be further extended to study conformationalswitch and oligomerization of other signaling proteins. [This work was sup-ported by grants from the NIH (R01GM112003 to Y.Z.) and the AmericanCancer Society (RSG 129848 to Y.Z.)]

478-Pos Board B243Structural Differences between Cardiac and Skeletal RyanodineReceptorsSonali Dhindwal, Joshua Lobo, Montserrat Samso.Physiology and Biophysics, VCU School of Medicine, Richmond, VA, USA.Cardiac and skeletal muscles have different mechanisms of excitation-contraction coupling that depend on a set of tissue-specific protein isoforms.In heart, ryanodine receptor isoform 2 (RyR2), cardiac dihydropyridine recep-tor (DHPR) and FKBP12.6 together with other proteins couple the plasmalem-mal depolarization to the release of Ca2þ from the sarcoplasmic reticulum (SR),via a calcium-induced calcium release mechanism. In skeletal muscle, RyR1,skeletal DHPR and FKBP12 together with other proteins couple these twoevents via an entirely different mechanism that requires a more direct RyR-DHPR coupling, and different long-range allosterism pathways.While the 3D structure of RyR1 has been solved at near-atomic resolution, the3D structure of RyR2 lags significantly in resolution (currently ~30 A). To startuncovering the different molecular mechanisms of these two main RyR iso-forms, we have determined the 3D structure of RyR2 in complex withFKBP12.6 in the closed state at nanometer resolution. This has enabled build-ing a pseudo-atomic model for RyR2. Comparative analysis between the RyR2and RyR1 isoforms shows significant structural differences localized to specificdomains, but not all structural differences can be explained by primarysequence divergence. These differences are interpreted in the context of thetwo modes of excitation-contraction coupling. The analysis of heterogeneityin the dataset allowed us to further define two conformations representingtwo functional states of RyR2.This work was supported by American Heart Association grant No.14GRNT19660003 and Muscular Dystrophy Association grant No.MDA352845 (to MS).

479-Pos Board B244Increased Density of SERCA Pumps at the Periphery of Cardiac PurkinjeCells after Myocardial InfarctionBruno Stuyvers1,2, Penelope Boyden3, Henk EDJ ter Keurs4, Yunbo Guo2,Wen Dun3, Kazi Haq1, Meleze Hocini2, Michel Haissaguerre2,Olivier Bernus2, Sebastien Chaigne2, Sabine Charron2, Caroline Cros2,Fabien Brette2.

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1Memorial University, St John’s, NL, Canada, 2Electrophysiology and HeartModeling Institute, Bordeaux, France, 3Columbia University, New York,NY, USA, 4Libin Cardiovascular Institute of Alberta, Calgary, AB, Canada.BACKGROUND. The trigger of ventricular tachyarrhythmias after myocar-dial infarction (MI) has been attributed to increased incidence of electrogenicCa2þ waves in cardiac Purkinje cells (Pcells). A change in Ca2þ release chan-nel (CRC) gating properties and overload of intracellular Ca2þ store are 2potential causes of this increase. We analysed through a model of intracellularCa2þ dynamics the spontaneous Ca2þ transients of Pcells after MI. Regionalprotein expression was assessed for potential remodelling associated withischemia.METHODS. Pcells were dispersed enzymatically from dog, pig, sheep, andhuman Purkinje fibers dissected from normal hearts and from hearts withMI. Spontaneous local Ca2þ transients were studied by high resolutionconfocal fluorescence microscopy techniques. Specific antibodies wereused to map proteins involved in Ca2þ release/uptake functions (ERCs,SERCAs).RESULTS. In Pcells of MI heart, the density and firing rate of Ca2þ-releasesites were increased by 20% [Normal: 3.0 5 0.3 sites /25mm (14 scans; 12cells) vs MI: 3.6 5 0.2 sites /25mm (9 cells); P<0.05] and 120%[Normal:0.77 5 0.10 sparks/s (42 sites, 12 cells) vs MI: 1.74 5 0.23 sparks/s (40 sites, 9 cells); P<0.05), respectively. A model fit of Ca2þ-waves in MIPcells revealed 50% increase in amplitude, 25% faster decay, and 35% eleva-tion of Ca2þ-uptake rate. No change was observed in CRC expression. How-ever SERCAs redistribution to the cell periphery was detected in all animalMI models and in human heart with large MI. Peripheral protein expressionand gene expression of SERCA2 showed 4.5 and 2.3 times increases post MI,respectively. Interestingly, no change was observed in regulatory proteinphospholamban.CONCLUSION. An unknown factor associated with myocardial ischemiamediates a dramatic increase in SERCA expression at the Pcell periphery,i.e. wherein the CRCs IP3R and RyR3 are specifically located. We propose amechanistic model of intracellular Ca2þ cycling demonstrating that augmenta-tion of peripheral SERCA activity is sufficient to promote pro-arrhythmic spon-taneous Ca2þ release in Pcells of post-MI human heart.

480-Pos Board B245Functional Effects of the RyR2

R420Q Catecholaminergic VentricularPolymorphic Tachycardia in Mouse CardiomyocytesRiccardo Rizzetto1, Miguel Fernandez-Tenorio2,Alexandra Zahradnikova Jr1, Simona Boncompani3, Elena Marques-Sule1,Yue Yi Wang1, Jean-Pierre Benitah1, Esther Zorio4, Feliciano Protasi3,Ernst Niggli2, Ana M. Gomez1.1UMR-S 1180, Inserm, Chatenay-Malabry, France, 2University of Bern,Bern, Switzerland, 3University G. d’Annunzio, Chieti, Italy, 4Hospital La Fe,Valencia, Spain.Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a geneticdisease characterized by stress-induced syncope and/or sudden death. CPVT1mutations are clustered in three regions: the N-terminal, the central, and theC-terminal domains of the cardiac Ca2þ release channel (type-2 ryanodine re-ceptor, RyR2). We have created a knock-in (KI) mouse model bearing theRyR2

R420Q mutation, which is located in the N-terminal region, found in threefamilies diagnosed with CPVT. Electrocardiographic measurements of wakeanimals showed bidirectional ventricular tachycardia after epinephrine/caffeineinjection in all the KI (heterozygous for RyR2

R420Q) but in none of the WT(wild type) animals, validating the model. Consistent with the in-vivo pheno-type, during action potential (AP) measurement by patch-clamp in the presenceof 100 nM isoproterenol all KI myocytes displayed delayed afterdepolariza-tions (DADs) and triggered activity (TA), while the incidence of pro-arrhythmic events was significantly lower in WT myocytes. AP characteristicsand potassium currents showed only minor differences between KI and WT.We measured ICa, [Ca

2þ]i transients and intra-SR [Ca2þ] by patch-clamp andconfocal microscopy. ICa density was similar between WT and KI cardiomyo-cytes, but the associated [Ca2þ]i transient amplitude was unchanged orenhanced depending on the protocol, as well as its response to beta-adrenergic stimulation. Intact cardiac myocytes showed more Ca2þ sparksoccurrence with a lowered SR Ca2þ threshold to initiate Ca2þ waves. Electronmicroscopy analyses indicate that Ca2þ release sites (or estimates indicate thatcouplons) in KI cardiomyocytes were fewer in number, significantly smallerand, hence, may contain a significantly lower number of RyR2. This morpho-logical changes in couplons could participate to the differences in Ca2þ sparkscharacteristics recorded in KI cells compared to WT. Taken together, we havefound a unique cellular alteration in this N terminal RyR2 mutation at structuraland functional levels.

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481-Pos Board B246Suppression of Arrhythmia by Enhancing Mitochondrial Calcium Uptakein Experimental Models of Catecholaminergic Ventricular TachycardiaMaria K. Schweitzer1, Fabiola Wilting1, Simon Sedej2, Lisa Dreizehnter3,Nathan J. Dupper4, Alessandra Moretti3, Ohyun Kwon4, Silvia G. Priori5,Karl-Ludwig Laugwitz3, Michael Mederos y Schnitzler1,Thomas Gudermann1, Johann Schredelseker1.1LMU M€unchen, Munich, Germany, 2Medial University of Graz, Graz,Austria, 3TU M€unchen, Munich, Germany, 4UCLA, Los Angeles, CA, USA,5IRCCS Salvatore Maugeri Foundation, Pavia, Italy.We have recently identified a critical role of mitochondria to shape intracel-lular Ca2þ signals and to regulate cardiac rhythmicity. Activation of mito-chondrial Ca2þ uptake by efsevin, an agonist of the voltage-dependentanion channel 2 in the outer mitochondrial membrane, restored rhythmiccardiac contractions in a zebrafish cardiac arrhythmia model. Here we inves-tigated the potential of pharmacological activation of mitochondrial Ca2þ

uptake as a novel pharmacological strategy for human cardiac arrhythmiain a translational approach. To this aim we first used a murine model ofryanodine receptor 2 (RyR2)-mediated catecholaminergic polymorphic ven-tricular tachycardia (CPVT). In freshly isolated cardiomyocytes ofRyR2R4496C/WT mice, harboring the human RyR2R4496C mutation associatedwith CPVT, efsevin restricted diastolic Ca2þ sparks and prevented theformation of propagating Ca2þ waves and spontaneous, diastolic actionpotentials. This anti-arrhythmic effect was abolished in the presence ofmitochondrial Ca2þ uniporter (MCU) blocker Ru360 , but could be repro-duced with the MCU activator kaempferol, demonstrating an immediaterole of mitochondrial Ca2þ uptake for the anti-arrhythmic effect ofefsevin. In RyR2R4496C/WT mice both mitochondrial Ca2þ uptake enhancers(MiCUps), efsevin and kaempferol, significantly reduced episodes of ven-tricular tachycardia after catecholaminergic stimulation by a bolus injectionof epinephrine and caffeine in vivo while baseline ECG was unaffected.Finally, we used stem cell-derived cardiomyocytes from a CPVT patientto show efficacy of MiCUps in a human model. Both MiCUps abolishedarrhythmogenic events in human CPVT cardiomyocytes. Our resultsdemonstrate that enhancement of mitochondrial Ca2þ uptake by MiCUpsis a promising pharmacological strategy for treatment and prevention ofCa2þ-triggered arrhythmias, such as CPVT.

482-Pos Board B247Antidepressant Drugs Citalopram and Escitalopram but not ParoxetineInduce Arrythmogenic Sarcoplasmic Reticulum Calcium ReleaseDaniel Blackwell, Bjorn Knollmann.Vanderbilt University, Nashville, TN, USA.The antidepressant citalopram, a selective serotonin reuptake inhibitor(SSRI), has been associated with increased risk of sudden cardiac death.Epidemiological data from the Danish population suggest that inpatients taking citalopram, co-administration of carvedilol reduced suddendeath risk compared to two other beta-blockers, metoprolol and bisoprolol.Since carvedilol is the only beta blocker that suppresses store overload-induced calcium release, we hypothesized that citalopram promotes calciumrelease from sarcoplasmic reticulum (SR) calcium stores. Left ventricularmyocytes were isolated from black 6 mice, permeabilized, and loadedwith the calcium indicator, Fluo-4. Incubation of citalopram or escitalopram(S-enantiomer) for 20 minutes significantly increased calcium wavefrequency and decreased calcium wave amplitude in a dose-dependentmanner. This response was more sensitive to escitalopram, indicatingselectivity for the S-enantiomer in the high nanomolar range. At 30 mM,calcium waves were no longer evident, however cytosolic calciumwas elevated approximately two-fold, indicating possible constitutivecalcium release from the SR stores. Incubation with a different SSRI,paroxetine, did not significantly alter calcium wave frequency oramplitude at concentrations as high as 30 mM. Calcium wave activationby citalopram and escitalopram resembled those seen in a calsequestrinknockout mouse model of catecholaminergic polymorphic ventriculartachycardia (CPVT), suggesting a possible similar mechanism forcitalopram. Pre-incubation of isolated myocytes with 1 mM carvedilol,but not metoprolol or bisoprolol, for 30 minutes significantly reducedcalcium wave frequency and increased SR calcium stores in myocytestreated with citalopram or escitalopram. Taken together, increased calciumleak from SR stores through ryanodine receptor may contribute to theincreased risk of sudden death associated with citalopram. Hence, our invitro findings support the existence of drug-induced CPVT caused by cita-lopram or escitalopram, which can be prevented by co-administration ofcarvedilol.

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483-Pos Board B248The Interplay between Length-Dependent Calcium Affinity of Troponinand X-ROS Signaling on Myoplasmic Calcium Levels in HeartSarita Limbu1, Benjamin L. Prosser2, W. Jonathan Lederer3,M. Saleet Jafri1.1School of Systems Biology and Krasnow Institute for Advanced Studies,George Mason University, Fairfax, VA, USA, 2Department of Physiologyand Pennsylvania Muscle Institute, University of Pennsylvania, Philadelphia,PA, USA, 3Department of Physiology and Center for BiomedicalEngineering and Technology, University of Maryland School of Medicine,Baltimore, MD, USA.The stretching of a cardiomyocyte leads to increased production of reactiveoxygen species and an elevation in ryanodine receptor open probabilitythrough a process termed X-ROS signaling. Stretching also increases thecalcium binding to the myofilaments by elevating troponin C calciumaffinity. Here an integrative experimental and modeling study is pursuedthat explains the interplay of length-dependent changes in troponin’scalcium affinity and stretch activated X-ROS calcium signaling. Thedecreased myoplasmic calcium concentration caused by increased calciumaffinity of troponin during myocyte stretching is offset by increased calciumrelease from the sarcoplasmic reticulum caused by X-ROS signaling. Thecombination of modeling and experiment also help with proper interpreta-tion of the length-dependent changes to the calcium binding affinity oftroponin in the presence of blebbistatin. The model suggests that in orderto explain all the experimental data, the decrease in calcium affinity oftroponin with increasing blebbistatin is accompanied by a reduction andeventual elimination in length dependent differences in calcium affinity.Therefore, this work suggests that X-ROS signaling may serve to maintainfree myoplasmic calcium concentrations during a change in myocyte length.Furthermore it defines the relative contributions of X-ROS signaling and thelength-dependent changes in calcium binding by troponin on myoplasmiccalcium levels.

484-Pos Board B249Mitochondrial Ca2D Uptake and Superoxide Generation RegulatesAngiotensin II-Induced Proliferation in Neonatal Cardiac FibroblastsJin O-Uchi1, Deming Fu2, Jyotsna Mishra2, Bong Sook Jhun1,Shey-Shing Sheu2.1Cardiovascular Research Center, Medicine, Rhode Island Hospital, BrownUniversity, Providence, RI, USA, 2Center for Translational Medicine,Medicine, Thomas Jefferson University, Philadelphia, PA, USA.Cardiac fibroblasts (CFs) are one o the most abundant cell types in theheart and play key roles in regulating myocardial physiological functionand pathophysiological remodeling especially for the cardiac fibrosis. Thelevels of Angiotensin II (AT-II) are increased in the remodeling heart andAngiotensin signaling participates in pathological CF proliferation. It hasbeen shown that CF proliferation may occur via the increased levels ofcellular reactive oxygen species (ROS), but the detailed signal transductionremains unclear. We previously reported that the enhancement of mito-chondrial Ca2þ uptake by mitochondrial Ca2þ uniporter (MCU) inducesmitochondrial superoxide (mtSO) generation in cardiac myofibroblast cellline H9C2 cells. Therefore, we hypothesize that Ang-II stimulation enhancesmitochondrial Ca2þ-induced mtSO generation in primary CFs, which canactivate ROS-dependent proliferation signaling in primary CFs. First, weconfirmed that AT-II (R1 mM) stimulation induces significant mitochon-drial Ca2þ uptake assess by mitochondria-targeted Ca2þ biosensor inresponse to the Ca2þ release from the endoplasmic reticulum inneonatal rat CFs (NCF). In addition, AT-II stimulation increases themtSO levels detected by a mtSO indicator MitoSOX Red. We alsoconfirmed that AT-II application activates proliferative pathway, includingERK1/2, p38 and JNK1/2 in time-dependent manner, which was abolishedby losartan pretreatment. Lastly, pretreatment of a mitochondria-targetedantioxidant, Mito-tempo significantly inhibited AT-II-mediated activationof the mtSO production as well as proliferative pathway withoutchanging the AT-II-induced the mitochondrial Ca2þ uptake profile. Ourresults indicate that mtSO generation induced by mitochondrial Ca2þ accu-mulation via MCU serves as an important regulator for the Ang II-inducedproliferation in CFs.

485-Pos Board B250TRPV4 Enhances Cardiomyocyte Calcium Transients and CardiacContractility Following Hypoosmotic Stress and Ischemia-ReperfusionJohn L. Jones, Deborah Peana, Michelle D. Lambert, Timothy L. Domeier.Medical Pharmacology and Physiology, University of Missouri, Columbia,MO, USA.

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The Transient Receptor Potential (TRP) ion channel family plays an impor-tant role in cardiomyocyte calcium homeostasis, particularly in diseasedstates. Here, we test the hypothesis that the osmotically-activated TRPVanilloid 4 (TRPV4) channel enhances cardiomyocyte calcium transientsin the aged heart following hypoosmotic stress. Expression of TRPV4 waslow in cardiomyocytes of Young (3-6 month) mice, but significantlyincreased in cardiomyocytes of Aged (24-26 month) mice. In cardiomyo-cytes of Aged, hypoosmotic stress (250 mOsm) induced an increase incalcium transient amplitude (F/F0:3.650.1 hypoosmotic versus 2.950.2isosmotic, P<0.05). This effect was prevented by the TRPV4 inhibitorHC067047 (1 mM, F/F0:2.950.1 hypoosmotic) and was absent in cardio-myocytes of Young mice (F/F0:3.250.1 hypoosmotic versus 2.950.2 isos-motic). However, cardiomyocytes of Young mice with cardiac-specificTRPV4 Overexpression exhibited enhanced calcium transient amplitudefollowing hypoosmotic stress (F/F0:3.550.2 hypoosmotic versus 2.450.1isosmotic, P<0.05) that was prevented by HC067047 (F/F0:2.650.1 hypo-osmotic). Ischemia-reperfusion (I-R) injury is a pathological scenario asso-ciated with pronounced osmotic stress on cardiomyocytes. We thereforemonitored left-ventricular pressure development in Langendorff-perfusedhearts of Young, Aged, Young TRPV4 Overexpressor, and Aged TRPV4knock-out mice subjected to 45 minute global ischemia followed by reper-fusion. Prior to ischemia, all hearts exhibited similar contractility (dP/dtMax

range: 19345206 to 2226562 mmHg/s). Following I-R, hearts of Agedmice exhibited enhanced contractile performance (dP/dtMax: 27705180mmHg/s) versus hearts of Aged TRPV4 knock-out mice (dP/dtMax:14005300 mmHg/s, P<0.05). Similarly, hearts of Young TRPV4 Overex-pressor mice exhibited enhanced contractile performance (dP/dtMax:27505180 mmHg/s) versus hearts of Young mice (dP/dtMax: 16505260mmHg/s, P<0.05). In conclusion, TRPV4 enhances calcium transientsfollowing hypoosmotic stress and contributes to hypercontractility followingI-R. Our findings have potential clinical implications in the treatment ofelderly populations at increased risk of myocardial infarction and reperfu-sion injury.

486-Pos Board B251Characterization of Intracellular Sodium Homeostasis in Murine AtrialMyocytesLibet Garber1, W. Jonathan Lederer2, Maura Greiser2.1Bioengineering, University of Maryland College Park, College Park, MD,USA, 2Center for Biomedical Engineering and Technology and Departmentof Physiology, University of Maryland School of Medicine, Baltimore,MD, USA.Intracellular sodium concentration ([Naþ]i) is an important regulator ofintracellular Ca2þ and provides insight into the activation of the sarco-lemmal sodium calcium exchanger (NCX) and the behavior of both Naþ

channels and the Naþ,Kþ-ATPase. In atrial diseases such as atrial fibrilla-tion (AF), intracellular Ca2þ signaling is profoundly altered. While themechanisms underlying altered intracellular Ca2þ homeostasis are wellcharacterized, the role that [Naþ]i may play and its dysregulation in diseasein atrial myocytes is less well understood. For example, we have previouslyshown that high rate (10 Hz) atrial activation for 5 days in a rabbit modelleads to an expected but significant reduction in [Naþ]i (Greiser et al, JCI,2014; 124: 4759-72).Here we characterize intracellular Naþ homeostasis in murine atrial myo-cytes using the Naþ indicator SBFI (Molecular Probes) in quiescent cells.Dual UV excitation (340 nm and 380 nm) was provided by a rapid switchingSutter DG5-plus illuminator using a 300 W xenon light source with wide-field imaging by an EMCCD camera (Princeton Instruments). Freshly iso-lated atrial myocytes were loaded with 10 mmol/l SBFI-AM for 90 min inthe presence of the non-ionic surfactant Pluronic F-127 (0.05 %w/v). Afterwashout of the extra cellular dye, SBFI-AM de-esterification was allowedfor 20 minutes before proceeding with [Naþ]i measurements. Fluorescenceimages (F340 and F380) were collected at 510 5 40 nm. The calibrationof [Naþ]i was performed in each cell separately after the measurements ofquiescent [Naþ]i by exposing the SBFI-loaded myocytes to various extracel-lular [Naþ] ([Naþ]o) in the presence of 10 mmol/l gramicidin D and100 mmol/l-strophanthidin. Resting [Naþ]i in mouse atrial myocytes was8.2 5 0.6 mM. External field stimulation (4 ms, 20 V, 1 Hz) increased[Naþ]i by 3.1 5 0.2 mM. These data show that [Naþ]i in murine atrial my-ocytes is lower than in murine ventricular myocytes (12 5 1 mM, Correllet al, Circ Res, 2014;114:249-56). These data and more will provide a broadunderstanding of atrial Naþ homeostasis and its implication for atrial myo-cyte Ca2þ signaling.

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487-Pos Board B252Genetic Ablation of Fibroblast Mitochondrial Calcium Uptake IncreasesMyofibroblast Transdifferentiation and Exacerbates Fibrosis in Myocar-dial InfarctionAlyssa A. Lombardi, Ehtesham Arif, Timothy S. Luongo, John W. Elrod.Temple University School of Medicine, Philadelphia, PA, USA.When the heart is injured, fibroblasts transition from a structural role into con-tractile, synthetic myofibroblasts. This is crucial for scar formation aftermyocardial infarction to prevent ventricular rupture, but excessive fibrosis ismaladaptive and leads to heart failure. While intracellular calcium (iCa

2þ)elevation has been shown to be necessary for myofibroblast transdifferentia-tion, the mitochondrial calcium (mCa

2þ) domain has not been explored. Mcuencodes the channel-forming portion of the mitochondrial calcium uniportercomplex and is required for acute mCa

2þ uptake. We generated a conditional,fibroblast-restricted Mcu knockout mouse by crossing Mcufl/fl mice with amouse expressing a tamoxifen-inducible Cre recombinase under control ofthe collagen1a2 promoter (fibroblast Mcu-cKO). Fibroblast Mcu-cKO miceand controls were subjected to ligation of the left coronary artery and cardiacfunction was examined weekly by echocardiography. Deletion of fibroblastMcu worsened left ventricular dysfunction and increased fibrosis. To examinecellular mechanisms responsible we isolated mouse embryonic fibroblasts(MEFs) from Mcufl/fl mice and deleted Mcu using Cre-adenovirus. Whenchallenged with pro-fibrotic ligands, Mcu-/- MEFs exhibited decreased mCa

2þ

uptake and enhanced iCa2þ transient amplitude. Mcu-/- MEFs displayed

enhanced myofibroblast transdifferentiation as evidenced by decreased mi-gration and proliferation as well as increased a-SMA expression and gelcontraction. Metabolically, Mcu-/- MEFs display a shift towards more glycol-ysis with less oxidative phosphorylation, correlating with increased phosphor-ylation (inactivation) of the pyruvate dehydrogenase subunit E3. These resultssuggest that alterations in mitochondrial buffering of pro-fibrotic iCa

2þ

signaling and changes in energetic pathways may be mechanisms driving my-ofibroblast transdifferentiation and fibrosis.

488-Pos Board B253Gain-of-Function Mutation in Ryanodine Receptor Type 1 Modulates Mu-rine Thymocyte Calcium Signaling and Autoimmune Response in MiceLukun Yang, Athena Soulika, Lillian Cruz-Orengo, Paul D. Allen,Alla F. Fomina.Univ. California, Davis, Davis, CA, USA.Gain-of-function mutations in gene encoding ryanodine receptor type 1(RyR1), an endo/sarcoplasmic reticulum Ca2þ release channel, are linked toa variety of skeletal muscular disorders. Other tissues, including cells of theimmune system, have been shown to express RyR1. A previous study demon-strated enhanced immune responses in mice with gain-of-function RyR1 muta-tion (Vukcevic et al., 2013, J Cell Sci, 126: 3485-92). Here we report that afterinduction of an experimental autoimmune encephalomyelitis (EAE), a T cell-mediated autoimmune disease, the mice carrying gain-of-function RyR1R163C mutation (R163C mice) displayed an earlier disease onset and augmen-tation of neurological symptoms compared with wild-type (WT) mice. In vitrostudies revealed that splenocytes from the R163C mice have altered Ca2þ

signaling compared with those from WT mice, which was revealed by chal-lenging the cells with different concentrations of extracellular Ca2þ andCa2þ mobilizing agent thapsigargin. No differences were found in the relativeabundances of immune cell subtypes in spleens and lymph nodes from R163Cand WT mice prior to the onset of the EAE. Nevertheless, immunostaining ofthe lumbar spinal cord sections after the onset of the clinical symptoms, re-vealed the larger pro-inflammatory infiltrate and axonal damage in theR163C mice compared with the WT mice, indicating that R163C mutationmay affect immune cell trafficking to the CNS. These data indicate that alteredCa2þ handling in R163C mice immune cells may enhance these cells autoim-mune responses and that gain-of-function mutations in RYR1 may represent apreviously unknown risk factor for autoimmune diseases.

489-Pos Board B254Regulation of Cardiac Pacemaker Activity by PDE4 IsoformsDelphine Mika1, Ana Maria Gomez1, Marco Conti2,Rodolphe Fischmeister1, Gregoire Vandecasteele1.1INSERM UMR-S 1180, Chatenay-Malabry, France, 2ObGyn departmentUniversity of California San Francisco, San Francisco,CA, USA.Background Numerous epidemiological and clinical studies have revealed apositive correlation between heart rate (HR) and cardiovascular morbimor-tality. The autonomic nervous system is the major extracardiac determinant

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of HR. During sympathetic stimulation, the activation of b-adrenergic re-ceptors (bAR) induces an increase in cAMP levels, leading to a positivechronotropic effect. Among the 5 cAMP-PDE families expressed in theheart, PDE4 is critical for controlling excitation-contraction coupling(ECC) during bAR stimulation in atrial and ventricular cells. PDE4 mayalso be important for automaticity. 3 genes encode for cardiac PDE4s:pde4a, pde4b and pde4d. Their respective contribution to the regulation ofpacemaker activity remains ill-defined. Methods The total enzymatic PDEactivity was determined in mouse sinoatrial node (SAN) tissue as thecAMP hydrolytic activity measured in the absence of PDE inhibitor andthe fraction corresponding to PDE4 activity was assessed by including thePDE4 inhibitor Ro-20-1724 (10 mM). The in vitro pacemaker activity wasassessed by measuring the spontaneous Ca2þ transients in Fluo4-loaded-SAN intact tissue. Images were obtained using confocal microscopy.Results Ro-20-1724 increased the beating rate of intact mouse SAN andincreased PKA-phosphorylation levels of key ECC actors (ryanodine recep-tor, phospholamban). PDE4 enzymatic activity was found to account for60% of the total cAMP-PDE activity in SAN. The 3 isoforms PDE4A, 4Band 4D were found to be expressed in mouse SAN. In PDE4D-, but notin PDE4B-deficient mice, Ca2þ homeostasis was altered in control condi-tions and after bAR stimulation. Indeed, ablation of PDE4D induced adecreased beating rate and an increased Ca2þ spark frequency in controland bAR-stimulated conditions. Conclusion Our preliminary results revealthat PDE4 controls pacemaker function in mice and that PDE4D ablationstrongly perturbs normal SAN activity.

490-Pos Board B255Functional Role of TRPC1 Channels in Neonatal CardiomyocytesAhmad A. Azmi, Chris Hunter, Hu Qinghua, Frank B. Sachse.CVRTI, University of Utah, Salt Lake City, UT, USA.Transient receptor potential cation (TRPC) 1 channels are thought to play arole in store-operated and receptor-operated calcium entry in variousmammalian cell types. However, our understanding of the functional roleof these channels in cardiomyocytes remains to be ill-defined. Here, wetest the hypothesis that the channels are involved in calcium leak fromthe sarcoplasmic reticulum (SR) and modulate the cytosolic calciumconcentration in cardiomyocytes. Our studies were performed on neonatalrodent ventricular myocytes. After cell isolations, cells were plated on glassslides. Two days after isolation cells were infected with adenoviral vectorsof TRPC1 tagged with eGFP. Measurements were performed 4-5 days afterinfection. Immunolabeling, three-dimensional scanning confocal micro-scopy and quantitative colocalization analysis revealed an abundant intracel-lular density of native TRPC1 and TRPC1 expressed via adenoviral vectors.TRPC1 was not associated with the sarcolemma, but the SR. We measuredthe rest decay and caffeine induced peak calcium release using rapidscanning confocal microscopy on infected cells loaded with the calciumsensitive dye Rhod-3. We found an increased SR calcium content in thepresence of the TRPC channel blocker SKF-96365. SR calcium contentexhibited a decreasing relationship with TRPC1 expression. In a computa-tional model, activated SR TRPC1 channels increased the systolic anddiastolic cytosolic calcium concentration with only minor effects onaction potential and SR calcium content. Our studies indicate that TRPC1channels are not involved in sarcolemmal electrophysiology of rodentventricular myocytes, but localized in the SR. The studies support ourhypothesis that the channels play a role as SR calcium leak channels. Thefindings could guide us to an understanding of TRPC channels as physiolog-ical modulators of intracellular calcium and contractility in cardiacmyocytes.

Excitation-Contraction Coupling

491-Pos Board B256Probing the Inter-Subunit/Subdomain Interactions Relevant to DiseaseMutations in the N-Terminal Domain of Ryanodine Receptors by Molecu-lar Dynamics SimulationWenjun Zheng.SUNY at Buffalo, Buffalo, NY, USA.The ryanodine receptors (RyR) are essential to calcium signaling in skeletaland cardiac muscles, and numerous disease mutations have been found intwo RyR isoforms (RyR1 and RyR2). A deep understanding of the activa-tion/regulation mechanisms of RyRs has been hampered by the shortage ofhigh-resolution structural and dynamic information for this giant tetramericcomplex in different functional states. Toward elucidating the molecular mech-anisms of disease mutations in RyRs, we performed molecular dynamics simu-

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lation of the N-terminal domain (NTD) which is not only the best resolvedstructural component of RyRs, but also a hotspot of disease mutations.First, we simulated the tetrameric NTD of wild-type RyR1 and three diseasemutants (K155E, R157Q, and R164Q) that perturb the inter-subunit interfaces.Our simulations identified a dynamic network of salt-bridges involvingcharged residues at inter-subunit/subdomain interfaces and disease-mutationsites. By perturbing this key network, the above three mutations result ingreater flexibility and unstable inter-subunit interfaces that favour channelopening.Next, we simulated the monomeric NTD of RyR2 in the presence or absence ofa central Cl anion which is known to stabilize the interfaces between the threeNTD subdomains (A, B, and C). We found that the loss of Cl restructures thesalt-bridge network near the Cl-binding site, leading to rotations of subdomainA/B relative to subdomain C and enhanced mobility between the subdomains.This finding supports a mechanism for some disease mutations in the NTD ofRyR2 via perturbation of the Cl binding.The structural and dynamic insights gained from this study will guide futurefunctional/mutational studies of the NTD of RyRs.

492-Pos Board B257Genotype-Phenotype Correlations of the Central Core Disease Mutationsin the C-Terminal Region of the RyR1 ChannelTakashi Murayama1, Nagomi Kurebayashi1, Haruo Ogawa2,Toshiko Yamazawa3, Takashi Sakurai1.1Department of Pharmacology, Juntendo University School of Medicine,Tokyo, Japan, 2Institute of Molecular and Cellular Biosciences, TheUniversity of Tokyo, Tokyo, Japan, 3Department of Molecular Physiology,Jikei University School of Medicine, Tokyo, Japan.Central core disease (CCD) is a congenital myopathy and has been linked tomutations in the type 1 ryanodine receptor (RYR1), a Ca2þ release channelof the sarcoplasmic reticulum. CCD has been classified into two groups interms of susceptibility to malignant hyperthermia (MH), i.e., MH/CCDand CCD alone. MH/CCD is frequently found in the cytoplasmic regions,and CCD alone mutations mostly reside in the C-terminal region. Functionalanalyses have proposed that MH/CCD is caused by gain-of-function of thechannel, whereas loss-of-function of the channel leads to the CCD alonephenotype. However, how specific mutations cause different phenotypes re-mains largely unknown. In this study, we investigated the channel activity of16 mutations (14 CCD alone and 2 MH/CCD) in the C-terminal region usinga heterologous expression system in HEK293 and myogenic C2C12 cells.Ca2þ-induced Ca2þ release (CICR) activity was evaluated with HEK293cells by caffeine-induced Ca2þ release, cytoplasmic Ca2þ ([Ca2þ]i) andER luminal Ca2þ ([Ca2þ]ER) measurements, and [3H]ryanodine bindingassay. MH/CCD mutants consistently exhibited a gain-of-functioneffect; an enhanced sensitivity to caffeine, a reduced [Ca2þ]ER, anincreased [Ca2þ]i and enhanced [3H]ryanodine binding. In contrast, CCDalone mutations showed divergent CICR phenotypes: gain-of-function,loss-of-function and no appreciable change. Effect of these mutations onthe excitation-contraction coupling is now in progress using C2C12 myo-tubes. Plausible mechanisms of how these mutations alter the channel gatingwill be discussed based on recent near-atomic structures of the RYR1channel.

493-Pos Board B258The Role of TRIC Channels in SR Countercurrent during SR Ca2D

Release and SERCA Re-UptakeVilmos Zsolnay, Claudio Berti, Michael Fill, Dirk Gillespie.Molecular Biophysics and Physiology, Rush University Medical Center,Chicago, IL, USA.In heart, the release of Ca2þ from the SR that generates sparks or AP-driventransients requires countercurrent to prevent large SR membrane potentialchanges and maintain normal excitation/contraction coupling. SR Kþ

channels (TRIC channels) have been proposed to be an important counter-current pathway during SR Ca2þ release. It has separately been proposedthat TRIC channels conduct countercurrent during diastole for SERCA re-uptake of Ca2þ. To probe the dependence of countercurrent on the numberand location of TRIC channels in the SR, we used an equivalent circuitmodel of a compartmentalized SR and the surrounding cytosol to simulatethe cycling of ions into and out of the SR during a heartbeat. We alteredthe total number of TRIC channels at heart rates ranging between 60 and600 bpm. Our results agree with experiments that show removal of half ofthe SR TRIC channels is not fatal in mice, whereas total removal is. Inour simulations, there is no physiological difference between having100% or 50% the normal number of TRIC channels, but if there were

735


0 TRIC channels, the model SR generated non-physiological a diastolicmembrane potential (~20 mV). We also explored the dependence ofcountercurrent on the location and distribution of TRIC channels on thejunctional SR (JSR)-sublemmal, JSR-cytosol, and nonjunctional SR(NSR)-cytosol membranes. The only qualitative difference occurred whenthe TRIC complement of the NSR-cytosol membrane (SERCA pumplocation) was varied. Overall, we found that TRIC channels provide coun-tercurrent both during SR Ca2þ release and SERCA uptake.

494-Pos Board B259A Method for Validating Mutations Associated with Malignant Hyper-thermia using CRISPR/Cas9 and Dual Integrase Cassette ExchangeKevin J. De Leon1, Shane Antrobus2, Paul Denney Allen2, Isabelle Marty1,David Segal1.1Genome Center, University of California, Davis, Davis, CA, USA, 2VMMolecular Biosciences, University of California, Davis, Davis, CA, USA.Malignant Hyperthermia (MH) is a pharmacogenetic disorder characterizedby a hypermetabolic skeletal muscle response to volatile anesthetics. Whilethere are hundreds of mutations in the Ryanodine Receptor 1 gene (RyR1)associated with MH, the current diagnostic protocol has only confirmed 35mutations to be causative. This study aims to develop a more efficientmethod for validating mutations. The CRISPR/Cas9 system has emergedas an efficient and targetable gene-editing tool used to assist inhomology-driven repair (HDR) for the insertion of donor templates in vitro.Serine integrase site-specific recombination offers a more efficient methodof gene editing but requires specific recognition sites. In this study, weuse CRISPR/Cas9 and Dual Integrase Cassette Exchange (DICE) to createRyR1-mutant cell lines. Using calcium imaging, myotubes consisting ofdifferentiated myoblasts are tested for their sensitivity to the calcium-releasing triggers KCl, caffeine, and 4-chloro-m-cresol. The sensitivitiesof RyR1-mutant myotubes are compared to wild-type myotubes. An in-crease in sensitivity is indicative of MH and is used to classify the mutationsas likely causative. The study is performed on primary murine myoblaststhat have been immortalized via lentiviral transduction of cyclin-dependent kinase 4 (CDK4). We have designed and tested guide RNAs(gRNAs) used in the CRISPR/Cas9 system, identifying three gRNAs withDNA-cleavage efficiencies of 25-30%. The combined use of CRISPR/Cas9 and DICE allows for the efficient and rapid creation of RyR1-mutant cell lines in which the mutations can subsequently be functionallyvalidated. In doing so, we hope to expedite the validation ofMH-associated mutations for the purpose of assisting the development ofa genetic screen. In addition, our methods can be applied to other regionsof the genome to create an improved, high-throughput method of genomicengineering.

495-Pos Board B260Role of STIM1 And Orai1 in the Formation of Tubular Aggregates inAgeing Skeletal Muscle FibersClaudia Pecorai, Antonio Michelucci, Laura Pietrangelo, Feliciano Protasi,Simona Boncompagni.University G. D’Annunzio, Chieti, Italy.Tubular aggregates (TAs), ordered arrays of sarcoplasmic reticulum (SR)tubes, form in ageing fast-twitch fibers of mice, preferentially in males.TAs are also found in biopsies from patients affected by TA Myopathy(TAM), a muscle disorder linked to mutations in STIM1 and Orai1 (proteinsinvolved in store-operated Ca2þ entry, SOCE) We have previously shownusing Electron Microscopy (EM) that tubes of TAs appear linked by smallbridges, resembling aggregated STIM1 molecules. Here, we comparedextensor digitorum longus (EDL) muscles from 2 groups of mice(~4 months, adults and R24 months, aged) to determine: a) presence ofSTIM1 (and Orai1) in TAs; b) the relative contribution of Ca2þ entry tomuscle function during repetitive stimulation in ageing muscle. Immunoflu-orescence indicates that ageing causes STIM1 (a SR protein), but notOrai (that resides in TTs) to accumulate in TAs. This finding is consistentwith: a) EM data showing that TTs are rarely seen in TAs; andb) western-bot analysis showing increased expression levels of bothSTIM1 splicing variants (arbitrary units: STIM1-short=0.8750.09 vs.1.1850.04; STIM1-long=0.8150.1 vs. 1.1150.05, respectively adult vs.aged). During a repetitive stimulation protocol (30 x 1s-60Hz pulses every5 seconds): i) in 2.5 mM Ca2þ external solution, EDL muscles from agedmice exhibit a decreased capability to maintain contractile (relative forceafter 10 tetani in adult vs. aged: 61.053.2%, and 49.952.4%); ii) inCa2þ-free external solution, aged EDL muscles display a lower decay in

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contractile force (relative force after 10 tetani in adult vs. aged:41.053.1%, and 53.451.5%). Our results indicate that TAs a) accumulateSTIM1, which is likely visible as electron-dense strands between tubes;b) while contain STIM1, do not functionally contribute to Ca2þ entry duringrepetitive stimulation.

496-Pos Board B261Mitsugumin 53 Regulates Extracellular Ca2D Entry and IntracellularCA2D Release via Orai1 and RyR1 in Skeletal MuscleMi Kyoung Ahn1, Keon Jin Lee1, Chuanxi Cai2, Mei Huang1,Chung-Hyun Cho3, Jianjie Ma4, Eun Hui Lee1.1Dept. of Physiology, College of Medicine, The Catholic Univ. of Korea,Seoul, Korea, Republic of, 2Center for Cardiovascular Sciences, Departmentof Molecular and Cellular Physiology, Albany Medical College, Albany, NY,USA, 3Dept. of Pharmacology, College of Medicine, Seoul NationalUniversity, Seoul, Korea, Republic of, 4Dept. of Surgery, Davis Heartand Lung Research Institute, The Ohio State University, Columbus,OH, USA.Mitsugumin 53 (MG53) participates in the membrane repair of variouscells, and skeletal muscle is the major tissue that expresses MG53. Exceptfor the regulatory effects of MG53 on SERCA1a, the role(s) of MG53 inthe unique functions of skeletal muscle such as muscle contraction havenot been well examined. Here, a new MG53-interacting protein, Orai1, isidentified in skeletal muscle. To examine the functional relevance of theMG53-Orai1 interaction, MG53 was over-expressed in mouse primary orC2C12 skeletal myotubes and the functional properties of the myotubeswere examined using cell physiological and biochemical approaches. ThePRY-SPRY region of MG53 binds to Orai1, and MG53 and Orai1 are co-localized in the plasma membrane of skeletal myotubes. MG53-Orai1interaction enhances extracellular Ca2þ entry via a store-operated Ca2þ en-try (SOCE) mechanism in skeletal myotubes. Interestingly, skeletal myo-tubes over-expressing MG53 or PRY-SPRY display a reduced intracellularCa2þ release in response to Kþ-membrane depolarization or caffeine stimu-lation, suggesting a reduction in RyR1 channel activity. Expressions ofTRPC3, TRPC4, and calmodulin 1 are increased in the myotubes, andMG53 directly binds to TRPC3, which suggests a possibility that TRPC3also participates in the enhanced extracellular Ca2þ entry. Thus, MG53could participate in regulating extracellular Ca2þ entry via Orai1 duringSOCE and also intracellular Ca2þ release via RyR1 during skeletal musclecontraction.

497-Pos Board B262Localization of Junctophilin-1 at the Junctional Sarcoplasmic ReticulumRequires a Sequence in the Transmembrane DomainDaniela Rossi1, Angela Maria Scarcella1, Stefania Lorenzini1, Enea Liguori1,Mirko Messa2, Pietro De Camilli2, Vincenzo Sorrentino1.1Molecular and Developmental Medicine, University, Siena, Italy,2Departments of Neuroscience and Cell Biology, Yale University School ofMedicine, New Haven, CT, USA.The endoplasmic reticulum of striated muscle cells, called sarcoplasmic re-ticulum (SR), is mainly dedicated to Ca2þ homeostasis and regulation ofmuscle contraction. The SR is organized in longitudinal (l-SR) and junc-tional domains (j-SR). In skeletal muscle, this latter domain is closely asso-ciated with sarcolemmal invaginations, the T-tubules, to form specificjunctions called triads, where proteins regulating the excitation-contraction coupling mechanism assemble. Junctophilin-1 (JPH1) proteintethers the membrane of the T-tubules with those of the j-SR. This interac-tion is mediated by eight phospholipid-binding modules (MORN) in theN-terminus and by a trans-membrane domain (TMD) in the C-terminus ofJPH1. The mechanisms that regulate targeting of JPH1 at triads arecurrently unknown. When expressed in muscle cells, GFP-JPH1 proteinwas localized selectively only to triads. Deletion of the TMD resulted inde-localization of this GFP-JPH1 deletion mutant on the surface sarcolemmaand on T-tubules. Phospholipase C-mediated PI(4,5)P2 hydrolysis decreasedthe association of the TMD-deleted JPH1 with the surface sarcolemma andT-tubules, indicating that MORN motifs bind PI(4,5)P2, but this interactionis not sufficient to selectively direct localization of JPH1 at triads. Incontrast, progressive deletion of the cytosolic sequence of JPH1 restrictedthe region for triadic localization to the TMD of JPH1. Fusion of theTMD of JPH1 to SEC61ß relocated this Sec61ß-JPH1TMD fusion proteinto triads. Finally, site-directed mutagenesis of selected residues of JPH1TMD identified a short amino acid sequence required for triadic localizationof JPH1.

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498-Pos Board B263Interaction of Junctophilins and the C-Terminus of Cav1.1 SubunitsRegulates Localization and Function of L-Type Calcium Channels inSkeletal MuscleTsutomu Nakada, Toshihide Kashihara, Mitsuhiko Yamada.Shinshu University School of Medicine, Matsumoto, Nagano, Japan.Junctophilins (JPs) are known to contribute to the stabilization of the junc-tional membrane (JM) complex by bridging the plasma membrane andsarcoplasmic reticulum; however, the role of JPs on the JM-targeting andfunction of L-type calcium channels is still unclear. Thus, we exploredstructural and functional consequences of JP knockdown (KD) inC2C12 and GLT myotubes. JP1 or JP2 KD significantly inhibited theJM-targeting of L-type calcium channels whereas JP2 but not JP1 KDsignificantly decreased the current density of L-type calcium channels. Cal-cium imaging assay showed that JP1 or JP2 KD significantly decreased thenumber of myotubes exhibiting calcium transient in response to electricalstimulation. Co-immunoprecipitation study showed that CaV1.1 interactedwith JP1 and JP2 in mouse skeletal muscle. Pull down assay withGST-fusion proteins bearing cytosolic regions of CaV1.1 indicated that theproximal C-terminus of CaV1.1 is necessary for the binding with JPs. ThisJP binding domain (JPD) was well conserved between CaV1.1 and cardiacCaV1.2, which also targeted into JM when expressed in GLT myotubes.However, JPD was not conserved in neuronal CaV2.1, which did notlocalize into JM when expressed in GLT myotubes. Reduced binding ofthe GST fusion proteins to JPs were observed by alanine substitutionsof several residues in JPD. We introduced same alanine substitutionsin JPD of CaV1.1, and transiently expressed it in GLT myotubes.Immunocytochemical analysis revealed that the JM-targeting rate of theJPD-mutated CaV1.1 was significantly reduced compared to the wildtype. These results suggested that interaction of CaV1.1 with JPs via JPDis important for the proper localization and function of L-type calciumchannels.

499-Pos Board B264Dynamics of Triad OrganizationMuriel Sebastien, Perrine Teyssier, Julie Brocard, Eric Denarier,Isabelle Marty, Julien Faure.Grenoble Insitut des Neurosciences, Inserm U1216, Grenoble, France.Skeletal muscle contraction results of massive intracellular calcium release af-ter stimulation. This calcium release is mediated by the calcium release com-plex (CRC) and occurs in a very specific sub-compartment of muscle cells, thetriads. Triads are formed by the close apposition of two sarcoplasmic reticu-lum (SR) terminal cisternae (TC) on both sides of an invagination of theplasma membrane, the transverse-tubule. All CRC proteins are exclusivelylocalized at the triad, but the molecular mechanisms leading to their trafficand localization at the triads are so far unknown. Among CRC proteins, tria-din was proposed to act as a triad anchor for the other reticulum proteins. Toinvestigate the mechanisms leading to triad protein targeting and to the orga-nization of triad membranes, we have recorded movements of fluorescent chi-meras of triadin expressed in primary myotubes from triadin KO mice. Themobility of GFP-triadin was recorded during cell differentiation. Althoughimmobile at late differentiation stages, GFP-triadin clusters can move atearlier stages. These movements require intact microtubule cytoskeleton andmay be necessary for triad organization at the A-I junctions along cell differ-entiation. What these moving structures precisely are is still under investiga-tions. A photoactivatable version of triadin was used to study the dynamics oflimited pools of activated molecules. At both early and late myotube differen-tiation stages, a continuous diffusion of triadin molecules in the whole SR wasrevealed. The progressive accumulation of triadin in the TC was also evi-denced, and we have shown that it depends on its C-terminal luminal domain.Overall, triadin clusters move along microtubules during early differentiationstage probably to reach their final localization along the sarcomeres. Duringall observed differentiation stages, a continuous diffusion of triadin allowsits traffic to the TC. Yet its retention is mediated by triadin C-terminaldomain.

500-Pos Board B265Assessment of Ca2D Sensitivity of SK Channels in Rat VentricularCardiomyocytes using Intrinsic CA2D Cycling MachineryIuliia Polina, Radmila Terentyeva, Karim Roder, Gideon Koren, Jin O-Uchi,Dmitry Terentyev.Cardiovascular Research Center, Rhode Island Hospital, Providence, RI,USA.Small conductance Ca2þ activated Kþ (SK) channels are thought to playsignificant roles in ventricular arrhythmias. It has been hypothesized that

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increase in Ca2þ sensitivity is the main underlying cause of functional up-regulation of SKs in diseased hearts. We developed the protocol to assessCa2þ sensitivity using simultaneous recordings of currents with intracellularCa2þ in voltage clamped cardiomyocytes. By converting Rhod-2 fluores-cence into [Ca2þ]i we estimated Ca2þ sensitivity of rat SK2 channels over-expressed in cells using Adenovirus gene transfer. We obtained apparentaffinity of rSK2 ~ 500 nM which is in consistence with published values.To test whether SK channels from diseased hearts are more sensitive toCa2þ we used rat model of cardiac hypertrophy induced by thoracic aorticbanding (TAB). In contrast to Shams, myocytes from TAB hearts exhibitedsignificant prolongation of APD upon application of SK inhibitor UCL-1684(1 mM). Voltage clamp experiments showed SK inhibitor-sensitive currentwith amplitude ~1 pA/pF. Simultaneous measurements of caffeine-inducedISK (10 mM) and [Ca]i at �30 mV before and after application of UCL-1684 demonstrated that sensitivity of SKs in TAB myocytes is low (Kd~900 nM). This result suggests that sensitivity to [Ca2þ]i in hypertrophyis rather reduced than increased. To explore potential mechanisms respon-sible for changes in SK sensitivity to [Ca2þ]i we treated cultured rat myo-cytes overexpressing SK2 with Phenylephrine (100 mM) and Propranolol(1 mM). We found that SK2 Ca2þ dependence in Phe/Pro was shifted tothe right (Kd ~850 nM), which was preventable by coexpressing CRNK,an inhibitory peptide for ROS and Ca2þ-dependent tyrosine kinase Pyk2.Western blot analysis demonstrated that Pyk2 levels were upregulated inTABs. These data implicate Pyk2 as a novel negative regulator of SKCa2þ sensitivity in diseased hearts.

501-Pos Board B266Modified Calcium Homeostasis in Aged Mouse Skeletal MuscleLaszlo Csernoch, Janos Fodor, Dana Al-Gaadi, Tamas Czirjak, Tamas Olah,Beatrix Dienes, Peter Szentesi.Department of Physiology, University of Debrecen, Debrecen, Hungary.In aging decreased physical activity and reduced muscle mass (sarcopenia)leads to impaired muscle force and increased fatigability accompanied by adecline in sarcoplasmic reticulum (SR) calcium release. As an essential traceelement selenium plays a significant role in muscle functions, as in seleniumdeficiency skeletal muscle disorders manifesting in muscle pain, fatigue,proximal weakness, and serum creatine kinase elevation could develop.Here in vivo physical activity of control and myostatin deficient (Cmpt)mice during 22 months were examined. Their performance in grip and volun-tary wheel tests reached its maximum 3 month after birth and declined after10 month. This decrease was faster in Cmptmice as the average daily runningdistance decreased to 37% in control and to 25% in Cmpt mice in old age.In vitro force was measured on soleus and extensor digitorum longus(EDL) muscles from 20-month-old control, Cmpt, and selenium supple-mented mice. Albeit the absolute force was higher in Cmpt than in controlmice, after normalization to cross section EDL was significantly stronger inthe latter (4.7050.88 vs 7.6150.96 mN/mm2, respectively). Seleniumsupplementation significantly increased the maximal force of EDL(10.8950.60 mN/mm2). Changes in intracellular calcium concentrationwere measured on enzymatically isolated intact flexor digitorum brevis mus-cle fibers using Fura-2. The rate of KCl depolarization-evoked SR calciumrelease was greater in selenium supplemented than in control animals(6915100 vs 481533 mM/s, respectively). Western-blot analysis revealedno change in the expression of the dihydropyridine receptor in the threeanimal groups while that of the ryanodine receptor declined with aging whichwas reversed by long-term training. Our results support the positive effects ofselenium and training on SR calcium release in old age associated muscleweakness. On the other hand, the increased muscle mass of Cmpt miceduring their lifespan doesn’t improve their physical performance in oldage. Supported by: NKFIH-K-115461

502-Pos Board B267Shear Stress Induces Transverse Ca2D Waves via Autocrine Activation ofP2X Purinoceptors in Rat Atrial MyocytesJoon-Chul Kim, Sun-Hee Woo.College of Pharmacy, Chungnam National University, Daejeon, Korea,Republic of.Atrial myocytes are exposed to high shear stress during blood regurgitationand high intra-atrial pressure due to valve diseases and heart failure, sincesuch disturbances disrupt endocardium. We have previously reported thatshear stress induces two types of global Ca2þ waves in atrial myocytes, lon-gitudinal and transverse Ca2þ waves (T-waves) (Biophys J 2012;102(3, Suppl 1);227a), and that the longitudinal wave is triggered by Ca2þ

release via P2Y1 purinoceptor-inositol 1,4,5-trisphosphate receptor signaling(J Physiol 2015;593:5091-5109). Here, we investigated cellular mechanism

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for the generation of T-wave in atrial cells under shear stress. Shear stress of~16 dyn/cm2 was applied onto single myocytes using micro fluid-jet, andtwo-dimensional confocal Ca2þ imaging was performed. Shear stress-induced T-waves were observed repetitively under 3-4 min intervals be-tween the stimuli, and occurred at ~1 event per 10 s. They were eliminatedby inhibition of the voltage-gated Naþ or Ca2þ channels, or ryanodine re-ceptors, suggesting that the T-wave is mediated by action potential-triggered Ca2þ release. Blockades of key stretch signaling molecules,stretch-activated channel, Naþ-Ca2þ exchange, and NADPH oxidase, didnot suppress T-wave generation by shear. However, shear-induced T-wavegeneration was abolished by pre-incubation of cells with external ATP-metabolizing enzyme apyrase, the gap junction blocker carbenoxolone, orwith P2X purinoceptor antagonist iso-PPADS. Inhibition of P2Y1 purinergicsignaling that mediates the longitudinal Ca2þ wave under shear did notattenuate the occurrence of T-waves. Our data suggest that shear stress in-duces activation of P2X purinoceptors via gap junction-mediated ATPrelease, thereby triggering action potential with subsequent T-wave in atrialmyocytes.

503-Pos Board B268Optical Mapping in Rat Models of Atrial DilationSamantha Cannazzaro1, Claudia Crocini1, Marina Scardigli1,Raffaele Coppini2, Ping Yan3, Leslie M. Loew3, Chiara Tesi2,Elisabetta Cerbai2, Francesco S. Pavone1, Corrado Poggesi2,Leonardo Sacconi1, Cecilia Ferrantini2.1LENS, Sesto Fiorentino, Italy, 2University of Florence, Florence, Italy,3University of Connecticut Health Center, Farmington, CT, USA.Atrial fibrillation (AF) is commonly associated with atrial dilatation causedby pressure or volume overload. Acute atrial stretch may create a myocar-dial substrate to promote AF via stretch-activated channels and mechano-electrical feedback mechanisms. However, enhanced AF vulnerability inchronic dilated atria occurs as a result of complex remodeling that includeschanges of myocyte membrane currents, altered intracellular Ca2þ homeo-stasis and alterations of the extracellular matrix (e.g. interstitial fibrosis).Here, we dissect the role of acute stretch-related changes versus those occur-ring as part of the long-term remodeling process. Spontaneous hypertensiverats (SHR) with chronic atrial dilation were compared to normotensiveWistar-Kyoto rats (WKY) and to WKY rats with acute biatrial dilation(dil-WKY) obtained with atrial balloons (inserted into both atria and inflatedwith controlled pressure after baseline recording). Briefly, the hearts wereperfused on a Langendorff’s apparatus and then the ventricles were excisedjust below the atrio-ventricular junction. To maximize atrial perfusion andstaining main coronary ventricular branches were cauterized. Atria werestained with a bolus injection of a red-shifted voltage-sensitive dye (di-4-ANBDQPQ). An ultrafast wide-field macroscope operating at 2 KHz(100 x 100 pixel) was developed to optically map action potential propaga-tion of Langendorff’s perfused-atria and to test AF vulnerability. AF wasinduced through burst pacing and occurred in 5.8% of WKY, 10.6% ofdil-WKY and 17.4% of SHR atria. Action potential duration at 90% repolar-ization (APD90) was prolonged in SHR as compared to WKY and dil-WKY.APD90 variability between contiguous regions was increased in both dil-WKY and SHR compared to WKY, potentially contributing to the increasedAF vulnerability. Next, we analyzed activation maps during AF episodes.Occasionally, they exhibited a stable reentry pattern characterized by similarorigin and propagation on a beat-to-beat basis. Alternatevely, the origins ofsuccessive reentrant waves varied randomly and the arrhythmia was perpet-uated by coexisting reentrant circuits, maintained through the continuousannihilation and creation of multiple wavelets. Voltage oscillations duringAF were analyzed in time and frequency domains, and the spectrogram(Fourier transform) revealed coexisting reentrant circuits at different fre-quencies. Analysis of the dispersion of the action potential durations andconduction velocity maps will clarify the mechanism of AF in chronicand acute models of atrial dilation.

504-Pos Board B269Epac2-Rap1 Signaling Influences Reactive Oxygen Species Productionand Susceptibility to Cardiac ArrhythmiasZhaokang Yang1, Hannah M. Kirton1, Moza Al-Owais2, Chris Peers2,Derek S. Steele1.1School of Biomedical Sciences, University of Leeds, Leeds, UnitedKingdom, 2Division of Cardiovascular Medicine, University of Leeds, Leeds,United Kingdom.In the heart, the inotropic and lusitropic effects of b1-adrenergic stimulationare known to involve the second messenger cAMP acting via proteinkinase A (PKA) to increase phosphorylation of intracellular targets

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including the L-type calcium channel, phospholamban and RyR2. In addi-tion, cAMP activates ‘exchange protein directly activated by cAMP’(Epac): a cAMP-activated guanine nucleotide exchange factor (GEF). How-ever, little is known about Epac signalling via its role as a GEF and effectsmediated via downstream effectors such as the small GTPase Rap1. In thepresent study on adult rat ventricular myocytes (ARVMs), the Epac2 inhib-itor ESI-05 induced prolongation of the [Ca2þ]i transient, followed by devel-opment of a plateau phase with [Ca2þ]i oscillations, characteristic of earlyafter depolarisations (EADs). This effect was blocked by pre-incubationwith (i) the late Naþ current (INalate) inhibitor ranolazine (ii) the mitochon-drial ROS scavenger mitoTEMPO or (iii) the CaMKII inhibitor KN93. Alleffects of ESI-05 were mimicked by downstream inhibition of Rap1GTPusing GGTI-298. Active Rap1 (Rap1GTP) was detectable under basalconditions in ARVMs and was reduced ~50% by ESI-05. These data suggest(i) that Epac2 regulates the level of active Rap1GTP in the heart and (ii)that active Rap1GTP may supress mitochondrial ROS production andsusceptibility to EAD arrhythmias, which involve activation of CaMKIIand INalate.

505-Pos Board B270Generation and Characterization of a Human iPSC Cardiomyocyte Modelof Troponin T I79N Linked Hypertrophic CardiomyopathyLili Wang1, Dmytro Oleksandrovych Kryshtal1, Kyungsoo Kim1,Shan Parikh1, Kevin Richard Bersell1, Jose R. Pinto2, Huan He3,Bjorn Christian Knollmann1.1Vanderbilt University Medical Center, Nashville, TN, USA, 2Department ofBiomedical Sciences, Florida State University College of Medicine,Tallahassee, FL, USA, 3Institute of Molecular Biophysics, Florida StateUniversity, Tallahassee, FL, USA.Objective: The aim of this study was to examine the functional conse-quences of a troponin T mutation (I79N) associated with hypertrophic car-diomyopathy (HCM) in cardiomyocytes derived from human inducedpluripotent stem cells (hiPSC). Method and Results: We generated aTnT-I79N hiPSC HCM model by inserting the 236T>A mutation into theTNNT2 gene of one of the alleles of hiPSC derived from a healthy donorusing CRISPR/Cas9. There was no off-target cleavage produced byCRISPR/Cas9. Nano-liquid chromatography mass spectrometry showedthat 43% of TnT protein was mutant in cardiomyocytes (CM) derivedfrom TnT-I79N hiPSCs. In 2D monolayers, the impedance amplitude(a measure of contractility) was increased and the Ca-contractility relation-ship leftward shifted in TnT-I79N hiPSC-CMs compared to isogenic controlhiPSC-CM. Results were confirmed using single hiPSC-CM cultured on amatrigel matress: The TnT-I79N mutation altered cytosolic Ca bufferingproperties by lowering the apparent dissociation constant Kd (0.4050.04mM in TnT-I79N, n=12 vs. 0.7250.10 mM in control, n=16, P<0.05)without changing the maximal binding capacity Bmax, indicating thatTnT-I79N mutation enhanced the Ca-binding affinity of myofilaments.Likely as a result of the increased cytosolic Ca-binding affinity, I79NhiPSC-CM paced at 0.2 Hz exhibited significantly reduced intracellularCa-transients (DF/Fratio 0.8550.03 in I79N vs. 1.0150.06 in control,P<0.05) and reduced caffeine transient (DF/Fratio 0.9250.06 in I79N vs.1.1050.06 in control, P<0.05), whereas Ca-decay kinetics where un-changed. Despite the reduced Ca-transient amplitude, TnT-I79N mutationenhanced contractility but delayed the relaxation in single hiPSC-CMs,indicating an increased sensitivity to Ca. Conclusion: The I79N hiPSC-CM model reproduces key findings from I79N transgenic mice: increasedmyofilament Ca-sensitivity, enhances contractility, impaired relaxationand enhanced cytosolic Ca-binding affinity. Human iPSC-CM could be apromising tool to model HCM in vitro.

506-Pos Board B271Cardiac Electromechanical Coupling Model of Myocardial ContractileFunction under Ischemic ConditionsYasser Aboelkassem, Natalia Trayanova.Biomedical Engineering, Johns Hopkins University, Baltimore, MD, USA.Hyperkalemia, acidosis, and anoxia are major pathophysiological conditionsof myocardial ischemia. Cardiac arrhythmias induced by these conditionsare widely investigated. However, the myocardium contractile functionduring acute ischemic perturbations is not fully explored. A fully coupledelectromechanical computational model is used to investigate cardiacinotropic response under various ischemic conditions. The coupling isaccomplished with a detailed myofilament model (MF) that allows for adynamic feedback between both electrophysiology and contractile modelcomponents. Additionally, an ATP-sensitive potassium channel (KATP)kinetics is incorporated in the model to account particularly for anoxia

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effects. Simulations are performed on a highly resolved cellular scale whereCa2þ sensitivity to ischemic perturbations is well predicted and fed to theMF model, thus inotropic alterations due to ischemia can be calculated.Both Ca2þ transient and twitch dynamics are calculated to establish anischemic-mechanics relationship. When compared with non-ischemiccontrol case, Ca2þ levels have shown to be attenuated as a result of bothanoxia and acidosis but not with hyperkalemia perturbations. Mechanical re-sponses represented by twitch dynamics as triggered by these changes inCa2þ behavior are computed. Simulations suggest that, the activation ki-netics of the KATP – channel (% f-ATP) is the main ischemic parameterthat significantly alters the cellular excitation-contraction coupling processin the form of; action potential shortening, reduced excitability, delayed re-covery, Ca2þ attenuation, significant decrease in the myocardium twitchproduction.

507-Pos Board B272Cardiomyocyte Functional Kinetic Reserve is Lost in an Ossabaw SwineModel of Heart Failure with Preserved Ejection FractionAdam B. Veteto1, John L. Jones1, Joel C. Robinett1, T. Dylan Olver2,Jenna C. Edwards2, Michelle D. Lambert1, Pamela K. Thorne2,Maike Krenz1, Kerry S. McDonald1, Jaume Padilla3, David A. Ford4,Christopher Baines2, R. Scott Rector3, Craig A. Emter2,Timothy L. Domeier1.1Medical Pharmacology and Physiology, University of Missouri Columbia,Columbia, MO, USA, 2Biomedical Sciences, University of MissouriColumbia, Columbia, MO, USA, 3Nutrition and Exercise Physiology,University of Missouri Columbia, Columbia, MO, USA, 4Biochemistry andMolecular Biology, Saint Louis University School of Medicine, St. Louis,MO, USA.Heart failure with preserved ejection fraction (HFpEF) comprises half of HFdiagnoses and is clinically characterized by comorbidities including aging,obesity, hypertension, and type 2 diabetes (T2D), with increased prevalencein females. Animal models accurately representing clinical HFpEF are lack-ing, thus limited data exists describing functional properties of myocardiumin HFpEF. We therefore examined cardiomyocyte function in an Ossabawswine model of HFpEF induced by combined T2D (Western-diet; WD)and pressure-overload (aortic-banding; AB). Female WD-AB animals ex-hibited T2D, including increased body weight, HOMA-IR, hyperlipidemia,and elevated liver enzymes (P<0.05) compared to control lean Ossabawswine (CTL). Pressure-overload increased LV diastolic wall thicknesswithout changing internal diastolic dimension (i.e. concentric hypertrophy)in WD-AB (P<0.05 versus CTL) with preserved ejection fraction. Enzymat-ically isolated cardiomyocytes were utilized to monitor intracellular Ca2þ

homeostasis (fura-2/AM) and contractile function in response to action-potential stimulation (0.5 Hz). Ca2þ transient and shortening amplitudewere similar between CTL and WD-AB, yet Ca2þ transient upstroke andcontraction kinetics were faster (P<0.05) in WD-AB compared to CTLwith a shorter time-to-peak Ca2þ and time-to-minimum sarcomere length.Spontaneous Ryanodine Receptor-mediated Ca2þ spark (fluo-4/AM) fre-quency and amplitude were also elevated (P<0.05) in WD-AB versusCTL. Following treatment with the beta-adrenergic agonist dobutamine (1uM), Ca2þ transient and shortening amplitude each increased (P<0.05)versus baseline conditions yet remained similar between CTL andWD-AB groups. However, dobutamine-induced changes in kinetic parame-ters (time-to-peak Ca2þ, time-to-minimum sarcomere length, Ca2þ transientrecovery tau, relaxation rate) were all blunted (P<0.05) in WD-AB versusCTL. In conclusion, WD-AB cardiomyocytes exhibited enhanced Ca2þ

and contractile kinetics under basal conditions, yet lost functional kineticreserve following beta-adrenergic challenge. Our findings suggest b-adren-ergic cardiomyocyte functional reserve is impaired in a novel translationalmodel of HFpEF.

508-Pos Board B273Heart Failure Re-Distributes Phospholamban between NuclearMembranes and Sarcoplasmic Reticulum in CardiomyocytesZhipeng Tian1,2, Yan Li1,3, Peng-Sheng Chen1, Steven Cala4,Zhenhui Chen1.1Medicine, Krannert Institute of Cardiology, Indiana University,Indianapolis, IN, USA, 2Cardiology, Central Hospital Affiliated to ShenyangMedical College, Shenyang, China, 3Geriatrics, Shengjing Hospital of ChinaMedical University, Shenyang, China, 4Physiology, Wayne State University,Detroit, MI, USA.

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We recently documented that phospholamban (PLB), a sarcoplasmic reticu-lum (SR) resident protein regulator of cardiac SR Ca-ATPase (SERCA2a),is localized to the nuclear envelope (NE) of cardiomyocytes (CMs). It iswell-known that heart failure (HF) remodeling involves altered proteinexpression of PLB and SERCA2a; however, with the demonstrated compart-mentalization of PLB accumulation, we asked whether HF alters traffickingbetween ER/SR subcompartments affecting steady-state intracellular distri-bution of PLB and SERCA. In this study, we examined heart tissue fromnon-failing and pacing-induced HF dogs with the monoclonal PLB (2D12or 1F1) and SERCA (2A7-A1) antibodies. Confocal immunofluorescencemicroscopy was used to compare subcellular concentration of PLB andSERCA. At least 3 regions from NE and SR in 10 CMs were analyzedfor each dog, n = 4 dogs). Both PLB and SERCA antibodies stained SRand NE in these CMs. Fluorescence intensity ratios between NE and SRfor PLB was 2.0550.82 (mean 5 SD) in control dog CMs, consistentwith results just reported. In HF CMs we found a highly decreased ratioof PLB between NE and SR to 1.26 5 0.34 (n = 8 dogs). In contrast tore-distribution of PLB, intensity ratios for SERCA in NE and SR remainedunchanged (1.0150.14 in Control, and 0.9850.26 in HF). Furthermore,while there was only minor levels of phosphoPLB in Control CMs, we foundthat phosphoPLB (S16) were very significantly higher in NE of failed CMs.The decreased PLB relative to SERCA in the NE of CMs suggests thatretention and accumulation of PLB in the NE is decreased in HF, leadingto a smaller, possibly hyper-phosphorylated steady-state of PLB concentra-tion in the NE.

509-Pos Board B274The Effect of Ovariectomy on Calcium (Ca2D) Handling in Guinea PigCardiomyocytesHsiang-Yu Yang1,2, Anita Alvarez-Laviada1, Jahn M. Firth1,Alice J. Francis1, Kenneth T. MacLeod1.1National Heart and Lung institute, Imperial College London, London,United Kingdom, 2Tri-Service General Hospital, National Defense MedicalCenter, Taipei, Taiwan.This study addressed the hypothesis that long term absence of ovarian hor-mones alters calcium handling in ventricular myocytes. Female guinea pigswere randomly assigned to have either a bilateral ovariectomy (Ovx) or asham operation. Pellets containing 17b-estradiol (E) (1mg, 60-day release),were placed subcutaneously in selected Ovx animals. Cardiac myocyteswere enzymatically isolated and action potential durations and L-typeCa2þ currents (ICa,L) were measured under current- or voltage-clamp respec-tively with a switch clamp system. Action potential morphology was inves-tigated and, while action potential duration at 90% repolarisation (APD90)remained unchanged, APD10 was 1.46 times longer and the rate of repolar-isation was slower in the Ovx group. In Fluo-4 loaded cells, Ca2þ transientswere 1.34 fold larger in Ovx group compared with sham along with a greaterfractional release. Sarcoplasmic reticulum Ca2þ stores were greater by 20%in the Ovx group and these cells also showed higher frequency of Ca2þ

sparks and waves during a quiescent period following 2 Hz field-stimulation. The changes occurring in the Ovx group did not take place inthe OvxþE group. Cardiac myocytes isolated from the Ovx group showedincreased peak ICa,L with the voltage dependence of activation and inactiva-tion shifting to more positive voltages which will increase the likelihood ofchannel opening. The protein kinase A inhibitor H-89, resulted in lessreduction in peak ICa,L in the Ovx group compared with sham. These find-ings suggest long term absence of ovarian hormones lead to potentiallydetrimental changes in Ca2þ handling mechanisms that may cause the for-mation of a more proarrhythmic substrate. Estradiol replacement preventedthese adverse effects.

510-Pos Board B275Biochemical and Mechanical Properties of Murine Extraocular MusclesJan Eckhardt1, Marijana Sekulic-Jablanovic1, Susan Treves1,2,Francesco Zorzato1,2.1Department of Anesthesiology, University Hospital, Basel, Switzerland,2University of Ferrara, Ferrara, Italy.Extraocular muscles (EOMs) are among the fastest and most fatigue resis-tant skeletal muscles. The distinct origin and innervation of EOMs are prob-ably responsible for their different gene and protein expression. Indeed,western blot analysis revealed that the expression of the protein isoformsforming the Excitation-Contraction Coupling Macromolecular Complex(ECC-MC) of human EOM is substantially different from human quadricepsmuscles. Human EOM muscles express, in addition to the skeletal muscle

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isoforms of proteins involved in ECC, Cav1.2, CASQ2 and RYR3. Becauseof the use of genetically modified mouse models to study human diseases wethought it important to investigate the biochemical and mechanical proper-ties of murine EOM. We found that the expression level of the ECC-MCproteins in mouse EOM is different from that of mouse hind limb musclesand from that of human EOM. We also studied the mechanical properties ofmouse EOM. In the presence of 1.8 mM CaCl2 in the extracellular mediumthe force induced by a single action potential and by a train of action poten-tials delivered at 100 Hz was 40,4535,7 mN (n=8), 3595213 mN (n=8 ),respectively . The half time to peak, the time to peak and the half relaxationtimes of force development evoked by a single action potential were2.5350.69 ms (n=8), 10.150.3 ms (n=8), 42.3527.8 ms (n= 8),respectively. Addition of 100 mM La3þ to the extrcellular medium causeda mean decrease of 75 % of the peak tetanic tension. The effect ofLa3þ was reversed by washing the muscle with Tyrode’s solution contain-ing 1.8 mM CaCl2 This study defines the biochemical and mechanicalproperties of preparations of mouse EOM under normal conditions andwill be used as reference to investigate the phenotype of EOM from micecarrying modifications of genes encoding key proteins encompassing theECC-MC.

511-Pos Board B276The Maintenance Ability and Ca2D Availability of Skeletal Muscle areEnhanced by SildenafilMei Huang1, Keon Jin Lee1, Kyung-Jin Kim2, Mi Kyoung Ahn1,Chung-Hyun Cho2, Do Han Kim3, Eun Hui Lee1.1Dept. of Physiology, College of Medicine, The Catholic Univ. of Korea,Seoul, Korea, Republic of, 2Dept. of Pharmacology, College of Medicine,Seoul National University, Seoul, Korea, Republic of, 3School of LifeSciences and Systems Biology Research Center, Gwangju Institute ofScience and Technology, Seoul, Korea, Republic of.Sildenafil relaxes vascular smooth muscle cells, and is used to treat pulmo-nary artery hypertension as well as erectile dysfunction. However, the effec-tiveness of sildenafil on skeletal muscle and the benefit of its clinical usehave been controversial, and most studies have focused primarily on tissuesand organs from disease models, without cellular examinations. Here, theeffects of sildenafil on skeletal muscle at the cellular level were examinedusing mouse primary skeletal myoblasts (the proliferative forms of skeletalmuscle stem cells) and myotubes along with single-cell Ca2þ imaging ex-periments and cellular and biochemical examinations. The proliferation ofthe skeletal myoblasts was enhanced by sildenafil, without dose-dependency. In the skeletal myotubes, sildenafil enhances the activity ofryanodine receptor 1, an internal Ca2þ channel, and Ca2þ movementsthat promote skeletal muscle contraction, possibly due to an increase inthe resting cytosolic Ca2þ level and a unique microscopic shape in themyotube membranes. Therefore, these results suggest that the maintenanceability of skeletal muscle mass and the contractility of skeletal musclecould be improved by sildenafil via enhancing the proliferation of skeletalmyoblasts and increasing the Ca2þ availability of skeletal myotubes,respectively.

512-Pos Board B277Dantrolene Shifts the Affinity of the Ryanodine Receptor for MG2D

Rocky H. Choi, F. Xaver Konig, Tanya R. Cully, Bradley S. Launikonis.School of Biomedical Sciences, University of Queensland, St Lucia,Australia.Dantrolene is the only approved drug for the treatment of life-threateningmalignant hyperthermia episodes. It has been shown by several studiesthat the voltage-dependent Ca2þ transient or force response is reducedby dantrolene; and indeed a binding domain for dantrolene on theryanodine receptor (RyR) has been identified. However, studies of isolatedRyRs have mostly failed to observe alterations in the conductance ofions through the channel in the presence of clinically relevant concentra-tions of dantrolene. This may be the case because the ionic conditions inbilayer studies typically do not match those occurring in the musclefibre. To examine the mechanism of dantrolene action on the RyR weused mechanically skinned fibres where the imposed ionic conditionsmimic those occurring in the body. The skinned fibre allowed us totrack the effect of dantrolene on Ca2þ transients in the cytoplasmelicited by action potentials; and a recently developed techniqueallowed the detection of the activity of the RyR under defined ionicconditions, including basal levels of activity, with a high degree of sensi-tivity. By varying the [Ca2þ] and [Mg2þ] of the cytoplasm, with and without

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dantrolene present, we observed that dantrolene reduced RyR activity byaltering its affinity for Mg2þ.

513-Pos Board B278MG53 Interacts with Cardiolipin to Protect Mitochondria from Ischemia-Reperfusion Induced Oxidative StressHanley Ma1, Xinyu Zhou1, Xinxin Wang1, Junwei Wu1, Kristyn Gumpper1,Tao Tan1, Timothy Ayodele Adesanya1, Chunlin Yang1, Yongqiu Zheng1,Heather Chandler1, Jingsong Zhou2, Jianjie Ma1, Hua Zhu1.1The Ohio State University, Columbus, OH, USA, 2Kansas City University ofMedicine and Bioscience, Kansas City, MO, USA.MG53 is a tripartite motif protein that is essential to plasma membraneregeneration. It binds to phosphatidylserine and facilitates the formationof repair patches at sites of plasma membrane disruption. Our previousstudies have reported MG53’s efficacy in ameliorating ischemia-reperfusion (I/R) injury to multiple organs including the heart, lungs, kid-ney, and brain. In I/R injury, mitochondria, however, are the primary victimof damage and the exposure of mitochondrial cardiolipin (CL) in response toI/R-induced oxidative stress procures cell death. Thus far, the role of MG53in the preservation of mitochondrial function has not been studied. Here, wetest the hypothesis that MG53 interacts with CL to protect mitochondriafrom oxidative stress. We find that mitochondria in the heart of MG53knockout mouse are more susceptible to I/R injury than those of wildtype mice. We also observe that MG53 targets mitochondria under chronicoxidative stress conditions, such as high-fat diet induced metabolic syn-drome and amyotrophic lateral sclerosis. We demonstrate that MG53 bindsto CL through a lipid dot-blot and a quantitative ELISA lipid-protein bind-ing assay. Additionally, we show that exogenous recombinant human MG53(rhMG53) protein can be up-taken by a variety of cells, including mesen-chymal stem cells, human corneal epithelial cells, and valvular interstitialcells. Once internalized, rhMG53 translocates to mitochondria inresponse to oxidative stress induced by anoxia-reoxygenation treatment asrevealed by live-cell imaging and 3D reconstruction of confocal micro-scopy. These data suggest that MG53 can target damaged mitochondriathrough a potential CL signaling mechanism and may implicate MG53 inmitochondrial membrane protection. To our knowledge, this study is thefirst to explore the interaction of MG53 with subcellular structures and pro-vides a novel mechanism for MG53-mediated amelioration of I/R-inducedtissue injury.

Voltage-gated Na Channels I

514-Pos Board B279Domain Specific Role of S4 for Stepping into and Recovering from theInactivated State as Obtained from Omega- and R4H Mutants in Nav1.2Nikolaus Guenter Greeff, Hansjakob Heldstab, Claudia Lehmann.Biophysics Institute Greeff, Uetikon am See, Switzerland.The macroscopic time courses of activation, inactivation and recovery arevoltage dependent. Control of these processes on the molecular level bythe voltage sensors S4 of each domain together with the DIII-IV loop (theinactivation particle) is not completely understood. With omega currentmutations (RR//QQ) along S4 we explore the position or state of S4 inthe gating-pore and also disturb the normal gating kinetics in a domainand state specific manner. In addition, we used the mutation S4/R4H ineither DIII or DIV which slowed recovery from inactivation about 10 times;the return of the loop into the recovered position releasing the a-pore par-allels the appearance of the omega current reflecting the arrival of S4 inthe resting state. This confirms that both domains DIII and DIV controlrecovery.Stepping from resting into inactivated state appears more complex. Previousdata for squid showed that the inactivation time constant tau-h has a kink atabout �10 mV. Combination with high-resolution gating currents showedthat below this voltage tau-h obtained its steeper voltage dependence bycoupling to activation; the smaller voltage dependence of tau-h above�10 mV appeared as result of a single inactivation voltage sensor (Greeffand Forster, 1991). In Nav1.2, we now see a similar kink at about�10 mV. Gating currents are too small to be resolved. Instead, we useomega mutations in DI to IV to study changes of voltage dependence ofinactivation. Modifying DIV/S4 with RR//QQ along 3 positions shifts thekink to the left and seems to reduce the voltage dependence indicating therole of DIV/S4 as voltage sensor of inactivation. In contrast, in domain Ito III the resting state RR//QQ mutation does not change tau-h. However,

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omega mutations in the inner region of DI, II and III, especially DII34QQ,increased tau-h substantially combined with a strong right shift of the kink.Conclusion for Nav1.2: Recovery from inactivation clearly depends on S4 inDIII and DIV. Going into the inactivated state, i.e. closing of the a-pore bythe DIII-IV loop, needs as prerequisite the activation of DI and DII at lowervoltages, while at higher voltages the slower DIV is rate limiting. The relationof DIII to a specific function when stepping up is less clear from theseexperiments.

515-Pos Board B280Divergence in Domain IV of an Electric Fish NaV Channel Tunes its FastInactivation to Support Rapid Firing Rates by Electro-MotorneuronsDaniel Thomas Infield1, Ammon Thompson2, Troy Smith3,Harold H. Zakon2, Christopher A. Ahern1.1Department of Physiology and Molecular Biophysics, University of Iowa,Iowa City, IA, USA, 2Department of Neuroscience, University of Texas,Austin, TX, USA, 3Department of Biology, University of Indiana,Bloomington, IN, USA.In some neuronal cell types, persistent or resurgent current through voltage-gated sodium channels enables the regular firing of ‘‘spontaneous’’action potentials from a few to a hundred Hz. However, the neuronswith the fastest spontaneous firing rates known are found in the nocturnalghost knifefish (family apteronotidae), which accomplish active electroloca-tion via a unique neuronal electrical organ that spontaneously fires actionpotentials at rates exceeding 1000 Hz. Voltage-clamping of electro-motorneurons from apteronotidae revealed voltage-gated sodium currentswith incomplete fast inactivation (i.e., the existence of a persistent sodiumcurrent). Unexpectedly, RT-PCR revealed that Nav 1.4b is the dominantNav isoform in the apteronotid spinal cord, and it is expressed here nearlyexclusively. We found that this gene contains five apteronotid-specificsubstitutions in the S4-S5 cytoplasmic linker of Domain IV, a region thathas been implicated in the process of fast inactivation of mammalian voltagegated sodium channels. Using two electrode voltage clamp electrophysi-ology, we assayed the effects of making combinations of these substitutionsin the human cardiac sodium channel Nav 1.5 (R1644W, L1647W, M1651R,I1660F, G1661S). Interestingly, when all five apteronotid substitutions areincorporated into DIV S4-5 of Nav 1.5, a persistent sodium current isobserved that is qualitatively similar to that of the apterotontid Nav 1.4bin native cells. In addition, the effects observed in some partial substitutions(for example, loss of voltage dependence of inactivation in R1644W/M1651R-Nav 1.5), were apparently lost in the context of the quintuple-mutant. Taken together, these results suggest that the evolution of rapidelectrical organ discharge in knifefish was driven by the tissue-specificexpression of and sequence divergence within the voltage gated sodiumchannel Nav 1.4b. Moreover, they help define the structural requirementswithin the DIV S4-S5 linker for normal inactivation in mammalian sodiumchannels.

516-Pos Board B281Role for Fast Inactivation in Domain I of Voltage Gated Sodium ChannelsJames R. Groome, Ryann Camp.Biological Sciences, Idaho State University, Pocatello, ID, USA.In sodium channels, the voltage sensing S4 segments move outward inresponse to membrane depolarization to promote activation and fast inactiva-tion of the channel. Domain-specific roles for S4 segments include activationand fast inactivation. In the present work we investigated the role of domain Iin activation, deactivation and fast inactivation of the skeletal muscle sodiumchannel hNaV1.4. Charge reversal at R1 (R219D) produced a significanthyperpolarizing shift of the activation curve, an effect confirmed in an inac-tivation deficient background (R219D/QQQ) and with gating currents. Chargereversal at residues lower in the S4 segment (R225D, K228D) produced a de-polarizing shift of the activation curve. R219D selectively enhanced fast inac-tivation from closed states (hyperpolarizing shift of the steady-state fastinactivation curve, and accelerated kinetics of closed-state fast inactivation.Fast inactivation from the open state was not affected by R219D. Lessereffects on closed-state fast inactivation were observed for charge reversal atDIS4 residues lower in that voltage sensor. Charge reversal at inner negativecountercharges (E171R, S2; D197R, S3) also produced a hyperpolarizing shiftof the steady-state fast inactivation curve and accelerated kinetics of closed-state fast inactivation. These results suggest that in the wild type NaV1.4channel, interactions of inner negative charges with the outer charge inDIS4 restrict outward movement of that voltage sensor in response to weakdepolarization, and limit fast inactivation elicited from closed states. Thiswork was supported by NIH 1R15NS093579-01A1 to JRG and NIHP20GM103408 to ISU.

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517-Pos Board B282Effects of Cysteine Substitutions In D1-S6 on Fast and Slow Inactivation inNav1.4John O’Reilly, Penny Shockett.Southeastern Louisiana University, Hammond, LA, USA.Previous studies in voltage-gated Na channels (Navs) have suggested a rolefor segment 6 of domain 1 (D1-S6) in slow inactivation of Navs. In thecurrent studies, we used scanning site-directed mutagenesis to substitutecysteine residues for the native amino acids in D1-S6 of the human skeletalmuscle Nav isoform (hNav1.4) spanning from the putative ‘‘gating hinge’’to the ‘‘bundle crossing’’ of S6 segments. Presently, we have obtaineddata from mutants I439C, N440C, L441C, I442C, and L443C. Comparedwith wild-type hNav1.4, the activation curve (G-V) was only affected(increased) in the slope factor (k) of L443C. The V1/2 of the steady-statefast inactivation curve (hN) in N440C was hyperpolarized and the slopewas increased. The slope factor for hN was also increased in I442C andL443C. In slow inactivation, all mutants differed from hNav1.4. Enhanceddevelopment of slow inactivation (faster entry) was observed in N440Cand I442C, while I439C, L441C, and L443C all showed resistance to devel-opment of slow inactivation. Steady-state slow inactivation (SN) was alteredin I439C (depolarized V1/2 and increased k) and N440C (hyperpolarized V1/2

and decreased k). We conclude that cysteine substitutions in D1-S6 ofhNav1.4 disrupt the normal molecular gating kinetics of slow inactivationin hNav1.4. Ongoing and future experiments include analysis of additionalcysteine mutants in D1-S6 and application of methanethiosulfonate (MTS)reagents to examine potential molecular rearrangements of D1-S6 duringslow inactivation gating.

518-Pos Board B283Molecular Mechanisms of Cardiacvoltage-Gated NaD Channel Regulationby Acidic pHBicong Li, Wandi Zhu, Jon Silva.Biomedical Engineering, Washington University in St. Louis, St. Louis, MO,USA.Background: During ischemic heart disease, pH drops from 7.4 to 6.0 within10 minutes of onset, severely affecting ion channel gating. The cardiac so-dium channel (Nav1.5) is particularly susceptible to this abrupt pH change,and its altered gating is thought to predispose patients suffering ischemia toarrhythmia and sudden cardiac death. We observed the voltage-sensing do-mains (VSDs) of NaV1.5 to discover molecular mechanisms of its regulationby pH.Methods: A cysteine mutation was made in each of the four VSDs (DI-DIV) ofNav1.5. Synthesized RNA from these constructs was injected into Xenopusoocytes. Once channels were expressing, a fluorophore was tethered to thecysteine via a disulfide bond. By measuring the kinetics and change in magni-tude of the fluorescence, we were able to track VSD conformational changesalong with the current-voltage relationship.Results:Reducing the pH of the extracellular solution from 7.4 to 6.0 causes INato decrease in magnitude by 50%, and shifts in both activation and fast inacti-vation rightward, consistent with previous results. At a pH of 6.0, time to peakwas reduced by 300% while inactivation was only 10% slower. Observation ofthe VSDs showed that the DII-VSD is not affected by pH, and the DIII-VSDshowed a small depolarizing activation shift ~6.65 mV. The DIV-VSDdisplayed a complex phenotype, not shifting after short pulses, but shiftingprominently (23.27 mV) after prolonged pulses. Its kinetics were also slowedby a factor of 2 at a pH of 6.0.Conclusions:These results suggest an important role for the DIV-VSD indetermining regulation of NaV1.5 by pH.

519-Pos Board B284Omega Mutations along S4 in Nav1.2 Channels Give Insight into DomainSpecific Contribution to Activation and Steady State InactivationClaudia Lehmann, Hansjakob Heldstab, Nikolaus Guenter Greeff.Biophysics Institute Greeff, Uetikon am See, Switzerland.We previously identified the resting state positions of the voltage sensor S4for each domain of Nav1.2 by means of omega mutations. We found that adouble gap is needed to open the omega pore (narrow part of thegating pore) resulting in detectable omega current, also known as gatingpore current. At hyperpolarizing conditions, the resting state of S4 wasfound for double gap RR1,2QQ in domain I, II and IV and for double gapRR2,3QQ in domain III. In this work we evaluated additional conforma-tional states of the voltage sensor S4 moving through the gating poreby further double gap mutations along S4 (second double gap 2,3QQand third double gap 3,4QQ). Two electrode voltage clamping on X. laevisoocytes expressing rat brain sodium channels Nav1.2 was used to measure

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macroscopic ionic current through the alpha pore and, if present, omegacurrent through the omega pore. In DI and DII we detected clearoutward omega current for S4 mutants RK3,4QQ at depolarizing conditions.Furthermore, we found that activation of sodium currents was right shiftedby about 30 mV towards higher potentials compared to the first and seconddouble gap mutants or wild-type sodium channel. These findingssuggest two sequential gating steps of S4 between resting and activated statein both domains DI and DII. In DIII and DIV no clear outward omegacurrent could be detected at depolarized potentials either for the second orfor the third double gap mutant. However steady state inactivation wasstrongly left shifted by about 50 to 100 mV to more hyperpolarized poten-tials for the second and third double gap mutant in both domains, consistentwith involvement in recovery from inactivation and immobilization,respectively.

520-Pos Board B285Characterization of a NaV1.5 Gain-of-Function Mutation (G213D) causingMultifocal Atrial and Ventricular Premature Ectopies and an IncreasedRisk of Dilated CardiomyopathyKirstine Calloe1, Anders K. Broendberg2, Alex H. Christensen3,Lisbeth N. Pedersen4, Morten S. Olesen5, Maria A. Tejada1, Soren Friis6,Morten B. Thomsen7, Henning Bundgaard3, Henrik K. Jensen2.1Department of Veterinary Clinical and Animal Science, University ofCopenhagen, Copenhagen, Denmark, 2Department of Cardiology, AarhusUniversity Hospital, Aarhus University Hospital, Aarhus, Denmark, 3Unit forInherited Cardiovascular Diseases, the Heart Centre, National UniversityHospital, University of Copenhagen, Copenhagen, Denmark, 4Department ofMolecular Medicine, Aarhus University Hospital, Aarhus, Denmark,5Laboratory for Molecular Cardiology, the Heart Centre, National UniversityHospital, University of Copenhagen, Copenhagen, Denmark, 6NanionTechnologies, Munich, Germany, 7Department of Biomedical Sciences,University of Copenhagen, Copenhagen, Denmark.Background: Mutations in SCN5A have been linked to different cardiacdiseases including multifocal ectopic Purkinje-related premature contrac-tions (MEPPC) characterized by ventricular ectopy and dilated cardiomyop-athy (DCM). Here we characterize the Nav1.5 G213D variant found in alarge Danish family with frequent premature ventricular and atrial contrac-tions, frequent nonsustained ventricular tachycardia and DCM. Methodsand Results: The family was clinically assessed and an MEPPC-likephenotype including complex atrial and ventricular arrhythmias and dilatedcardiomyopathy was found. Genetic screening revealed a G213D Nav1.5missense mutation in the link of segment 3 and 4 in domain 1 of theNav1.5 protein. The MEPPC-like phenotype co-segregated with G213D.Electrophysiological studies of wild type (WT) hNav1.5 and hNav1.5_G213D expressed in CHO-K cells was performed using conventionalpatch clamp and a SynchoPatch 384PE (Nanion Technologies) highthroughput automatic patch clamp. The half-maximal inactivation (V½)was significantly more negative for hNav1.5_G213D compared to WT andthe V½ of steady-state inactivation was shifted towards more positivevalues for hNav1.5_G213D, resulting in increased window-currents. Thissuggests that hNav1.5_G213D activates at more negative potentials and alarger fraction of channels will recover from inactivation during the diastoleresulting in a gain-of-function phenotype. A combination of wild type andG213D mimicking the heterozygote state exhibited an intermediate pheno-type. Flecainide or amiodarone resulted in a markedly reduced number ofpremature atrial and ventricular contractions in patients. Conclusions:The G213D Nav1.5 variant is associated with a gain-of-function and isassociated with multifocal atrial and ventricular ectopy and dilatedcardiomyopathy.

521-Pos Board B286Voltage-Gated Sodium Channel Mutations can Exert Dominant-NegativeSuppression by Coupled GatingJerome Clatot1, Aurore Girardeau2, Celine Marionneau2,Isabelle Deschenes1.1Case Western Reserve University, Cleveland, OH, USA, 2Universite deNantes, Nantes, France.Introduction: Mutations in voltage-gated sodium channels have been linkedto channelopathies such as cardiac arrhythmias, epilepsy and myotonia.We previously demonstrated that trafficking-deficient mutant channels couldlead to a dominant-negative effect by impairing trafficking of the wild-typecardiac sodium channel (WT). We and others have also reported that sodiumchannel polymorphisms present on one construct can affect the gating of the

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WT on a separate construct suggesting gating cooperation betweenchannels. Therefore, our objective was to investigate whether trafficking-efficient but gating-deficient channels could also lead to a dominant-negative effect by impairing the gating of the WT. Methods: Cell surfacebiotinylation was used to assess trafficking efficiency of the channel andits presence at the cell surface. Current density was measured using thewhole-cell configuration of the patch-clamp technique. Open probabilityof sodium channels was measured using single-channel recordings in thecell-attached configuration. Results: Co-expression of WT with thedominant-negative mutant Nav1.5-L325R led to a dominant-negative effect.However, the presence of difopein, a 14-3-3 protein inhibitor, was able toabolish this effect leading to restored current density. Surprisingly, cellsurface biotinylation results showed that the channel density at the cell sur-face was not altered when WT and L325R mutant were coexpressedtogether, even though we observed a dominant-negative effect when cur-rents were measured. Interestingly, our single channel recordings showedthat Nav1.5 alpha-subunits display coupled gating properties. Importantly,this effect was abolished in presence of difopein, the 14-3-3 protein inhib-itor. Hence, this uncoupling of the WT and mutant channels seen at thesingle-channel level could explain the suppression of the dominant-negative effect observed at the whole-cell level. Conclusions: Our datastrongly support that dominant-negative suppression exerted by dominantnegative mutant is not only due to trafficking deficiency but could also bedue to impairment of the WT gating probability.

522-Pos Board B287Open and Closed States of the NaVAb Activation GateMichael Lenaeus, Tamer M. Gamal El-Din, Karthik Ramanadane,Ning Zheng, William A. Catterall.University of Washington, Seattle, WA, USA.The bacterial voltage-gated sodium channel NavAb is a structural model foreukaryotic voltage-gated sodium channels, and it has been crystallized inboth the pre-open and inactivated states (Payanadeh et al 2011, 2012).These structures revealed the general architecture of bacterial voltage-gated sodium channels and provided insight into mechanisms of voltage-sensing, electromechanical coupling, and the selective conductance ofsodium ions. Comparison with structures of other bacterial sodium channels(Zhang et al 2012, Bagneris et al 2013) has also informed hypotheses ofactivation gating, an opening transition now thought to involve twistingmovements of the S6 helix that ultimately result in an iris-like dilation ofthe intracellular ends of the four S6 segments. We sought to capture closedand open states in the same channel protein through x-ray crystallographyand electrophysiological studies of mutations in the S6 of NavAb. Ourstudies show that paired mutations of hydrophobic residues in the S6 helixcan uncouple voltage-sensor movement from pore opening and thereby lockthe channel in a permanently closed state in which the intracellular ends ofthe four S6 helices interact tightly with each other. On the other hand, trun-cation of the intracellular extensions of the S6 segments results in an openconformation of the activation gate. These analyses allow for comparison ofthe closed, open, and inactivated states of NavAB’s activation gate, a firstfor a voltage-gated sodium channel. Our results help confirm and sharpenthe previously hypotheses for the pore-opening transition of sodium chan-nels and reveal key conformational changes that connect the closed, open,and inactivated states of NavAb.

523-Pos Board B288In Vitro Single-Molecule Study of Nachbac using Planar Lipid BilayerDeviceHiofan Hoi1,2, Andrew Jo1,2, Manisha Gupta2,3, Carlo D. Montemagno1,2.1Chemical and Materials Engineering, University of Alberta, Edmonton, AB,Canada, 2Ingenuity Lab, Edmonton, AB, Canada, 3Electrical and ComputerEngineering, University of Alberta, Edmonton, AB, Canada.Voltage-gated sodium channels (Navs) are essential component for the gen-eration and propagation of electric signals in excitable cells. The successesin the biochemical, biophysical and crystallographic studies on prokaryoticNav in recent years has greatly promote the understanding of the molecularmechanism underlie these proteins and their eukaryotic counterparts. In thispaper, we aim to investigate the conductance and ionic selectivity of theprokaryotic Nav NaChBac at single-molecule level. Purified NaChBac pro-tein was first reconstituted into lipid vesicles to form proteoliposome, whichis subsequently incorporated into lipid bilayer with known lipid compositionby proteoliposome fusion and studied using a planar bilayer device. Wewere able to insert a single NaChBac into the bilayer by using a high

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lipid-to-protein ratio for the proteoliposome preparation. At single-moleculelevel, we observed three distinct conductance sub-states, 26 5 6 pS(mean 5 SEM, n=4), 92 5 18 pS (n=3), and 268 5 45 pS (n=2), respec-tively. The first two values are comparable to previous reported ones thatwere obtained by single-channel patch-clamp and multi-channel recordingwith planar bilayer device, respectively. But the third conductance sub-state is reported for the first time. In addition, our data at single-moleculerecording shows similar permeability among Naþ, Kþ and Ca2þ, indicatingthat the reconstituted NaChBac is non-selective in the artificial membraneenvironment. Study of NaChBac at single-molecule level in an artificialenvironment reveals new properties that were not observable in in vivostudies. Our results provide novel insight to understanding the biophysicalproperties of Navs.

524-Pos Board B289Control of Slow, Use Dependent Inactivation of NaVAb by its C TerminalTailTamer M. Gamal El-Din, Michael J. Lenaeus, Karthik Ramanadane,Ning Zheng, William A. Catterall.Pharmacology, University of Washington, Seattle, WA, USA.Bacterial voltage-gated sodium channels are composed of four identicalsubunits. They share major biophysical features with eukaryotic counter-parts. Crystallization of full-length bacterial sodium channels (Payandehet al. 2011, 2012; Zhang et al. 2012) makes them invaluable models forstudying the structural basis of ion conductance, activation, inactivation,and drug interaction. Mammalian sodium channels have two main typesof inactivation: fast inactivation on the order of milliseconds, which ismediated by the IFM motif in the intracellular loop between domains IIIand IV; and slow inactivation on the order of hundreds of milliseconds toseconds, which is believed to occur through pore collapse. The bacterial so-dium channel NavAb activates at very negative membrane potentials, and ithas a late use-dependent phase of slow inactivation that reverses veryslowly (Gamal El-Din et al. 2013). We studied the effect of the NavAbC-terminal cytoplasmic domain on use-dependent slow inactivation. Dele-tion of 40 residues of the cytoplasmic tail (a40) abolished late use-dependent inactivation. Progressively smaller deletions to yield a28, a10,a7, and a3 caused graded effects. However, deletion of only 10 residueswas sufficient to abolish most of the late, use-dependent inactivation ofNavAb. In addition to modulating the extent of use-dependent inactivation,the progressively truncated constructs showed decremental slowing of thedecay of sodium current during depolarizations. NaVAb-a40 has voltage-independent kinetics of current decay, while other constructs have differen-tial profiles of decay kinetics that are correlated with C-terminal length.Our experiments reveal a surprisingly crucial role for the C-terminaldomain, which forms a coiled-coil structure unique to bacterial sodiumchannels, in both early and late phases of use-dependent slow inactivationof NaVAb.

525-Pos Board B290Role of Channel Fluctuations in Ion Transport and Selectivity in BacterialSodium Channel NavAbChristopher Ing1,2, Nilmadhab Chakrabarti1, Ning Zheng3,4,William A. Catterall3, Regis Pomes1,2.1Molecular Structure and Function, Hospital for Sick Children, Toronto, ON,Canada, 2Biochemistry, University of Toronto, Toronto, ON, Canada,3Pharmacology, University of Washington, Seattle, WA, USA, 4HowardHughes Medical Institute, Seattle, WA, USA.The elucidation of high-resolution structures of voltage-gated sodium chan-nels has opened the way to elucidating the mechanism of sodium permeationand selectivity. Molecular simulation studies of bacterial sodium channelNavAb (Chakrabarti et al., PNAS 110, 11331-11336, 2013) suggested thatNaþ binding and permeation through the selectivity filter are coupled tothe conformational isomerization of the Glu177 side chains of the EEEEring from an outfacing conformation to a lumen-facing conformation, result-ing in a high rate of Naþ diffusion through the selectivity filter. To clarifythe role of channel dynamics on ion permeation and selectivity, we examinethe mechanism of ion permeation in various systems in which either the na-ture of the EEEE ring or the extent of channel fluctuations have been modi-fied. Specifically, we study how the molecular mechanism of Naþ and Kþ

permeation in NavAb is affected by protonating a single Glu177 side chain,by replacing all four Glu177 side chains by Asp, by preventing conforma-tional isomerization of Glu177 side chains, by removing the voltage-sensing domains, and by introducing artificial structural restraints on thetransmembrane helices of the pore domain. The analysis of unbiased equi-librium simulations totalling over 200 microseconds, including simulations

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with competitive binding of Naþ and Kþ, shows how modifying the struc-ture and fluctuations of the selectivity filter, either directly or indirectly, al-ters both the ion conduction mechanism and the ionic selectivity of thechannel. These findings have implications for the study of cation permeationand selectivity in a large class of tetrameric ion channels containing acidicside chains in the conduction pore.

526-Pos Board B291An Open State Model of the Navab Channel Explored by Rosetta andMolecular Dynamics SimulationPhuong T. Nguyen1, Kevin R. DeMarco1, Igor Vorobyov2,Coleen E. Clancy2, Toby W. Allen3,4, Vladimir Yarov-Yarovoy5.1Biophysics Graduate Group, UC Davis, Davis, CA, USA, 2Department ofPharmacology, UC Davis, Davis, CA, USA, 3School of Applied Sciences,RMIT, Melbourne, Australia, 4Department of Chemistry, UC Davis, Davis,CA, USA, 5Department of Physiology and Membrane Biology, UC Davis,Davis, CA, USA.Voltage-gated sodium (Nav) channels play a pivotal role in propagatingelectrical signals in excitable cells and key targets for development of noveltherapeutics. Despite recent progress in determining x-ray structures of bac-terial Nav channels in closed, inactivated, and partially open states, we arestill missing a stable open state structure of a Nav channel that will be usefulto study ion conduction, channel gating and drug - channel interactions. Weused Rosetta molecular modeling software to build the full-length open statemodel of the bacterial NavAb channel. The fully activated state of theNavAb voltage sensor domain (VSD) was modeled using the crystal struc-ture of NavRh channel VSD as a template. The initial open state of NavAbpore was modeled using the crystal structure of NavMs with partially openpore. Rebuilding the interacting regions of NavAb (S3, S4, S5, S6, and S4-S5 linker) using Rosetta loop modeling and relax approaches was essentialto generate stable NavAb open state model. Output models were evaluatedusing RosettaMembrane energy function and Rosetta clustering approach.The top Rosetta models represented different alternative open states of theNavAb channel. We have conducted a set of molecular dynamics (MD) sim-ulations on the NavAb channel open state model using the Anton supercom-puter to examine its stability. The simulations revealed that the NavAbchannel open state model reproducibly sustained a fully hydrated openpore that conducted sodium ions under an applied membrane potential forperiods of hundreds of nanoseconds. These results suggest that our structuralmodeling approach could be useful for modeling multiple states of other ionchannels and for rational design of novel therapeutics targeting ionchannels.

527-Pos Board B292Simulating the Access and Binding of Subtype Selective Sodium ChannelInhibitorsBen Corry.Research School of Biology, Australian National University, Acton, ACT,Australia.Sodium channel blockers are commonly used as local anaesthetics, anti-arrhythmics and anti-epileptics, however they generally cannot distinguishbetween the different sodium channel subtypes expressed in humans. Thedevelopment of highly selective channel inhibitors will allow for a range ofnew clinical applications and a decrease in side effects. To help achieve thisaim we here examine how two recently discovered subtype selective voltagesensor inhibitors interact with a bacterial sodium channel (NavAb) and withthe eukaryotic channel Nav1.7 (as part of a Nav1.7/NavAb chimera). Using arange of advanced sampling techniques and tens of ms of molecular dynamicssimulations we show both where these compounds bind and how they accessthis site. Our simulations help unravel how subtype selectivity is achievedand are able to accurately predict the binding affinity in each channel. Thishighlights that for these compounds binding is much weaker for bacterialchannels than for Nav1.7. The simulations also answer the puzzle as to whythe channels have to be held in the inactivated state for prolonged periods inorder to measure inhibition, as the compounds face large barriers to accessthe binding site due to specific interactions with the protein. These resultswill assist in developing strategies to speed up the kinetics of inhibition andto improve selectivity of the inhibitors for specific sodium channel subtypes.

528-Pos Board B293Propofol is a Potent Gating Modifier of Voltage-Gated Sodium ChannelsElaine Yang1, Daniele Granata2, Roderic Eckenhoff3, Vincenzo Carnevale2,Manuel Covarrubias1.1Vickie and Jack Farber Institute and Department of Neuroscience,Thomas Jefferson University, Philadelphia, PA, USA, 2Institute forComputational Molecular Science, Temple University, Philadelphia, PA, USA,

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3Department of Anesthesiology and Critical Care, University of PennsylvaniaSchool of Medicine, Philadelphia, PA, USA.Voltage-gated Naþ channels (Navs), which play a pivotal role in the electri-cal excitability of the central nervous system, are inhibited by clinicallyrelevant concentrations of many general anesthetics and are thus highly rele-vant anesthetic targets. The molecular mechanisms of this inhibition, how-ever, remain unclear. Here, we investigated the electrophysiologicalresponse of NaChBac and NaVMs, two anesthetic-sensitive bacterial homo-logs of eukaryotic Navs, to the intravenous anesthetic propofol at 2, 5, and10 mM. In both NaChBac and NaVMs, propofol induced hyperpolarizingshifts of the pre-pulse inactivation and conductance-voltage (G-V) relation-ships, reduced the time constant of inactivation, and increased the time con-stant of deactivation in a dose dependent manner, without significant effectson recovery from inactivation. Previous investigations suggested that gen-eral anesthetics might inhibit NaChBac by open channel block, much likelocal anesthetics. Contrary to predictions based on a mechanism of poreblockade, however, propofol induced hyperpolarizing shifts in the G-Vcurve with minimal effects on peak current and current decay in a non-inactivating NaChBac mutant at both 2 and 5 mM. Propofol may bind tothe channel to stabilize the open and inactivated-open states, a mechanismsupported by kinetic modeling. Guided by molecular dynamics simulations,we are evaluating putative propofol binding sites in the pore and voltage-sensing domains with electrophysiology and mutational analysis to identifystructural determinants of Nav gating involved in modulation by propofol.Furthermore, using the propofol analog ‘‘fropofol’’, which contains a fluo-rine substitution that selectively weakens its hydrogen bonding capability,we are also evaluating the contribution of the propofol 1-hydroxyl to molec-ular recognition.

529-Pos Board B294The Sodium Ion Binding Region at the Focus of P1 Helices Attracts bothCharged and Electroneutral Ligands of Sodium ChannelsDenis B. Tikhonov1, Boris S. Zhorov1,2.1Sechenov Institute, RAS, St. Petersburg, Russian Federation, 2Biochemistry,McMaster University, Hamilton, ON, Canada.The inner pore of eukaryotic voltage-gated sodium channels is targetedby ligands of dramatically different chemical structures. These includeorganic cations such as a local anesthetic lidocaine and electroneutral drugssuch as an anticonvulsant carbamazepine. Mutations of the critical phenyl-alanine residue in helix IVS6 and some other inner pore-facing residues areknown to affect action of both charged and neutral ligands. The structuralcause of this ligand-binding promiscuity of sodium channels is unclear.Here we used the X-ray structure of a prokaryotic sodium channel NavMsto model the pore domain of the Nav1.x channels. We further employedthe Monte Carlo energy-minimization method to perform intensive dockingof lidocaine and carbamazepine from thousands starting points in theinner-pore region. The sodium ion NaIII, which is located between thefour backbone carbonyls at the C-ends of P1 helices and does not makedirect contacts with the channel protein, attracted carbamazepine, butrepelled lidocaine. Therefore we further docked electroneutral ligands(lamotrigine, carbamazepine, phenytoin, lacosamide and bisphenol A) andcationic ligands (lidocaine, QX-314, cocaine, quinidine, and sipatrigine)in the channel models, respectively, with and without NaIII. In our modelsall the ligands interacted with the phenylalanine residue in IVS6 and mostof the ligands also interacted with the tyrosine residue in IVS6. Someligands extended their moieties in the III/IV sidewalk (fenestration). Theelectroneutral ligands bound the sodium ion with their electronegativegroups and lacosamide chelated this ion. The ligand-bound ion remainedclose to the NaIII position due to attraction to the pore-facing backbonecarbonyls. The same region attracted the charged group of the cationic li-gands. In the predicted binding modes even small-size ligands would blockthe ion permeation by the electrostatic and steric mechanisms. Our studyproposes a common pharmacophore for the diverse ligands. It includes acation (the ligand’s ammonium group or the ligand-bound sodium ion)and an aromatic moiety, which are usually linked by four bonds. Supportedby NSERC and RFBR.

530-Pos Board B295Comparison of Ion Selectivity Mechanisms in Bacterial and MammalianSodium ChannelsEmelie Flood, Celine Boiteux, Toby W. Allen.RMIT University, Melbourne, Australia.Voltage-gated sodium (Nav) channels play essential roles in electrical sig-nalling in the body and are associated with many physiological disorders.

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The availability of recent high-resolution structures for bacterial Nav chan-nels makes them ideal tools for exploring functional mechanisms. Bacterialand mammalian channels have been proposed to select for Naþ over Kþ viadifferent mechanisms, owing to their distinct selectivity signature se-quences, with bacterial channels making use of a 4-fold ring of Glu sidechains, in place of Asp, Glu, Lys and Ala (DEKA) in mammalian channels.We have investigated the underlying causes of Naþ selectivity by carryingout multi-ms fully atomistic simulations that capture long time-scale proteinmovements essential for ion permeation. Our previous bacterial channelsimulations demonstrated that the ability of the high-field strength Gluring to accommodate 2 Naþ ions provides more efficient conduction forNaþ than for Kþ (for which only 1 ion binds to this ring). To probeconduction mechanisms in mammalian channels, a model of Nav1.2 wasconstructed by grafting residues of its selectivity filter and externalvestibular regions into bacterial NavAb and NavRh channels. We haveobserved similarities in the multiple ion mechanisms, enabled bycarboxylate-ion complexes involving DEKA and carboxylates of the vestib-ular region, previously thought to play passive roles in modulating conduc-tion. Our simulations also demonstrate that the DEKA Lys is an activeparticipant in permeation, with its protonation state influencing the conduc-tion mechanism. Our findings suggest a possible common mechanism forNaþ-selective conduction across bacterial and mammalian Nav channels,and provide insight into the origins of Naþ-selective binding across theproteome.

531-Pos Board B296How C-Terminal Domain Stabilize the Gate of Voltage-Gated SodiumChannelsSong Ke1, Bonnie Ann Wallace2, Jakob Ulmschneider1,Martin Ulmschneider3.1Institute of Natural Sciences, Shanghai Jiaotong University, Shanghai,China, 2Department of Biological Sciences, Birkbeck College, University ofLondon, London, United Kingdom, 3Department of Materials Science andEngineering, Johns Hopkins University, Baltimore, MD, USA.Sodium ion conduction plays pivotal role in action potential of excitable cells;this selective, rapid and transient process is regulated by the opening, inactivat-ing and closing of voltage-gated sodium channels (Navs).A wide variety of life-threatening diseases such as epilepsy, cardiacarrhythmia, and chronic pain syndrome were attributed to pathogenic muta-tions of human sodium channels; hence these channels are subject to extensivestructural and functional studies and enlisted as key targets for drugdevelopment.Several crystal structures of bacterial sodium channels (BacNavs) have beenpublished recently (J. Payandeh et al. 2011; Jian Payandeh et al. 2012; Zhanget al. 2012; McCusker et al. 2012; Bagneris et al. 2013; Bagneris et al. 2014;Shaya et al. 2013), which serve good starting points in understanding structuraland mechanistic details of Navs. Although the proteins are present in differentfunctional states, these studies render a consensus transmembrane architecturefor BacNavs. Transmembrane (TM) helices 1 to 4 form the voltage sensordomain (VSD) to sense the voltage changes a cross the cell membrane. In addi-tion, TM5 and TM6 form the pore domain (PD) to selectively allow Naþ ionpassage across membrane.Besides that, the cytoplasmic C-terminal domain (CTD) that follows thepore-lining TM6 transmembrane helices has two parts: a membrane proximalcharged region termed the ‘‘neck’’ and a unique ‘‘coiled-coil’’ region forBacNaVs. Structural and electrophysiological studies suggest this CTD hasfundamental role in maintaining channel activity and recovery from channelinactivation (Tsai et al. 2013; Bagneris et al. 2013; Shaya et al. 2013)Here, molecular dynamics (MD) simulations elucidate key interplays betweenCTD and other domains such as, VSD and PD, and shed lights on the mecha-nistic roles of this domain during channel gating.

532-Pos Board B297Molecular Modeling of Mammalian Nav1.4 ChannelAli O. Acar1, Esra Korpe1, Murat Cavus2, Serdar Kuyucak3, Turgut Bastug4.1Micro and Nanotechnology, TOBB University of Economics andTechnology, Ankara, Turkey, 2Faculty of Education, Bozok University,Yozgat, Turkey, 3School of Physics, Sydney University, Sydney, NSW,Australia, 4Material Science and Nanotechnology Engineering, TOBBUniversity of Economics and Technology, Ankara, Turkey.Cell membranes consist of two layers of lipid molecules and are impermeableto ions. Ion transportation across the membrane is mediated via membrane pro-teins which are pumps, transporters and ion channels. Voltage gated sodium(NaV) channels are essential elements in ion channels family that responsible

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for the rapid upstroke of the action potential. The crystal structures of bacterialNaV channels that have been determined recently, have made way for studiesof mammalian NaV channels through homology modeling. However, homol-ogy modeling is not straightforward because of the differences betweenmammalian and bacterial NaV channels. The Selectivity Filter (SF), whichplays a key role in ion permeation, has EEEE residues in bacterial channelbut has DEKA residues in mammalian channels. We have constructed a homol-ogy model for the NaV1.4 channel from the crystal structure of NaVMs (PDBID: 4CBC) bacterial channel. A recent study indicate that there are four stableinter-domain links between SF and neighboring domains. We additionally real-ized that there are extra residues making inter-domain links around the SF inNaVMs channel which are also conserved in the alignment between NaV1.4and NaVMs channel. The distances between the linked residues were used asrestraints in our homology model. The time series of N-O distances havebeen obtained between linked residues from a 100 ns MD simulation whichis performed using NAMD with the CHARMM36 force field. The N-O dis-tances of extra links were found to be stable. We also studied channel stability,sodium ion hydration and coordination as well as the permeation mechanism.Comparison of the our simulation results with experiments show that our ho-mology model provides a realistic representation of the mammalian NaV1.4structure.

533-Pos Board B298Mechanism and Energetics of Ion and Tetrodotoxin Binding to NavMsChannelEsra Korpe1, Ali Osman Acar1, Murat Cavus2, Serdar Kuyucak3,Turgut Bastug4.1Micro and Nanotechnology, TOBB University of Economics andTechnology, Ankara, Turkey, 2Faculty of Education, Bozok University,Yozgat, Turkey, 3School of Physics, Sydney University, Sydney, NSW,Australia, 4Material Science and Nanotechnology Engineering, TOBBUniversity of Economics and Technology, Ankara, Turkey.Voltage-gated sodium channels (Nav) are important targets for treatingvarious diseases. Crystal structure of the bacterial voltage-gated sodium chan-nel NavMs in the open conformation has been obtained by Ulmschneider et alrecently. We used this structure in our simulation work in order to study chan-nel stability, sodium ion coordination as well as the ion permeation mecha-nism. We have employed free energy techniques to calculate the potentialof mean force (PMF) for ion movement through the NavMs channel. ThePMF calculations revealed the ion-binding sites in the channel and the mech-anism of ion conductance. We also studied tetrodotoxin binding to the NavMschannel. Using docking and molecular dynamic simulations, we have con-structed a model for the NavMs-tetrodotoxin complex. The toxin binds tovarious parts of the channel and occludes the ion-conducting pore. Our resultshelp to explain experimental data and provide insights into the Nav inhibitionprocess. The complex structures we have found provide templates for devel-oping new sodium channel blockers with improved affinity and selectivityproperties, which will be useful in the design of novel drugs targeting sodiumion channels.

Voltage-gated Ca Channels I

534-Pos Board B299Modeling the Effects of Volatile Anesthetics on L-Type Ca2D Channelsand Ca2D Induced Ca2D Release in CardiomyocytesNeeraj Manhas.Physiology, MCW, Milwaukee, WI, USA.Studies have shown that volatile anesthetics (VA; e.g. isoflurane) exert negativeinotropic effects on cardiac cells, which could be one way in mediating cardi-oprotection against ischemia-reperfusion (IR) injury. Specifically, VA is shownto decrease the rate of Ca2þ entry into the dyadic space via the L-type Ca2þ

channels (LCC), while also activating the ryanodine receptors (RyR) andCa2þ induced Ca2þ release (CICR) process. However, the kinetic mechanismsof VA actions in these two processes and the consequent effects on the dyadicspace Ca2þ dynamics, which also regulate these two processes, are notwell-understood. We developed here a computational model to investigatethe effects of VA on Ca2þ dynamics in the dyadic space, by extending andintegrating contemporary kinetic models of the LCC and RyR, incorporatingtheir regulations by VA. The extended kinetic model of the LCC systematicallyreproduces the various aspects of the LCC and its regulations by VA, observedexperimentally; specifically, the VA inhibition of the LCC open probability andVA modulation of the LCC activation and inactivation gates that are mechanis-tically inferred through additional VA bound states of the LCC. Also, theextended kinetic model of the RyR is able to simulate the increased activity

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the RyR during VA exposure, as observed experimentally. The integratedcomputational model predicts the combined actions of the LCC and RyR andtheir modulations by VA on the dyadic space Ca2þ dynamics and how thetwo factors collectively regulate the LCC and RyR kinetics duringexcitation-contraction (EC) coupling in cardiac cells during VA exposure.This mechanistic computational model provides a strong foundation for awhole-cell level model of cardiomyocytes Ca2þ dynamics and electrophysi-ology during EC coupling and VA exposure during cardioprotection againstIR injury.

535-Pos Board B300Electrophysiological Characterization of T-Type Calcium Channels inCentral Medial Nucleus of the Rat ThalamusTamara Timic Stamenic, Slobodan M. Todorovic.Anesthesiology, CU Anschutz Medical Campus, Aurora, CO, USA.Central medial nucleus (CeM) is a part of intralaminar thalamus that isinvolved in the control of arousal but mechanisms that regulate its activityare not well studied. It is well known that low-voltage-activated T-typecalcium channels (T-channels) are abundantly expressed in the thalamuswhere they regulate neuronal excitability but their role in CeM was not pre-viously investigated. Here, we investigated properties of T-channels in CeMusing patch-clamp technique in acute coronal brain slices of adolescent rats.We used voltage-clamp recording with three different internal solutions: tet-ramethylammonium hydroxide (TMAOH) without ATP, cesium hydroxide(CsOH) with ATP and CsOH ATP-free solution. TMAOH internal solutioninduced a profound hyperpolarizing shift in steady-state inactivation curvesof about 20 mV when compared to CsOH with ATP (p<0.001, one-wayANOVA). Smaller but significant shift of about 8 mV was achieved withCsOH ATP-free internal solution (p<0.001). In contrast, voltage-dependent steady-state activation kinetics of T-currents were not differentunder identical recording conditions. Additionally, we found that pan-selective T-channel blocker TTA-P2 at 5 mM decreased T-current densityby about 70% (p<0.001, two-way RM ANOVA) and induced hyperpolariz-ing shift of 4 mV (p<0.001, t-test) in steady-state inactivation curves after10 minutes of application. Moreover, we used current-clamp recordings toinvestigate the effects of TTA-P2 on firing patterns and passive membraneproperties of CeM neurons. We found that TTA-P2 reduced tonic actionpotential frequency by 23% (p<0.05, two-way RM ANOVA), completelyabolished rebound burst firing, reduced low-threshold spike (LTS) ampli-tude by 78% (p<0.001), slightly increased input resistance by 15%(p<0.001) and latency to LTS by 18% (p<0.05). Our results stronglysuggest that T-channels are important regulators of neuronal excitabilityin CeM, which may be finely tuned by voltage-dependent phosphorylation.Supported by GM102525 to SMT.

536-Pos Board B301Unmasking the Molecular Determinants Important for Ca2D -DependentRegulation of CaV2.2Jessica R. Thomas, Jussara Hagen, Amy Lee.University of Iowa, Iowa City, IA, USA.(CDI) and facilitation (CDF), respectively, which contribute to short-term syn-aptic plasticity. Both CDI and CDF are mediated by calmodulin (CaM) bindingto sites in the C-terminal domain (CT) of the Cav2.1 a1 subunit, including aconsensus CaM-binding IQ-domain. Cav2.2 (N-type) channels display CDIbut not CDF but the underlying mechanism that blocks CDF in Cav2.2 is un-known. Here, we tested the hypothesis that Cav2.2 does not undergo CDF sinceit lacks essential molecular determinants for CDF that are present in Cav2.1.We find that alternative splicing of exons in the proximal and distal CT, whichregulates CDF of Cav2.1, has no effect on CDF of Cav2.2. However, replace-ment of the entire CT of Cav2.2 with that of Cav2.1 produces robust CDF ofthe chimeric channel. Further analyses reveal that transfer of the Cav2.1EF-hand, Pre-IQ- IQ domains, and a downstream CaM-binding domain(CBD) are sufficient to support CDF in chimeric Cav2.2 channels. Our resultshighlight the importance of the CT in distinguishing Ca2þ feedback regulationof Cav2.2 and Cav2.1, and underscore how molecular distinctions may underliethe unique contributions of these channels in regulating neurotransmitterrelease.

537-Pos Board B302C-Terminal Splice Variation Reveals New Insights into CalmodulinRegulation of CaV1.4 ChannelsBrittany Williams, Vasily Kerov, Daniel Soh, Amy Lee.University of Iowa, iowa city, IA, USA.In synaptic terminals of retinal photoreceptors, Cav1.4 (L-type) Ca2þ chan-nels mediate Ca2þ influx that promotes neurotransmitter release. Mutations

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in Cav1.4 are associated with multiple vision disorders including congenitalstationary night blindness (CSNB2). Cav1.4 does not undergo Ca2þ-depen-dent inactivation (CDI) – a negative feedback mechanism seen for otherL-type channels mediated by calmodulin (CaM) binding to a consensusIQ-domain in proximal C-terminal domain (pCT) of the pore-forming sub-unit. The lack of CDI in Cav1.4 is due to a C-terminal automodulatorydomain (CTM), located in the distal CT. The CTM is thought to suppressCDI of Cav1.4 channels by competing with CaM-binding to sites in thepCT. A CSNB2-causing mutation (K1591X) in Cav1.4 that deletes theCTM promotes CaM-binding and CDI, but also causes channel activationat more negative potentials than full-length channels (Cav1.4FL). Here,we demonstrate that similar properties are exhibited by a naturallyoccurring human Cav1.4 splice variant lacking exon 47 (Cav1.4Dex47),and characterized an unexpected role for CaM in the regulation of thischannel. By qPCR, we found that Cav1.4Dex47, which lacks the initial 43amino acids of the CTM, is expressed in primate but not mouse retina. Inelectrophysiological recordings of transfected HEK293T cells, Cav1.4Dex47Ca2þ currents activate at more negative voltages and display stronger CDIthan Cav1.4FL, similar to K1591X. These effects were blunted by IQ-domain mutations known to disrupt CaM-binding to Cav1.4. Mutationsthat prevent Ca2þ-binding to either N- or C-terminal CaM lobes suppressCDI of Cav1.4Dex47. However, mutations in the N-terminal but not theC-terminal lobe of CaM abolish the effect of exon 47 deletion on channelactivation. We conclude that exon 47 contains key molecular determinantswithin the CTM for regulating CDI and activation, and that CaM playsdistinct roles in these processes.

538-Pos Board B303Structural Characterization of Calmodulin Disease MutationsKaiqian Wang1, Jocelyn Lu1, Kamilla T. Larsen2, Michael T. Overgaard2,Filip Van Petegem1.1University of British Columbia, Vancouver, BC, Canada, 2AalborgUniversity, Aalborg, Denmark.Calmodulin (CaM) is a ubiquitous calcium-sensing protein involved in thepropagation of intracellular calcium signals and the regulation of eventsranging from muscle contraction to cell excitability. The human genomecontains three CaM genes (CALM 1-3) which encode for protein with iden-tical primary sequences. Despite the redundancy of CaM, single missensemutations in even one of the six alleles are associated with disease pheno-types such as catecholaminergic polymorphic ventricular tachycardia(CPVT) and early-onset severe long QT syndrome (esLQT). CPVT canlead to stress- and exercise-induced arrhythmias and sudden cardiac death;esLQT is characterized by a prolonged QT interval which can also resultin ventricular fibrillation. Despite the devastating genetic disorders associ-ated with CaM mutations, the molecular mechanisms by which these muta-tions manifest into dominant disease phenotypes have yet to be elucidated.Here, we present the crystal structures of several CaM disease mutants, oneof which represents a novel CaM conformation not previously characterized.Significant structural changes are observed in both EF-hands III and IV ofthe C-lobe. In particular, the mutation disrupts the calcium coordinationnetwork in EF-hand III and results in abolished calcium binding, leadingto CaM that resembles an intermediate between the Ca2þ-CaM andapo-CaM states. In contrast, structures of other disease mutants revealedCaM conformations that closely resemble the wild type structure, with littlepositional shift in the EF-hand helices of either the N- or C-lobe. Thesestructures can help explain the diverse effects of CaM mutations and theassociated disease mechanisms, especially as CaM mutations have differen-tial effects on the function of ryanodine receptor 2 and calcium-dependentinactivation of L-type calcium channels.

539-Pos Board B304Strontium and Barium in Aqueous Solution and an Ion ChannelBlocking SiteMangesh Chaudhari, Susan Rempe.Sandia National Labs, Albuquerque, NM, USA.Ion hydration structure and free energy establish criteria for understandingselective ion binding in potassium (Kþ) ion channels, and may be significantto understanding blocking mechanisms as well. Recently, we investigated thehydration properties of Ba2þ, the most potent blocker of Kþ channels amongthe simple metal ions. Here, we use a similar method of combining ab initiomolecular dynamics simulations, statistical mechanical theory, and electronicstructure calculations to probe the fundamental hydration properties ofSr2þ, which does not block bacterial Kþ channels. The radial distribution of

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water around Sr2þ suggests a stable 8-fold geometry in the local hydrationenvironment, similar to Ba2þ. While the predicted hydration free energy of�331.8 kcal/mol is comparable with the experimental result of �334 kcal/mol,the value is significantly more favorable than the �305 kcal/mol hydrationfree energy of Ba2þ. When placed in the innermost Kþ channel blockingsite, the solvation free energies and lowest energy structures of both Sr2þ

and Ba2þ are nearly unchanged compared with their respective hydration prop-erties. That result suggests that differences in blocking behavior may arise dueto kinetic properties associated with exchange of water ligands for channel li-gands instead of equilibrium thermodynamic properties.

540-Pos Board B305The Calcium Channel A2D Subunit Increases the Gating Charges ofCav1.2 ChannelsGustavo F. Contreras1, Nicoletta Savalli2, Antonios Pantazis2,Carlos Gonzalez1, Riccardo Olcese2, Alan Neely1.1Centro de Neurociencias de Valparaiso, Valparaiso, Chile, 2Division ofMolecular Medicine, Department of Anesthesiology, David Geffen School ofMedicine at UCLA, LA, CA, USA.Optically tracking the movement of individual voltage sensors (VSD) of humanCaV1.2 revealed that association of a2d-1 with a1C resulted in a substantialchange in the intrinsic voltage-sensing properties of VSDs I-III (Savalli et al2016, JGP 142, 147-159). Co-expression of a2d-1 increased the sensitivity ofactivation for several VSDs indicating that this mostly extracellular subunitincreased the electrical distances traversed by voltage-sensing charges. Inthis study we measured the slope of the voltage-dependence of the channel’sprobability of being open (Po) at extremely low probability using marcro-patches of Xenopus oocytes expressing CaV1.2/CaVb3 by themselves or incombination with a2d-1. Patches containing from 50 to several 100 channelswere recorded in 75 mM Ba2þ and in the presence of 1 mM (-) Bay K 8644and held at several voltages ranging from �70 to �20 mV for 10 to 90 s. Potimes the number of channels (NPo) were obtained by finding the most likelycombination of normal distributions shifted by the single channel currentsamplitude that described the data. The relative weight of the different distribu-tions was then contrasted with what should be expected from a Poisson distri-bution of opening levels. The number of channels (N) was estimated from thenoise analysis of hundreds of tail current traces at �40 mV following a depo-larizing pulse to þ80 mV. In the presence of a2d-1, the limiting slope wasabout 4 elementary charges, close to the sum of the voltage-dependencies ofVSDII and VSDIII activation and clearly less than the sum of all VSDs andthus consistent with the idea that only a subset of VSDs contribute to the effec-tive charges for channel opening. When the a2d-1 subunit was absent thelimiting slope was reduced by about one elementary charge. This modestchange can be accounted by a reduction in the electrical distance that gatingcharges need to cross for channel opening.Funding: FONDECYT 3140590 (GC) and 1120864 (AN), R01GM110276(RO) and 16POST27250284 (NS).

541-Pos Board B306Calmodulin and Stac3 Enhance Functional Expression of CaV1.1Jacqueline Niu, Manu Ben Johny, David T. Yue, Takanari Inoue.Johns Hopkins University, Baltimore, MD, USA.CaV1.1 is a prominent L-type voltage-gated calcium channel (VGCC) thatplays an integral role in mediating skeletal muscle excitation-contractioncoupling. Unlike other homologous L-type channels (e.g. CaV1.3), in depthbiophysical analysis of CaV1.1 is challenging as functional expression ofthese channels has been generally restricted to cell types with a muscular line-age. Interestingly, in contrast to CaV1.3, the carboxy terminus of CaV1.1 has alow affinity for the calcium binding protein, calmodulin (CaM) suggestingthat the loss of CaM pre-association may be responsible for the poor func-tional expression of CaV1.1 in recombinant systems. Here, we explicitlydemonstrate that restoration of CaM to the channel complex enables func-tional expression of CaV1.1 in HEK293 cells. Further mechanistic analysisshows that CaM substantially enhances surface membrane trafficking ofCaV1.1. In conjunction with recent studies that showed Stac3 (SH3 andcysteine rich domain 3) adaptor proteins also enable CaV1.1 currents in re-combinant systems (Polster et al (2015) PNAS 112:602), our results arguethat multiple cell signaling molecules can evoke similar functional outcomesand points to redundancy in molecular pathways that orchestrate CaV1.1 sur-face expression. These results also suggest there may be an overlap betweenchannel regulation and trafficking of L-type VGCC with Stac3 and CaM. Inall, our findings furnish a convenient platform to probe CaV1.1 function andrelated pharmacology.

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542-Pos Board B307CaV1.3 (Cacna1d) Gain-of-Function De Novo Missense Mutations areAssociated with CNS DisordersAlexandra Pinggera1, Luisa Mackenroth2, Jorg Striessnig1.1Pharmacology and Toxicology, University of Innsbruck, Innsbruck, Austria,2Institute for Clinical Genetics, Technical University Dresden, Dresden,Germany.Cav1.3 belongs to the family of L-type voltage gated Ca2þ channels. It isinvolved in many physiological functions, like hearing, sinoatrial node pace-making and hormone secretion. Moreover, it is expressed postsynaptically inneurons where it shapes neuronal firing, mediates gene transcription, and con-trols synaptic morphology and pruning. Recently we described two differentde novo Cav1.3 gain-of-function missense mutations in two patiens with autismspectrum disorders (ASD) suggesting that they present a strong risk for thedisorder. Here we strenghten this hypothesis by reporting the discovery andcharacterization of a third Cav1.3 de novo missense mutation in a patientwith ASD and epilepsy, localized in a highly conserved region within the chan-nel’s activation gate.The mutation was identified by sequencing of the coding exons of 4813 genesassociated with known Mendelian disorders. We introduced the mutation intotwo functionally distinct C-terminal long (Cav1.3L) and short (Cav1.343s) splicevariants of the Cav1.3 a1-subunit. We co-expressed wild-type or mutant a1-sub-units with auxiliary b3 and a2d-1 subunits in tsA-201 cells and performedwhole-cell patch-clamp recordings.The mutation resulted in a pronounced gain-of-function in both splicevariants, which, next to other gating changes, was evident by enhanced cur-rent densities and a negative shift of steady-state activation and inactivationleading to an increased window current at more negative voltages. Moreover,it significantly reduced Ca2þ-dependent inactivation in both splice variantswhereas voltage-dependent inactivation was only affected when introducedinto Cav1.343s. The observed gating changes are expected to affect neuronalsignaling and excitability. Together with previous findings we identifyrecurrent Cav1.3 gain-of-function mutations as a strong risk factor for CNSdisorders presenting as ASD, with and without epilepsy. Existing L-typeCa2þ channel blockers used as antihypertensives may provide a therapeuticoption for such patients.Supported by Austrian Science Fund (FWF F44020, W11010).

543-Pos Board B308Dual Effect of Palmitate on Voltage-Gated Calcium Channels and InsulinSecretion in Pancreatic Beta Cells of RatsNeivys Garcıa-Delgado1, Myrian Velasco-Torres1,2, Carmen Sanchez-Soto1,Marcia Hiriart2,3.1Inst Fisiologia Celular, Neuroscience Division, Universidad NacionalAutonoma de Mexico, Mexico DF, Mexico, 2Centro de Ciencias de laComplejidad, Universidad Nacional Autonoma de Mexico, Mexico, Mexico,3Inst Fisiologia Celular, Neuroscience Division, Universidad NacionalAutonoma de Mexico, Mexico, Mexico.A variety of signaling molecules modify voltage-gated calcium channels ac-tivity in pancreatic beta cells, among them are, free fatty acids (FFA). Acuteexposure to FFA increases glucose-stimulated insulin secretion, whilechronic exposure increases basal insulin secretion, but decreases glucose-stimulated (Olofsson et al., 2004; Zhou and Grill, 1994). Preliminary studiesin our laboratory showed that pre-incubation with 1 mM palmitate during48-72 hours reduced barium currents in beta cells. In this work, we analyzethe effect of different palmitate concentrations on calcium currents and in-sulin secretion in beta cells of adult male Wistar rats. Electrophysiologicalrecordings were performed using whole-cell voltage clamp technique.Reverse hemolytic plaque assay measured insulin secretion by isolatedbeta cells. Pre-incubation with 0.25 and 0.5 mM palmitate during 48-72 hincreased the high voltage-activated (HVA) calcium current withoutaffecting the LVA current. An acute 5 min pre-incubation with 0.5 mMpalmitate also increased HVA current. In contrast, 24 h pre-incubationwith 1 mM decreased both, LVA and HVA currents. A similar effect wasobserved during an acute incubation. Chronic pre-incubation with 0.25,0.5 and 1 mM palmitate decreased the percentage of insulin secretory cells,immunoplaque area and insulin secretion index in a glucose-stimulated con-dition (15.6 mM). Chronic pre-incubation with 0.5 and 1 mM palmitate, alsodecreased the secretion index at basal glucose (5.6 mM). However, acutepre-incubation with 1 mM palmitate increased the immunoplaque area in5.6 mM glucose. Together these results show that palmitate has a dual ef-fect, depending on time and concentration on the calcium currents and insu-lin secretion in beta cells. Partially supported by DGAPA PAPIIT-UNAMGrants: IN213114, M Hiriart; IV100116 to A. Frank and M Hiriart, andIN211416 to M Velasco-Torres.

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Voltage-gated K Channels and Mechanisms ofVoltage Sensing and Gating I

544-Pos Board B309Examining the Role of Phosphorylation on Interactions between theCardiac Potassium Channel Alpha-Subunits hERG and KVLQT1Medeea C. Popescu1, Louise E.O. Darling2.1Biochemistry Program, Wellesley College, Wellesley, MA, USA,2Biological Sciences Department and Biochemistry Program, WellesleyCollege, Wellesley, MA, USA.KvLQT1 and hERG are the voltage-gated Kþ channel a-subunits of thechannels which carry the cardiac repolarizing currents IKs and IKr, respec-tively. These currents function in vivo with some redundancy to maintainappropriate action potential durations (APDs) in cardiomyocytes. As such,protein-protein interactions between hERG and KvLQT1 may be importantin normal cardiac electrophysiology, as well as in arrhythmia and suddencardiac death. Previous phenomenological observations of functional,mutual downregulation between these complementary repolarizing currentsin transgenic rabbit models and cell culture have motivated our investiga-tions into interactions between hERG and KvLQT1. These data suggestthat a dynamic physical interaction between hERG and KvLQT1 modulatesthe respective currents. However, the mechanism by which HERG-KvLQT1interactions are regulated is still poorly understood. Phosphorylation isthought to play a regulatory role in this process: modifying the phosphory-lation state of each the proteins has been shown to alter channel kinetics,and both hERG and KvLQT1 are targets of the Ser/Thr protein kinasePKA, activated by elevated intracellular cAMP concentration. Throughclassic biochemical assays and quantitative FRET approaches, we aim tocharacterize the effects of phosphorylation in regulating interactions be-tween KvLQT1 and hERG in cellular model systems. We have developedion channel fusions to fluorescent proteins, which include hERG andKvLQT1 phosphonull and phosphomimetic mutants. We hypothesize thatphosphorylation abrogates protein-protein interactions, as suggested by find-ings that increased cAMP levels leads to decreased hERG-KvLQT1 interac-tion. This work potentially furthers our understanding of hERG-KvLQT1interactions and may elucidate mechanisms that underlie many types ofarrhythmia as well as characterize novel interactions between two distinctpotassium channel families.

545-Pos Board B310Monitoring Structural Reorganization of Calmodulin in Complex with theC-Terminus of KCNQ ChannelsCarolina Gomis-Perez1, Eider Nunez-Viadero1, Ganeko Bernardo-Seisdedos1, Covadonga Malo1, Pilar Areso2, Alvaro Villarroel1.1Instituto Biofisika (CSIC, UPV/EHU), Leioa, Spain, 2Dpt. Farmacologıa(UPV/EHU), Leioa, Spain.Calmodulin (CaM) is an essential component of the non-inactivatingvoltage-dependent potassium channels conformed by Kv7 subunits, and me-diates current suppression upon intracellular calcium elevation. Despiterecent atomic-level information on CaM complexed to Kv7 domains, thestructural consequences upon calcium binding remains elusive. To obtain in-sights on the structural changes caused by calcium, we have monitoredFRET between the AB module tagged with the blue protein mTFP1 andCaM tagged with the yellow protein Venus using purified recombinant pro-teins. In addition, FRET has been monitored in CaM/AB complexes inwhich the AB module was tagged with both fluorescent proteins. Significantchanges in energy transfer were observed in the presence of calcium, beingmore prominent for Kv7.1 than for Kv7.2 subunits. Thus, these data suggestthat calcium causes structural changes to different extent on each Kv7isoform.Financed by grant BFU2015-66910-R from MINECO.

546-Pos Board B311Novel Insights from Structural Analysis of Interactions of KCNQ KD

Channels with CalmodulinCrystal R. Archer1, Akash Bhattacharya1, Benjamin T. Enslow2,Alex B. Taylor1, Dmitri N. Ivanov1, Mark S. Shapiro3.1Biochemistry, University of Texas Health Science Center, San Antonio, SanAntonio, TX, USA, 2School of Medicine, University of Texas Health ScienceCenter, San Antonio, San Antonio, TX, USA, 3Cellular and IntegrativePhysiology, University of Texas Health Science Center, San Antonio, SanAntonio, TX, USA.Voltage-gated M-type Kþ channels, made by KCNQ subunits, regulate excit-ability in nerve and muscle. The gating of these channels are modulated by

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receptors coupled to Gq/11 G proteins via several signals, such as calmodulin(CaM) and the membrane phospholipid phosphatidylinositol 4,5-bisphosphate(PIP2), and protein kinases. Here, focusing on KCNQ4, we studied the struc-tural and biochemical mechanisms of channel regulation by CaM, usingisothermal titration calorimetry, microscale thermophoresis and nuclear mag-netic resonance. As for a number of other ion channels, CaM directly interactswith the C-terminus of KCNQ channels, at their A & B helices. Using similarbiochemical and structural methods, we also studied the interaction of KCNQchannels with PIP2 analogues and its cross-talk with CaM actions. Such cross-talk is made more likely by two loci of PIP2 interactions with the C-terminusnear the A&B helices. Since it is still unclear how CaM and PIP2 binding causephysical rearrangements of the channels that affect gating, we probed the affin-ities of CaM for the A & B helices, asked whether CaM is constitutively boundto the channels, and utilized our crystal structure of CaM bound to the KCNQ4A & B helices to gain insight into CaM-induced structural rearrangements ofthe C-terminus. To probe for potential cross-talk, we asked if Ca2þ bindingto the CaM/C-terminal complex affects PIP2 interactions and thus, PIP2 regu-lation of channel gating. Supported by NIH grants R01 NS094461-01 andR01NS043394-11 to M.S.S. and NRSA training grant F31 NS090887-02/NIH to C.R.A.

547-Pos Board B312KCNE1 and KCNE3 Modulate KCNQ1 Channels by Affecting DifferentGating TransitionsRene Barro-Soria1, Rosamary Ramentol1, Sara I. Liin1, Marta E. Perez1,Robert S. Kass2, H Peter Larsson1.1Physiology & Biophysics, University of Miami, Miami, FL, USA,2Pharmacology, College of Physicians & Surgeons, Columbia University,New York, NY, USA.KCNE b subunits assemble with and modulate the properties of voltage-gated Kþ channels. In the heart, KCNE1 associates with KCNQ1 to generatethe slowly activating, voltage-dependent IKs current that controls the repo-larization phase of cardiac action potentials. By contrast, in epithelial cellsfrom the colon, stomach and kidney, KCNE3 coassembles with the a-sub-unit KCNQ1 to form apparent voltage-independent Kþ channels importantfor controlling water and salt secretion. How KCNE1 and KCNE3 subunitsmodify KCNQ1 channel gating so differently is largely unknown. Differentmolecular mechanisms have been proposed to explain the effects of KCNE1and KCNE3 on KCNQ1 channels. Here, we use voltage clamp fluorometryto determine how KCNE1 and KCNE3 affect the voltage sensor and the gateof KCNQ1. By separating S4 movement and gate opening by a mutation, weshow that KCNE1 affects both the S4 movement and the gate, whereasKCNE3 directly affects the S4 movement and only indirectly affects thegate in KCNQ1. Further, we show that a triple mutation in KCNE3 convertsKCNQ1/KCNE3 channels into KCNQ1/KCNE1-like channels by intro-ducing KCNE1-like effects on the KCNQ1 gate. Our results suggest thatthe difference between the effects of KCNE1 and KCNE3 on KCNQ1 isthat KCNE1 affects both the voltage-sensing domain and the gate, whereasKCNE3 primarily affects the voltage-sensing domain and only indirectlyaffects the gate.

548-Pos Board B313CP1 Opens IKs Channels by Substituting PIP2

Moawiah M. Naffaa1, Xianjin Xu2, Hongwu Liang1, Guohui Zhang1,Hong Zhan Wang3, Junyuan Goa3, Ira S. Cohen3, Xiaoqin Zou4,Jianmin Cui1.1Biomedical Engineering, washington university in st louis, st louis, MO,USA, 2Department of Physics and Astronomy, University of Missouri,Columbia, MO, USA, 3Department of physiology and Biophysics, StonyBrook University, Stony Brook, NY, USA, 4Dalton Cardiovascular ResearchCenter, University of Missouri, Columbia, MO, USA.The voltage-gated potassium channel KCNQ1 co-assembles with the auxiliarysubunit KCNE1 to form the IKs channel in the heart. The IKs current, a slow de-layed rectifier Kþ current, plays a key role in repolarization of cardiac actionpotentials. Mutations in the KCNQ1 gene are associated with long QT(LQT) syndrome, atrial fibrillation and short QT syndrome, which lead to car-diac arrhythmias. It was shown that KCNQ1 and IKs channels require PIP2(Phosphatidylinositol 4,5-bisphosphate) for activation. This lipid acts as acofactor of the channel for the movements of the voltage sensor domain totrigger the pore gate domain to open. Using the previously identified PIP2binding site in a homology structural model of KCNQ1 as the target, anin-silico screening of chemical compounds in the Available Chemical Database(ACD, Molecular Design, Ltd.) and a subsequent experminetal testing on theIKs channels expressed in Xenopus oocytes identified CP1 as a novel IKs opener.

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CP1 increases IKs currents by two-fold and alters voltage dependence and ki-netics of activation and deactivation of IKs channels. The potentiation effectof CP1 on IKs currents has an EC50 of 7.4 mM. Consistent with targeting thePIP2 binding site,CP1 rescues the IKs current with the coexpression of CiVSP,which depletes PIP2 and inhibits the currents. The KCNQ1/IKs specific blocker,Chromanol 293B inhibits the enhanced current due to CP1 application, suggest-ing that CP1 substitutes for PIP2in activating IKs channels. In preliminary ex-perimetns with Guinea-pig ventricular myocytes, CP1 caused similar changesin chromanol sensitive IKs while also shortening action potential duration.CP1 as an opener of the IKs channels may provide a novel therapy to treat con-gential and acquired LQT syndromes.

549-Pos Board B314Pi(4,5)P2 Modulates Hysteresis and Pharmacology of KV7 ChannelsCarlos A. Villalba-Galea.Physiology and Pharmacology, University of the Pacific, Stockton, CA, USA.KV7 channels are critical components of the plasma membrane in many excit-able cells. The main function of KV7 channels is to contribute to the mainte-nance of the resting potential, thus regulating cellular electrical excitability.In Central Nervous System, the activity of the heteromeric KV7.2/KV7.3 chan-nel gives rise to M-currents. These Kþ-currents are subject to muscarinicmodulation as the phosphoinositide PI(4,5)P2, a signaling lipid dephosphory-lated during muscarinic activity, is required by KV7.2/KV7.3 channels to befunctional. On the other hand, Corbin-Leftwich and colleagues (JGP, 2016)have recently reported that the deactivation rate of KV7.2/KV7.3 channelsdepends on the duration of activation. This observation revealed that this het-eromeric channel displays a remarkable hysteretic behavior. In addition, it wasshown that these channels have, at least, two open modes and that these modesdiffer in stability, as deactivation from one mode is slower than the other, and inpharmacology, as the anticonvulsant Retigabine preferentially acts on theslower-deactivating mode. Since, (1) the hysteretic behavior of KV7.2/KV7.3depends of activity and, in turn, (2) activity depends on PI(4,5)P2, an clearnext step was to determine whether PI(4,5)P2 can modulate the effect of theanticonvulsant Retigabine. In addressing this question, here, it is presentedthat each of the open modes of KV7.2/KV7.3 channels displays distinct apparentaffinities for PI(4,5)P2. Also, it was found that decreasing the PI(4,5)P2 concen-tration either pharmacologically or enzymatically, reduces the effect of Retiga-bine on channel activity when applied in low doses. Furthermore, here it isshown that the hysteretic behavior of the KV7.2/KV7.3 channels does notemerge from being heteromeric, as the individual components display hyster-etic behavior as well. The work presented here provides evidence the leadsto propose that muscarinic modulation of M-current is likely dependent onthe hysteretic behavior of KV7.2/KV7.3 channels.

550-Pos Board B315Molecular Mechanism of the Kv7.1-Channel Activator N-ArachidonoylTaurineSara I. Liin1, Rene Barro-Soria2, H Peter Larsson2.1Clinical and Experimental Medicine, Linkoping University, Linkoping,Sweden, 2University of Miami, Miami, FL, USA.The cardiac IKs channel - formed by KV7.1 and KCNE1 - is important for thetiming of the cardiomyocyte repolarization. About 300 loss-of-function muta-tions in the KV7.1/KCNE1 channel have been identified in patients with LongQT syndrome and cardiac arrhythmia. These arrhythmias are today primarilytreated with drugs that prevent the triggering of arrhythmic activity, such asbeta-blockers, or with arrhythmia-terminating implantable defibrillator.A different treatment strategy for Long QT syndrome caused by loss-of-function mutations in the KV7.1/KCNE1 channel would be to pharma-cologically augment the channel function of these mutants, thereby shorteningthe prolonged QT interval and lower the risk of arrhythmia development.We have in previous studies observed that the fatty acid analogue N-arachido-noyl taurine activates the wild-type KV7.1/KCNE1 channel and KV7.1/KCNE1channels with diverse Long QT syndrome mutations by shifting the conduc-tance versus voltage (G(V)) curve towards negative voltages. Thissuggests that N-arachidonoyl taurine may function as a general activator ofKV7.1/KCNE1 channels with diverse mutational defects. The molecularmechanism of how N-arachidonoyl taurine activates the KV7.1/KCNE1channel is however not fully understood. In this project, we therefore usevoltage-clamp fluorometry and kinetic modeling to study the effect of N-arach-idonoyl taurine on voltage sensor movement and channel opening in KV7.1/KCNE1 channels. We also test the importance of specific residues in thechannel’s voltage sensing domain for the ability of N-arachidonoyl taurine toactivate the KV7.1/KCNE1 channel. An understanding of the molecular mech-anism of N-arachidonoyl taurine is critical to further improve this and related

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fatty acid analogues, which may inspire development of future KV7.1/KCNE1channel activators to treat congenital Long QT syndrome caused by mutationsin KV7.1/KCNE1.

551-Pos Board B316ML277 Opens KCNQ1 Channels by Selectively Enhancing the AO StatePanpan Hou, Ling Zhong, Powei Kang, Zachary Beller, Kelli McFarland,Jingyi Shi, Jianmin Cui.Biomedical engineering, Washington University in St Louis, St. Louis,MO, USA.The KCNQ1 Kþ channel a subunit and the auxiliary subunit KCNE1 form theIKs channel in the heart that is important in controlling heart rhythm. Inresponse to membrane depolarization, the KCNQ1 channel undergoes interme-diate open (IO) and activated open (AO) states that correspond to the stepwisemovement of the voltage sensor to the intermediate and full activation. IO andAO states showed different properties in voltage dependence of gating, perme-ation, and pharmacology. The association of KCNE1 suppresses IO but en-hances AO, thereby radically alters properties of the channel to suit thephysiological role of IKs in terminating the action potential. In this study, wefound that a recently identified KCNQ1 opener, ML277, increased theKCNQ1 current by selectively enhancing the AO state without affecting theIO state. ML277 effects on KCNQ1 channels showed a time and voltage depen-dence that coincide with that of the AO state. Voltage clamp fluorometrystudies revealed that ML277 did not change the voltage sensor movementbut enhanced channel opening in the AO state, indicating an enhanced VSD-pore coupling selectively in the AO state. Consistently, ML277 was found toincrease the sensitivity of channel activation to PIP2, which is required formediating the VSD-pore coupling as shown in our previous studies. These re-sults suggest that ML277 enhances the VSD-pore coupling specifically whenthe VSD is at the fully activated state to increase the AO state occupancy,thereby increasing the KCNQ1 current. Given that the IKs channel primarilyopens at the AO state, ML277 provides an example of drugs that can modulateIKs channels with a high specificity. ML277 may also be used as an excellenttool to study the molecular mechanisms of how the IO and AO states aredetermined in KCNQ1 channels.

552-Pos Board B317Comprehensive Assessment of Disease Mutant Forms of the HumanKCNQ1 Potassium ChannelHui Huang, Keenan Taylor, Charles Sanders.Biochemistry, Vanderbilt University, Nashville, TN, USA.The voltage-gated potassium channel KCNQ1 is critical for the cardiac actionpotential. Mutations in KCNQ1 and its accessory protein KCNE1 are the mostcommon cause of congenital long-QT syndrome (LQTS). There are a variety ofmechanisms by which a given mutation may cause KCNQ1 channel dysfunc-tion and prolonged activation potentials. The ideal treatment of patientsharboring a KCNQ1 mutation is dependent on which specific mechanismscause loss of function. In the present study, we have employed a multidisci-plinary approach to systematically investigate the specific effects of 51KCNQ1 mutations on the channel structure, stability, trafficking, and (i.e.through collaboration) electrophysiological properties. The 51 mutations,located in the voltage sensor domain (VSD), are disease causing, benign, orof unknown significance. High quality NMR spectra of the isolated wild typeVSD and of its mutant forms that are locked in fully activated state or in restingstate serve as reference spectra. The 1H-15N TROSY spectrum of each mutantwas collected and compared with the reference spectra to determine whetherthe mutation destabilizes the protein, or shifts the basal activated vs. restingstate equilibrium. We have also expressed each mutant full length KCNQ1in HEK293 cells and quantitatively assessed its total protein expression andcell surface expression using flow cytometry. These results will help elucidatethe exact defects of each mutant associated with LQTS, potentially providinginformation that can be used to inform personalized treatment of LQTS subjectsharboring KCNQ1 mutations.This work was supported by NIH Grant RO1 HL122010. We also thank the labof Prof. Alfred George at Northwestern University for providing the cDNA forthe KCNQ1-VSD mutants.

553-Pos Board B318A Voltage- to Ligand- Gated Switch in Voltage-Gated Potassium ChannelsXiaoping Pi, Qiang Ding, Zhaobing Gao.SIMM, Shanghai, China.To open voltage-gated ion channels (VGIC), the changes of membranepotential, usually depolarization, is prerequisite. In contrast, ligand-gatedion channels (LGIC) open when ligands bind. VGIC often stay in theclose state under hyperpolarize membrane potentials. Small chemical li-gands have been found to be able to allosterically modulate VGIC,

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but none of them can open VGIC in spite of membrane potential. Here,we report a cysteine is a ‘voltage- to ligand- gated’ switch for KCNQ2 chan-nel. Substitutions with alanine (A) or other smaller volume amino acids inthis position endow a ligand-gating to KCNQ2 channel in a measurablerange of membrane potential. Under constant �120 mV or even lowermembrane potentials, the mutant channels can be opened by small chemicalligands, such as ztz240 and ZnPy . Whereas, the wild type channel kept inclose under the identical conditions. Further mechanism investigationsrevealed that the volumn rather than other properties in this position isessential for the ligand induced opening, inferring that this position mayfunction as a baffle plate that restricts the upward movement of the S4segment. Our study reports a ligand-gated opening of KCNQ2 channel forthe first time. The identification of the ‘voltage- to ligand-gated’ switch inKCNQ2 channels breaks through the traditional barrier between VGICand LGIC.

554-Pos Board B319Voltage and Ca2D Sensor Coupling Modulation by b Subunits in the BKChannelYenisleidy Lorenzo1,2, Karen Castillo1, Gustavo Contreras1,Willy Carrasquel-Ursulaez1, Carlos Gonzalez1, Ramon Latorre1.1Centro Interdisciplinario de Neurociencia de Valparaiso, Facultad deCiencias, Universidad de Valparaiso, Valparaiso, Chile, 2Doctorado enCiencias Mencion Neurociencia, Facultad de Ciencias, Universidad deValparaiso, Valparaiso, Chile.BK channels are modulated by b-subunits (b1-b4) in a tissue-specificmanner. The b1- and b2-subunits increase in the apparent Ca2þ sensitivityof BK can be explained by a stabilization of the voltage sensor domain(VSD) in its active configuration. However, whether b1 modifies the numberof charges associated with the voltage sensor activation is still a matter ofcontroversy. The purpose of our study is to determine the effects of thepresence of b-subunits on the gating charge in the presence of Ca2þ, andto evaluate the coupling between Ca2þ-binding and VSD activation. Thenumber of gating charges per channel was measured in BK channels formedby a-subunit alone and with the different b-subunits in Ca2þ-free internalsolutions. The maximum gating charge displaced was obtained from thecharge-voltage (Q-V) curve and the total number of channels in the patchwas determined using noise analysis. Furthermore, we evaluated the effectof b-subunits on the interaction between Ca2þ sensors and VSD in differentCa2þ concentrations. We found that the total number of charges perchannel was 4.4, 3.0 and 4.2 e0 for BKa, BKa/b1 and BKa/b3b channelsrespectively. Increasing intracellular [Ca2þ] in BKa (100 mM) promotes asignificant leftward shift (~-140 mV) of the Q-V curve. The calcium effecton voltage sensor in BKa becomes apparent at [Ca2þ] R 1 mM (~V�30 mV). However, the leftward shift of the Q-V curve in BKa/b1 channelsbecomes evident in the nanomolar [Ca2þ] range (100-500 nM). BKa/b3bchannels behave as BKa channels. We conclude that: a) b1-subunit notonly modifies the resting-active equilibrium of the voltage sensor but alsodecreases the total number of apparent gating charges; b) there is a strongcoupling between voltage and Ca2þ sensors, this coupling is increased inthe presence of b1-subunit.

555-Pos Board B320Alpha-B Helix of RCK1 is a Major Transduction Pathway for Ca2D

Activation of BK ChannelsYanyan Geng1, Zengqin Deng2, Gonzalo Budelli3, Alice Butler Butler2,Jianmin Cui2, Peng Yuan2, Lawrence Salkoff2, Karl Magleby1.1University of Miami, Miami, FL, USA, 2Washington University, St. Louis,MO, USA, 3Brandeis University, Waltham, MA, USA.BK (Slo1) type Kþ selective channels are activated by both depolarizationand intracellular Ca2þ. BK channels are comprised of an integral-membrane core consisting of four lateral voltage sensor domains surround-ing a central pore-gate domain (S5-S6), and a large cytosolic domain (CTD)assembled from 8 RCK domains. The CTD (gating ring) is attached to thecore through four short S6-RCK1 peptide linkers. Ca2þ binding to theCTD activates the channel. Crystal structures (Yuan et al. 2010, 2012) sug-gest that Ca2þ binding elevates the alpha-B helix in each RCK1 domainwhere it could push upwards against the core while simultaneously movinga rigid protein arm in RCK1 laterally and downward to pull on S6 throughthe S6-RCK1 linkers. Ca2þ activation thus may involve a synergistic push-pull mechanism. We examined the contribution of the alpha-B helix to thishypothetical mechanism by introducing several discrete mutations into thealpha-B helix. The mutation L390P in the alpha-B helix of mSlo1 greatlydecreased Ca2þ activation. The crystal structure of the hSlo1 CTD withthe L390P mutation indicated a discrete alteration of the alpha-B helix

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alone: Ca2þ still bound to the Ca2þ bowl and there were no other notabledifferences in the CTD structure. L390P decreased Ca2þ activation throughboth high affinity Ca2þ binding sites, and also increased the voltage requiredfor activation in 0 Ca2þ. Lengthening the RCK1-S6 linker of L390P by oneresidue in 0 Ca2þ decreased voltage activation, indicating that L390P didnot remove all of the tension in the linker. Other alpha-B helix mutationswere also consistent with push-pull activation. These observations suggestthat an intact and rigid alpha-B helix plays a major role in transducingCa2þ binding to pore opening in BK channels. Supported by R01GM114694.

556-Pos Board B321Carbon Monoxide May Regulate BK slo1 Channel Activity by PartiallyDisrupting Heme CoordinationTaleh Yusifov1, Nicoletta Savalli1, Antonios Pantazis1,Stefan H. Heinemann2, Toshinori Hoshi3, Riccardo Olcese1,4.1Anesthesiology, Division of Molecular Medicine, UCLA, Los Angeles, CA,USA, 2Biophysics, Friedrich Schiller University of Jena, Jena, Germany,3Physiology, University of Pennsylvania, Philadelphia, PA, USA,4Physiology, UCLA, Los Angeles, CA, USA.The putative gasotransmitter CO is reported to activate large-conductanceKþ channels (BK); however, the mechanism remains controversial. Werecently found evidence that M691, within the RCK1-RCK2 linker of theBK C-terminus, coordinates heme together with the distal, conservedheme regulatory motif (HRM), acting as a second axial ligand to theheme Fe atom. A similar binding configuration is observed in cytochromec (CytC). Based on this and other lines of evidence, we have proposedthat BK channels possess a CytC-like domain (BKCytCD). In CytC, CO as-sociates with heme by cleaving the Met80 S-Fe2þ bond; in this work, we aretesting the hypothesis that BK channels bind CO by a similar mechanism.We expressed and purified the intracellular C-terminal portion of the humanBK channel (gating ring, GR). We first strengthened the evidence for theparticipation of M691 in heme coordination by detecting an absorptionpeak at 690nm, characteristic of the Met-Fe2þ bond, which was eliminatedby either mutation M691A or HRM disruption (C615S/H616R). We thenprobed for CO-induced GR conformational rearrangements using the 8-ani-lino-1-naphthalenesulfonic acid (ANS) fluorescent label in the presence ofheme: the wild-type GR/heme complex exhibited strong ANS fluorescencereduction in response to CO, reporting structural transitions likelyunderlying CO regulation of BK conductance. The GR-M691A/heme com-plex was unresponsive up to 1.2 mM of CO. However, in the presence ofCO-releasing CORM-2 ([RuCO3CI2]2), the GR-M691A/heme complex ex-hibited b/a absorption bands (540, 570nm) characteristic of CO binding.We propose that CO regulates BK conductance by inducing conformationalrearrangements in its GR ligand-sensing domain. Specifically, CO substi-tutes M691 as the second heme axial ligand; cleavage of the M691-hemebond partially unfolds the BKCytCD, generating the optically-trackedconformational changes. This action may relieve heme-induced inhibitionof BK opening.

557-Pos Board B322Role of the slo1 CRAC4 Motif in BK Channel’s Ethanol SensitivityMaria N. Simakova, Dasha Zaytseva, Shivantika Bisen, Alex M. Dopico,Anna N. Bukiya.Pharmacology, UTHSC, Memphis, TN, USA.Cholesterol (CLR) has been recognized for its ability to interact with ionchannels. Among ion channels that sense CLR presence are large conduc-tance, voltage/Ca2þ-gated Kþ (BK) channels. Functional BK channels resultfrom tetrameric association of pore-forming slo1 subunits. CLR inhibitionof slo1 current may result from CLR-sensing by the slo1 cytosolic taildomain (CTD). CTD contains seven CLR-recognition amino acid consensus(CRAC) motifs that contribute to the overall CLR sensitivity of the channel,with the contribution by CRAC4 being studied (Singh et al., 2012). Alcohol(ethanol) also modifies BK channel function, this effect resulting from anorchestration of several factors that include [Ca2þ]i. In native membranes,ethanol increases slo1 channel open probability (Po) at [Ca2þ]i<20-30 mMwhereas reduces Po at [Ca2þ]i<30 mM. Both CLR-enrichment and CLR-depletion of native membranes modify ethanol action on BK channels.Thus, we set to determine the role of CLR-sensing by the CRAC4 motifin the slo1 channel’s ethanol sensitivity. Slo1 cloned from rat cerebral arterymyocytes (cbv1) and the cbv1CRAC4T450F were incorporated into POPE:POPS (3:1 w/w) bilayers in 300/30 mM Kþ

i/Kþo gradient. At 0.3 mM Ca2þi,

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ethanol increased cbv1 Po by z3-fold. At 300 mM Ca2þi, ethanol failed tomodify cbv1 Po in CLR-free bilayers but increased cbv1 Po in CLR-containing (33 mol%) bilayers. The discrepancy in ethanol effect betweennative membranes and planar bilayers may likely arise from their differencein proteo-lipid, including CLR, composition. Cbv1CRAC4T450F did notrespond to ethanol in CLR-free bilayers, but was activated by ethanol inpresence of 33 mol% CLR. Collectively, the similarity of ethanol modula-tion of BK channel activity studied with cbv1 vs. cbv1CRAC4T450Fsuggests that CRAC4 does not contribute to CLR modulation of ethanol ac-tion on slo1 at high [Ca2þ]i. Support: R01AA023764 (AB), R37AA11560(AD).

558-Pos Board B323Differential Expression of BK Channel Beta1 Subunits in Rat Mesenteric,Coronary and Cerebral ArteriesGuruprasad Kuntamallappanavar, Shivantika Bisen, Anna Bukiya,Alex Dopico.Pharmacology, University of Tennessee Health Center, Memphis, TN,Memphis, TN, USA.Large conductance, Ca2þi/voltage-gated Kþ (BK) channels regulate severalphysiological processes (Yang et et al., 2015). In smooth muscle, BK com-plexes include channel-forming a and auxiliary b1 subunits (Brenner et al.,2000). BK b1 increases the channel’s apparent Ca2þ sensitivity (Brenner etal.,2000) and is required for channel activation by lithocholate (LCA)(Bukiyaet al., 2009). Here, we studied the expression of BK a and b1 subunits inmesenteric, coronary and cerebral (anterior, middle, posterior and basilar)arteries, and the contribution of such differential expression to LCA-inducedartery dilation. Thus, we conducted quantitative real-time PCR, fluorescenceconfocal imaging and patch-clamp electrophysiology on freshly isolatedcerebral artery myocytes, and diameter determination of arteries pressurizedin vitro. Data show that: 1) BK a mRNA is higher in cerebral anterior andbasilar arteries. In turn, b1 mRNA is higher in basilar and coronary arteries;2) BK a protein levels at the myocyte membrane surface are similar acrossthe different arteries whereas b1 levels are higher in basilar and coronary ar-teries; 3) BK channel basal activity is also higher in basilar and coronary arterymyocytes; 4) Likewise, BK channels from basilar and coronary artery myo-cytes are more responsive to LCA; 5) Finally, LCA-induced dilation of basilarand coronary arteries is higher than dilation of other arteries. Our resultsdemonstrate a differential protein expression and functional impact on bothbasal channel activity and its response to LCA, of BK b1 subunits acrossarteries from cerebral and systemic vasculatures. In particular, basilar andcoronary artery myocytes show an increased biochemical and functionalpresence of BK b1 subunits, suggesting that these vascular territoriesare more susceptible to modulation via BK b1. Support: HL104631,R37AA11560 (AMD); AHA Pre-doctoral Fellowship (GK).

559-Pos Board B324Functional Coupling of BK Channels to NMDAReceptors in HippocampalDentate GyrusXin Guan, Jiyuan Zhang, Qin Li, Hui-Lin Pan, Jiusheng Yan.Department of Anesthesiology and Perioperative Medicine, The Universityof Texas MD Anderson Cancer Center, Houston, TX, USA.The large conductance calcium- and voltage-activated potassium channel (BKchannels) is widely expressed in the central nervous system. The physiologicalroles of BK channels and the calcium sources of their activation in mammalianbrain remain not well-understood. Our proteomic and biochemical analyses ofBK channels and the calcium-permeable NMDA receptors and have found thatthey form protein complexes in whole brain and various brain regions, consist-ing of the obligatory BK channel alpha-subunit and GluN1 subunits and at leastthe regulatory GluN2A and GluN2B subunits. We examined the functionalcoupling of BK channels to NMDA receptors in mice hippocampal dentategyrus. We observed that glutamate evoked outward currents of BK channelsin dentate gyrus granule cell soma at potentials more positive than �20 mVwhich were sensitive to BK channel-specific blocker paxilline and abolishedby NMDA receptor antagonist AP-5. We also found that blockade of BK chan-nels by paxilline increased the amplitude of excitatory postsynaptic potential(EPSP) of granule cells evoked by stimulation in the perforant path-granulecell synapses, which was prevented by the presence of AP-5. We thus inferredthat BK channels were activated by NMDA receptor-mediated Ca2þ influx inhippocampal dentate gyrus granule cells and the calcium-mediated functionalcoupling between these two types of channels plays a role in regulation ofsynaptic transmission.

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TRP Channels I

560-Pos Board B325Binding of Extracellular Ca2D to the Specific Amino Acids is Required forHeat-Evoked Activation of TRPA1Erkin Kurganov1, Shigeru Saito1, Claire T. Saito1, Makoto Tominaga2.1Division of Cell Signaling, National Institute for Physiological Science,Okazaki-shi, Japan, 2Division of Cell Signaling, National Institute forPhysiological Science, OIIB, Okazaki-shi, Japan.Transient receptor potential, ankyrin 1 (TRPA1) is a homotetrameric nonselec-tive cation- permeable channel with six transmembrane domains handed bycytoplasmic N and C termini. Although many TRP channel family memberscontain the range of 3-6 ankyrin repeats (AR) within the N-terminal region,TRPA1 is distinguished by having an unusually large number of such repeats(16-17 ARs). A number of studies have shown that both intracellular and extra-cellular Ca2þ is a key regulator of many TRP channels, including TRPA1. Inthe previous study, we found that extracellular Ca2þ, but not intracellularCa2þ plays an important role in heat-evoked activation of green anoleTRPA1 (gaTRPA1). In this study, we focus on extracellular Ca2þ-dependentheat sensitivity of gaTRPA1 by comparing gaTRPA1 with other heat-activatedTRPA1s from rat-snake (rsTRPA1) and chicken (chTRPA1). We found thatrsTRPA1 and chTRPA1s are activated by heat with small inward currents inthe absence of extracellular Ca2þ. We identified several negatively chargedamino acid residues (glutamate and aspartate) near outer pore vestibule ingaTRPA1, chTRPA1 and rsTRPA1 for activation by heat in the presence ofextracellular Ca2þ. These results suggest that the neutralization of the acidicamino acids by extracellular Ca2þ is important for the heat-evoked activationof gaTRPA1, chTRPA1 and rsTRPA1.

561-Pos Board B326Oligomeric and Thermal Stability of TRPA1 Coiled-Coil Domain by Poly-phosphatesGilbert Q. Martinez, Luke D. Cody, Sharona E. Gordon.Physiology and Biophysics, University of Washington, Seattle, WA, USA.Human Transient Receptor Potential Ankyrin 1 (TRPA1) is a cation channelinvolved in the sensation of pain that is regulated by multiple stimuli includingtemperature, a wide range of irritants, and oxidative stimuli. The importance ofTRPA1 is underscored by the numerous studies of the channel as a target fornovel pain therapies. The recent cyro-EM structure (Paulsen et al, 2015) pro-vides a strong framework in which to understand the mechanisms underlyingfunctional activation. The structure of TRPA1 revealed the presence of a C-ter-minal coiled-coil domain that was hypothesized to require polyphosphates foroligomeric structural stability. This is consistent with the requirement of poly-phosphates for functional channel expression assessed via electrophysiology(Kim and Cavanaugh, 2007). Furthermore, it has been speculated that the un-folding of the TRPA1 coiled-coil helices in response to temperature could beinvolved in thermal gating, as has been observed in bacterial sodium channels(Arrigoni et al, 2016). Here we examine the biochemical and biophysical prop-erties of the coiled-coil of TRPA1 to determine the role of polyphosphates inoligomerization and their contributions to thermal stability.

562-Pos Board B327Toward Elucidating the Heat Activation Mechanism of the TRPV1 Chan-nel Gating by Molecular Dynamics SimulationHan Wen, Feng Qin, Wenjun Zheng.SUNY at Buffalo, Buffalo, NY, USA.As a key cellular sensor, the TRPV1 cation channel undergoes a gating transi-tion from a closed state to an open state in response to various physical andchemical stimuli including noxious heat. Despite years of study, the heat acti-vation mechanism of TRPV1 gating remains enigmatic at the molecular level.Toward elucidating the structural and energetic basis of TRPV1 gating, wehave performed extensive molecular dynamics (MD) simulations (with cumu-lative simulation time of 3 microseconds), starting from the high-resolutionclosed and open structures of TRPV1 solved by cryo-electron microscopy. Inthe closed-state simulations at 30�C, we observed a stably closed channel con-stricted at the lower gate (near residue I679), while the upper gate (near resi-dues G643 and M644) is dynamic and undergoes flickery opening/closing. Inthe open-state simulations at 60�C, we found higher conformational variationconsistent with a large entropy increase required for the heat activation, andboth the lower and upper gates are dynamic with transient opening/closing.Through ensemble-based structural analyses of the closed state vs. the openstate, we revealed pronounced closed-to-open conformational changesinvolving the membrane proximal domain (MPD) linker, the outer pore, andthe TRP helix, which are accompanied by breaking/forming of a network of

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closed/open-state specific hydrogen bonds. By comparing the closed-state sim-ulations at 30�C and 60�C, we observed heat-activated conformational changesin the MPD linker, the outer pore, and the TRP helix that resemble the closed-to-open conformational changes, along with partial formation of the open-statespecific hydrogen bonds. Some of the residues involved in the above keyhydrogen bonds were validated in comparison with previous mutationalstudies. Taken together, our MD simulations have offered rich structural anddynamic details beyond the static structures of TRPV1, and promising targetsfor future mutagenesis and functional studies of the TRPV1 channel.

563-Pos Board B328The Emerging Role of Human TRPV1 S1 S4 Sensing Domain in ChannelGatingMinjoo Kim, Nicholas Sisco, Jacob Hilton, Wade Van Horn.Arizona State University, Tempe, AZ, USA.Transient potential receptor ion channel vanilloid member 1, TRPV1, wasinitially identified as the capsaicin receptor, but is also modulated by variouschemical and physical stimuli including elevated temperature, acidic pH,endogenous ligands and regulatory proteins. TRPV1 functions as a primaryheat sensor in humans. Recently determined structures of rat TRPV1 have pro-vided a better understanding of channel architecture and the ligand gating.However, the mechanism of how thermal stimulus is integrated and convertedto channel gating remains poorly understood. The isolated S1-S4 sensingdomain of human TRPV1 (hV1-SD) was probed for its role in ligand gatingand thermosensing using biophysical techniques such as solution nuclear mag-netic resonance (NMR) spectroscopy and far-UV circular dichroism. Here, weinvestigate the direct binding of capsaicin to the hV1-SD. In addition, we iden-tify that the hV1-SD undergoes reversible conformational change as a functionof temperature. Thermodynamic studies suggest that the hV1-SD provides thedominant TRPV1 driving force that underlies thermosensitivity. Further NMRstudies probe temperature dependent changes in this domain and suggest sim-ilarities between TRPV1 ligand and temperature based activation.

564-Pos Board B329Inactivation in TRPV1 Ion ChannelsLeon D. Islas1, Ana Sanchez-Moreno1, Gisela Rangel-Yescas1,Ernesto Ladron de Guevara1, Tamara Rosenbaum2.1Physiology, School of Medicine, National Autonomous University ofMexico, Mexico City, Mexico, 2Instituto de Fisiologia Celular, NationalAutonomous University of Mexico, Mexico City, Mexico.TRPV1 are cation-permeable ion channels involved in several physiologicalprocesses. TRPV1 can be activated by several, very different, stimuli. Chiefamong these is a characteristic activation by temperatures in the noxious rangeand activation by several ligands, including capsaicin, the pungent compoundthat gives chilies their characteristic spiciness. Depolarizing voltages canalso activate these channels, although with a very small voltage-dependence(the associated valence of activation is at most 1 elementary charge, eo). Inthe absence of any other ligands, voltage behaves as a ‘‘partial agonist’’, pro-ducing a maximal open probability of less than 0.5 at extremely high voltages.Activation by voltage is more effective when combined with other activationmodes, including activating by capsaicin and temperature. It has been previ-ously shown that allosteric coupling models can describe this behavior, with in-dependent voltage, temperature and ligand gating modules allostericalycoupled to a pore module. Here we show that in the presence of these activators,TRPV1 channels show a marked inactivation behavior induced by high volt-ages, which can be seen as time- and voltage-dependent reductions in themagnitude of the outward currents and can be explained by models with inac-tivation happening after the open state. High activating temperatures (47-60 oC)can also induce inactivation by itself, even at moderate voltages and this tem-perature-dependent inactivation seems to be an irreversible process. Inactiva-tion of TRPV1 has not been previously described; although carefulinspection of previously published TRPV1 current records show evidence ofthis phenomenon. This work supported by grants PAPIIT-DGAPA-UNAMNo. IN209515 to LDI and No. IN200314 to TR and CONACYT Fronterasde la Ciencia No.77 to LDI and TR.

565-Pos Board B330Kinetic Analysis of Vanilloid-Induced Activation in TRPV1 ChannelSimon Vu, Bo Hyun Lee, Jie Zheng.University of California, Davis, Davis, CA, USA.The capsaicin receptor, TRPV1 ion channel, is a polymodal nociceptor andattractive drug target. Recent advances in cryo-electron microscopy revealedthe vanilloid-binding pocket in high resolution, which allowed atomic interac-tions mediating capsaicin binding to be proposed. In the current model, capsa-icin adopts a ‘‘tail-up, head-down’’ pose; key interactions include two

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hydrogen bonds formed between the amide of capsaicin and Thr551 and be-tween the hydroxyl on the vanillyl group and Glu571 (of mouse TRPV1).Guided by this model, the vanilloid-insensitive TRPV2 channel has been suc-cessfully transformed to be vanilloid-sensitive with minimal alterations. In thisstudy, we provide further evidence from kinetic and equilibrium analyses insupport of the capsaicin binding model. Eliminating the key hydrogen bondsby point mutations right-shifted the concentration-response relationship, whichwas achieved by accelerating the current OFF rate and decelerating the ONrate. These observations are consistent with the expectation that mutationswould weaken capsaicin binding. Surprisingly, though resiniferatoxin (RTX)has been proposed to bind in a similar fashion as capsaicin, kinetic and equilib-rium analyses revealed that removing these crucial hydrogen bonds did not alterRTX activation kinetics as they did capsaicin, suggesting that RTXmay behavedifferently.

566-Pos Board B331Rational Design and Validation of a Vanilloid-Sensitive TRPV2 IonChannelFan Yang, Simon Vu, Vladimir Yarov-Yarovoy, Jie Zheng.Physiology and Membrane Biology, University of California, Davis, Davis,CA, USA.Vanilloids activation of TRPV1 represents an excellent model system forligand gating of ion channels. Recent studies using cryo electron microcopy(cryo-EM), computational analysis and functional quantification revealed thelocation of capsaicin-binding site and critical residues mediating ligand-bind-ing and channel activation. To validate our current working model for capsaicinactivation, here we have successfully converted the vanilloid-insensitiveTRPV2 channel to be resiniferatoxin-activated, using a rationally designedminimal set of four point mutations (F467S-S498F-L505T-Q525E, termedTRPV2_Quad). We found that resiniferatoxin binds to TRPV2_Quad withsub-micromolar affinity even though the ligand-induced open state is relativelyunstable. Using Rosetta-based molecular docking, we observed a commonstructural mechanism underlying vanilloids activation of TRPV1 andTRPV2_Quad, where the ligand serves as a molecular ‘‘glue’’ that bridgesthe S4-S5 linker to the S1-S4 domain to open these channels. Furthermore,our analysis revealed previously unrecognized structural requirements neededfor capsaicin binding to TRPV1. These results should help guide the designof drug candidate compounds for this important pain sensor.

567-Pos Board B332Localization of the Temperature Sensors to the Pore Domain of the TRPV1ChannelFeng Zhang1, Sonya Hanson1,2, Andres Andres Jara-Oseguera1,Kenton Swartz1.1NINDS, NIH, Rockville, MD, USA, 2Computational Biology Program,Memorial Sloan Kettering Cancer Center, New York, NY, USA.Temperature detection is key to animal survival, and thermo-TRP channelsplay important roles in sensing environmental temperature in mammals. TheTRPV1 channel is a classic thermo-TRP channel that is also a detector ofnoxious stimuli, including acidosis, vanilloid compounds, venom toxins, mem-brane lipids and heat. Despite extensive investigation, the mechanisms of tem-perature sensing and the location of the temperature sensor in TRPV1 channelsremains enigmatic. One candidate for housing the temperature sensor is theouter pore of TRPV1, as mutants in this region, as well as occupancy by toxinsand sodium ions, have been shown to alter temperature-dependent activation.Here we engineered chimaeras between TRPV1 and the Shaker Kv channel us-ing available cryo-EM and X-ray structures to see if functional chimeras couldbe generated. Our biochemical analysis shows that a chimera containing thepore domain of TRPV1 in the background of the Shaker Kv channel can berobustly expressed in oocytes and traffic to the membrane surface. Electrophys-iological recordings show that these chimeric channels can be activated by aTRPV1-selective pore targeting double-knot toxin and are permeable to cal-cium ions similar to TRPV1. Remarkably, these chimeric channels are highlytemperature sensitive, being activated by temperatures above 40�C, similar tothe WT TRPV1 channel. Taken together, our results suggest that the poredomain of TRPV1 houses the sensory apparatus underlying steep tempera-ture-dependent activation.

568-Pos Board B333Transfer of TRPV1 Sodium Binding Site into TRPV2Katherine E. Huffer, Andres Jara-Oseguera, Kenton J. Swartz.National Institute of Neurological Disorders and Stroke, National Institutes ofHealth, Bethesda, MD, USA.The transient receptor potential vanilloid 1 (TRPV1) channel is a nonselectivecation channel activated by a wide variety of agonists, including acid, toxins,

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capsaicin, and noxious temperatures above 42�C. While recent cryo-EM struc-tures have revealed the binding pockets for capsaicin, double-knot toxin(DkTx), and resiniferatoxin (RTx), no consensus has been reached on the struc-tural basis of temperature activation. It was recently discovered that removal ofsodium from the extracellular side of the membrane shifts a temperature-depen-dent opening transition such that TRPV1 is open at room temperature, indi-cating that sodium binding modulates activation by temperature.TRPV1’s closest relative, TRPV2, is similar in sequence and structure, and canbe rendered sensitive to the TRPV1-selective agonist RTx by only two pointmutations, suggesting that the two channels have similar underlying gatingmechanisms. In spite of these similarities, TRPV2 fails to respond to mostTRPV1 agonists, including temperatures near 42�C, and TRPV2 is not acti-vated by removal of extracellular sodium. To probe the link between tempera-ture dependence and sodium sensitivity, and to localize the sodium ion bindingsite, we generated a series of chimeras in which transmembrane regions ofTRPV1 were transferred into TRPV2. Transfer of the entire transmembranedomain and TRP box of TRPV1 into TRPV2 resulted in chimeras that becameactivated upon sodium removal, supporting the feasibility of our strategy.Although none of chimeras in which small regions of TRPV1 were transferredinto TRPV2 were activated upon sodium removal, several exhibit potentiationwhen sodium is removed in the presence of subthreshold concentrations ofagonist, indicating the successful transfer of the sodium binding site. Ongoingexperiments are focused on determining the minimal TRPV1-like sodium bind-ing site and characterizing the temperature-activation thresholds of thesechimeric proteins to determine whether TRPV1-like temperature sensitivityis conferred by the sodium-binding domain.

569-Pos Board B334Investigating the Putative Activation Gate in the Selectivity Filter of theTRPV1 ChannelAndres Jara-Oseguera, Kenton J. Swartz.Molecular Physiol/Biophys, NINDS, NIH, Bethesda, MD, USA.The TRPV1 cation channel functions as heat sensor and an integrator of painfulstimuli in sensory neurons. The mechanism through which any of the multipleknown TRPV1 modulators induce the opening and closing of the pore is un-known. In order to understand this, it is necessary to identify the regions ofthe channel that directly open and close the pore, known as ‘‘activation gates’’.CryoEM structures of the TRPV1 channel in different closed and activatedstates revealed that the conduction pathway in the closed state presents twoconstrictions, or activation gates, that have to expand in the activated state toenable cation conduction. The first constriction is formed by Ile679 at the intra-cellular half of the pore-lining S6 helices. The second constriction is located atthe selectivity filter, and its function as a gate has not been tested experimen-tally. In order to determine whether the conducting and non-conducting confor-mations of the selectivity filter are associated with the activation or deactivationof the channel, we have investigated the accessibility to external cadmium ionsof cysteines introduced along the conduction pathway. We have introduced sin-gle cysteine substitutions starting at positions above the selectivity filter and allthe way to the internal gate at Ile679. We have determined which introducedcysteines can coordinate externally applied cadmium ions leading to rapid cur-rent block. We found that surprisingly few positions can lead to high-affinitymetal coordination. Moreover, those cysteines that coordinate metals canalso form spontaneous disulfide bridges that are consistent with the structuraldata. Finally, preliminary experiments suggest that in the closed state, cadmiumions can still access cysteines below the selectivity filter, suggesting that the fil-ter does not function as a gate. We are now investigating the effects of themulti-ion binding properties of the TRPV1 pore on the kinetics of cadmiumcoordination.

570-Pos Board B335The Mechanism of Regulation of TRPV6 Channels by PI(4,5)P2

Marina Kasimova1, Aysenur Yazici2, Chike Cao2, Phanindra Velisetty2,Vincenzo Carnevale1, Tibor Rohacs2.1Temple University, Philadelphia, PA, USA, 2Rutgers - New Jersey MedicalSchool, Newark, NJ, USA.Transient Receptor Potential Vanilloid 6 (TRPV6) is a Ca2þ selective ion chan-nel playing important roles in intestinal Ca2þ absorption, male fertility and can-cer development. TRPV6 is a member of the highly diverse TRP ion channelfamily. The only known common functional feature among TRP channels istheir dependence on, and modulation by phosphoinositides, mostly phosphati-dylinositol 4,5-bisphosphate [PI(4,5)P2]. Most TRP channels require factorsother than PI(4,5)P2 to open, which are often in complex interaction withPI(4,5)P2 regulation, which hinders understanding of the molecular mechanismof how PI(4,5)P2 opens these channels. TRPV6 is constitutively active, thusdevoid of these complexities, and therefore is an ideal model to gain molecular

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insight into regulation by PI(4,5)P2. We computationally docked PI(4,5)P2 toboth TRPV6, and the heat and capsaicin sensitive TRPV1. We find thatPI(4,5)P2 binds to TRPV1 mainly in the proximal C-terminus and in the S4-S5 linker, consistent with earlier predictions, and experimental data. InTRPV6, however, the closest interacting residues were in the proximal N-ter-minus, close to the first transmembrane region and, similar to TRPV1, in theS4-S5 linker. Introduction of a positive charge in the S4-S5 linker that ismissing from TRPV6, but present in TRPV1, markedly increased the affinityof TRPV6 to PI(4,5)P2. Neutralizing a proximal N-terminal positively chargedresidue predicted to interact with PI(4,5)P2 markedly reduced current TRPV6amplitudes, but neutralizing neighboring positively charged residues had no ef-fect. Our data will provide mechanistic insight into PI(4,5)P2 regulation ofTRPV6.

571-Pos Board B336The Role of the Sensing Domain (S1-S4) in TRPM8 Temperature andMenthol Dependent GatingParthasarathi Rath.School of Molecular Sciences, Arizona State University, TEMPE, AZ, USA.The human transient receptor potential melastatin 8 (hTRPM8) ion channel is aprimary sensor of environmental cold temperature. hTRPM8 is gated in a poly-modal manner by voltage, lipids, modulatory proteins, and chemical ligands,including menthol and icilin. TRPM8 is also involved in human health and dis-ease, playing roles in pathophysiologies including cancer, pain, obesity, anddiabetes. A better understanding of the molecular mechanism of channel acti-vation may prove useful in unlocking the TRPM8 therapeutic potential. Here,we analyze the sensing domain (transmembrane helices S1-S4) as a function oftemperature and menthol with circular dichroism (CD), and compare it with theS1-S6 transmembrane domain and full-length hTRPM8. Additionally, planarbilayer electrophysiology measurements of the S1-S6 transmembrane domainin presence of PIP2 and menthol indicate similar thermosensitivity whencompared to the full-length protein. The temperature and menthol dependentconformational change of the sensing domain was further investigated by solu-tion nuclear magnetic resonance (NMR) spectroscopy. Narrowing down fromthe full-length protein to the sensing domain, our data suggest that the S1-S4sensing domain is important for both TRPM8 temperature- and menthol-depen-dent gating of the ion channel.

Skeletal Muscle Mechanics, Structure, andRegulation

572-Pos Board B337Modulation of Fast Transients by Compliance of Crossbridges andMyofilamentsSrboljub M. Mijailovich1, Djordje Nedic2, Boban Stojanovic2,Thomas C. Irving3, Michael A. Geeves4.1Mechanical Engineering, Wentworth Institute of Technology, Boston, MA,USA, 2Mathematics and Informatics, University of Kragujevac, Kragujevac,Serbia, 3Department of Biological Sciences, Illinois Institute of Technology,Chicago, IL, USA, 4School of Biosciences, University of Kent, Canterbury,United Kingdom.Understanding the coupling between actomyosin cycle and compliance ofcrossbridges and the myofilaments in the 3D sarcomere lattice is essential forinterpretation of fast transient data and for extracting key parameters fromthe observations. This mechano-chemical coupling is complex due to thethree-dimensional nature of myosin binding to actin, as well as its effect onthe strain-dependence of transition rates between actin-myosin states. In orderto assess the effect of crossbridge compliance and myofilament elasticity onsarcomere contraction we implemented both linear and nonlinear crossbridgecompliance in the computational platform MUSICO (MUscle SImulationCOde). In the model simulation we included nine state myosin cycle (Smithet al., Ann. Biomed. Eng.36(10):1624-40, 2008), up to 20 sarcomeres in seriesand at least 500 myosin filaments per sarcomere. Stochastic binding in the 3Dsarcomere lattice generates large variation of strains in time at any locationalong the filaments and up to 20% between the filaments. Overall the modelpredictions are modulated by nonuniform distributions of crossbridge strainsalong the filament overlap and by variation of forces between the filamentsdue to the stochastic myosin binding process. The predicted T1-T2 transient re-sponses of the multi-sarcomere system strongly depend on the number ofattached crossbridges. This dependence is strongly evident in traces of T1 vs.the imposed length change, y, and to a much lesser degree of the T2-y responses.After matching the observed value of isometric tension before release, the sim-ulations closely recapitulate the rapid tension recovery, after the rapid drop intension to T1, to the early plateau, T2,followed by a slow tension redevelopment

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phase to isometric tension. The predicted rates, r, of quick recovery phasefollowing the length step, were similar to observations. However, to obtaingood fits to the observed T1-y nonlinear relationship required inclusionnonlinear crossbridge compliance (Kaya et al., Science 329:686-688, 2010).In summary, the precise coupling between nonlinear strain dependent transitionrates of the actomyosin cycle, nonlinear crossbridge compliance and elasticityof myofilaments provides excellent quantitative description of the molecular in-teractions and muscle fiber transient responses.Supported by: R01 AR048776, HD048895 and W.Trust 085309

573-Pos Board B338Increased Non-Uniformity in In Vivo Sarcomere Length during a TetanicContractionEng Kuan Moo, Timothy R. Leonard, Walter Herzog.Human Performance Laboratory, University of Calgary, Calgary, AB,Canada.IntroductionThe maximal, steady-state, isometric force produced by a muscle depends on itssarcomere length (SL). In previous studies in single myofibrils, it was foundthat sarcomere length non-uniformities increased during activation and forceproduction. However, single myofibrils lack much of the structural proteinsthat provide stability to entire muscles, thus the observed SL non-uniformitiesin myofibrils might not occur in whole muscles. This study was aimed at inves-tigating the change in SL distribution during tetanic contractions in an intactmuscles of live mice.MethodsMice were anaesthetized using isoflurane. The proximal femur and foot of theleft lower limb were clamped. The skin over the left tibialis anterior (TA) mus-cle was opened and stretched to form a bath for a saline solution that allowedfor imaging using a water-immersion objective. The TA was supra-maximallystimulated using a nerve cuff electrode on the sciatic nerve using 0.1ms squarewave pulses at 60Hz for 1s. SL were measured using second harmonic gener-ation microscopy for the fully stretched TA. Measurements were made for thepassive and activated TA over an area of 160x3 mm2 in the mid-belly of themuscle.ResultsSLs for the passive muscle were 2.5350.06mm (mean5sd). During contrac-tion, sarcomeres shortened by ~12% to 2.2450.12mm. The coefficient of vari-ation of the SLs doubled from 2.4% at rest to 5.2% during tetanic contractions.The range of SLs increased from the passive (2.36-2.71mm) to the active state(1.85-2.62mm).ConclusionSL non-uniformity doubled during muscle activation and differed by more than0.7 mm. The functional implications of these massive SL non-uniformities needto be explored, and the common practice of representing muscles with a singleSL value needs to be reconsidered.

574-Pos Board B339Myofibrillar Regulatory Mechanisms of Stretch Activation in MammalianStriated MuscleJoel C. Robinett, Laurin M. Hanft, Kerry S. McDonald.Medical Pharmacology and Physiology, University of Missouri, Columbia,MO, USA.Stretch activation is described as a delayed increase in force after an imposedstretch. Stretch activation is an essential process in the flight muscles of manyinsects and is observed, to some degree, in mammalian striated muscles. Themechanistic basis for stretch activation remains uncertain, although appearsto involve cooperative activation of the thin filaments (Campbell, K.S., Bio-physical Journal, 2006). The purpose of this study was to address myofibrillarregulatory mechanisms of stretch activation in mammalian striated muscle. Forthese studies, permeabilized rat slow-twitch and fast-twitch skeletal muscle fi-bers were mounted between a force transducer and motor, and a slack-re-stretchmaneuver was performed over a range of Ca2þ activation levels. Followingslack-re-stretch there was a stretch activation process that often resulted in atransient force overshoot (PTO), which was quantified relative to steady-stateisometric force. PTO was highly dependent upon Ca2þ activation level andthe magnitude was greater in slow-twitch fibers than fast-twitch fibers. Inboth slow-twitch and fast-twitch fibers, force development involved a fast,Ca2þ activation dependent process (k1) and a slower, less Ca2þ activationdependent process (k2). Interestingly, the two processes converged at low levelsof Ca2þ activation in both fiber types. PTO also contained a relaxation phase,which progressively slowed as Ca2þ activation levels increased and wasmore Ca2þ activation dependent in slow-twitch fibers. We next investigatedthe effects of PKA-induced phosphorylation of MyBP-C on stretch activationin slow-twitch fibers. Following PKA treatment, the magnitude of PTO more

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than doubled, however, this only occurred at low levels of Ca2þ activation(i.e., ~25% maximal Ca2þ activated force). Overall, these results are consistentwith a model whereby stretch transiently increases the number of cross-bridgesmade available for force generation and PKA phosphorylation of MyBP-C en-hances these stretch activation processes.

575-Pos Board B340Effect of Calcium on Myosin Binding to a Regulated Thin Filament fromSingle Molecule to EnsembleThomas Longyear1, Sam Walcott2, Edward P. Debold1.1Kinesiology, University of Massachusetts, Amherst, Amherst, MA, USA,2University of California, Davis, Davis, CA, USA.Muscle contraction is regulated via two processes that both contribute to acti-vation of the thin filament (actinþtroponinþtropomyosin). First, calcium(Caþþ) displaces tropomyosin, which increases the availability of myosinbinding sites on actin. Second, myosin binding accelerates the attachment ofneighboring myosin molecules to the thin filament. The relative importanceand underlying mechanisms of these two processes remain unclear, thereforewe devised assays to separately quantify their contribution . Myosin bindingonly activates nearby myosins, therefore by varying myosin density we variedthe contribution of this process. Myosin-induced activation was eliminated byusing a single myosin molecule in the laser trap assay to directly measureCaþþ’s effect on myosin’s interaction with a thin filament. Slightly increasingthe myosin density generated mini ensembles of myosin that experienced weakcoupling . Increasing the myosin density further using a motility assay createdlarge myosin ensembles with strong myosin-induced activation, and thin fila-ment speed was measured at increasing ATP and Caþþ levels. The datawere analyzed with a mathematical model that de-convolves the effects ofCaþþ-induced and myosin-induced activation. This analysis revealed (1)how Caþþ affects myosin’s attachment to a thin filament; and (2) that myosinmolecules are coupled over a Caþþ-independent distance L=400nm. Scalingup the model generated accurate predictions of isometric force productionand Caþþ binding to the thin filament previously observed in muscle fibers,providing a molecular basis for how Caþþ and myosin contribute to filamentactivation.

576-Pos Board B341Coordinated Variations in Myosin Light Chain and Troponin T Isoformsand in Maximal Shortening Velocity in Human Slow Skeletal MuscleFibersPeter J. Reiser.Biosciences, Ohio State University, Columbus, OH, USA.Recent measurements revealed a 1.7-fold range in maximal shortening velocity(Vmax) among a set of slow fibers from adult human skeletal muscle. The fiberswere examined on multiple gel formats to determine whether there were differ-ences in myofibrillar protein isoform composition that might explain the vari-ation in Vmax. All of the fibers in this set were determined to expressexclusively slow-type myosin heavy chain (MHC). The fibers were then exam-ined on a gel format to reveal potential differences in myosin light chain (MLC)isoform composition. The faster fibers in this set expressed substantial amountsof fast-type MLC1 (MLC1F), along with slow type MLC1 (MLC1b) and slowtype MLC2, and their mean Vmax was significantly higher (24%) than theslower fibers in this set, which had only trace or no detectable amounts ofMLC1F. The slower fibers had greater amounts of a protein that is tentativelyidentified as MLC1Sa, which others have reported to be expressed in humanskeletal muscle. The same gel format also indicated differences in troponin T(TnT) isoform composition among the same fibers. Regression analysis re-vealed that there are strong associations between the expression of specificTnT isoforms and the expression of specific MLC 1 isoforms among humanslow skeletal muscle fibers. Conclusion: coordinated variations in MLC iso-form and TnT isoform appear to provide a mechanism to expand the rangeof contractile properties of human slow muscle fibers that express exclusivelyslow-type MHC.

577-Pos Board B342Computational Simulations of Load-Dependent Myosin Kinetics duringMuscle Shortening and LengtheningAxel J. Fenwick, Bertrand C.W. Tanner.Integrative Physiology and Neuroscience, Washington State University,Pullman, WA, USA.Muscle contraction results from myosin heads binding to the actin filament toform cross-bridges, with contractile force dependent on the total number ofcross-bridges, the force that each cross-bridge produces, and the myosin attach-ment time (ton). Muscle shortening and lengthening also influences contractileforce, though the direct effect of dynamic length changes on cross-bridge ki-

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netics is unclear. Here, we used a spatially explicit, computational model ofmuscle force generation to examine how cross-bridge characteristics, such asforce per cross-bridge and ton, are affected by various rates of shortening andlengthening (0.1-1 muscle lengths (ML) per second). We found that the forceper cross-bridge increased by ~15% when the sarcomere was lengthened at0.1 ML/s, and by ~40% when lengthened at 1.0 ML/s. Conversely, force percross-bridge decreased during shortening, by ~25% at 0.1 ML/s and by~70% at 1.0 ML/s. Concurrent with these measurements, we found that myosinton decreased by ~50-70% as the sarcomere was lengthened at 0.1-1 ML/s,potentially due to increased cross-bridge strain or forced detachment. However,myosin ton increased by ~50% when the sarcomere was shortened at 0.1 ML/s,but decreased by ~50% when shortened at 1 ML/s. These data suggest thatmyosin attachment time is sensitive to length-dependent changes in cross-bridge strain, and that the kinetics may be more sensitive to the rate of short-ening than the rate of lengthening. While these results are model dependentand rely upon parameter estimation to fit model predictions with empiricaldata from skinned fibers, these computational models help illustrate complexsystem behavior underlying dynamic muscle contraction. These simulation re-sults indicate that sarcomere lengthening may increase the strain borne by in-dividual cross-bridges and decrease myosin ton, and conversely, shorteningmay decrease cross-bridge strain and prolong myosin ton.

578-Pos Board B343Cleavage of Skeletal Muscle Myosin Loops 1 and 2 Leads to a DecreasedFunctionYu-Shu Cheng, Oleg Matusovskiy, Dilson Rassier.Department of Kinesiology and Physical Education, McGill University,Montreal, QC, Canada.Introduction: There is controversy over the roles of loops 1 and 2 in skeletalmuscle myosin. The goals of this study were to investigate the effects of cleav-age of loops 1 and 2 on the amino-acid structure of the myosin and to correlatethese changes with myosin function. Methods: Heavy meromyosin (HMM)was isolated from myosin purified from rabbit psoas, and treated with trypsinfor periods of time ranging between 0 and 30 minutes, which caused a cleavageof loops 1 and 2. Specific cleavage of loop 1 was performed by adding actin thatbinds to myosin and protects loop 2, and cleavage of both loops was achievedby an ATP-free treatment. A motility essay with a frictional load was used byadding different concentrations of a-actinin (0.5 to 1.5mg/mL), and actin veloc-ity was recorded at 30�C in 1% nitrocellulose-treated coverslips. The myosinbands were cut from sodium dodecyl-sulfate polyacrylamide gel after trypsinproteolysis and subjected to tryptic cleavage. Myosin peptides of each bandwere identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A model was developed to interpret the results and showed that theloop 2 was damaged before loop 1 on myosin during actin motility. Resultsand Discussion: Sliding velocity of actin filaments decreased consistentlywith increasing time during trypsin treatment. The decrease was dependenton the treatment: cleavage of loops 1 and 2 decreased the velocity from4.05mm/sec to 2.24mm/sec after 4 minutes of enzymatic treatment, and cleav-age of loop 1 decreased the velocity from 3.82mm/sec to 1.22mm/sec after 2minutes of enzymatic treatment. These results suggest that loops 1 and 2 regu-late the velocity of the myosin motor and the kinetics of the actomyosininteractions.

579-Pos Board B344Force and Power of a Synthetic Myosin II-Based MachinePasquale Bianco1, Irene Pertici1, Luca Melli2, Giulia Falorsi1,Danut-Adrian Cojoc3, Tamas Bozo4, Miklos Kellermayer4,Vincenzo Lombardi1.1Department of Biology, University of Florence, Sesto Fiorentino, Italy,2National Heart, Lung and Blood Institute, Bethesda, MD, USA, 3IOM-CNR,Trieste, Italy, 4Semmelweis University, Budapest, Hungary.The function as a collective motor of skeletal muscle myosin II is studiedin vitro with a synthetic bio-machine, consisting of an ensemble of myosin mo-tors interacting with a single actin filament attached with the correct polarity toa bead trapped in the focus of a Dual Laser Optical Tweezers (DLOT, Bianco etal. Biophys. J.101:866-874, 2011). The motor ensemble provides the conditionfor cyclic interactions with the actin filament, allowing the development ofsteady force and filament sliding. The mechanical outputs of the machine aremeasured by means of the DLOT, which acts as a force transducer (range0.5-200 pN and resolution ~0.3 pN), and a piezoelectric nano-positioner car-rying the support for the myosin motors, which acts as a length transducer(range 1-75.000 nm and resolution ~1 nm). Here is reported the performanceof a first version of the machine, consisting of an ensemble of myosin motorspurified from frog skeletal muscle randomly adsorbed on the surface of a chem-ically etched single-mode optical fibre with diameter 4 mm. Isometric and

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isotonic contractions are reproduced by the motor ensemble in solution withphysiological [ATP] (2 mM) and temperature 21 �C. Following a drop in forcefrom the maximum isometric value (F0) to a lower value (F), the actin filamentslides at a constant velocity (V) which is larger the smaller the force, as ex-pected from the in vivo force-velocity relation. Up to five F-V points foreach interaction can be determined, allowing the definition of the maximum po-wer at F ~0.3 F0 (V ~2 mm/s) and demonstrating the unequalled ability of thesynthetic machine to define the power of native and engineered myosin II mo-tors from striated muscle. Supported by IIT-SEED, Genova and ECRF, 2015(Italy).

580-Pos Board B345Dissecting ActomyosinMechanochemistry using Blebbistatin as a Pharma-cological ToolMohammad A. Rahman1, Dilson Rassier2, Alf Mansson1.1Department of Chemistry and Biomedical Sciences, Linnaeus University,Kalmar, Sweden, 2Department of Kinesiology and Physical Education,McGill University, Montreal, QC, Canada.Muscle contraction results from cyclic interactions between actin and myosinunder turnover of ATP. Here, we use the myosin II-specific inhibitor blebbis-tatin as a pharmacological tool to elucidate this interaction. Particularly weconsider the hypotheses that: 1. the effects of blebbistatin depend on the levelof myosin regulatory light chain phosphorylation1 and 2. blebbistatin stabilizesthe start of the actomyosin power-stroke state, characterized by an absence ofinorganic phosphate (Pi) in the active site2. In vitro motility assays (IVMAs)and skinned muscle fiber studies were performed using preparations fromfast rabbit muscle. Blebbistatin (1mM) inhibited sliding velocity in the IVMA(~50%) independently of the RLC phosphorylation status, suggesting that thephosphorylation dependence in muscle cells is fully attributed to myosinhead-backbone interactions. The inhibitory effect was approximately half inconditions of reduced ionic strength from 130 to 60 mM, whereas the effectwas similar at different MgATP concentrations (0.01 �1 mM). Muscle fiberdata showed a reduced isometric force development by 36 5 11% and 89 55% at 2mM and 10mM blebbistatin, respectively. The force enhancementduring an imposed stretch to the fiber during activation (10% of fiber length;1 length/s) was unaltered. An interpretation of the blebbistatin effects linkedto a mecahano-kinetic model of muscle contraction is consistent with anincreased population of a start-of-power stroke actomyosin state without Piin the active site, and it is necessary for the assumption that blebbistatin stabi-lizes actomyosin states with bound Pi.

581-Pos Board B346Development and Phenotype Studies of a Slow Skeletal Troponin T E180Nonsense Mutation Knock-In Mouse LineHan-Zhong Feng, J.-P. Jin.Physiology Department, Wayne State University, Detroit, MI, USA.A nonsense mutation in codon Glu180 of TNNT1 gene encoding the slow skel-etal muscle isoform of troponin T (ssTnT) causes a recessively inherited severenemaline myopathy (Amish nemaline myopathy, ANM). Muscle biopsies ofANM patients showed a total loss of ssTnT. The present study developedand characterized mice with the E180 nonsense mutation knocked-in theTnnt1 gene (ssTnT-KI). Homozygotes of ssTnT-KI mice survived to adult-hood. Histology studies revealed significant increases of centralized nuclei inthe soleus muscle of ssTnT-KI when compared with age matched wild typecontrols. Immunohistochemistry staining showed atrophic slow type 1 fibersin ssTnT-KI mouse soleus muscle with severe inflammatory cells infiltration.Western blots confirmed the absence of full length ssTnT with no ssTnT frag-ments detectable, mimicking that seen in the muscle of ANM patients. Contrac-tile properties of ssTnT-KI mouse soleus muscle measured in situ demonstrateda decreased fatigue resistance. Significant expression of cardiac TnT is de-tected in soleus muscle of ssTnT-KI mice, consistent with the presence ofactive muscle regeneration. Decrease of slow type isoforms and increase offast type isoforms of other myofilament proteins with a significant hypertrophyof fast fibers in ssTnT-KI mouse soleus muscle indicate a potentially compen-satory adaptation to the loss of slow fiber functions. The ssTnT-KI mouse lineprovides a model of ANM for pathogenesis, pathophysiology and therapeuticstudies.

582-Pos Board B347Skeletal Myosin-Binding Protein CModulates Actomyosin Contractility inan Isoform-Dependent MannerAmy Li1, Shane Nelson1, Kyounghwan Lee2, Samantha Previs1,Karen Brack1, Michael Previs1, Suresh Govindan3, Sakthivel Sadayappan4,Roger Craig2, David Warshaw1.1Molecular Physiology & Biophysics, University of Vermont, Burlington,VT, USA, 2Cell and Developmental Biology, University of Massachusetts

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Medical School, Worcester, MA, USA, 3Oregon Health and ScienceUniversity, Portland, OR, USA, 4Molecular Physiology & BiophysicsDivision of Cardiovascular Health and Disease, University of Cincinnati,Cincinnati, OH, USA.Myosin-binding protein C (MyBP-C) is a sarcomeric protein localized todiscrete regions (C-zones) of myosin thick filaments in all striated muscles. Un-like cardiac muscle with its specific MyBP-C isoform, mass spectrometryshows that rat slow-twitch soleus (SOL) muscle expresses two differentslow MyBP-C isoforms (sMyBP-C), having one (sMyBP-C1) or two N-termi-nal inserts (sMyBP-C2), while fast-twitch extensor digitorum longus (EDL) ex-presses these same sMyBP-C isoforms and a fast MyBP-C isoform (fMyBP-C).Do these MyBP-C isoforms differentially regulate skeletal actomyosin contrac-tility in a muscle-specific manner? To address this, we characterized Ca2þ-regulated motion of SOL and EDL native actin-thin filaments (NTFs) overtheir corresponding native myosin-thick filaments, having the physiologicalcomplement of MyBP-C in the C-zone. At low calcium (pCa7.5) whereNTFs should be inhibited, motion occurred within the C-zone of both SOLand EDL native thick filaments. Whereas, at high Ca2þ (pCa5), SOL andEDL NTFs had initial fast velocities followed by ~30% slower velocitieswithin the C-zone. These data suggest that one or more of these MyBP-Ccan sensitize NTFs to Ca2þ and modulate contractility at high Ca2þ. To deter-mine how each isoform contributes to these modulatory capacities, we bacte-rially-expressed N-terminal fragments (sMyBP-C1, sMyBP-C2, fMyBP-C)and assessed their individual impact on SOL or EDL NTF motility over theirrespective monomeric myosins. For the EDL where all three MyBP-Cs are ex-pressed, at pCa7.5 sMyBP-C1 was most effective at activating NTFs, while atpCa5 all three reduced velocity by ~50%. For SOL, where only sMyBP-C iso-forms are expressed, at pCa7.5 sMyBP-C1 activated NTFs while at pCa5sMyBP-C2 reduced velocity ~40%. Our data suggest that the distinctivemixture of MyBP-C isoforms expressed in the SOL and EDL is matched tothe muscle’s physiological demands. Each MyBP-C isoform possesses specificmodulatory capacities that may be called upon under different contractilestates. Specifically, to fine-tune actomyosin contractility within the C-zoneby Ca2þ-sensitizing the thin filament and/or modulating velocity at maximalactivation.

583-Pos Board B348Expression of Myosin StorageMyopathyMutations inDrosophilaDisruptsMuscle Function, Myofibrillar Structure and Causes Defects in ThickFilament AssemblyMeera C. Viswanathan1, Rick Tham2, William A. Kronert2,William Schmidt3, Floyd Sarsoza2, Adrianna S. Trujillo2,Sanford I. Bernstein2, Anthony Cammarato3.1Johns Hopkins University, Baltimore, MD, USA, 2Molecular BiologyInstitute, San Diego State University, San Diego, CA, USA, 3School ofMedicine, Johns Hopkins University, Baltimore, MD, USA.Myosin storage myopathy (MSM) is a congenital skeletal muscle disordercaused by missense mutations in the slow muscle/b-cardiac myosin heavychain (MHC) rod and is characterized by subsarcolemmal accumulations ofb-MHC that have a hyaline appearance. Cases of MSM with cardiomyopathyhave been reported. The mutations disrupt hydropathy or charge of residuesin the heptad repeat, altering interactions that stabilize the rod’s coiled-coil di-mers and thick filaments. This potentially affects ordered myofibrillogenesisand/or myofibrillar integrity causing myosin aggregation. We generatedDrosophila MSM models to perform biochemical, biophysical, physiologicaland structural analysis of the effects of L1793P, R1845W, and E1883K muta-tions. Flight and jump ability were highly compromised in mutant homozy-gotes and heterozygotes. Confocal and electron microscopy revealed severedisruption of indirect flight muscle (IFM) morphology and myofibrillardisarray and degeneration with hyaline-like inclusions in pupal muscles,respectively. Mutant heterozygotes exhibited restrictive cardiac physiologyand diastolic dysfunction, which worsened with age. Lifespans of MSM mu-tants were reduced relative to control. Thus, our Drosophila models pheno-copy afflicted patients’ muscle structure and function. To help identify themolecular basis of the disease, in vitro studies were performed using MHC pu-rified from homozygous IFMs to evaluate the mutations’ effects without theconfounding influence of wild-type myosin. Assembly assays demonstrateda decreased ability of mutant MHC to polymerize, which resulted in shorterL1793P filament lengths. In addition, proteolysis experiments indicatedreduced stability of L1793P and E1883K filaments. We are currently deter-mining if similar thick filament aberrations are present in heterozygotes,in vivo. Our results indicate that diminished ability of the mutant myosinto form stable thick filaments likely contributes to the dystrophic musclephenotype observed in humans. Supported by R01HL124091 (AC) andR21OD01561 (SIB).

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584-Pos Board B349A Simplified Flexible Chain Model of Calcium Regulated Myosin-ActinInteractionLeonard P. Heinz, Rainer H.A. Fink.Medical Biophysics Unit, Institute for Physiology und Pathophysiology,Heidelberg University, Heidelberg, Germany.While the process of muscle contraction is relatively well understood at themolecular level, the collective behavior of many molecular motors hasbecome an interesting topic for computer simulations in recent years. Duke’smodel has proven to provide a good description of the myosin-actin interac-tion and the Flexible Chain Model can be used to describe the regulatorybehavior of the troponin-tropomyosin complex. Based on these models,we developed and implemented a simple computational motility assay thatcovers calcium-regulated muscle activation and allows to study the coopera-tive effects of many motormolecules working in parallel as well as the rela-tionships between contraction speed and isometric force versus calciumconcentration. Like in Duke’s model, myosin heads are modeled using a sim-ple 3-state system corresponding to detached, attached and strongly attachedto actin. The behavior of the troponin-tropomyosin complex, located at every7th actin binding site, is treated using a simple 2-state model, allowingtroponin to be attached or detached to actin. Both troponin and myosin controla continuous energy landscape that models the steric blocking of actin bindingsites by tropomyosin. This troponin-potential is computed using radial basisinterpolation. It is locally reduced, if a myosin head binds to actin andincreased, if a binding site is occupied by troponin, causing the system toact cooperatively.

585-Pos Board B350Reconstruction of Functional Insect Flight Muscle Fibers with RabbitSkeletal Muscle ActinHiroyuki Iwamoto.Res. & Util. Div., SPring-8, JASRI, Sayo-gun, Hyogo, Japan.Stretch activation (SA, delayed rise of force after stretch) is a mechanismessential for the fast wing-beat of insects with asynchronous flight muscles.To determine which constituent protein(s) is (are) indispensable for SA, wehave been conducting experiments to replace the endogenous actin ofbumblebee flight muscle with rabbit skeletal muscle actin, and we havedemonstrated that actin filaments can be regenerated as judged from X-raydiffraction patterns (2016 annual meeting). In this experiment, the endoge-nous actin is removed by gelsolin, and after this, rabbit G-actin is polymer-ized in situ. To prevent spontaneous contraction, a myosin inhibitor mustbe added to the solutions. For vertebrate skeletal and cardiac muscles, thepreferred inhibitor is BDM (butanedione monoxime, Fujita et al., 1996).However, BDM is not an effective inhibitor for insect flight muscle. Forthis reason, we used blebbistatin instead, and the actin filaments were suc-cessfully restored. The problem is that the inhibition by blebbistatin is irre-versible, and it is not reversed by extensive washout or irradiation withblue light. Here we used a high concentration of BDM (100 mM) andrepeated the experiments. The actin filaments were restored, and unlike inthe case of blebbistatin, actin-based layer line reflections were intensified af-ter washout of BDM and ATP. This indicates that the ability of the endoge-nous myosin to form rigor complexes is not compromised by the use ofBDM. We are currently trying to activate the flight muscle fibers preparedin this way.

586-Pos Board B351The Skeletal Muscle Molecular Clock Regulates Titin Isoform ExpressionLance A. Riley1,2, Xiping Zhang1,2, Karyn A. Esser1,2.1Physiology and Functional Genomics, University of Florida, Gainesville,FL, USA, 2Myology Institute, University of Florida, Gainesville, FL, USA.The circadian clock transcription factors, BMAL1 and CLOCK, are funda-mental transcriptional regulators of cell time keeping and critical cellspecific genes important for homeostasis. To determine the specific role ofthe molecular clock in adult skeletal muscle, our lab developed the inducible,skeletal muscle specific Bmal1 knockout (iMSBmal1-/-) mouse. Notably,skeletal muscles from these mice exhibit decreased specific tension andreduced unstimulated tension developed during a fatigability test. To beginto discern the molecular mechanisms that link the changes in the molecularclock with changes in muscle function, we tested whether iMSBmal1-/- musclewould also exhibit changes in sarcomeric protein expression. We determinedthat the tibialis anterior muscle of the iMSBmal1-/- mice exhibits a significantincrease (38% in iMSBmal1-/- mice vs. 19% in iMSBmal1þ/þ mice) in ex-pression of a longer isoform of titin. It is established that titin, the giant fila-mentous protein that maintains sarcomeric structure, underlies passive

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tension, and contributes to active tension development. To determine if thisincreased heterogeneity of titin isoforms within the TA had an effect onsarcomere length, we performed immunohistochemistry. An antibody to a-ac-tinin was used to demarcate Z-lines in longitudinal sections of the tibialisanterior muscle and sarcomere length was measured as the distancebetween Z-lines. While average sarcomere length was not different betweeniMSBmal1þ/þ and iMSBmal1-/- muscle, variation in sarcomere length wasincreased following Bmal1 knockout in skeletal muscle. Further studieswith our model are currently ongoing and will lead to increased knowledgeof the importance of titin isoform maintenance in a variety of pathologicalconditions.

587-Pos Board B352Cacium-Induced SR Calcium Leak in Dysferlin-Null Murine MuscleFibersValeriy Lukyanenko, Joaquin Muriel, Robert J. Bloch.Physiology, University of Maryland, Baltimore, MD, USA.Osmotic shock injury (OSI) decreases the amplitude of voltage-induced Ca2þ-transients (VICTs) in dysferlin-null (A/J) but not control (A/WySnJ) myofibersand also markedly increases sarcoplasmic [Ca2þ] (Kerr et al., Proc. Natl. Acad.Sci. USA, 2013). We compared the effects of drugs that target L-type Ca2þ-channels (LTCC: diltiazem, nifedipine, verapamil) and ryanodine receptors(RyR1: dantrolene, tetracaine, S107) on A/WySnJ and A/J FDB myofibers inculture to assess their effect on VICTs following OSI. We also examinedA/J fibers transfected to express N-terminal Venus chimaeras of dysferlin(V-Dysf). All Ca2þ antagonists inhibited VICTs in A/J and A/WySnJ fibersat high concentrations, but 1-10 mM diltiazem specifically increased VICT am-plitudes by ~15% in A/J fibers, restoring them to values close to controls. Allthe inhibitors at low concentrations improved recovery of VICTs in A/J fibersafter OSI. The fact that inhibitors of the LTCC and of the RyR1 protect A/Jfibers from OSI-induced loss of VICTs strongly suggests that the damagecaused to the VICTs by OSI is mediated by Ca2þ-induced SR Ca2þ leakthrough the RyR1. Consistent with this, injured A/J fibers produced Ca2þ

waves, indicative of Ca2þ-induced Ca2þ release (CICR). Treatment of A/J fi-bers with 10 mM S107 (stabilizer of RyR1-FKBP coupling that reduces Ca2þ

leak) or expression of V-Dysf both protected A/J fibers against the loss inamplitude of VICTs following OSI and prevented OSI-induced Ca2þ waves.Our data suggest that, in the absence of dysferlin, OSI causes increased leakof SR Ca2þ through the RyR1, leading to CICR. We conclude that dysferlinstabilizes the coupling of the LTCC and RyR1 to reduce Ca2þ leak when fibersare mechanically stressed. Supported by the Jain Foundation, MDA and NIH(RO1 AR064268).

588-Pos Board B353MG29 Interacts with Bin1 for Maintaining T-Tubule Structure in SkeletalMuscle Physiology and RegenerationXinyu Zhou1, Kristyn Gumpper1, Xinxin Wang1, Junwei Wu1, Tao Tan1,Miyuki Nishi2, Hiroshi Takeshima2, Jianjie Ma1, Hua Zhu1.1Department of Surgery, Davis Heart and Lung Research Institute, The OhioState University, Columbus, OH, USA, 2Department of BiologicalChemistry, Kyoto University, Kyoto, Japan.Mitsugumin 29 (MG29), a member of the synaptophysin-like family proteins,is a transmembrane protein mainly expressed in the t-tubule membranes ofskeletal muscle. Electron microscopy analysis of skeletal muscle derivedfrom mg29-/- mice revealed morphological defects of t-tubules, indicatingimportance of MG29 in maintenance of muscle structure and function. Todetermine the function of MG29 in muscle physiology and regeneration, weperformed co-immunoprecipitation and found that MG29 can bind to Bin1,another t-tubular protein. Two-color STORM super-resolution imaging anal-ysis confirmed co-localization of MG29 and Bin1 on t-tubules of skeletalmuscle. Interestingly, organized distribution Bin1 is severely disrupted inmg29-/- muscle. For testing the role of MG29 in muscle regeneration, weinjured gastrocnemius muscle with cardiotoxin (CTX) and tracked musclerepair and regeneration. Western blot showed that following CTX injury,MG29 protein levels were transiently reduced from day 1 to day 3, followedby recovery associated with muscle regeneration. Protein level of Bin1 in wildtype muscle remained unchanged during the CTX-injury and recover process.Compared with wild type muscle, the mg29-/- muscle displayed delayedregeneration with significantly reduced levels of Bin1 following CTX-inducedinjury. Together our data suggest that functional interaction between MG29and Bin1 contribute to maintenance of t-tubule network and its remodelingprocess associated with muscle injury and regeneration. Targeting theMG29/Bin1 complex might provide a potential effective approach for treat-ment of muscle diseases.

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589-Pos Board B354Phosphorylation of Myosin Increases the ATPase Activity of RelaxedSkeletal Muscle FibersNariman Naber, Roger Cooke.Biochem/Biophys, Univ california, San Francisco, CA, USA.Myosin in skeletal muscle is phosphorylated by a calcium-calmodulin depen-dent kinase during periods of sustained activity. Phosphorylation of myosinis known to destabilize the array of myosin heads bound to the core of the thickfilament in relaxed muscle, known as the interacting heads motif. Myosin headsin this array are associated with a state with a highly inhibited ATPase activity,known as the Super Relaxed State (SRX). To determine whether phosphoryla-tion of myosin increases the ATPase activity, we measured the ATPase activityof relaxed, skinned skeletal fibers in the presence and absence of myosin phos-phorylation. Fibers were phosphorylated, >50%, by including phosphatase in-hibitors in the glycerinating solution. Control fibers had an ATPase activity of0.040 5 0.004 s�1. Phosphorylated fibers had an ATPase activity of 0.060 50.002 s�1, a 50 % increase. We have recently found a small molecule, piperinethat destabilizes the SRX, increasing relaxed fiber ATPase activity. In thepresence of 100mM piperine the control fibers had an ATPase activity of0.064 5 0.004 s�1, the phosphorylated fibers had an ATPase activity of0.057 5 0.001 s�1. Thus both phosphorylation and piperine increase theATPase activity of resting fibers by destabilizing a fraction of myosin headsin the SRX, 30-50%, but their effects are not additive, suggesting that theyare each affecting the same population of myosin heads. One candidate forthis population may be the ‘‘free’’ head of the interacting heads motif, whichis known to be less stable than the other head. As myosin remains in a phos-phorylated state for several minutes following a period of activity, the effectdescribed above will contribute to the energetic cost of physical activity, partic-ularly during light activity.

Cardiac Muscle Mechanics and Structure I

590-Pos Board B355Impact of Para-Nitroblebbistatin on Human Beta-Cardiac Myosin at theMolecular and Tissue LevelsWanjian Tang1, Cheavar Blair2, Kenneth S. Campbell2,Christopher M. Yengo1.1Cellular and Molecular Physiology, Pennsylvania State University Collegeof Medicine, Hershey, PA, USA, 2Physiology, University of Kentucky,Lexington, KY, USA.Inherited cardiomyopathies are a common form of heart disease caused by mu-tations in sarcomeric proteins with beta cardiac myosin being one of the mostfrequently affected genes. Since the discovery of the first cardiomyopathy asso-ciated mutation in beta-cardiac myosin, a major goal has been to correlate the invitro myosin motor properties with the contractile performance of cardiac mus-cle. Mutations that cause hypertrophic cardiomyopathy are the most commonform of the disease and are often associated with increased isometric forceand hyper-contractility. Therefore, the development of drugs designed todecrease isometric force by reducing the duty ratio (the proportion of ATPasecycle time myosin spends bound to actin) has been proposed for the treatmentof hypertrophic cardiomyopathy. There is still no consensus about whether hy-pertrophic mutations in beta-cardiac myosin increase myofilament calciumsensitivity as is commonly found in troponin mutations. We examine theimpact of a small molecule drug proposed to decrease duty ratio (para-Nitro-blebbistatin) using purified human beta cardiac myosin motor assays andstudies of permeabilized human cardiac muscle mechanics. We find that thisdrug reduces actin-activated ATPase and in vitro motility while not changingthe ADP release rate constant. Thus, the reduction in the in vitro sliding veloc-ity is likely due to a slowing of attachment rate. In muscle fiber studies wefound that para-Nitroblebbistatin reduces steady-state force and calcium sensi-tivity but does not change maximum contractile velocity and the rate of forcedevelopment. These results support a model in which the drug reduces the dutyratio without altering the kinetics of attached crossbridges. Thus, small mole-cule drugs that target the crossbridge attachment rate are an attractive strategysince they are capable of reducing the force generating capacity and calciumsensitivity without altering contractile velocity.

591-Pos Board B356Modulation of Cardiac Myosin by a Small Molecule Agent that Targets theRegulatory Light ChainAnu R. Anto, Raja Kawas, Robert Anderson, Marcus Henze,Hector Rodriguez, Danielle Aubele, Jacques Mauger.MyoKardia, South San Francisco, CA, USA.

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A small molecule modulator of cardiac myosin enzymatic function has beenidentified (refered to as compound-A) and our characterization demonstratesthat this agent exerts its effect through the regulatory light chain (RLC) ofmyosin. Compound A inhibited the actin activated ATPase activity of cardiacheavy meromyosin (HMM, derived from bovine heart), which contains both theessential and regulatory light chains, whereas no inhibition was seen withbovine cardiac myosin subfragment-1 which does not contain the regulatorylight chain in our preparations. The regulatory light chain from full length car-diac myosin was removed and the extent of inhibition was dramaticallyreduced. Binding of this small molecule to the RLC was assessed using differ-ential scanning fluorimetry (DSF) and found that binding occurs to the RLC.Compound A was further characterized in permeabilized rat cardiac musclefibers and a dose-dependent decrease in maximal tension was observed. Ithas been demonstrated in animal studies that a small molecule modulator ofmyosin has applicability in treating hypertrophic cardiomyopathy, the identi-fied regulatory light chain modulator described here suggests that the RLCmay be a potential new target for therapeutic applications in hypertrophiccardiomyopathies.

592-Pos Board B357Cardiac Myosin Strutural Kinetics Modulated by Small MoleculesJohn A. Rohde, Hyun Cho, Lien Phung, David D. Thomas,Joseph M. Muretta.Department of Biochemistry, Molecular Biology and Biophysics, Universityof Minnesota, Minneapolis, MN, USA.Myosin’s lever arm undergoes structural changes that are coupled to itsbiochemical state during ATPase cycling in muscle. Structural states of myo-sin’s lever can be detected directly using the high-precision, high-throughputtechnique of transient time-resolved FRET, (TR)2FRET, revealing kinetics,thermodynamics, and allosteric coupling. We have previously used this tech-nique to investigate mechanochemical coupling in skeletal myosin and cardiacmyosin perturbed by the heart failure drug omecamtiv mecarbil. Here we inves-tigate other small molecule modulators of cardiac myosins mechanochemistry:arachidonic acid, EMD 57003, and compounds developed by MyoKardia, un-der investigation for treating hypertrophic cardiomyopathy. We find that oneMyk compound dramatically decreases the amplitude of the fast phase of thepower stroke, but does not significantly affect the rate of this transition. Thesedata suggest that the compound shifts the equilibrium constant for hydrolysistoward the pre-hydrolysis state. This is in sharp contrast to the effect of ome-camtiv mecarbil (Cytokinetics) under investigation for systolic heart failure,which shifts the equilibrium constant toward the post-hydrolysis states. Thiswork was supported by NIH AR032961 & AR057220 (DDT), American HeartAssociation Scientist Development Grant (JMM), and Graduate ExcellenceFellowship-University of MN (JAR).

593-Pos Board B358Force-Dependent Recruitment of Cross-Bridges from the Myosin Off-State can Contribute to Length-Dependent ActivationKenneth S. Campbell1, Paul Janssen2, Stuart G. Campbell3.1Physiology and Cardiovascular Medicine, University of Kentucky,Lexington, KY, USA, 2Physiology and Cell Biology, The Ohio StateUniversity, Columbus, OH, USA, 3Department of Biomedical Engineering,Yale University, New Haven, CT, USA.Cardiac muscle cells develop more force if they are stretched before they areactivated. The Ca2þ concentration that is required to produce half-maximalforce also decreases. These effects are known as length-dependent activationand are thought to contribute to the Frank-Starling Mechanism. The precisemechanisms underlying length-dependent activation remain unclear. Kam-pourakis et al. (PMID 27162358) recently showed that increasing the lengthof cardiac muscle causes myosin head domains to adopt orientations thatare more perpendicular to the thick filament axis. (There is an experimentaltrend at low Ca2þ and a statistically significant effect at high Ca2þ.)Increasing the extent of phosphorylation of cardiac regulatory light chain in-duces a similar but synergistic effect. These structural changes may representmyosin heads shifting from a super-relaxed myosin OFF state towards a con-ventional detached cross-bridge state. We developed a computer model of thissystem using freely-available MyoSim software (http://www.myosim.org).Our initial simulations established the model parameters that govern cooper-ative Ca2þ activation of the thin filament. Parameters were fitted to data byassuming a proportional relationship between the number of available bindingsites (as predicted by the model) and the movement of troponin C in theabsence of bound cross-bridges (as assessed experimentally by Kampourakiset al.). Further calculations then fitted predictions for tension-pCa curvesmeasured at short and long lengths and before and after treatment with myosin

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light chain kinase to complementary experimental data. We discovered thatthe simulations could reproduce Kampourakis et al.’s experimental data ifthe rate constant governing myosin’s transition from the OFF super-relaxedstate to the conventional detached state increased with myosin light chainphosphorylation and also increased linearly with force. Simulations withoutthe force-dependent transition did not exhibit length-dependent change inCa2þ sensitivity. These simulations demonstrate that a mechanism of force-sensitive myosin activation is capable of producing realistic length-dependent activation.

594-Pos Board B359Differences in Actomyosin Function in the Left and Right Ventricles ofHuman HeartsSebasian Requena.University of North Texas, Fort Worth, TX, USA.In both ventricles of the heart, actin is expressed from the same genes [1].There are no differences in twitch duration, work performance and poweramong the right (RV) and left (LV) ventricles in animals [2] So there is noexpectation that the properties of actin or myosin isolated from either ventriclewould be different. Nevertheless, the situation is more complex in humanhearts. The LV must pump more powerfully, because it has to overcome alarger resistance presented by the systemic system than the RV, which has toovercome a lower resistance offered by the pulmonary system. The questionarises whether stronger pumping action of the LV is partially caused by theLV actomyosin developing more force than the RV actomyosin. Such a ques-tion is impossible to answer by making macroscopic measurements, (such astension or ATPase activity) because the number of molecules involved insuch processes are enormous (of the order of 1011). To obtain molecular infor-mation, measurements must be taken from a few molecules. We measured var-iations in the polarization of fluorescence of a few actomyosin moleculesduring the contraction cycle and obtained molecular information by calculatingits autocorrelation function using R (version 3.3.1). The autocorrelation curvewas fitted with a bi-exponential decay model in order to extract the rate con-stants using XPFIT (version 1.2.1) Alango Ltd. XPFIT employs the InverseLaplace algorithm in order to numerically invert the decay time domaindata. The goodness of fit was assessed by chi-squared. The results suggestthat actomyosin function is identical in both ventricles. Refs: 1. Wessels,M.W. and P.J. Willems, Mutations in sarcomeric protein genes not only leadto cardiomyopathy but also to congenital cardiovascular malformations.Clin Genet, 2008. 74(1): p. 16-9. 2. Wikman-Coffelt, J., C. Fenner, A. Smith,and D.T. Mason, Comparative analyses of the kinetics and subunits of myosinsfrom canine skeletal muscle and cardiac tissue. J Biol Chem, 1975. 250(4):p. 1257-62.

595-Pos Board B360The Frank-Starling Mechansim is Attenuated by a DilatedCardiomyopathy-Associated Tropomyosin MutationJoseph D. Powers1, Farid Moussavi-Harami1,2, Maria Razumova1,Jil Tardiff3, Michael Regnier1.1Department of Bioengineering, University of Washington, Seattle, WA,USA, 2School of Medicine, Division of Cardiology, University ofWashington, Seattle, WA, USA, 3Department of Medicine, University ofArizona, Tucson, AZ, USA.Sarcomere length-dependent activation (LDA) of force development in cardiacmuscle is the cellular basis of the Frank-Starling mechanism, and is oftenblunted in heart failure. In patients with dilated cardiomyopathy (DCM),LDAmay be affected differently depending on the sarcomeric protein mutationunderlying the disorder, thus complicating strategies for therapeutic interven-tion. We investigated the effects of the DCM-associated tropomyosin mutationD230N (TpmD230N) on LDA by measuring force generation and twitch kineticsat short (~2.0mm) and long (~2.3mm) sarcomere lengths (SL) of intact and de-membranated trabeculae/papillary muscles from hearts of a transgenic murinemodel (>6 months of age) containing the TpmD230N mutation. Both types ofpreparations were mounted between a force transducer and linear motor. Intacttrabeculae were perfused with oxygenated physiological solution (30�C) andstimulated at 2Hz. Demembranated muscle preparations were bathed in phys-iological solutions (15�C) containing varying Ca2þ concentrations and allowedto generate steady-state force. As expected, for intact WT trabeculae, the twitchforce (Tp) and maximum rate of force development increased and the time topeak tension decreased when muscles were stretched from short to long SL(p<0.02). In contrast, TpmD230N trabeculae did not show significant changesin these parameters between short and long SL. There were no significantlength-dependent changes in twitch relaxation kinetics for either WT orTpmD230N trabeculae. In demembranated muscle preparations, pCa50 (the

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[Ca2þ] required to generate half-maximal force) decreased for TpmD230N prep-arations at both SL compared with WT preparations (with a larger effect at longSL), and the increase in pCa50 with stretch from SL ~2.0mm to ~2.3mm wasgreater for WT compared to TpmD230N preparations (p<0.05). Together, theseresults demonstrate an attenuated LDA in cardiac muscle of DCM TpmD230N

hearts and suggest a role for tropomyosin in modulating LDA. Funding:HL111197.

596-Pos Board B361Cardiomyopathy Mutations in the Converter Domain of Human Beta-Cardiac Myosin Impairs Mechanochemistry in the Presence and Absenceof LoadWanjian Tang, Shane D. Walton, William C. Unrath, Christopher M. Yengo.Department of Cellular and Molecular Physiology, Penn State College ofMedicine, Hershey, PA, USA.Missense mutations in human b-cardiac myosin are associated with inheritedcardiomyopathies, a leading cause of heart failure worldwide. The mecha-nisms for how mutations alter myosin’s motor performance are still unclear.We expressed and purified human beta-cardiac myosin subfragment 1(M2b-S1) containing point mutations that are associated with hypertrophic(R723G) or dilated (F764L) cardiomyopathy. We demonstrate that both theR723G and F764L mutations slow down the maximum actin-activatedATPase activity (20-30%). Direct measurements of the ADP release rate con-stant in the presence of actin using mant labeled ADP demonstrate R723G hasa 35% faster ADP release while F764L has a 20% slower ADP releasecompared to wild-type. Since the actin-activated ATPase is reduced in bothmutants, it suggests a slower actin-activated phosphate release rate constantor slower transition between actomyosin.ADP states. The in vitro motilityassay demonstrates relatively small differences compared to wild-type, witha 5-10% higher actin sliding velocity for R723G and 5-10% slower velocityfor F764L. We also examined the sliding velocity as a function of ADP con-centration, which provides information about the strain sensitivity of the mu-tants. We found that both mutants were less sensitive to ADP inhibition of invitro actin sliding velocity suggesting reduced strain sensitivity. Therefore, wepropose that both the hypertrophic R723G mutation and dilated F764L muta-tion have slower ATPase kinetics in the absence of load, while the mechano-chemistry in the presence of load indicates a reduced ability to adapt to higherloads.

597-Pos Board B362Maximal Force Increases at Physiological Temperature in MyocardialStrips from Non-Failing and Failing Human HeartsPeter O. Awinda1, Cheavar A. Blair2, Maya E. Guglin2,Kenneth S. Campbell2, Bertrand C.W. Tanner1.1Integrative Physiology and Neuroscience, Washington State University,Pullman, WA, USA, 2Division of Cardiovascular Medicine, Department ofPhysiology, University of Kentucky, Lexington, KY, USA.Heart failure contributes to 1 in 9 deaths in the United States. Contractile def-icits at the myofilament level may contribute to the heart inadequately pump-ing blood throughout the body. Yet, few studies have investigated forceproduction and cross-bridge kinetics at physiological temperature in humanmyocardium from non-failing and failing hearts. Using skinned myocardialstrips from the left ventricle free wall, we found that maximal Ca2þ-activated,isometric force was ~35% greater in non-failing vs. failing tissue at both 17and 37�C (p<0.001 for condition effect at pCa 4.8 and 2.3 mm sarcomerelength). Moreover, increasing temperature from 17 to 37�C increasedmaximal force by roughly 30% and 40% in non-failing and failing myocardialstrips, respectively (p<0.001 for temperature effect). Ca2þ-sensitivity of theforce-pCa relationship was reduced for non-failing vs. failing myocardialstrips (p=0.03 for condition effect), with differences in pCa50 being greatestat 17�C between non-failing and failing samples. These decreases in maximalforce and increases in Ca2þ-sensitivity of the force-pCa relationship thatoccurred with heart failure were not driven via differences in cross-bridgeattachment or detachment kinetics, as kinetics measures were largely consis-tent among non-failing and failing samples at each temperature. However,cross-bridge kinetics were 12-13 times faster at physiological temperaturevs. 17�C in both non-failing and failing samples (p<0.001 for temperature ef-fect), thereby representing a Q10 of ~3.5 for cross-bridge attachment anddetachment rates in human myocardial strips. Thus, faster cross-bridgecycling rates accompanied greater force production at physiological tempera-ture in both non-failing and failing samples. In addition, these data suggestthat heart failure compromises force production without significantly alteringcross-bridge kinetics, both at sub-physiological and physiological tempera-tures in human myocardium.

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598-Pos Board B363Structure and Nanomanipulation of the Titin M-Line ComplexZsolt Martonfalvi, Dominik Sziklai, Marton Kovacs, Zsombor Papp,Miklos S. Kellermayer.Semmelweis University, Budapest, Hungary.Titin is a giant protein spanning between the Z- and M-lines of the sarcomere.In the M-line the C-terminal region of titin overlaps with that of oppositely ori-ented titin from the other half of the sarcomere. Furthermore, titin-binding pro-teins such as myomesin and M-protein localize in the M-line so as to form acomplex. The M-line complex appears as a globular head-like structure in titinpreparations. The exact structure and the molecular arrangement within thisM-line complex is unknown. We analyzed the structure and stability of theM-line complex by using atomic force microscopy (AFM). We mechanicallydissected the M-line complex of single surface-adsorbed titin oligomers byusing AFM-based nanolithographic procedures. Titin was first deposited onmica so that the oligomers conformationally equilibrated on the surface.Then the M-line complex was dissected by pressing the cantilever tip intothe center of the globular head and moving the tip sideways in predetermineddirections and with constant velocity (1 um/s) and pressing force (1 nN).Finally, we scanned the surface so as to reveal the evoked changes. Loops offilaments with lengths up to 400 nm were pulled out of the M-line complex.The thickness of the filaments was one half that of native titin, suggestingthat partial unfolding may have taken place as a result of nanodissection.Our results suggest that the titin M-line complex may have a higher order threedimensional structure involving the packaging of participating filamentousmolecules. Nanodissection may be used as a tool to investigate the internalstructure of stable biomolecular complexes.

599-Pos Board B364Pre-Activation of Cardiomyocytes Determines Speed of Contraction: Roleof TitinMichiel Helmes1,2, Aref Najafi1, Martijn van der Locht1, Maike Schuldt1,Ilse AE Bollen1, Max Goebel1, Coen Ottenheijm1, Jolanda van der Velden1,3,Diederik WD Kuster1.1Physiology, VU Medical Centre, Amsterdam, Netherlands, 2Ionoptix Llc,Westwood, MA, USA, 3ICIN Netherlands Heart Institute, Utrech,Netherlands.The giant myofilament protein titin has an extendable region that functions as amolecular spring. Cardiomyocytes have exquisite control over the length of ti-tin, through splicing, enabling it to regulate passive stiffness. We hypothesizedthat titin as it sets the preload on the cardiomyocyte when stretched, togetherwith diastolic Ca2þ pre-activates the cardiomyocyte during diastole and thatthis pre-activation is a major determinant for force production in the subsequentsystolic phase. Via this route titin is thought to play an important role in activeforce development. Mutations in the splicing factor RNA binding motif protein(RBM20) results in the expression of large, highly compliant titin isoforms. Inthe present study we aimed to investigate the effect of long, highly complianttitin on the contractile properties of single cardiomyocytes. Wemeasured singlecardiomyocyte work-loops that mimic the cardiac cycle, in wildtype (WT) andheterozygous (HET) RBM20 deficient rats. In addition we studied detergent-permeabilized human patient samples that had known variations in titin basedpassive stiffness. At low pacing frequencies, myocytes isolated from HET leftventricles were unable to produce normal levels of work (55% of WT), but thisdifference disappeared when diastolic calcium increased at high pacing fre-quencies (>6 Hz). HET myocytes operated at higher SL to achieve the samelevel of work (2.1mm vs. 1.94mm at 6 Hz). In detergent-permeabilized cardio-myocytes isolated from human and rat heart we simulated cardiac twitches bytransiently (0.5 s) exposing the cell to a physiological calcium concentration ofpCa 5.7. Increasing pre-activation by bathing the cells in pCa 6.7 or pre-stretching the myocyte increased the kinetics of force development and thusthe total force development within a transient activation. This is consistentwith our hypothesis that pre-activation can increase force development in atime limited contraction such as a cardiac twitch. Pre-activation was pre-loaddependent as the sarcomere length to which the myocytes had to be stretchedfor equivalent levels of pre-activation varied with the compliance of titin.

600-Pos Board B365A Novel Approach to Identify the Role of Single Molecule Titin Mechanicsin Human Heart FailureMei-pian Chen1,2, Nancy S. Saad1,3, Benjamin D. Canan1,2, Ahmet Kilic4,Peter J. Mohler1,2, Paul M.L. Janssen1,2.1Department of Physiology and Cell Biology, The Ohio State University,Columbus, OH, USA, 2Dorothy M. Davis Heart and Lung Research Institute,The Ohio State University Wexner Medical Center, Columbus, OH, USA,

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3Department of Pharmacology and Toxicology, Helwan University, Cairo,Egypt, 4Department of Surgery, The Ohio State University Wexner MedicalCenter, Columbus, OH, USA.Diastolic dysfunction occurs not only in diastolic heart failure, but alsoubiquitously in systolic heart failure. A main determinant of diastolic passivetension is the elastic sarcomeric protein titin. The role of titin mechanicsin diastolic dysfunction is not clearly understood. Here we developed anovel approach to detect mechanical properties of the elastic domain of singletitin molecules from failing and non-failing human hearts. We further inves-tigate the role of titin mechanics in heart failure by correlating clinical andbiometric data from the corresponding human hearts, in conjunction withdiastolic function of isolated human cardiomyocytes. Using atomic forcemicroscopy, antibody specific tethering of the human native titin PEVK-distal Ig domain and the distal Ig domain alone was identified. Titin domainswere stretched and released at different frequencies based on ventricularvolume changes in the cardiac cycle. The nonlinear force tracings recordedfrom a series of linear stretch and release steps reflected titin viscoelasticity.Mean titin domain passive tension was measured in the range of 21.2 to213.8 pN. The mechanical properties of the testing domains were significantlycorrelated with both the diastolic parameters measured in field-stimulated sin-gle cardiomyoyctes, and the biometric data of the corresponding humanhearts. Our approach identified the mechanical properties of human nativecardiac titin domains, and their relationship to the diastolic function ofhuman hearts, up to single cell level. The current study provided insightsinto our understanding of cardiac relaxation kinetics in health and disease,contributing to the discovery of novel diagnostic and therapeutic strategiesfor heart failure.

601-Pos Board B366Modeling and Experiment to Determine the Role of Passive Stiffness onMechanical (Strain Rate) Control of RelaxationCharles S. Chung.Department of Physiology, Wayne State University, Detroit, MI, USA.Diastolic dysfunction is increasingly linked to exercise intolerance and heartfailure-like symptoms. The serendipitous discovery of a rat with a sponta-neous mutation in the RNA Binding Motif-20 (RBM20) has led to new studiesshowing the effects of reduced titin-based passive tension. Decreased passivestiffness in the heart is associated with increased exercise tolerance, but alsoslowed crossbridge attachment and detachment rates. The influence on relax-ation, and specifically mechanical control of relaxation by fast end systolicstretch, is not yet known. We utilized MyoSim, a computational modelingenvironment, and experiments using intact trabeculae to investigate the rela-tionship between titin-based passive stiffness and mechanical control ofrelaxation. We have previously obtained physiological myofilament parame-ters by fitting a 2-state crossbridge model to experimental data using MyoSim.These model parameters were held fixed except for passive stiffness, whichwas reduced by 50 and 75% to match passive stiffness in heterozygous andhomozygous RBM20 mutant tissues. Isometric relaxation was slowed by 4and 5%, respectively, but relaxation rate became 19 and 23% more sensitiveto mechanical control of relaxation (slope of relationship between relaxationrate and end systolic strain rate). These data predict that titin basedstiffness may modify relaxation rate. Trabeculae experiments using wild-type, heterozygous, and homozygous RBM20 mutant rats, show no significantchange in the mechanical control of relaxation. However, reduced titin stiff-ness was associated with a later time to peak contraction (shortening).Reducing crossbridge attachment and detachment rates in the computationalmodel parameters replicates the experimental results. These data suggestthat passive stiffness may play a role on mechanical control of relaxationand confirm that crossbridge properties significantly contribute to early dia-stolic relaxation.

602-Pos Board B367Direct Observation of Strain Transmission through the MicrotubuleNetwork of CardiomyocytesMatthew A. Caporizzo1, Brandon Kao2, Patrick Robison1,Alexey I. Bogush1, Benjamin L. Prosser1.1Department of Physiology, The University of Pennsylvania, Philadelphia,PA, USA, 2Department of Material Science, The University of Pennsylvania,Philadelphia, PA, USA.The stable microtubule (MT) network plays a significant role in regulating car-diomyocyte contractility and acts as a critical component for stretch-dependentactivation of intracellular Ca2þ release. Our previous studies suggest that forcetransmission though the MT network enhances Ca2þ sparks in response to me-chanical stress, yet stress transmission through the MT network has not been

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observed or characterized. To this end we have combined high-speed sub-diffraction limit microscopy and atomic force microscopy (AFM) to directlyobserve deformation of the MT network in response to localized stress. Theradial spread of MT deflection and the timescale of network relaxation wasquantified while directly probing myocyte mechanical properties. We findthat the normalized radial propagation of strain in the MT network is inverselyproportional to cardiomyocyte stiffness. Further, MT strain propagation isdecreased by a post-translational modification of the MT network, detyrosina-tion, which is increased in heart disease. To determine if MT deflections may betransmitting forces capable of enhancing Ca2þ release, the length and timedependence of calcium sparks were measured before, during and after localizedmechanical stress. During the application of stress, a localized upregulation incalcium sparks occured over the same length-scale that deformations in the MTnetwork propagated. These findings suggest that the stable MT-network maytransmit mechanical signals through the cardiomyocyte that correlate withCa2þ release and depend on MT detyrosination.

603-Pos Board B368Novel Roles for Obscurin Proteins in Cardiac MusclePatrick F. Desmond, Stephanie Myers, Anush Velmurugan, Matthew Klos,Yusu Gu, Nancy Dalton, Eric Devaney, Kirk Peterson, Ju Chen,Stephan Lange.Medicine, University of California San Diego, San Diego, CA, USA.Obscurin is a giant myofibrillar protein important for sarcomere structure,signaling, and maintenance of sarcoplasmic reticulum (SR) organization. Sur-prisingly, obscurin knockout mice develop normally and only show signs of amild skeletal muscle myopathy. However, little is known about cardiac func-tions of obscurin. We characterized cardiac roles of obscurin and found nochanges to cardiac physiology. One explanation for the lack of a cardiac pheno-type in obscurin knockout mice is that other obscurin protein family memberspossess a functionally redundant role. We hypothesized that obscurin-like 1(Obsl-1) is responsible for this redundancy, in part due to its shared ability tobind the sarcomeric proteins titin and myomesin. We generated doubleknockout (dKO) mice by crossing the obscurin-KOmice with a cardiac specificObsl1-KO line to investigate the roles for these proteins in cardiac developmentand function. Although a majority of the dKO-mice died after 12 months of age,microscopic and ultrastructural analysis failed to reveal overt changes tosarcomere organization. Dramatic changes were observed, however, in SRstructure and function. Specifically we observed: 1.) loss of SR structural integ-rity and apparent loss of SR content, and 2.) alterations to Ca2þ transients and inthe levels of Ca2þ handling proteins in KO animals. The majority of thesechanges were more severe in dKO-mice. Furthermore, we employed serialblockface TEM technology to precisely measure the volume of the SR andmitochondria and noticed significant differences between KO and control ani-mals. Physiologically, these mice develop a late onset cardiomyopathy charac-terized by increased systolic left ventricular internal dimension and alterationsin hemodynamic properties at ~12 mo. of age. Our data suggests that obscurinand Obsl1 are not critical for sarcomerogenisis, however are important forjointly organizing SR organization, cellular Ca2þ handling, and long-term sur-vival in mice.

604-Pos Board B369Diffusion in the Transverse-Axial Tubule System of Cardiac MyocytesCherrie H.T. Kong1, Eva A. Rog-Zielinska2, Clive H. Orchard1, Peter Kohl2,Mark B. Cannell1.1Physiology, Pharmacology and Neuroscience, University of Bristol, Bristol,United Kingdom, 2Cardiac Biophysics and Systems Biology Group, ImperialCollege, London, United Kingdom.Excitation-contraction (E-C) coupling in cardiac ventricular myocytes is criti-cally dependent on the structure of transverse-axial (t-) tubules, which are in-vaginations of the surface sarcolemma. Many currents have been shown tobe preferentially located at the t-tubular membrane, including the L-Type cal-cium current, Na-Ca exchange, tetrodotoxin-sensitive Na and steady-state Kcurrents (Orchard, Pasek, and Brette. 2009. Exp. Physiol. 94: 509-519). Ithas been suggested that membrane folding within the t-system may introducea slow diffusion zone, which may have implications for ion balance for thesecurrents during the E-C coupling cycle (Hong et al. 2014. Nat. Med. 20:624-632.). To investigate diffusion in the t-tubule system, we examined t-tu-bule structure in rabbit and mouse ventricular myocytes as two common exper-imental models, using electron tomography. Cells were also superfused withsolutions containing solutes of varying molecular weight to examine their pene-tration into the t-system. Fluorescence Recovery After Photobleaching (FRAP)was used to examine transport within the t-tubules of quiescent myocytes. Wefound that despite marked differences in t-tubule diameter and complexity in

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the two species, there were relatively small differences in solute penetrationbelow ~70kDa, and in the time-course of fluorescence recovery duringFRAP. These data suggest that gross differences in t-tubule structure do nothave marked effects on the rate of diffusion within t-tubules. This work wassupported by the BHF and MRC

Cell Mechanics, Mechanosensing, and Motility I

605-Pos Board B370From Elasticity to Inelasticity in Cancer Cell Mechanics: A Loss of ScaleInvarianceFrancoise Argoul.Laboratoire Ondes et Matiere d’Aquitaine, Talence, France.Soft materials such as polymer gels, synthetic biomaterials and living biolog-ical tissues are generally classified as visco-elastic or visco-plastic materials,because they behave neither as pure elastic solids, nor as pure viscous fluids.When stressed beyond linear viscoelastic regime, cross-linked biopolymergels can behave nonlinearly (inelastically) up to failure. In living cells thesenetworks exhibit high sensitivity to stress and propensity to local failure (plas-ticity). However, unlike synthetic passive gels, living cells can dynamicallycompensate these failures thanks to ATP driven reparation mechanisms andrecover a sustainable networked cytoskeleton architecture, as long as theyare kept in a suitable culture environment. When the failure events are toofrequent or too strong or when the reparation mechanisms are not efficient, irre-versible losses of mechanical homeostasis and chronic diseases such as cancermay ensue. Here, we concentrate on chronic myelogenous leukemia (CML) asa model for stem cell cancer transformation. We compare the mechanical abil-ity of both primary (healthy donors and patients) and immature cells (TF1)transduced by the CML oncogene BCR-ABL to respond to mechanical stresses(nano-indentation experiments). Our experimental study shows that primaryCML (CD34þ) hematopoietic cells encounter more frequent brittle failureevents than normal ones. This failure enhancement is also accompanied by amodification of the scale-invariant rheologic property of these cells. Interest-ingly, stiffening induced failure events are more visible on primary than onTF1 model cells, pointing out that their cross-talk with their native microenvi-ronment may also affect their transformation. Finally, combining these exper-imental studies with RNA-seq analysis on both control and BCR-ABLtransduced cells, we highlight the actin cytoskeleton signaling pathway assignificantly altered during CML transduction.References:B. Laperrousaz, et al. Physical Biology 13 (2016) 03LT01.C. Martinez-Torres et al. Applied Physics Letters 108 (2016) 034102.

606-Pos Board B371Migration Behavior of Normal and Metastatic Human Mammary CellsJosiah Low, Keith Bonin, Hyunsu Lee, Amanda Smelser, Martin Guthold.Physics, Wake Forest University, Winston-Salem, NC, USA.Metastasis is the process by which cancer spreads from a primary tumor siteto other sites in the body. Once a cancer has metastasized it is much moredifficult to treat, and a better understanding of metastasis is, thus, a criticalgoal in cancer research. Metastasis involves cell movement through thebody, and the aim of our study was to explore the differences in motility be-tween metastatic cells and noncancerous cells. Toward this end, we investi-gated cell motility using migration assays, in which cells are grown in twoadjacent, but separate zones to confluency. After confluency is reached, thebarrier separating the two cell zones on a glass bottom dish is removed,and the motion of the two edges of the cells separated across the septumgap is recorded. We hypothesized that cancerous human breast cells (MDA-MB-231 cells) would exhibit a higher rate of migration and a lower degreeof coordinated movement than normal breast cells (HMEC cells). Migrationassays were performed on both types of cells, and the migration into thegap between cell populations was recorded using time-lapse DIC microscopy.The resulting videos were processed using areal analysis and particle imagevelocimetry (PIV), which allowed extraction of areal migration rates and ve-locity vector fields. An average migration rate of 242mm2/min was measuredfor HMEC cells, and a lower degree of coordination was observed in MDAcells compared to HMEC cells.

607-Pos Board B372Adhesion-Controlled Proliferation Revealing Anti-Cancer Drug Resis-tance of Breast Cancer CellsSoyeun Park.College of Pharmacy, Keimyung University, Daegu, Korea, Republic of.Mechanical interactions of cancer cells with surrounding environments playan important role in cancer progression. More evidence suggests that the

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mechanical interplays between cancer cells and the extracellular matrix triggeradhesion-mediated signaling pathways that affect not only the cells’ mechan-ical properties such as motility and rigidity but also the cell’s viability suchas proliferation and apoptosis. In this study, we interrogated whether theanti-cancer drug resistance of breast cancer cells can be discerned by moni-toring the proliferation of breast cancer cells seeded on nanoscaffolds. Thenanoscaffolds help systematically control the maturation of focal adhesionson well-defined nano-sized areas and distances. They were fabricated astwo-dimensional arrays of gold nanoislands on glass substrates using ourbottom-up procedures combining nanosphere lithography and orthogonalchemistry. By varying the size of nanospheres (300 - 1,000 nm) used for nano-lithography, the size and spacing of nanoislands were controlled. The MCF-7and MCF-7/ADR cells were investigated as the drug-sensitive and the drug-resistant breast cancer cell lines, respectively. The difference in thedoxorubicin-sensitivity of the two cell lines was confirmed by the MTT assay.The cell proliferation was determined from the phase contrast images takenevery 24 hours after the initial cell seeding on the nanoscaffolds. We foundthat the proliferation of the drug-sensitive breast cancer cells was highlyaffected by the geometrical characteristics of the underlying nanoscaffolds.Unlike the MCF-7 cells, the proliferation of the drug-resistant breast cancercells was not noticeably affected by the nanoscaffolds for the first two daysof observation. We postulate that MCF-7/ADR cells showed the abnormalmaturation of focal adhesions beyond the restricted area and thus continuouslyproliferated. We also observed a faster wound closure for MCR-7/ADR cellsthan MCF-7 cells. The increase in 2D motility of MCF-7/ADR could be re-sulted from higher levels of the traction force generated by the enhanced focaladhesions. In conclusion, we found that the abnormal maturation of focal ad-hesions via vinculin overexpression caused the uncontrolled proliferation andresulted in the acquisition of the drug resistance in breast cancer cells. We sug-gest that the restoration of the normal maturation of focal adhesions might be apromising way to sensitize the anti-cancer drug response from breast cancercells.

608-Pos Board B373Division Induced Dynamics in Non-Invasive and Invasive Breast CancerAnn-Katrine Vranso West1, Lena Wullkopf2, Amalie Christensen1,Natascha Leijnse1, Jens Magelund Tarp1, Joachim Mathiesen1,Janine Terra Erler2, Lene Broeng Oddershede1.1The Niels Bohr Institute, University of Copenhagen (UCPH), Copenhagen,Denmark, 2Biotech Research & Innovation Centre (BRIC), University ofCopenhagen (UCPH), Copenhagen, Denmark.Cancerous cells pose a great threat when they gain the ability to metasta-size, but little is known on why some cells gain the ability to invadeadjacent tissue while other cells are restricted to the primary tumor. Wehave approached this topic by experimentally characterizing the divisioninduced dynamics of invasive and non-invasive breast cancer monolayersusing human and murine model systems. Particle image velocimetrymeasurements of intrinsic velocities surrounding a dividing cell reveal astrong relation between tissue dynamics, such as vorticity and divergence,and the invasive potential of the cell type. When a cell divides weobserve two distinct vortex pairs in the vorticity field surrounding thedividing cell. Analyzing images over longer time scales reveals no longrange interactions between the cancerous cells, and the interactionsobserved surrounding a dividing cell are constricted to under one cell diam-eter away from the point of cytokinesis. This is to be expected as cancercells are known to have decreased cell-to-cell interactions compared tohealthy cells. An increased intensity in the dynamics (velocity, vorticityand divergence) of invasive monolayers compared to their non-invasivecounterparts, is apparent for both human and murine cell lines. These dy-namics can be simulated using a continuum model, and from this we extractthe characteristic force exerted by a dividing cell on the neighboring cells.These values reveal a correlation between the force and the invasiveness ofthe breast cancer cells. Together, the model and experimental data suggest acorrelation between the dynamical properties of cells and their invasivepotential. Further study of the difference in dynamics between invasiveand non-invasive cancers could help us understand the mechanisms govern-ing metastasis.

609-Pos Board B374Inhibition of a DNA Repair Kinase ATMLeads to Cell Death in 3DMigra-tion Independent of DNA DamageYuntao Xia, Jerome Irianto, Charlotte Pfeifer, Roger Greenberg,Dennis Discher.University of Pennsylvania, Philadelphia, PA, USA.

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The DNA damage response (DDR) is a collection of processes by which a cellidentifies and repairs a site of DNA damage. Many factors can cause DNA dam-age and initiate the repair machinery, such as UV, reactive oxygen species nu-cleases, or even drug treatments like etoposide. Recent studies of several cancerlines as well as immortalized epithelial cells (RPE-1) and primary dendriticcells have convincingly characterized nuclear envelope rupture and cell deathin migration through narrow channel. ATM kinase phosphorylates H2AX (togH2AX) as well as 53BP1 among other targets. A high dose of an ATM inhib-itor (ATMi: KU-55933) increases migration-induced death of RPE-1 andHT1080 fibrosarcoma cells, particularly when combined with perturbationsto the nuclear lamina or envelope repair. However, ATMi at high doses affectsother pathways and ATM’s roles beyond DNA repair now include a migrationphenotype independent of DNA damage in MDA-MB-231 cells treated withsiATM. Such results motivate dose-response assays here of ATMi in assessingrelations between DNA damage and survival in pore migration of U2OS cells.Foci counts of phospho-ATM and of gH2AX (a target of ATM kinase) in 2Dcultures were reduced by 50% at just 0.01 mM, consistent with pharmacologicalstudies of ATM. Surprisingly, comet assays showed no DNA breaks even at10-32 mM. Only at these very high ATMi concentrations did cells die: in 2Dcultures, 50% of cells die at 66 mM ATMi, and this decreases to 37 mM and14 mM in migration through 8 mm and 3 mm pores, respectively. However,ATM knockdown does not increase cell death in constricted migration, sug-gesting that high dose effects of ATMi involves pathways independent ofDNA damage.

610-Pos Board B375Genomic Variation in an Osteosarcoma Cell Line Caused by PoreMigrationJerome Irianto1, Yuntao Xia1, Charlotte R. Pfeifer1, Avathamsa Athirasala1,Jiazheng Ji1, Cory A. Alvey1, Manu Tewari1, Rachel R. Bennett2,Shane M. Harding3, Andrea J. Liu2, Roger A. Greenberg3,Dennis E. Discher1.1SEAS, University of Pennsylvania, Philadelphia, PA, USA, 2GraduateGroup/Department of Physics & Astronomy, University of Pennsylvania,Philadelphia, PA, USA, 3Cancer Biology, Abramson Family CancerResearch Institute, Perelman School of Medicine, University ofPennsylvania, Philadelphia, PA, USA.Normal and diseased cells in vivo sometimes squeeze their nucleus throughtissues and basement membrane matrices. Recent studies has shown that con-stricted migration give rise to an increase in GFP-53BP1 marked DNA dam-age and to die more frequently, but any effect on genome integrity isunknown. Similar studies here with one of the cancer cell lines, U2OS oste-osarcoma cells, show that such migration increases gH2AX foci, increaseselectrophoretic displacements of DNA from isolated nuclei, and changeschromosome copy numbers. DNA breaks decrease over time and the cellsproliferate normally on rigid plastic, but lasting changes in chromosomecopy number are revealed by standard DNA array methods. DNA repair fac-tors tend to be more cytoplasmic after migration, which is consistent with re-ports of migration-induced nuclear envelope rupture as is cell death due toinhibition of repair. Accumulation of mutations in cancer progression is oftenassociated with the replication stress during cell division. The repair ofmigration-induced DSBs through the mutation prone non-homologous endjoining, could potentially provide an alternative path toward mutationaccumulation, increasing intra- and inter-tumor heterogeneity. Genomic insta-bility is closely related to the development and progress of cancer, which is amajor disease in the US and around the world. New cures to cancer willbenefit from new insights into basic mechanisms of nuclear processes inmigration.

611-Pos Board B376Diffusive Behavior of Mismatch Repair ProteinMSH2 in Cells at DifferentStages of CancerKeith D. Bonin1, Justin Sigley1, John Jarzen2, Karin Scarpinato3,Martin Guthold1, Tracey Pu1, Daniel Nelli1, Josiah Low1.1Physics, Wake Forest University, Winston-Salem, NC, USA, 2MedicalUniversity of South Carolina, Charleston, SC, USA, 3OVPR, University ofMiami, Miami, FL, USA.Cellular interiors present media that are very complex as they encompassa mixture of proteins, organelles, viscoelastic filaments, and chromatinstructures in the nuclear interior. The behavior of proteins in such environmentsreflects the cellular complexity. Here we present the results of studies on themobility, or diffusion, of free eGFP proteins, and of the important nuclearmismatch repair protein, MSH2. MSH2 was labeled with eGFP and the diffu-sion of both the free eGFP and MSH2-eGFP was measured using Fluorescence

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Recovery After Photobleaching (FRAP) on four distinct cell types that act as amodel for the neoplastic cancer transformation of human mammary epithelialcells from non-cancerous to metastatic. We obtained several interesting results,such as a significant change in behavior for the first stage of cancer (immortal)compared to other stages. We also observed a significantly lower value forMSH2 diffusion (in both cytoplasm and nucleus) than what would be expectedby scaling from the eGFP values (using appropriate size ratios of the two pro-teins). Diffusion coefficients ranged from 14 to 24 mm2/s for EGFP and from 3to 7 mm2/s for EGFP-MSH2. This material is based upon work supported by theNational Science Foundation under Grant Number 1106105 (KB, KS, andMG). We also thank Glen Marrs, director of the WFU Microscopic ImagingCore Facility, and Anita McCauley, for their help with the confocal microscopysetup.

612-Pos Board B377Chemotherapy Impedes In Vitro Microcirculation and Promotes Migra-tion of Leukemic Cells with Impact on MetastasisSruti V. Prathivadhi-Bhayankaram1, Jianhao Ning2, Michael Mimlitz1,Carolyn Taylor1, Erin Gross2, Michael Nichols1, Jochen Guck3,Andrew E. Ekpenyong1.1Physics, Creighton University, Omaha, NE, USA, 2Chemistry, CreightonUniversity, Omaha, NE, USA, 3Biotechnology Center, TechnischeUniversit€at Dresden, Germany.Although most cancer drugs target the proliferation of cancer cells, it is metas-tasis, the complex process by which cancer cells spread from the primarytumor to other tissues and organs of the body where they form new tumors,that leads to over 90% of all cancer deaths. Thus, there is an urgent needfor anti-metastasis therapy. Surprisingly, emerging evidence suggests thatcertain anti-cancer drugs such as paclitaxel and doxorubicin can actually pro-mote metastasis, but the mechanism(s) behind their pro-metastatic effectsare still unclear. We used a microfluidic microcirculation platform whichmimics the capillary constrictions of the pulmonary and peripheral microcir-culation, to determine if in-vivo-like mechanical stimuli can evoke differentresponses from cells subjected to various cancer drugs. We found thatleukemic cancer cells treated with doxorubicin and daunorubicin, commonlyused anti-cancer drugs, have over 100% longer transit times through the de-vice, compared to untreated leukemic cells. Such delays in the microcircula-tion are known to promote extravasation of cells, a key step in the metastaticcascade. Furthermore, there was a significant (p < 0.01) increase in thechemotactic migration of the doxorubicin treated leukemic cells. Bothenhanced retention in the microcirculation and enhanced migration followingchemotherapy, are pro-metastatic effects which can serve as new targets foranti-metastatic drugs.

613-Pos Board B378The hERG1/Beta1 Interaction Compromises the Mechano-Reciprocity ofPancreatic CancerStefano Coppola1, Annarosa Arcangeli2, Thomas Schmidt1.1Physics of Life Processes, Huygens-Kamerlingh Onnes Laboratory, LeidenUniversity, Leiden, Netherlands, 2Department of Experimental and ClinicalMedicine, University of Florence, Florence, Italy.Kþ channels encoded by the human ether-a-go-go related gene (Kv11.1, orhERG1) are frequently overexpressed in human cancers, including the highlymetastasizing pancreatic ductal adenocarcinoma (PDAC). HERG1 controlsthe neoplastic cell biology by modulating intracellular signaling cascades,coupled to cell adhesion, due to a functional cross-talk with integrin receptors.The integrin-hERG1 interplay is bidirectional. Integrins both activate hERG1channels and alter hERG1 electrophysiology and, conversely, hERG1 channelsmodulate integrin-mediated signaling. We hypothesized that hERG1 channelscompromise the PDAC mechano-reciprocity, the ability to dynamicallyrespond to externally applied forces by exerting forces, which enhances inva-sion and compromises treatment.A combination of single-molecule microscopy and force measurements givesus access on mechanical information at focal adhesion level, simultaneouslyto hERG1/b1 complex dynamics and localization. The correlated motion ofhERG and integrins suggest that both are localized in the same structure, prob-ably the focal adhesion as suggested by staining with vinculin. Force measure-ments further suggest that the mechanical properties of PDAC cells are alteredby the integrin-hERG1 interaction leading to an increase in cellular forces by20% on coexpression of both proteins. Our results on how cancer-related over-expression of hERG1 potassium channels perturbs the force sensing machineryprovides a new view on earlier findings which explain the chemo- and radio-resistance of PDAC mostly with the increased density of extracellular matrix

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(ECM) leading to reduced diffusion and convection of molecules through thetumor micro-environment (TME).

614-Pos Board B379Mechanical Properties of Normal Breast Cells andMetastatic Cancer Cellsin Co-CultureHyunsu Lee, Amanda Smelser, Josiah Low, Martin Guthold, Keith Bonin.Physics, Wake Forest Univserity, Winston Salem, NC, USA.The mechanical properties of cells play an important role in cellular processessuch as cell migration, division, and mechanotransduction. Stiffness is animportant mechanical property and can be measured using an atomic forcemicroscope (AFM) (to determine the Young’s modulus). Our previous studiesshowed that normal breast cells (human mammary epithelial cells, HMEC) arestiffer than highly metastatic breast cancer cells (MDA-MB-231). Also, normalbreast cells in the center of a monolayer micro-colony are stiffer than cellson the periphery and isolated cells which means that the microenvironmentsignificantly affects the mechanical properties of cells. However, the effecton stiffness when normal cells and cancer cells grow together has notbeen well established. In this study, we investigate how the mechanicalproperties of HMEC and MDA-MB-231 cells change in co-culture. Ourpreliminary results show that both HMEC and MDA-MB-231 cells getsignificantly stiffer in co-culture (HMEC 1.3 kPa -> 15 kPa; MDA-MB-2310.59 kPa -> 1.48 kPa). This study will offer an understanding of normal-to-cancer cell interactions affect stiffness, and consequently how it might be corre-lated to metastasis.

615-Pos Board B380Kappa-Actin Alters Hepatocellular Carcinoma Physiology in CirrhoticMicroenvironmentChi-Shuo Chen, Cheng-Yi Lin, Chi-Hung Ho, Wei-Chi Wu.Biomedical Engineering and Environmental Sciences, National Tsing HuaUniversity, Hsinchu, Taiwan.Hepatocellular carcinoma (HCC) is the fifth most common cause of cancer-related mortality over the world, and liver cirrhosis was reported as themost important risk factor for HCC development. However, the roles of me-chanotransduction in HCC have not been fully explored yet. Kappa-actin(k-actin), a novel class of actin correlated with poor postoperative survival,was selected as our study model. We investigated how k-actins regulateHCC cells using stiffness adjustable polymeric matrixes, which mimic thecirrhotic microenvironment. Different cellular physiology, such as prolifera-tion, contact topography and 3-dimensional invasion, were observed inmicroenvironment with different stiffness. Interestingly, with high k-actinexpression, we noticed the significantly decrease of focal adhesions (FAs) for-mation while substrate’s stiffness > 16 kPa. Photoactive microscopy showedlower stability of k-actin structures, and the unstable actin organization cancontribute to the decrease of FAs/Adherence junction (AJs) formations.Furthermore, the instability of AJs (E-cadherin) may correlate to the observedhigher invasion of k-HCC in vitro. Traction force microscopy (TFM) wasdeveloped to further quantify the mechanical interactions at the ECM-cell/cell-cell interfaces. By measuring the deformation of polymeric gel substrates,the force balance at the contact surfaces can be reconstructed. We observed theECM-cell traction force increased with the increasing substrate stiffness, andthe traction force decreased with the k-actin expression, which consisted withthe spatial pattern of FAs. In summary, we showed the expression of k-actinalters the formation of FAs/AJs in HCC, which can contribute to the observedhigh cell proliferation and invasion of HCC. Furthermore, using photoactivemicroscopy, we demonstrated the actin dynamics play the essential roles inFAs and AJs formation of HCC. These findings may contribute to our under-standing about the influence of actins on HCC through mechanotransductionperspective.

616-Pos Board B381Is Shape of Cancer Cell Correlated with its Invasiveness?Elaheh Alizadeh1, Samanthe M. Lyons2, Jordan M. Castle3,Jacqueline Irene Foss2,4, Ashok Prasad1,2.1Chemical and Biological Engineering, Colorado State University, FortCollins, CO, USA, 2School of Biomedical Engineering, ColoradoState University, Fort Collins, CO, USA, 3Department of Biology,Colorado State University, Fort Collins, CO, USA, 4Mechanical Engineering,Colorado State University, Fort Collins, CO, USA.This work is based on the hypothesis that shape of the cell on substrate is deter-mined by the active mechanical properties of the cytoskeleton. Since invasivecancer cells are believed to have altered mechanical properties comparedwith non-invasive cells, it follows that changes in shape of cancer cells may

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correlate with the acquisition of invasive capacity. We try to understandwhether there is a correlation between cancer cell shape and its phenotypicstate. To proceed, we first need to capture cell shape and quantify it. Cellswere cultured on glass substrates and then were fixed and stained for actincytoskeleton. Images were processed to eliminate background noise. In orderto quantitatively describe cell’s shape, we chose a set of Zernike moments,which are widely used in image recognition and are calculated from a setof orthogonal basis function. Since we are dealing with high dimensionaldata we used principal component analysis (PCA) to examine changes incell shape. We also used a machine learning neural network classifier todistinguish between different cell populations. We use paired osteosarcomacancer lines which consist of a parental line that rarely undergoes metastasisand a line derived from parental line that almost always does. Comparingshape changes of invasive cancer line versus their paired low invasive cancerline gives insight to correlation between cell shape and invasiveness. To under-stand cytoskeletal mechanism behind this relation we perturbed cytoskeletonwith different pharmaceutical drugs which each targets different elementof the cytoskeleton and looked at the patterns of drug effect on cell shape.We also characterize shape changes of normal cells during their geneticstep-wise transformation to cancer cells. [1]S. M. Lyons et. al. BiologyOpen 2016, 5, 289-299. [2]E.Alizadeh et. al. Integrative Biology, 2016,http://dx.doi.org/10.1039/C6IB00100A.

617-Pos Board B382Stability on the Edge: Probing the Biophysical Mechanisms of PolarityMaintenance at the Leading Edge of Motile Neutrophil-Like Hl-60 CellsRikki M. Garner1, Elena Koslover2, Julie Theriot3.1Biophysics Program, Stanford University, Stanford, CA, USA, 2PhysicsDept., The University of California, San Diego, San Diego, CA, USA,3Biochemistry Dept., Microbiology and Immunology Dept., and the HowardHughes Medical Institute, Stanford University, Stanford, CA, USA.Cell motility is an essential cellular function. With motility being such a ubiq-uitous process, cells are outfitted with a vast yet well-conserved set of proteinsthat self-organize in order to form protrusions with a surprising range of shapesand sizes. These protrusions are formed by the coordinated force of thousandsof actin polymers growing against the membrane at the leading edge. How arethousands of nanometer-sized actin filaments able to form a stable leading edgeseveral orders of magnitude larger, in time and space, than the assembly of itsindividual components? I seek to answer this question by analyzing protrusionfluctuations at the leading edge of lamellipodia, thereby quantifying the limitsof self-correction of the actin network. Using a cycle of theoretical modelingand experimental perturbation, I will tease apart the possible mechanisms forself-correction and the subsequent effects on the stability of the leading edgeof motile cells.

618-Pos Board B383Understanding the Mechanics of Neutrophil Migration in Three-Dimen-sional Extracellular MatricesJoshua Francois1, Ruedi Meili2, Juan Carlos del Alamo3, Richard Firtel2,Juan C. Lasheras3.1Bioengineering, University of California, San Diego, La Jolla, CA, USA,2Section of Cell and Developmental Biology, University of California, SanDiego, La Jolla, CA, USA, 3Mechanical and Aerospace Engineering,University of California, San Diego, La Jolla, CA, USA.While much research has been dedicated to the identification of the cascade ofspecific biochemical processes involved in the recruitment of neutrophils,much less is known about the mechanical events driving their migration; inparticular, how they generate the necessary traction forces to migrate acrossthree-dimensional (3-D) extravascular spaces and the importance of formingcell-substrate adhesions during this process is unclear. In this study, we inves-tigate the importance of cell-substrate adhesions on the mechanics of 3-Dneutrophil motility in collagen gels using Elastographic 3D Force Microscopy(E3DFM). We used wild type neutrophil-like differentiated human promyelo-cytic leukemia (dHL-60) cells and talin 1 knockout dHL60 cells, which wereunable to engage their integrins, as our model systems. Both cell lines wereembedded in collagen matrices containing fluorescent micro-beads. Neutrophilmotility was induced via the introduction of the neutrophil chemokine formyl-Methionyl-Leucyl-Phenylalanine (fMLP) in a custom build device. BothConfocal and Fluorescent microscopy techniques were used to image the move-ment of the embedded micro-beads as well as fluorescently labeled cells. Par-ticle Image Velocimetry (PIV) and Finite Deformation Theory were used tocompute displacement fields in the collagen matrices. Stress fields in thematrices were computed using our E3DFM method. We will present datashowing that morphological changes and migratory patterns differed depending

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on the cell’s ability to form cell-substrate adhesions. We will also provide datashowing a clear relationship between the aforementioned migratory character-istics and computed displacement and stress fields around migrating neutro-phils in collagen matrices. The results from our study show that neutrophilsmigrating in 3-D environments employ distinct mechanical mechanisms thatdepend on their ability to form adhesions.

619-Pos Board B384Novel Mechanism for Driving Amoeboid-Like Motility of Human Neutro-phils under an Electric Field, Based on Intracellular Proton Currents andCytoplasm StreamingHagit Peretz Soroka1, Reuven Tirosh2, Murray Alexander3, Jolly Hipolito1,Francis Lin1.1Physics and Astronomy, University of Manitoba, Winnipeg, MB, Canada,2Chemistry, Tel - Aviv University, Tel - Aviv, Israel, 3Physics, University ofWinnipeg, Winnipeg, MB, Canada.Experimental studies have shown that actin filaments motion, cytoplasmstreaming and muscle contraction can be reconstituted under actin-activatedATP hydrolysis by soluble non-filamentous myosin fragments. Thus, biologicalmotility was demonstrated without requiring conventional contractile proteinfunction. A possible alternative mechanism based on the integration ofdirectional proton current and cytoplasm streaming is proposed to driveamoeboid-like cell motility and muscle contraction. To test the cell motilityaspect of this mechanism, in the current study we employ a microfluidic deviceto quantitatively characterize human blood neutrophil electrotaxis. Severalinteresting predictions from this new model were confirmed in these experi-ments. We demonstrated firstly, the direct correlation of cytoplasm streamingto neutrophil motility profiles; secondly, the electric field and cell activationstage dependent directional neutrophil orientation and migration; and thirdly,the migration memory of neutrophil electrotaxis. These results support theimportance of active intracellular force for driving amoeboid-like cell motility,with implications for physiological processes such as cell-cell migratory inter-actions and metabolic activation. This basic integrative working hypothesis isexpected also to give raise to new applications in bioenergetics and immuno-logical diagnosis and therapy.

620-Pos Board B385Macrophages are Sensitive to Substrate Elasticity during PhagocytosisWolfgang Gross, Franziska Zecherle, Kathrin Weidner-Hertrampf,Holger Kress.University of Bayreuth, Bayreuth, Germany.Phagocytosis, the internalization of objects like living bacteria and dead cellsby macrophages is a main function of the innate immune system. After thedetection of foreign objects by membrane receptors, this process is driven bythe reorganization of the actin cytoskeleton, which leads to a protrusion ofthe membrane around the target. Although many molecular players havebeen identified in the past, there is still little known about the role of mechanicsduring this process in general and about the role of the mechanical cellularenvironment in particular.In this work we investigate the influence of the underlying substrate rigidity onthe phagocytic uptake efficiency and the uptake velocity. We cultured murineJ774 macrophages on thin polymer gels with different stiffnesses in the phys-iological range. The uptake efficiency of antibody-coated microparticles wasquantified with secondary antibody staining and the uptake speed was measuredin live cell experiments by using optical tweezers. We found that the uptake ef-ficiency as well as the uptake velocity depend on the rigidity of the substrate.Furthermore, we observed that cells were able to adapt to the various substratestiffnesses over time. In addition to these cellular measurements, we also pre-sent a novel calibration technique that enables the simultaneous characteriza-tion of the elastic modulus and the Poisson’s ratio of thin gel layers. Thetechnique requires only a standard epifluorescence microscope and sphericalindenters.Our results on the phagocytic uptake velocity and efficiency support the hy-pothesis that phagocytosis is a mechanosensitive process. Our findings mightcontribute to an understanding of the complex interplay between the immunesystem and disease states that come along with changes in tissue rigidity likecancer and atherosclerosis.

621-Pos Board B386Investigating Actin Mechanics during Phagocytic Uptake and TransportKonrad Berghoff, Steve Keller, Holger Kress.University of Bayreuth, Bayreuth, Germany.Phagocytosis, the internalization and consecutive digestion of biological mate-rial by macrophages is a major part of the innate mammalian immune response.

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Fc gamma receptor-mediated uptake is driven by actin recruitment to the inter-nalization site and consecutive formation of membrane protrusions around thetarget object. Molecular motors and cytoskeletal elements contribute to the sub-sequent transport of phagosomes inside the cells. Although a large number ofmolecules that are involved in the phagocytic uptake and the phagosomal trans-port are identified by now, the mechanics of these processes are largely un-known. We therefore investigated the mechanics of phagocytic uptake andphagosomal transport by using live cell microscopy in combination with opticaland magnetic tweezers with a focus on the role of actin filaments. We hypoth-esize that the actin recruitment during phagocytic uptake leads to a transientand localized increase of the stiffness of the uptake region.To test this hypothesis we induced Fc gamma receptor-mediated phagocytosisin J774A.1 mouse macrophages by offering the cells target microbeads coatedwith immunoglobulin-G. During the uptake we measured local cell stiffnesschanges with a ‘blinking optical traps’ technique executed via periodic inten-sity modulation of the optical force. Preliminary data indicates that that thestiffness of the uptake region increases temporarily during the uptake. In addi-tion, we investigated the influence of actin filaments on the phagosomal trans-port by magnetic tweezers-based bead displacement assays and cytochalasintreatment of the cells.Our measurements are expected to identify characteristic length- and time-scales for the variation of cell mechanical properties during phagocytosis andto contribute to a more comprehensive understanding of this medically relevantprocess.

622-Pos Board B387B Cell Antigen Extraction is Regulated by Physical Properties of AntigenPresenting CellsKatelyn M. Spillane, Pavel Tolar.The Francis Crick Institute, London, United Kingdom.B cells play a critical role in immune responses by producing antibodies againstthe foreign pathogens that they encounter. Their responses are initiated whenthe B cell receptor (BCR) binds antigen on the surface of an antigen-presenting cell (APC) in a cell-cell contact known as the immune synapse.This event triggers the B cell to internalize, process, and present the antigento helper T cells, which provide signals that are required for full B cell activa-tion and clonal selection. High affinity B cells receive more T cell help than lowaffinity B cells do, suggesting that affinity discrimination during antigen extrac-tion and internalization is essential for high-affinity antibody responses. Themechanisms of antigen recognition, discrimination, and uptake remain a topicof debate, with physical extraction through mechanical forces and enzymaticliberation through lysosome secretion proposed as efficient ways for B cellsto acquire antigen. To investigate B cell extraction mechanisms, we developedDNA-based nanosensors to interrogate antigen extraction from both artificialsubstrates and live APCs. We show that B cells acquire antigen primarilythrough mechanical force, and resort to enzymatic liberation only if force-dependent extraction fails. The use of mechanical force renders B cells sensi-tive to the physical properties of the APCs. We find that stiff APCs promotestrong B cell pulling forces and stringent affinity discrimination, while flexibleAPCs allow B cells to extract low-affinity antigen using weak forces. Thus, Bcell antigen extraction is regulated by the physical properties of immune syn-apses, suggesting that distinct physical properties of APCs may supportdifferent stages of B cell responses.

623-Pos Board B388Revealing the Mechanical Basis of T Cell SignalingKhalid Salaita.Chemistry, Emory University, Atlanta, GA, USA.Because T cells are highly migratory and antigen recognition occurs at an inter-membrane junction where the T cell physically contacts another cell, there arelong-standing questions of whether T cells transmit defined forces to their re-ceptors and whether chemo-mechanical coupling influences immune function.I will discuss the development of DNA-based gold nanoparticle tension sensorsto provide, to our knowledge, the first pN tension maps of individual T cell re-ceptor forces during T-cell activation. We show that naıve T cells harness cyto-skeletal coupling to transmit 12-19 pN of force to their receptors withinseconds of ligand binding and preceding initial calcium signaling. CD8 core-ceptor binding and lymphocyte-specific kinase signaling are required forantigen-mediated cell spreading and force generation. Lymphocyte function-associated antigen 1 (LFA-1) mediated adhesion modulates TCR-pMHC ten-

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sion by intensifying its magnitude to values >19 pN and spatially reorganizesthe location of receptor forces to the kinapse, the zone located at the trailingedge of migrating T cells, thus demonstrating crosstalk between TCR andLFA-1 receptor signaling. Finally, T cells display a dampened and poorly spe-cific response to antigen agonists when forces are chemically abolished orphysically ‘‘filtered’’ to a level of 12 pN using mechanically labile DNAtethers. Therefore, we conclude that T cells tune receptor mechanics with pNresolution to create a checkpoint of agonist quality necessary for specific im-mune response.

624-Pos Board B389Mechanics of Blood Cells with Marginal Band: Competition betweenCortical Tension and RigiditySerge A. Dmitrieff, Adolfo Alsina, Mathur Aastha, Nedelec Francois.CBB Nedelec Lab, EMBL, Heidelberg, Germany.The fast blood stream of animals is associated with large shear stresses. Conse-quently, blood cells have evolved a special morphology and a specific internalarchitecture allowing them to maintain their integrity over several weeks. Non-mammalian red blood cells, mammalian erythroblasts and platelets in particularhave a peripheral ring of microtubules, called the marginal band, that flattensthe overall cell morphology by pushing on the cell cortex.We modeled how the shape of these cells stems from the competition betweenmarginal band elasticity and cortical tension. We predict that the diameter ofthe cell scales with the total microtubule mass, and verify the predicted lawacross a wide range of species. Our analysis also shows that the combinationof the marginal band rigidity and cortical tension increases the ability of thecell to withstand forces without buckling. Eventually, we show that the buck-ling of the marginal band observed during platelet activation is caused by arapid increase of the cortical tension.

625-Pos Board B390Multiscale Modeling of Red Blood Cells Squeezing through SubmicronSlitsHuijie Lu, Zhangli N. Peng.Aerospace and Mechanical Engineering, University of Notre Dame, NotreDame, IN, USA.A multiscale model is applied to study the dynamics of healthy red blood cells(RBCs), RBCs in hereditary spherocytosis, and sickle cell disease squeezingthrough submicron slits. This study is motivated by the mechanical filtrationof RBCs by inter-endothelial slits in the spleen. First, the model is validatedby comparing the simulation results with experiments. Secondly, the deforma-tion of the cytoskeleton in healthy RBCs is investigated. Thirdly, the mecha-nisms of damage in hereditary spherocytosis are investigated. Finally, theeffects of cytoplasm and membrane viscosities, especially in sickle cell disease,are examined. The simulations results provided guidance for future experi-ments to explore the dynamics of RBCs under extreme deformation.

626-Pos Board B391Rolling Adhesion of Malaria-Infected Red Blood CellsAnil Kumar Dasanna.Heidelberg University, Heidelberg, Germany.The clinical symptoms of the malaria disease appear when healthy red bloodcells are invaded by the malaria parasites during the blood stage of the lifecycle. The whole infection of the blood cell by the malaria parasite Plasmo-dium falciparum takes about 48 hrs and proceeds through the three stages ofring, trophozoite and schizont. During this process, the infected red bloodcells (iRBCs) increasingly develop adhesive protrusions, so-called knobs, ontheir surface. These knobs cause iRBCs to adhere to endothelial cells in themicrovasculature, preventing their clearance by spleen and liver, but alsoleading to capillary obstruction. We first present how exactly the shape ofiRBCs change during the time course along with their geometrical featuressuch as volume and surface area using confocal microscopy and image pro-cessing. We then discuss how these changes in shape and knob details throughout the blood stage affect the rolling adhesion of iRBCs on endothelial cellsusing both flow chamber experiments and adhesive dynamics simulations. Inparticular, we will discuss how the spatial organization of the knobs on thesurface of the iRBCs lead to different dynamics states, such as flipping mo-tion, transient or stable rolling adhesion and firm adhesion, and how thesestates are related to parameters such as kinetic rates of ligand-receptor bondsand cell elasticity.

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Transporters and Exchangers I

627-Pos Board B392Markov State-Based Quantitative Kinetic Model of Sodium Release fromthe Dopamine TransporterAsghar Razavi1, George Khelashvil1, Harel Weinstein1,2.1Physiology and Biophysics, Weill Cornell Medical College of CornellUniversity, New York, NY, USA, 2Physiology and Biophysics, Institute forComputational Biomedicine, New York, NY, USA.The dopamine transporter (DAT) belongs to the neurotransmitter/sodium sym-porter (NSS) family of membrane proteins that are responsible for reuptake ofneurotransmitters from the synaptic cleft to terminate a neuronal signal andenable subsequent neurotransmitter release from the presynaptic neuron.The release of one sodium ion from the crystallographically determined so-dium binding site Na2 had been identified as an initial step in the transportcycle which prepares the transporter for substrate translocation by stabilizingan inward-open conformation. We have constructed Markov State Models(MSMs) from extensive molecular dynamics simulations of human DAT(hDAT) to explore the mechanism of this sodium release. Our results quantifythe release process triggered by hydration of the Na2 site that occurs concom-itantly with a conformational transition from an outward-facing to an inward-facing state of the transporter. The kinetics of the release process arecomputed from the MSM, and transition path theory is used to identify themost probable sodium release pathways. An intermediate state is discoveredon the sodium release pathway, and the results reveal the importance ofvarious modes of interaction of the N-terminus of hDAT in controlling thepathways of release.

628-Pos Board B393Oligomerization of Human Dopamine Transporter (hDAT)Kumaresan Jayaraman, Harald H. Sitte, Thomas Stockner.Institute of Pharmacology, Medical University of Vienna, Vienna, Austria.Monoamine transporters of the SCL6 family are found on presynaptic neu-rons and terminate the neurotransmission by the reuptake of neurotransmit-ters. The family of monoamine transporters includes the transporters fordopamine (DAT), serotonin (SERT) and norepinephrine (NET) whichcouple substrate transport with ion gradients of sodium and chloride.Dysfunction of these transporters can lead to clinically important diseasestates, for instance to depression. It has been shown that monoamine trans-porters and other members of the family exist in oligomeric form in cellsand studies reported that different oligomeric sizes are present at the levelof the plasma membrane. There is a wealth of reports regarding the residuescritical for oligomerization, however, the binding orientation and possiblehierarchies of the many putative oligomerization interfaces are poorlyunderstood. In this study, we extensively scrutinized the oligomeric formsof the human DAT using molecular dynamics simulations and applyingthe MARTINI coarse grained force field. Two monomers of DAT were in-serted into a POPC membrane in random orientations. Subsequently, the as-sembly of the monomers were analyzed over 2 ms time for 500 independentsystems resulting in a total of 1ms simulation time. The convergence of thewhole ensemble is quantified by the interaction energy plot, the number ofconformers and their orientations is shown by the density plot. This studyindicates that DAT forms dimers through four distinct orientations. Theseresults allowed us to propose a testable hypothesis of residues located withinthe oligomeric interface.

629-Pos Board B394Two Mechanisms in One Family: Packaging of Glutamate into SynapticVesicles against a Proton Gradient, a Transporter Driven by MembranePotential, versus a Homolog Driven Downhill with Proton GradientsRobert M. Stroud.Biochemistry & Biophysics, University of California, San Francisco, SanFrancisco, CA, USA.Vesicular transporters for glutamate depend on membrane potential asdriving force. The crystal structure of a close homolog (23% identity) inthe same superfamily has been determined, and transport assays used toconfirm that it conducts its anionic substrate driven by proton gradients asa proton symporter. How then can the vesicular cousin drive its anionic sub-strate against the normal proton gradient? Transmembrane channels and fa-cilitators can be highly selective in mediating the transport of nutrients andions across membranes ‘downhill’, down their concentration gradient.Transporters can drive and concentrate nutrients or ions ‘uphill’ (energeti-cally) across membranes achieving gradients of >1000:1 driven by ‘down-

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hill’ movement of other coupled ions, protons, metabolites, -orelectrochemical gradients. The focus is on how two variations, driven byelectric potential, and proton gradients, can drive anion transport, in highlyhomologous family members?

630-Pos Board B395Substrate Binding to Serotonin Transporters Reduces MembraneCapacitanceVerena Burtscher, Matej Hotka, Walter Sandtner.Center for Physiology and Pharmacology, Medical University of Vienna,Vienna, Austria.Monoaminergic signal transmission is terminated by reuptake of monoaminesvia the closely related transporters for dopamine (DAT), norepinephrine (NET)and serotonin (SERT). SERT is a secondary active transporter that harnessesthe chemical energy stored in the gradients of Naþ, Cl- and Kþ to transport5-HT uphill against an opposing concentration gradient.The transport cycle of SERT is complex and encompasses several partial-reactions, some of which can directly be monitored utilizing measurementsof currents mediated by SERT. However, these measurements are limited toionic conditions that support currents but fail to provide insights at conditionsat which SERT is electrically silent.We therefore performed capacitance measurements during rapid serotoninapplication and observed that substrate binding to SERT decreases the mem-brane capacitance. This effect was absent in control cells.Our analysis suggests that the reduction in membrane capacitance occurs as aconsequence of the adsorption of 5-HT to a charged residue in the binding siteof SERT. This process eliminates negative extracellular surface charges.Surface charge elimination is accompanied by a change in the surface potential,concomitantly affecting the transmembrane potential. The change of the latterthen gives rise to a change in the measured membrane capacitance. Thesubstrate-induced decrease in membrane capacitance was still present, whenomitting co-substrates. This is consistent with our hypothesis that the reducedcapacitance resulted from serotonin binding (adsorption). At sodium-freeconditions, the affinity for serotonin was decreased which was reflected by asmaller capacitance reduction. Our findings suggest random order binding re-actions of substrate and co-substrates and that serotonin and Naþ bind in acooperative manner.Financial support was given to WS by the Austrian Funds FWF (P28090-B27).

631-Pos Board B396Exploring Ligand-Binding Kinetics in the S2 Site of MhsT by AtomisticSimulations and Markov ModelsAra M. Abramyan1, Cathy Xue2, Lei Shi1.1Molecular Targets and Medications Discovery Branch, NIDA-IRP/NIH,Baltimore, MD, USA, 2Department of Computer Science & Department ofMathematics, Yale University, New Haven, CT, USA.Neurotransmitter:Sodium Symporters (NSS) terminate neurotransmissionthrough sodium-driven reuptake of cognate neurotransmitters, andtraverse between outward-open and inward-open states. NSS include seroto-nin and dopamine transporters, which are targets for antidepressants andabused psychostimulants. Crystallographically, whereas both substratesand inhibitors have been found to bind the central binding site (denotedS1) of the NSS proteins, it has been shown that inhibitors for LeuT andSERT can also bind to a binding cavity in the extracellular vestibule(denoted S2). Based on computational and experimental studies in LeuT,it has been found that substrates can bind to S2 as well, and such bindingtriggers the intracellular release the substrate and Naþ from S1. However,whether such a role of S2 substrate is common for other NSS remainsunclear.The newly available crystal structure of MhsT, a bacterial NSS homolog,which was solved in an inward-occluded state, provides a more suitablestarting point to study the substrate induced conformational changes,including the S2 substrate induced transition from inward-occluded toinward-open state transition. Here, we use extensive molecular dynamicssimulations combined with hidden Markov model (HMM) analysis toinvestigate the feasibility and kinetics of the substrate L-tryptophan bindingto S2 of MhsT. Based on HMM analysis, we identified transition binding po-sitions in the extracellular vestibule that form potential substrate bindingpathways, and the conformational changes near the S2 site associatedwith the two most populated bound states. Our findings shed light on theS2 substrate binding kinetics and the induced conformational changes,which may be critical in triggering the subsequent events in the transportmechanism.

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632-Pos Board B397Computational Investigation of the Serotonin Transporter Conformationand Reset MechanismEmily M. Benner, Jeffry D. Madura.Chemistry and Biochemistry, Duquesne University, Pittsburgh, PA, USA.The aid of monoamine transporter (MAT) proteins in terminating the effectof neurotransmitters in the synaptic cleft is crucial for the maintenance ofproper neurotransmitter concentrations. Termination of effect is achievedvia the process of reuptake, where the proteins bind neurotransmitters to acentral substrate binding site and transport the substrate to the other sideof the membrane through a permeation pathway. The MAT family of pro-teins includes the serotonin transporter (SERT), which is responsible forthe reuptake of serotonin from the synaptic cleft. SERT, as well as the otherMAT proteins are implicated in several psychological disorders, includingdepression, anxiety, and addiction. Treatments for these disorders arefocused on the inhibition of the MAT system, especially SERT. Thesedrugs are known as selective serotonin reuptake inhibitors, which actby binding to the substrate site of SERT, preventing the reuptake of seroto-nin. Recent work in our lab has focused on identifying the completetransport mechanism of SERT computationally, employing both a singlebilayer and dual bilayer system to investigate this phenomenon. The workpresented here represents the data collected from our most recent dualbilayer system. Herein we utilized the newly crystallized human SERT pro-tein (PDB 5I6X), embedded in POPE lipid membranes. The dual bilayersetup allows the system to maintain ion concentrations on either side ofthe membrane when periodic boundary conditions are implemented.Ions were added to either side of the membrane at physiological concentra-tions, keeping the membrane potential at around �70mV, representative ofresting potential. Understanding more fully the complete transport process,with specific attention paid to the reset mechanism, can aid in drug discov-ery and design for treatments that better alleviate symptoms of depressionand related disorders. Presented here are the molecular dynamics andconformational data from our ongoing simulations of the dual bilayerSERT system.

633-Pos Board B398Characterizing Outward- to Inward-Facing Transition Pathway ofDopamine TransporterZhiyu Zhao1, Emad Tajkhorshid2.1Center for Biophysics and Quantitative Biology, UIUC, Champaign, IL,USA, 2UIUC, Champaign, IL, USA.The dopamine transporter (DAT) belongs to the family of neurotransmittersodium symporters (NSSs), which harness transmembrane electrochemicalionic gradients to transport neurotransmitters against their chemical gradi-ents. Dysregulation of DAT is associated with serious neurological disor-ders, such as Parkinson’s disease, depression, anxiety and epilepsy. Aswith all transporters, substrate translocation through DAT follows thealternating-access mechanism in which the protein swiches betweenoutward-facing (OF) and inward-facing (IF) states. The details of thesestructural changes and their coupling to chemical events such as substrateand ion binding events remain elusive. In the present study, we charactrizedlarge-scale transition from the OF state to IF state using all-atom moleculardynamics simulations of membrane-bound models of DAT. As all crystalstructures of DAT are in the OF state, the initial phase of the study includedmodeling of a stable IF structure of DAT in the context of a membrane usingthe bacterial sodium-coupled leucine symporter (LeuT) as a template.Furthermore, equilibrium simulations performed in this phase revealed anovel sodium binding site located between TM3 and TM8 helices, whichare elements involved in coupling of protein structural changes to substratebinding and translocation. Using the orientations of helics TM1e/TM8e andTM1i/TM8i as collective variables, and employing two dimensional bias-exchange umbrella sampling and string method with swarms of trajectories,we characerize a structural transition pathway between the OF and IF statesof DAT. The results of this study provide a deeper understanding of thefunctional mechanism of DAT, wiht implications to all members of theNSS family.

634-Pos Board B399Structural Insights into Sodium-Dependent Sugar Transporters and theirInhibition MechanismPaola Bisignano1, Chakrapani Kalyanaraman2, Chiara Ghezzi3,Ernest M. Wright3, Jeff Abramson3, Aviv Paz3, Matthew P. Jacobson2,Rosmarie Friemann4,5, Michael Grabe1.

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1Cardiovascular Research Institute and Department of PharmaceuticalChemistry, UCSF, San Francisco, CA, USA, 2Department of PharmaceuticalChemistry, UCSF, San Francisco, CA, USA, 3Department of Physiology,UCLA, Los Angeles, CA, USA, 4Department of Chemistry and MolecularBiology, University of Gothenburg, Goteborg, Sweden, 5Department ofStructural Biology, Stanford, Stanford, CA, USA.Sodium-dependent glucose transporters (SGLTs) are members of thelarge solute carrier (SLC) family of proteins that exploit the sodium ionconcentration gradient to transport a myriad of small molecules acrossthe plasma membrane. In humans, there are six SGLT subtypes labeled1-6 that are expressed widely in the small intestine, kidney, lung, muscle,and brain. Due to their role in sugar reabsorption, SGLTs are currentlyexploited as drug targets for the treatment of type 2 diabetes, especiallyhSGLT2, which is responsible for 98% of glucose reabsorption in thekidneys. Current inhibitors are chemical derivatives of the naturallyoccurring small molecule phlorizin, which is expressed in the bark offruit trees, such as apple and pear. The structural basis of binding isnot known, in part, because high-resolution structures of mammalianSGLTs do not exist. However, the inward-facing structure of the bacterialhomologue from vibrio parahaemolyticus (vSGLT) has been solvedboth in apo and in complex with galactose, and our collaborators recentlysolved the outward-facing structure of a closely related homologue (unpub-lished). Here we combine homology modeling, virtual screening techniquesand molecular dynamics simulations to achieve two goals: 1) modelthe outward-facing state of SGLTs, and 2) predict the binding mode ofphlorizin and its derivatives hSGLT1 and 2. As a result, the spectroscopicdata (double electron-electron resonance) probing outward facing stateof SGLTs validate our homology model and mutagenesis studies testingbinding to hSGLT1 and 2 are in agreement with our predicted bindingmodes.

635-Pos Board B400Uptake Dynamics in the LacY Membrane Protein TransporterDari Kimanius1, Stephen White2, Erik Lindahl3, Ronald Kaback4,Magnus Andersson5.1Stockholm University, Stockholm, Sweden, 2University of California Irvine,Irvine, CA, USA, 3KTH Royal Institute of Technology/StockholmUniversity, Stockholm, Sweden, 4University of California Los Angeles,Los Angeles, CA, USA, 5KTH Royal Institute of Technology, Stockholm,Sweden.Membrane protein transporters govern important cellular processes andare therefore central to human health. To accomplish transport, theseproteins rearrange their structures to alternatively expose an internalbinding site to either side of the membrane. Recent advances in proteinstructural determination methods have resulted in a steadily increasingnumber of high-resolution structures of membrane transporters trappedin different intermediate states. However, to understand the under-lying transport mechanism, the molecular details of uptake and releaseneed to be determined. We have used specialized simulation hardware tosimulate uptake of galactoside sugar into the Lactose permease (LacY) ofEscherichia coli. The extended brute-force simulation revealed large-scalestructural rearrangements, lipid and amino acid interactions, and hydrationassociated with sugar uptake. The free energy landscape of sugar entrywas determined by parallel bias-exchange metadynamics simulations andidentified a global free energy minimum that coincided with the crystallo-graphic binding site and also a local free energy minimum in the largerperiplasmic cavity of LacY. Together, our observations show a putativemolecular mechanism for sugar uptake in this prototype membranetransporter.

636-Pos Board B401Elevator-Like Mechanism of Transport in the EIIC Glucose Superfamilyof TransportersZhenning Ren1, Yin Nian1, Jumin Lee2, Jason McCoy3, Wonpil Im2,Ming Zhou1.1Biochemistry andMolecular Biology, Baylor College of Medicine, Houston,TX, USA, 2University of Kansas, Lawrence, KS, USA, 3Broad Institute,Cambridge, MA, USA.The phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) iscrucial for sugar uptake in bacteria. It has a membrane embedded compo-nent, EIIC, that translocates a sugar from the extracellular to the intracel-lular side of the cell. Before the sugar is released into the cytosol, a

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cytosolic EIIB protein binds to EIIC and transfers a phosphate group to theincoming sugar that prevents the sugar from escaping the cell and at thesame time primes the sugar for entering metabolic cycles. Little is knownconcerning how EIICs recognize and transport carbohydrates or how anEIIC coordinates with EIIB to achieve phosphate transfer. Crystal structuresof a maltose transporter bcMalT[1] and a N-diacetylchitobiose transporterbcChbC[2] were solved recently, and the two structures appear to be indifferent states of a transport cycle: bcMalT in an outward facing stateand bcChbC in an inward facing state. The bcChbC structure provides atemplate to build a model of bcMalT in an inward facing state, and viseversa. To examine the models, we designed pairs of cysteine residues thatare distant in the crystal structures but are predicted to move close toeach other in the alternate conformation. Several pairs of cysteines in inboth bcMalT and bcChbC can be crosslinked by micromolar concentrationsof mercury, indicating that these residues can move close to each other. Wethen solved the structure of the T280C/E54C bcMalT double cysteinemutant in the crosslinked state to 3.6 A resolution, and the structure isindeed in an inward-facing conformation. The new structure illustrates thelarge-scale movement of a structurally conserved domain in bcMalT, andshows that EIIC employs an elevator-like mechanism for substrate translo-cation. Further analyses suggest how the inward facing conformation couldinteract with an EIIB protein to achieve phosphate transfer. The structuresalso provide a solid starting point for investigating the dynamics of theEIIC protein using spectroscopic approaches.Reference[1]. Mccoy JG, Ren Z, Stanevich V, et al. The Structure of a Sugar Transporterof the Glucose EIIC Superfamily Provides Insight into the Elevator Mechanismof Membrane Transport. Structure. 2016;24(6):956-64.[2]. Cao Y, Jin X, Levin EJ, et al. Crystal structure of a phosphorylation-coupled saccharide transporter. Nature. 2011;473(7345):50-4.

637-Pos Board B402Substrate-Induced Conformational Change in LeuTYuan-Wei Zhang1, Lucy R. Forrest2, Gary Rudnick1.1Pharmacology, Yale University, New Haven, CT, USA, 2NINDS, NIH,Bethesda, MD, USA.LeuT is a prokaryotic amino acid transporter that has been used extensivelyas a model for neurotransmitter transport. We recently demonstrated thatthe conformational change induced by Naþ ions requires the Na2 siteobserved in LeuT crystal structures. We observed this conformationalchange using LeuT in E. coli membranes, in the absence of detergent,by a decrease in reactivity of a single cysteine (Y265C) in the cytoplasmicpermeation pathway. We now show that this effect of Naþ is observedwhether Kþ or NMDGþ is used as a control ion. In the presence of Naþ,addition of alanine, a substrate, induces the reverse conformationalchange, opening the cytoplasmic pathway and increasing Cys-265 reactivity.Three mutations in the substrate binding site each altered the affinity ofLeuT for leucine and alanine, but did not interfere with the conformationalchange induced by Naþ. One of the mutations also blocked the substrate-dependent conformational change. The results suggest a mechanism bywhich substrate interactions with the central binding site of LeuT reversethe ability of Naþ to stabilize outward-facing conformations of this modeltransporter.

638-Pos Board B403Membrane Remodeling by GltPh in the Inward- and Outward-FacingConformations Explains Lack of Protomer CooperativityWenchang Zhou1, Claudio Anselmi1, Horacio Poblete1, Ali Karimi2,Lucy Forrest2, Jose Faraldo-Gomez1.1National Heart, Lung and Blood Institute, NIH, Bethesda, MD, USA,2National Institute of Neurological Disorders and Stroke, Bethesda,MD, USA.Membrane-embedded proteins can induce the remodeling of the adjacentlipid bilayers by promoting curvature, altering the membrane thicknessand/or exposing hydrophobic groups. When these proteins undergo afunction-related conformational transition, the energy cost associatedwith these membrane perturbations adds up to the free energy of each ofthe protein states and can therefore modulate their functional mechanisms.Here, we study the case of the Naþ-coupled aspartate transporter fromPyrococcus horikoshii (GltPh). GltPh has been crystallized as a trimer bothin an inward- and outward-facing conformations. In this outward-to-inward conformational exchange, the so-called transport domainmoves ~20 A across the membrane, relative to the seemingly rigid trimeri-zation/scaffold domains, so as to expose the substrate and Naþ-binding sitesto either the cytoplasm or the extracellular space. Using large-scale coarse-

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grained and all-atom molecular dynamics (MD) simulations, we character-ized the membrane deformation induced by GltPh trimers in all possiblepermutations of inward-facing and outward-facing protomer states. The sim-ulations show that when a protomer is in the outward-facing state, the sur-rounding membrane is largely unperturbed. However, in the inward-facingstate, the transport domain induces a strong deformation on the lipid bilayer,bending its average plane by ~10 A along the direction perpendicular to themembrane plane. This perturbation extends radially for ~50 A, but remark-ably, it is largely localized around each of the transport domains in thevicinity of the protein, i.e. the membrane shape is restored near theprotomer-protomer interfaces. The specific protein-lipid contacts thatexplain this local deformation are identified from all-atom MD simulations.In summary, MD simulations demonstrate that the lipid membranefavors the outward-facing state of GltPh over the inward-facing state, owingto the long-range curvature deformation induced by the latter. However,these simulations also show that this large deformation does not implya membrane-mediated protomer cross-talk, explaining the mystifyingabsence of measurable cooperativity among protomers in this trimerictransporter.

639-Pos Board B404Protein-Protein Interaction between Sodium-Coupled MonocarboxylateTransporter 1 (SMCT1) and PDZ Domain-Containing Ring FingerDomain 3 (PDZRN3)Yusuke Otsuka.Pharmacology, Chiba University Graduate School of Medicine, Chiba-shi,Japan.Sodium-coupled monocarboxylate transporters (SMCTs) are membraneproteins which transport lactate on the apical side of kidney. In additionto renal lactate reabsorption, it is known that both SMCTs and urate/aniontransporter1 (URAT1) bind to PDZK1 and that SMCTs and URAT1 func-tionally cooperate to transport urate. This indicates that these transportersreabsorb urate by exchange for sodium. Our recent study revealed thatSMCT1 (SLC5A8), a higher-affinity transporter than SMCT2 (SLC5A12),bound to PDZ domain-containing RING finger domain 3 (PDZRN3) in ayeast two hybrid screening. But there is less information available on theinteraction between SMCT1 and PDZRN3. In this study, we elucidatedthis protein-protein interaction between them to resolve the regulationmechanism of serum urate level. We performed coimmunoprecipitationstudy to confirm the binding between SMCT1 and PDZRN3, and performed[3H] nicotinate uptake study to reveal whether there was a functional changeby this binding, using HEK293 cells transiently transfected with SMCT1, itsmutant lacking the PDZ motif and PDZRN3. Coimmunoprecipitation studyrevealed that the wild type SMCT1, but not its mutant lacking the SMCT1C-terminal PDZ motif, directly bound to PDZRN3. In uptake study, therewas no significant difference in amount of nicotinate uptake via SMCT1and its mutant lacking the PDZ motif, in the presence of PDZRN3 or not.These results showed the protein-protein bind between SMCT1 andPDZRN3, but no functional interaction. This suggests that PDZRN3 regu-lates urate reabsorption via URAT1 by preventing SMCT1 from bindingto PDZK1.

640-Pos Board B405It Runs in the Family: Determining the Transport Mechanism of Sodium/Dicarboxylate Symporter hNaDC3Alissa J. Becerril1, Cristina Fenollar-Ferrer2, Lucy R. Forrest2,Joseph A. Mindell1.1Membrane Transport Biophysics Unit, NINDS, NIH, Bethesda, MD, USA,2Computational Structural Biology Unit, NINDS, NIH, Bethesda, MD, USA.Members of the divalent anion:Na(þ) symporter (DASS) family play im-portant roles in mammalian physiology, transporting divalent anions,including Krebs cycle intermediates and sulfate, across the plasma mem-brane. These transporters may be key contributors to determining urinarycitrate levels, which, in turn, may affect kidney stone formation; they alsohave been implicated in metabolic regulation in both drosophila andmammals. It is therefore important to understand the relationships in theseproteins between their structure and functional mechanisms. Though nostructures are yet available for mammalian DASS family members, acrystal structure of a bacterial homolog, vcINDY, has been determined.We recently demonstrated that vcINDY, a Naþ-coupled succinate trans-porter, utilizes a dramatic ‘‘elevator’’ mechanism to transport substrate,involving a large-scale vertical movement of a protein domain perpendicularto the plane of the lipid bilayer membrane. Here, we sought to determinewhether a mammalian family member utilizes a similar transport

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mechanism, specifically human NaDC3. With ~30% identity between thevcINDY and hNaDC3, we hypothesized that a similar mechanism couldbe responsible for the functioning of hNADC3. Homology modeling withvcINDY suggested that several pairs of residues are brought into proximityupon substrate translocation. We prepared a series of double mutantsintroducing cysteines at positions predicted to be brought together in theoutward-facing state of the protein and expressed them in Xenopuslaevis oocytes. Using two-electrode voltage clamp and disulfide cross-linking, we investigated whether inhibition of transport activity observedupon formation of disulfides is consistent with the proposed mechanism oftransport.

Mitochondria in Cell Life and Death I

641-Pos Board B406Cyclophilin D Acetylation Regulates Cardiac Myocyte DifferentiationGeorge A. Porter, Jr., Morgan L. Albert, Kathia R. Fantauzzi-Nieves,Gisela Beutner.Pediatrics, University of Rochester Medical Center, Rochester, NY, USA.INTRODUCTION: Protein acetylation is an important regulator ofcellular function. Acetylation of cyclophilin D (CyPD) at lysine 166 increasesits ability to open the mitochondria permeability transition pore (PTP), whichis an important regulator of mitochondrial metabolism and myocyte differen-tiation. HYPOTHESIS: Acetylation of CyPD controls PTP activity, mito-chondrial structure and function, and myocyte differentiation in thedeveloping heart. METHODS: We use immunoblotting and immunoprecipita-tion to determine the global protein acetylation state and that of CyPD duringembryonic cardiac development in wild type (WT) and CyPD-null mice. Wealso re-expressed WT and mutant CyPD in CyPD-null embryonic andneonatal cardiac myocyte cultures to determine effects on mitochondrialstructure and function and myocyte differentiation. RESULTS: Immunoblot-ting revealed that global protein acetylation was high in the embryo but fellduring later development. In contrast, CyP-D was highly acetylated in theearly embryonic heart, but this decreased dramatically during subsequentdevelopment. When re-expressed in cultures of CyPD-null myocytes, WTCyPD recapitulated the WT cell phenotype of fragmented mitochondrialstructure, low mitochondrial membrane potential (Dcm), and decreased differ-entiation. Re-expression of a CyPD acetylation mimic mutant (K166Q) hadsimilar or greater effects on these parameters than WT CyPD, while an inac-tivating (R96G) and de-acetylation mimic (K166R) CyPD mutant did not.CONCLUSION: These data suggest that myocyte differentiation during car-diac development is associated with a decrease in overall protein acetylation,perhaps because of the changes that occur in metabolism as the heart develops.However, specific acetylation of CyPD appears to be particularly importantfor maturation of mitochondrial function and regulation of cardiac myocytedifferentiation.

642-Pos Board B407The Role of MICU1 in Neuronal Mitochondrial Calcium Homeostasisand FunctionAdam Bartok1, Melanie Paillard1, Valentina Debattisti1, David Weaver1,Ashley Tyburski2, Lianteng Zhi3, Hui Zhang3, Melanie Elliott2,Gyorgy Hajnoczky1.1Pathology, Anatomy and Cell Biology, Thomas Jefferson Unversity,Philadelphia, PA, USA, 2Neurological Surgery, Thomas Jefferson Unversity,Philadelphia, PA, USA, 3Neuroscience, Thomas Jefferson Unversity,Philadelphia, PA, USA.Mitochondrial Ca2þ homeostasis controls intracellular calcium signals,cellular energy production and mechanisms of cell death. We and othershave shown that MICU1 is an intermembrane space protein that providesCa2þ sensitivity to Ca2þ uptake by the mitochondria through the mitochon-drial Ca2þ uniporter. Specifically, MICU1 is required both for protectingmitochondria from Ca2þ uptake when the cytoplasmic [Ca2þ] is low andfor proper transmission of the short-lasting Ca2þ signal from the ER/SR tothe mitochondria. Human loss of function mutation of MICU1 has been linkedto learning difficulties, skeletal muscle weakness, fatigue and motoricimpairment.In this study, we describe and validate a neuron-specific MICU1 KOmodel in mice. Proteomic analysis of the mitochondria confirms lossof MICU1 and indicates a possible compensatory mechanism for the lossof MICU1 by other constituents of the uniporter. Behavioral testsperformed on the animals showed decreased motoric function progressingwith age. Measurements of cytoplasmic and mitochondrial Ca2þ signalsin cultured cortical neurons and Ca2þ uptake by isolated mitochondria re-

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vealed altered Ca2þ homeostasis which might underlie the functionalimpairments.Our results reveal the specific contributions of MICU1 in neuronal Ca2þ ho-meostasis and provide some clues to the neuronal pathogenesis in MICU1loss of function patients.

643-Pos Board B408IP3 Receptor Isoform Dependence of the ER-Mitochondrial CA2D-Transfer in Mammalian CellsMate Katona1, Kamil Alzayady2, David I. Yule2, Gyorgy Hajnoczky1.1Department of Pathology, Anatomy and Cell Biology, Thomas JeffersonUniversity, Philadelphia, PA, USA, 2Department of Pharmacology andPhysiology, University of Rochester, Rochester, NY, USA.IP3 receptors (IP3Rs) mediate Ca2þ release from the ER to the cytosol and canrelay Ca2þ signals locally to mitochondria. The IP3R family is composed bythree isoforms (type 1,2 and 3) showing heterogeneous structure, function andtissue distribution. In some paradigms specific IP3R isoforms were shown tobe enriched close to and support Ca2þ transfer locally to the mitochondria.Until recently, a mammalian system allowing systematic evaluation of eachisoform has remained unavailable. The establishment of IP3R triple knockout (TKO) HEK cells and rescue with different IP3R isoforms tagged witha common biochemical tag provided new opportunities to test the hypothesisthat IP3R isoforms are equal in terms of coupling to the mitochondria. Tostudy Ca2þ coupling between ER and mitochondria we used WT HEK cellsand TKO cells acutely and stably rescued by the different isoforms ofIP3Rs. Ca

2þ-signaling was induced by an IP3-generating agonist, carbachol.To measure changes in mitochondrial Ca2þ ([Ca2þ]m) we transfected cellswith the genetically-encoded Ca2þ sensor CEPIA targeted to the mitochon-drial matrix (mtCEPIA), changes in cytosolic Ca2þ ([Ca2þ]c) were measuredby FURA-2 AM simultaneously. WT HEK cells showed a marked increase in[Ca2þ]c followed by a fast rise in [Ca2þ]m with the coupling time of0.94 50.22 s (n=56) while TKO cells showed no changes in [Ca2þ]c and[Ca2þ]m to agonist stimulation. Acute and stable type 1, 2 or 3 IP3R rescueTKO cells regained [Ca2þ]c responses and also showed a [Ca2þ]m increase.Quantitative analysis of the Ca2þ transfer efficiency to the mitochondriaand the localization of each IP3R isoform relative to the mitochondria arecurrently pursued. In summary, we have validated a new model for the studyof IP3R-mediated local Ca2þ signaling between the ER and mitochondria, al-lowing us to determine the specific function of each isoform in theseprocesses.

644-Pos Board B409Mitochondrial Calcium Uptake and Matrix Calcium Buffering in SkeletalMuscleValentina Debattisti, Gyorgy Csordas, Erin Seifert, Gyorgy Hajnoczky.MitoCare, Thomas Jefferson University, Philadelphia, PA, USA.Many clues suggest that mitochondrial calcium uptake plays an essentialrole in muscle function. Mitochondria can contribute to shaping ofthe sarcoplasmic [Ca2þ] transients by their Ca2þ handling and are providingenergy for muscle contraction and relaxation by their ATP production.Mitochondrial calcium handling involves uptake, release and Ca2þ chelationin the matrix, which allows storage of vast amounts of Ca2þ. Recent iden-tification of the molecular machinery of the Ca2þ uniporter (mtCU) createdan opportunity to target specific mechanisms of Ca2þ handling and addresstheir function in Ca2þ homeostasis and contractile function. To elucidatethe role of mitochondrial Ca2þ uptake in skeletal muscle (SM) functionwe ablated MICU1, the Ca2þ sensing regulator of mtCU in mouse.MICU1 ablation in SM resulted in impaired gatekeeping and attenuatedCa2þ uptake and resulted in less endurance exercise performance whenchallenged with fatigue protocols. However, in this mouse and in cell lines,upon MICU1 ablation protein levels of other components of the mtCU andCa2þ chelation in the matrix were also altered. To overcome the compensa-tory effects we decided to apply two strategies. First, we induce acuteMICU1 knock out using a tamoxifen-inducible CRE-system. Secondly, wealso targeted the mitochondrial phosphate (Pi) carrier (PiC) in a similarmanner. Pi transport into mitochondria is crucial for both mitochondrialATP synthesis and Ca2þ chelation in the matrix. To quantitatively andsimultaneously measure Ca2þ uptake and matrix buffering, mitochondriaare isolated from SM for fluorometry are loaded with furaFF/AM andare incubated in the presence of rhodFF. SM mitochondria acutelydepleted for PiC show a decreased Ca2þ chelation capacity in the matrixand unexpectedly, take up more Ca2þ than the control. When challengedin an incremental exercise test, PiC-ablated mice could only run for8.551.7 min, whereas control mice ran for the full 20 min, showing

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impaired performance. The effects of acute MICU1 depletion in SM onmtCU composition, mitochondrial Ca2þ handling and SM function arecurrently evaluated. Thus, our results provide genetic evidence for thecontribution of mitochondrial Ca2þ homeostasis to SM function and are ex-pected to determine the function and mechanism dependent on specificallyon MICU1 and PiC.

645-Pos Board B410Mitochondrial Calcium Flux Contributes to Arrhythmia in Mouse Heartduring Acute Myocardial InfarctionAn Xie, Hong Liu, Anyu Zhou, Guangbin Shi, Samuel C. Dudley, Jr.Rhode Island Hospital, providence, RI, USA.Introduction: Acute myocardial infarction (AMI) is associated withlethal ventricular arrhythmias. One important mechanism for these arr-hythmias is delayed afterdepolarizations resulting from Ca2þ overload inborder zone. Mitochondria participated in Ca2þ homeostasis, and themitochondrial Ca2þ uniporter (MCU) mediates mitochondrial Ca2þ entry.We tested whether mitochondrial Ca2þ flux played a role in ventricular ar-rhythmias during AMI. Methods: Hearts and ventricular cells were isolatedfrom CD1 wild type (Wt) and MCU knock down (MCUþ/-) mice. Whole cellcurrent-clamp was employed to record action potentials (APs). CytoplasmicCa2þ and mitochondrial Ca2þ were sampled by loading Indo-1 AM andRhod-2 AM (recorded synchronously with APs) respectively. Electricpacing was used to induce arrhythmia. MCU mRNA and protein expressionwere evaluated by Western blotting & Real-Time PCR. Optical mappingwas used to detect arrhythmias in the intact heart. Results: In MCUþ/�

mice, the mRNA (from 1.00 5 0.02 to 0.42 5 0.03 ) and protein expression(from 0.73 5 0.07 to 0.33 5 0.04) of MCU was substantially reduced, withpeak mitochondrial Ca2þ transients (F/Fo) prominently decreased from0.21 5 0.02 to 0.14 5 0.03. Infarct size was similar in the two models:Wt (48% 5 4%) and MCUþ/- (44% 5 2%) mice. Two of eight of Wtmice died suddenly within 1 h after AMI, but none of MCUþ/- mice diedsuddenly in this period. Compare to the Wt mice, pacing induced signifi-cantly less ventricular arrhythmias in MCUþ/- mice during the firststage of coronary artery ligation as VT/VF duration decreased from0.38 5 0.09 s in Wt mice to 0.12 5 0.05 s in MCUþ/- mice. Consistentwith a role of mitochondria in ischemic ventricular rhythms, inhibition ofmitochondrial NCX by CGP-37157 (i.p. 0.4mg/100g) in both Wt andMCUþ/� mice could abolish ventricular arrhythmias evoked by electric pac-ing during AMI. Furthermore, VT/VF was detected only in Wt mice atinfarction border zone by optical mapping experiment. Finally, after AMI,action potential durations were longer in Wt mouse hearts/cardiomyocytesthan those in MCUþ/- mouse hearts/cardiomyocytes. Conclusion: Ourstudy suggest a role for mitochondrial Ca2þ handling in arrhythmicrisk during AMI. Reducing mitochondrial Ca2þ cycling improvedarrhythmic risk.

646-Pos Board B411Regulation of Carbon Substrate Utilization by Cardiac MitochondriaSantosh K. Dasika, Sunil M. Kandel, Daniel A. Beard.MIP, University of Michigan, Ann Arbor, MI, USA.Mitochondria play key roles in central metabolism, not only by synthesizingATP via oxidative phosphorylation, but also by synthesizing key intermedi-ates that serve as anaplerotic entry points in numerous pathways. Becausereactions producing and/or consuming several of the intermediates in thetricarboxylic acid cycle that are transported across the mitochondrialinner membrane (pyruvate, a-ketoglutarate, citrate) are regulated in partby calcium concentration, we hypothesize the calcium is a key regulatorof entry and exit of substrates into and out of the TCA cycle. We set outto first refine this hypothesis through following time courses of consumptionof various carbohydrate based substrates (including complex I substratessuch as pyruvate and citrate, and the complex II substrate succinate) andmatching data on rates of oxidation and NADH production to simulationsof mitochondrial metabolic kinetics. Specifically, model simulations werefit to data on total NAD(P)H and oxygen consumption flux at differentrespiratory states and with varying inorganic phosphate concentrations.The resulting identified model is used to predict how calcium regulatescarbon substrate utilization by mitochondria. These hypotheses are testedusing in vitro experiments with purified mitochondria. By comparing theexperimental data with citrate alone to combination of pyruvate, citrateand malate as the substrates, we conclude that transport of citrate into thematrix is limited in the absence of malate. The rate of reduction ofNAD is slower with pyruvate alone as substrate compared to when malateis present. The role of glutamate dehydrogenase in the context of cardiac en-ergetics is investigated.

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647-Pos Board B412A Theoretical Study on the Roles of Ca2D in the Energy MetaboliteStability during Cardiac Workload TransitionAyako Takeuchi1, Ryuta Saito2, Yukiko Himeno3, Satoshi Matsuoka1.1University of Fukui, Fukui, Japan, 2Mitsubishi Tanabe PharmaCorporation, Saitama, Japan, 3Ritsumeikan University, Ninamikusatsu,Japan.The heart is a continuously working pump that is energetically driven byhydrolysis of ATP. Since the amount of ATP in the heart is small, ATPsynthesis must increase so as to adapt to a rise in cardiac ATP demand.Therefore, the cardiac energy metabolites such as ATP, phosphocreatine,ADP and NADH are kept relatively constant during physiological cardiacworkload transition. Ca2þ has been implicated to be one of the regulatorsof the constancy, though its detailed roles are not yet clarified. Here, weconstructed and analyzed a detailed mathematical model of cardiac mito-chondria which includes oxidative phosphorylation, substrate metabolismand ion/substrate transporters, based on experimental literatures, for the pur-pose of elucidating the roles of Ca2þ-dependent regulation mechanisms inthe metabolite constancy.The model well reproduced the experimental data on Ca2þ- and inorganicphosphate (Pi)-dependencies of oxygen consumption, NADH level, and mito-chondrial membrane potential, under the in vitro condition of isolated mito-chondria with malate/glutamate used as substrates. When the model wasincorporated into a simple cardiac cell model and malate/glutamate wereused as substrates, the metabolite constancy could not be maintained at higherworkload with cytosolic Ca2þ below physiological range ( ~ 0.1 mM). Pi andNADH levels became stable during workload transition with higher cytosolicCa2þ. Interestingly, the Ca2þ dependency of NADH was influenced by thecomposition of cytosolic mitochondrial substrates. That is, the Ca2þ-depen-dency of NADH almost disappeared and energy metabolites became morestable during workload transition under the in vivo condition that malate,glutamate, pyruvate, citrate and 2-oxoglutarate were used as substrates. Itwas revealed that composition of mitochondrial substrates is important formetabolite constancy during cardiac workload transition, and Ca2þ hasonly a minor role under physiological conditions. These results help us under-standing the regulation mechanisms of cardiac energy metabolism under thephysiological conditions.

648-Pos Board B413Reciprocal Polarization of the Mitochondrial Ca2D Uniporter andNAD-CA2D Exchanger in Cardiac MuscleSergio De la Fuente1, Shey-Shing Sheu2, Gyorgy Csordas1.1MitoCare Center, Pathology Anatomy and Cell Biology, Thomas JeffersonUniversity, Philadelphia, PA, USA, 2Center for Translational Medicine,Thomas Jefferson University, Philadelphia, PA, USA.Control of myocardial energetics by Ca2þ signal propagation to the mito-chondrial matrix includes local Ca2þ delivery from sarcoplasmic reticulum(SR) ryanodine receptors (RyR2) to the inner mitochondrial membrane(IMM) through the Ca2þ uniporter (mtCU). Ca2þ is extruded by the Naþ-Ca2þ exchanger (NCLX), predominantly active in excitable tissues suchas heart, and/or the Hþ-Ca2þ exchanger preferentially active in non-excitable cells. We have recently reported that the mtCU is strategicallypositioned in cardiac muscle at the mitochondria-junctional SR associationin order to support more efficiently the local Ca2þ signals. In those studieswe established a comparative abundance profile for a range of mitochondrialproteins between the mitochondrial and SR fractions of mouse and rat hearthomogenates. While mtCU constituents were equally distributed (MCU,MICU1) or enriched in the SR fraction (EMRE), the NCLX was strongly(multiple fold) enriched in the mitochondrial fraction. We thus hypothe-sized, that NCLX was excluded from the mtCU hot spots at the dyad junc-tions and that it would be localized at areas where mitochondria associatewith the network SR. Indeed, percoll purification of the crude mitochondrialfraction that decreased the abundance of MCU and EMRE lead to anenrichment of NCLX. Mitochondrial 45Ca2þ uptake assays using isolatedmitochondria or SR fraction with vs. without NCLX inhibition (pharmaco-logical or Naþ removal) revealed a robust Naþ-dependent efflux workingagainst the uptake in the mitochondrial but not the SR fraction. Directmeasurement of the fractional Ca2þ release from the two membranefractions also pointed toward much more efficient NCLX-mediated Ca2þ

extrusion in the mitochondrial fraction than in the SR. Thus, mitochondrialCa2þ uptake and Naþ dependent extrusion seem to be reciprocally polarizedto optimize both the efficacy of receiving local Ca2þ signals at thedyadic interface and of feeding back the uptake Ca2þ to the SERCA pumpsof the network SR.

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649-Pos Board B414Effect of Arsenic on Intracellular Calcium & Redox HomeostasisRafaela Bagur Quetglas1, Peter Varnai2, Gyorgy Csordas1,Gyorgy Hajnoczky1.1Pathology, Thomas Jefferson University, MitoCare Center, Philadelphia,PA, USA, 2Physiology, Faculty of Medicine, Semmelweis University,Budapest, Hungary.A range of environmental agents causes tissue injury that has been attributedto reactive oxygen species (ROS). Altered ROS production is commonlyoriginated from the mitochondria and is associated with dysregulationof calcium (Ca2þ) homeostasis and mitochondrial apoptosis. However,the causative pathways remain largely unknown because the fundamentalroles of mitochondria in cell survival, cell signaling and dynamics arecommonly mediated through local communication between mitochondriaand other organelles, which have been difficult to directly monitor. Ouraim is to develop fluorescent protein-based tools allowing sensitiveand specific measurements of ROS and [Ca2þ] at the mitochondria-endoplasmic reticulum (ER) interface. Applying these novel tools, wehave studied the effects of environmental stress caused by arseniconlocal mitochondria-ER communication and its impact for cell signaling,and cell physiology in primary mouse hepatocytes. Either acute addition(30 mM) or prolonged, overnight exposure (3 mM) to sodium arsenite hasinduced an increase in ROS. Upon prolonged arsenic exposure, these alter-ations were accompanied by a significant decrease in the cytoplasmiccalcium signal to an IP3-linked agonist: a decrease was observed in the frac-tion of responsive cells, in the amplitude of the response as well as in thesustained phase. Regarding the acute effect of arsenic, we observed a pro-gressive increase of the basal cytoplasmic [Ca2þ] with a decrease in theamplitude of the IP3-linked calcium signal. Neither acute nor prolongedarsenic exposure impaired mitochondrial calcium uptake. Nevertheless,we documented increased mitochondrial calcium content in the prolongedarsenic exposure group. In conclusion, the results suggest that aninitial component of the arsenic-induced hepatocyte injury is increasedROS, which is associated with impaired local mitochondria-ER calciumcommunication.

650-Pos Board B415Effect of Rutin on Mitochondrial Respiration and Cellular Energy StatusJeong-Soo Park, Icksoo Lee.Dankook University, Cheonan, Korea, Republic of.Rutin, a phenolic compound found in many plants is known to have anti-oxidative and anti-inflammatory properties. It has been reported that rutin res-cues the cells from the challenges such as ethanol, methylmercury, radiation,hydrogen peroxide, and other cytotoxic chemicals. It is also suggested toenhance mitochondrial biogenesis in obese rats.Cytochrome c oxidase is a rate limiting enzyme in mitochondrial electron trans-port chain and consequently influences on mitochondrial membrane potentialand therefore the ATP production. Also its electron donor cytochrome c iswell known to participate in type 2 apoptosis in cells.Here it is shown that rutin elevates the activity of cytochrome c oxidase in hu-man breast cancer cells. The mitochondrial respiration and cellular energylevels also increase by rutin treatment. We propose that the beneficial cytopro-tective effects of rutin might due to, at least in part, the upregulated mitochon-drial enzyme, cytochrome c oxidase which would enhance mitochondrialrespiration and therefore cellular energy levels.*This research was supported by Basic Science Research Program through theNational Research Foundation of Korea(NRF) funded by the Ministry of Edu-cation(NRF-2014R1A1A2058722).

651-Pos Board B416Detection of a Bithionol Sensitive Potassium Channel in CardiacMitochondria Absent in Slo2.1 KnockoutsCharles O. Smith.Biochemistry, University of Rochester, Rochester, NY, USA.Mitochondrial Kþ channels are important mediators of cell protectionagainst stress, however less is known about their physiologic roles. Usingmouse knockout models our lab has recently reported the absolute require-ment of the Naþ-activated-Kþ channel Slo2.1 for volatile anesthetic precon-ditioning against cardiac ischemia reperfusion injury [PMID: 26845140].Functional examination of this channel in isolated mitochondria using aTlþ flux assay demonstrated the presence of a bithionol-sensitive mito-chondrial Tlþ uptake in wildtype cardiac mitochondria that was absent inthose from Slo2.1 knockout. Herein we combine pharmacologic activation

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(bithionol) and inhibition (quinidine) of Slo2 channels with patch clampof isolated mitochondrial inner membranes (mitoplasts) from wildtype andSlo2.1-/- mice, to demonstrate the presence of Slo2.1 activity in cardiacmitochondria at the single channel level. Additional analysis of theseSlo2.1-/- mice at the whole animal, heart, cardiomyocyte, and mitochondriallevel has revealed a metabolic phenotype comprising differential substratepreference under stress conditions, indicating a functional relationship be-tween mitochondrial Kþ channels and the regulation of mitochondrial meta-bolism. These data demonstrate a role for Slo2.1 in the regulation of cardiacmitochondrial function.

652-Pos Board B417Mitochondrial Respiration and ROS Emission From b-Oxidation in theHeart: An Experimental Computational StudyMiguel A. Aon, Steven J. Sollott, Sonia Cortassa.Laboratory of Cardiovascular Science, National Institute on Aging/NIH,Baltimore, MD, USA.Lipids are main fuels for cellular energy and mitochondria their majoroxidation site. Yet unknown is to what extent the fuel role of lipids is influ-enced by their intrinsic uncoupling effects, and how this affects mitochon-drial energetics, redox balance and the emission of reactive oxygenspecies (ROS). b-oxidation of palmitoyl CoA (PCoA) in isolated heart mito-chondria from Sham and streptozotocin-induced type 1 diabetic (T1DM)guinea pigs (GPs) were comparatively analyzed. Parallel high throughputmeasurements of the rates of oxygen consumption and H2O2 emission asa function of PCoA concentration were performed. We found that PCoAconcentration < 200 nmol/mg mito protein resulted in low H2O2 emission,increasing thereafter in Sham and T1DM GPs under both states 4 and 3respiration with diabetic mitochondria releasing higher amounts of ROS.In both control and diabetic mitochondria, respiratory uncoupling andROS overflow in both states 4 and 3 respiration occurred at PCoA > 600nmol/mg mito prot., leading to extensive matrix swelling, NAD(P)H pooloxidation, impairment of the state 4->3 transition concomitantly withdecreased activity of matrix glutathione/thioredoxin ROS scavengingsystems, and enhanced H2O2 emission. The experimental data could besimulated with a computational model of mitochondrial b-oxidation oflong-chain fatty acids, including main energy-redox and ionic processes.Results indicate that in a demand-led organ like the heart, mitochondriacan increase ROS release as a function of the rate of b-oxidation dependentrespiration, but impairment of mitochondrial function would only happenafter a certain threshold of lipid concentration is attained. Keeping lowlevels of intracellular lipid appears to be crucial for mitochondria and cellsto maintain ROS within physiological levels compatible with signaling andreliable energy supply.

653-Pos Board B418Detection of Reactive Oxygen Species in Live Cell MitochondriaZhen Luo, Jixiang Liu, Qin Zhao, Yunting Xi, Ruogu Peng, Jinfang Liao,Zhenjun Diwu.AAT Bioquest Inc., Sunnyvale, CA, USA.Oxidative stress in mitochondria has received intensive attentions recentlybecause it is the most important source of intracellular reactive oxygen species(ROS) and is considered as a major contributor to some public health-relateddiseases, such as asthma, atherosclerosis, diabetic vasculopathy, osteoporosis,a number of neurodegenerative diseases and Down’s syndrome. However,the existing probes have difficulties to target live cell mitochondria and distinctthe individual ROS.We have developed a new family of mitochondrial ROS fluorescenceprobes, which not only can specifically localize in mitochondria, but alsocan selectively detect the different ROS species, including superoxide,hydroxyl radical and hydrogen peroxide in multiple Ex/Em wavelengthschannels.Cancer cells stained with the novel fluorescent probes showed negligible fluo-rescence in absence of ROS stimulants. In contrast, OxiVision Blue-loadedcells with hydrogen peroxide treatment displayed strong blue fluorescence inmitochondria. MitoROS OH580-loaded cells with Fenton reaction (to inducehydroxyl radical) showed strong red fluorescence in mitochondria. MitoROS520-loaded cells exhibited strong green fluorescence in mitochondria aftertreated with superoxide stimulants. All these probes have also been success-fully used with a flow cytometer for the quantitative detection of ROS in livecells.In conclusion, these probes are mitochondrion-targeted ROS probes, which canbe used for monitoring the exogenous and endogenous changes of ROS levels inliving cells in real time by fluorescence imaging and or with a flow cytometer.

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654-Pos Board B419A Standardized Method to Quantify ER-Mitochondrial Interfaces inElectron MircographsDavid Weaver, Adam Bartok, Gyorgy Csordas, Gyorgy Hajnoczky.Thomas Jefferson University, Philadelphia, PA, USA.The interaction sites between endoplasmic reticulum (ER) and mitochondriaare signaling hubs in the cell implicated in calcium transfer, lipid meta-bolism, autophagy, and cell death among other functions. Transmissionelectron microscopy (TEM) is commonly used to visualize the geometryof these interfaces, but metrics used to describe and compare between con-ditions, as well as the techniques of measurement are not standardized. Herewe describe a pair of scripts for ImageJ that allow for rapid, reproducibleand flexible quantification of interface geometries. One script is focusedon measuring the physical apposition of the membranes while the otherconsiders the potential exposure of the outer mitochondrial membrane(OMM) to calcium released from the ER. In both cases, the user simplytraces the OMM and nearby ER membranes; the script bins the interface dis-tances and returns the length of the OMM that participates in an interface ofa given gap width, as well as the total OMM and ER lengths. A ‘score’based on the inverse-square of the distance is also generated as anattempt to make a single value representing ‘interface-ness’. The additivenature of these measures allows cell-wise totals to be calculated for agiven TEM section. While this approach standardizes the measurementtechnique for a number of parameters of ER-mitochondrial interfaces, it re-mains to be seen which parameters best correspond with physiologicalchanges in different models.

655-Pos Board B420Control of Mitochondrial Structure and Antioxidant Response by theATPase InhibitoryFactor 1Define aNovel PotentialOncogenicMechanismDanilo Faccenda1, Junji Nakamura2, Gurtej K. Dhoot1, Mauro Piacentini3,Masasuke Yoshida2, Michelangelo Campanella1.1Department of Comparative Biomedical Sciences, Royal VeterinaryCollege, London, United Kingdom, 2Department of Molecular Biosciences,Kyoto Sangyo University, Kyoto, Japan, 3Department of Biology, Universityof Rome Tor Vergata, Rome, Italy.The ATPase inhibitory factor 1 (IF1) is an ubiquitously expressed mitochon-drial protein that blocks the reversal of the F1Fo-ATPsynthase, preventingdissipation of cellular ATP and ischaemic damage. Many human cancers ex-press high levels of IF1, which suppress cell death and enhance tumour cellinvasion and chemoresistance. In this study, we assessed the effect of IF1over-expression on mitochondrial redox balance and apoptotic cristae remod-elling. We found that IF1 maintains ATP levels under apoptosis and reducesglutathione (GSH) loss and inactivation of peroxiredoxin 3 (Prx3). This cor-relates with inhibition of the metallopeptidase OMA1-mediated processing ofthe pro-fusion dynamin related protein optic atrophy 1 (OPA1), impedingcristae remodelling and apoptosis completion. IF1 therefore has a pivotal anti-oxidant activity that hinders the OMA1/OPA1-dependent deconstruction ofmitochondrial morphology and cellular demise. The data presented here high-light a dual regulatory activity of IF1 on both mitochondrial bioenergetics andstructure, resulting in increased proliferative capacity of tumour cells andsignificantly contributing to the molecular signalling of mitochondria incancer.

Emerging Techniques and Synthetic Biology

656-Pos Board B421Site Saturation Mutant Viruses Evolve Neutralizing Antibody Resistancein a Microfluidic Cell Culture SystemJared D. Evans1, Audrey E. Fischer2, Susan Wu1, Peter M. Thielen1,Thomas S. Mehoke1, Ashok Sivakumar1, Joshua T. Wolfe1.1REDD, JHUAPL, Laurel, MD, USA, 2AOS, JHUAPL, Laurel, MD, USA.Viruses constantly evolve to overcome host and environmental pressures dur-ing their replication process. These evolutionary adaptations are generallyconsidered to be limited to a particular subset of beneficial mutations. Weconstructed a series of replication-competent mouse norovirus (MNV-1)site-saturation libraries to test how these viruses evolve in response to aneutralizing antibody. Residues in the A’-B’ (E296) and E’-F’ (V378,A382, D385) loops of the MNV-1 capsid protruding (P) domain were mutatedusing site directed mutagenesis. Viruses were evolved in the presence andabsence of monoclonal neutralizing antibody A6.2 in a droplet microfluidicdevice such that starting virus populations at low multiplicity of infectioncould replicate over several passages in the absence of large population effectssuch as purifying selection. Droplet evolved viruses were subsequently

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passaged in bulk to determine whether unique combinations of mutationsbecame fixed in the population. Using high depth RNA sequencing, wedemonstrate the distribution of all possible amino acid substitutions frominitial production in the laboratory, replication in the host, and passaging inthe presence or absence of neutralizing antibody. These results demonstratea method for surveying fitness landscapes that may be broadly applicable tovirus-host evolution.

657-Pos Board B422Chiral Vortex Dynamics on Membranes is an Intrinsic Property of FtsZDriven by GTP HydrolysisDiego A Ramirez-Diaz1, Daniela Garcia-Soriano1, Ana Raso2,Mario Feingold3, German Rivas2, Petra Schwille1.1Max Planck Institute of Biochemistry, Munich, Germany, 2Centro deInvestigaciones Biologicas, Consejo Superior de Investigaciones Cientıficas(CSIC), Madrid, Spain, 3Department of Physics, Ben Gurion University, BeerSheva, Israel.The primary protein of the bacterial Z ring guiding cell division, FtsZ, hasrecently been shown to engage in intriguing self-organization together withone of its natural membrane anchors, FtsA. When co-reconstituted on flat sup-ported membranes, these proteins assemble into dynamic chiral vorticeswhose diameters resemble the cell circumference. These dynamics are dueto treadmilling polar FtsZ filaments, supposedly destabilized by the co-polymerizing membrane adaptor FtsA, thus catalysing their turnover. Herewe show that FtsA is in fact dispensable and that the phenomenon is anintrinsic property of FtsZ alone when supplemented with a membrane anchor.The emergence of these chiral dynamic patterns occurs at intermediate FtsZsurface densities, in agreement with theoretical predictions, and beyond athreshold GTP concentration. The interplay of membrane tethering, GTPbinding, and hydrolysis promotes both, the assembly and the destabilizationof FtsZ polymers, leading to the observed treadmilling dynamics. Notably,the vortex chirality is defined by the position of the membrane targetingsequence (mts) and can be inverted when attaching it to the opposite end ofFtsZ. This reveals the vectorial character of the filament-supported membranesystem consisting of three orthogonal directions, filament polarity, curvature,and membrane attachment.

658-Pos Board B423Engineering of Chimeric Proteins to Enhance Immunogenicity for theProduction of High-Affinity Specific Monoclonal AntibodiesSienna Wong, M. Moazzem Hossain, Rong Liu, J.-P. Jin.Wayne State University, Detroit, MI, USA.A wide range of biomedical research and diagnostic applications utilize high-affinity antibodies, the production of which depends on effective immuno-gens. However, some proteins are intrinsically weak immunogens incapableof eliciting a strong immune response for the production of useful antibodies.To remedy this problem, weak immunogens are conjugated to exogenous car-rier proteins to increase immunogenicity and provide an adjuvant effect. In thepresent study, we tested an alternative strategy of using chimeric fusion pro-tein constructs to enhance immunogenicity. We first applied the N-terminaldomain of glucose-regulated protein 96 (GRP-NTD), a protein with newlyidentified activities in autoimmune diseases, as a carrier to present severalexperimentally identified weak immunogens: cardiac troponin C (cTnC), theN-terminal peptide of cardiac troponin T (N69), and the C-terminal peptideof calponin 2 (C85). Antigen peptides were engineered to the N-terminus ofthe carrier, expressed in E. coli, large scale purified, and injected intramuscu-larly into adult mice without adjuvants. The effects of immunization were as-sessed by ELISA titration of serum samples after three immunizations and/orthe outcome of high-affinity specific monoclonal antibodies (mAb) in hybrid-oma development. The results demonstrate significantly enhanced immunoge-nicity for cTnC and N69, but not C85. Based on an observation that clonedmouse ApoE protein served as an effective carrier for the production ofmAb against a small peptide, we constructed a C85-ApoE fusion proteinfor hybridoma development. Surprisingly, C85-ApoE immunization was noteffective for C85 but yielded high-affinity mAbs against mouse ApoE, anendogenous protein abundant in host plasma. The results suggest a potentiallynew strategy of using chimeric immunogens to improve the production ofspecific antibodies.

659-Pos Board B424A Multiscalar Framework describes Fluorescence and FRET of Fluctu-ating Molecular Species and Resolves Kinetic NetworksThomas-Otavio Peulen1, Oleg Opanasyuk1, Suren Felekyan1,Stanislav Kalinin1, Hugo Sanabria2, Claus A.M. Seidel1.1Physical Chemistry II, Heinrich Heine University, D€usseldorf, Germany,

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2Physics and Astronomy, CU School of Health Research, ClemsonUniversity, D€usseldorf, Germany.A combination of multi parameter fluorescence detection (MFD) withstructure based fluorescence models is presented, to capture and describefluorescence and FRET between a donor (D) and acceptor (A) dye offluctuating macromolecules over more than five orders of magnitude frompicoseconds to seconds with Angstrom resolution. The presented topdown approach combines molecular models with established fluorescencetechniques such as time correlated single photon counting, burst integratedfluorescence lifetime analysis, filtered fluorescence correlation spectroscopy(fFCS), and photon distribution analysis in a joined framework and thusfacilitates the analysis and interpretation of fluorescence experiments.Fluorescence and FRET on the picoseconds to nanosecond regime isdescribed by combining atomistic models with a coarse grained representa-tion of the dyes. Their conformational space is quantified by coarse grainedaccessible volume simulations while Brownian dynamics simulationscapture transient effects of FRET and fluorescence quenching. Assumingdynamic quenching and FRET are decoupled, the first two modes of theDA-distance distribution are determined for single molecules to conve-niently reveal macromolecular kinetics on the milli- and sub-millisecondkinetics by MFD histograms. Structural models are projected to yieldparametric equations of single-molecule observables. This serves as avisual guide to analyse MFD- histograms. Numeric integration of the chem-ical master equation analyses MFD-histograms and quantifies in com-bination with fFCS kinetic networks of dynamically exchangingmacromolecular conformations in the sub-microsecond to millisecondregime. A joint analysis of multiple fluorescence decays by structure basedpatterns resolved chemical equilibria in live cell. In future, such holistic ap-proaches may exploit the information contained in the fluorescence signaland connect dynamic molecular structural models with kinetic and equilib-rium networks of biomolecules to picture molecular machines in livingcells.

660-Pos Board B425Sub-Microsecond-Scale Dynamics in the Type-1 Ryanodine ReceptorObserved with CMOS-Integrated ElectrophysiologyAndreas J.W. Hartel1, Peiji Ong1, Indra Schroeder2, Oliver Clarke3,Siddharth Shekar1, Hunter M. Giese3, Andrew R. Marks4,Wayne A. Hendrickson4, Ken L. Shepard1.1Electrical Engineering, Columbia University, New York, NY, USA,2Membrane Biophysics Group, Dept. of Biology, Technische Universit€atDarmstadt, Darmstadt, Germany, 3Biochemistry and Molecular Biophysics,Columbia University, New York, NY, USA, 4Physiology and CellularBiophysics, Columbia University, New York, NY, USA.Conventional electronics typically limit the available temporal resolution forsingle ion-channel measurements, making fast channel gating events(<10ms) undetectable. Our group recently developed a platform that allowshigh-bandwidth conductance measurements by directly interfacing a sus-pended membrane containing the ion-channel under study on a customcomplementary-oxide-semiconductor (CMOS) preamplifier chip. A passiv-ation layer with a 20 mm aperture is deposited on the CMOS-chip surfaceand a small suspended lipid bilayer is formed within the aperture. Thisallows us to decrease the total capacitances at the input of the amplifierto less than 3pF, enabling us to measure high conducting ion-channels atbandwidths up to 1MHz. Here we present single ion-channel conductancemeasurements of the skeletal muscle ryanodine receptor (RyR1) at up to500kHz bandwidth. We observe microsecond current states which we asso-ciate with very fast channel gating events. Further analysis of the recordeddata by extended beta-distribution-based analysis reveals closed-stateflickering with time scales as short as 50ns. New high-speed CMOS ampli-fiers provide a new tool for high-bandwidth studies of ion-channel dynamicsin vitro.

661-Pos Board B426Droplet Interface Bilayers as a Platform for a Spatially SegregatedNanoreactorMaxwell P. Allen-Benton.Chemistry, King’s College London, London, United Kingdom.Compartmentalisation is a defining feature of biological processes. Withineukaryotic cells, organelles serve as segregated microenvironments forspecific biochemical processes. These microenvironments allow distinctchemical environments to be maintained within the cell and concentrateenzymes involved in catalytic cycles in the same space. This concentrationof enzymes and substrates within a small segregated environment contrib-utes to the great efficiency of biochemical processes. I aim to investigate

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the potential of a novel artificial lipid membrane system, the droplet in-terface bilayer (DIB), as a platform for a spatially segregated compart-mentalised nanoreactor. DIB technology serves as a simple method forproducing small aqueous compartments bounded by lipid membranes. Theincorporation of membrane transport proteins between the aqueous com-partments will allow distinct chemical environments to be maintainedand for the controlled separation of substrates and products. The nanoreac-tor design will aim to recreate the segregated microenvironments andenhanced catalytic efficiencies found in biological processes in a syntheticsetting.

662-Pos Board B427Cell Line Phenotypic Enrichement based on Migration and MorphologyJoannie Roy1, Loıc Binan1, Javier Mazzaferri1, Camille Lehuede2,Sebastien Tabaries2, Giuseppina Ursini-Siegel3, Peter Siegel2,Claudia Kleinman3, Santiago Costantino1.1Maisonneuve-Rosement Hospital, Montreal University, Montreal, QC,Canada, 2Goodman Cancer Research Centre, McGill University, Montreal,QC, Canada, 3Lady Davis Institute for Medical Research, McGill University,Montreal, QC, Canada.Cell Labeling via Photobleaching (CLaP) is an optical techniquerecently developed to cross-link fluorescent molecules to the surface ofindividual cells, by focusing a low-power laser on their membrane.Using CLaP, it is possible select single cells or a sub-population based onvisual criteria without the need of molecular markers. This technologycan also be exploited for inducing transient cell adhesion to culture sub-strates, which are resistant to dissociation agents such as EDTA or trypsin.Since only the illuminated cells remain attached to the dish surface, CLaPcan be exploited to tailor the spatial distribution of cells in a culture dish,and also to enrich the proportion of a chosen phenotype within proliferatingcells.Breast cancer cell line MDA-MB-231 is known for its potential to me-tastasize to secondary sites. Among the various factors influencingtheir metastatic ability, epithelial to mesenchymal transformation and itsreverse, mesenchymal to epithelial transformation, are required forcells to leave the tumor primary site, migrate and establish metastases atsecondary sites. Here we assess the heterogeneity in motility andmorphology of these cancer cells via time-lapse microscopy, automatedsegmentation and tracking. We use CLaP as a way to select clones ofproliferating cells based on morphological and behavioral characteristics,to enrich cell cultures of specific phenotypes. We validate which ofthese traits are preserved after cell division, and which ones are affectedby exposure to different conditioned media promoting mesenchymal toepithelial transformation.

663-Pos Board B428Zlock, a Broadly Applicable Optogenetic Method, Controls Cofilin inLiving CellsOrrin J. Stone1, Neha Kaul1, Hui Wang1, Ved P. Sharma2, Robert J. Eddy2,John S. Condeelis2, Klaus M. Hahn1.1UNC Chapel Hill, Carrboro, NC, USA, 2Anatomy and Structural Biology,Albert Einstein College of Medicine, New York City, NY, USA.The spatial and temporal organization of protein activity controls the flowof information through signaling networks. Optogenetics, the regulation ofprotein activity with light, has proven very useful to dissect the role ofspatio-temporal control by inducing specific protein activities with secondsand microns resolution in living cells. However, current methods are notapplicable to many important proteins. Here we describe Zlock, a broadlyapplicable optogenetic method to control protein or small-peptide activityin living cells using modifications of Zdark (Zdk), a small protein that bindsonly to the dark state of the LOV2 domain from Phototropin. The LOV2domain undergoes a reversible conformational change induced by 400-500 nm light. Zdk, derived by screening a library of Z domain variants ofprotein A, binds selectively to the dark state of LOV2 with dark Kd <100nanomolar, and lit Kd > 10 micromolar. In the ‘‘Zlock’’ system, Zdk andLOV2 are fused to the N- and C- termini of a target protein or small-peptide. In the dark, they bind to one another and cover or distort the activesite. Upon irradiation they release one another in less than a second, freeingthe active site for target interaction. We have engineered Zdk forthe reversible intramolecular binding needed in the Zlock design. Wehave successfully applied Zlock to generate photoactivatable analogs ofimportant cytoskeletal regulators including cofilin and Alpha Tubulin Ace-tyltransferase 1 (ATAT). Photoactivation of cofilin and ATAT in live cellscontrols cell protrusion and microtubule acetylation, and has demonstrateda clear role for cofilin in regulating directionality during cancer cell

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migration. Current work is focused on ‘caging’ photoactivatable small-peptide inhibitors of Cdc42, PP1, JNK and Gqa, to control endogenous pro-teins with light.

Neuroscience: General, Computational, andExperimental Approaches and Tools I

664-Pos Board B429Noise Induced Hearing Enhancement: Clinical and Machine LearningStudiesKang-Hun Ahn, Woo Seok Lee.Department of Physics, Chungnam National University, Daejeon, Korea,Republic of.The addition of a certain amount of background noise has been shown toimprove the hearing of pure-tone sounds. The underlying mechanism ofthis counter-intuitive phenomenon is believed to be stochastic resonance,where the threshold level of a pure-tone signal is lowered by adding anappropriate amount of background noise. Here, to investigate whether back-ground noise can aid the hearing of more complex sound such as the humanvoice, we perform hearing tests with Korean syllables, and find that syllablerecognition is enhanced with noise for rarely used syllables. Similar testswith a machine-learning speech recognition shows that the same enhance-ment arises only when the system is insufficiently trained, correspondingto hearing a rare syllable. The overall phenomenon looks similar to thestochastic resonance but any successful model should explain that theenhancement arises in perceptual processes as it depends on the level oftraining and the syllables.

665-Pos Board B430Stress-Induced Differential Regulation Leads to Decoupling of the Activitybetween mPFC and AmygdalaMohammed Mostafizur Rahman1,2, Sumantra Chattarji1,2.1Centre for Brain Development and Repair, Institute for Stem Cell Biologyand Regenerative Medicine, Bangalore, India, 2National Centre forBiological Sciences, Bangalore, India.Exposure to severe stress leads to the development of psychiatric disorders.Clinical studies have shown that three brain areas involved in learning andmemory—the hippocampus, amygdala and medial prefrontal cortex(mPFC)—undergo distinct changes with stress disorders. While the hippo-campus and mPFC show impairment in structural and functional changes,the amygdala shows an enhancement. Despite these three brain regionshaving strong anatomical connections, most of these studies focus on indi-vidual brain regions. However, recent studies have shown that these connec-tions between regions have strong functional implications. The connectivitybetween the mPFC and the amygdala has recently been shown to be crucialfor fear expression (Likhtik et al., 2014). The effect of stress on the func-tional connections between these regions is poorly understood. Therefore,we performed in-vivo local field potential recordings from the mPFC andthe amygdala in awake behaving rats during fear expression. We foundthat stress differentially regulates the activity in the mPFC and the amygdaladuring fear expression. Consistent with cellular findings, the activity in theamygdala is upregulated by stress during fear expression. However, the ac-tivity of the mPFC is unaffected by stress during fear expression. We alsofound that stress causes a decoupling between the activity in the amygdalaand mPFC. Interestingly, an earlier study showed that stress strengthens thecoupling between the hippocampus and the amygdala (Ghosh et al., 2013).Therefore, although chronic stress impairs structure and function in both thehippocampus and mPFC, the interactions of these two areas and the amyg-dala appear to be affected in a contrasting fashion. Functional connectivitygets stronger from amygdala to hippocampus but it gets disrupted betweenmPFC and amygdala. Future studies need to focus on mechanisms involvedin these connectivity changes.

666-Pos Board B431Temperature Sensation and Integration in the Drosophila Circadian ClockChang Jiang, Swathi Yadlapalli, Andrew Bahle, Pramod Reddy,Edgar Meyhofer, Orie Shafer.University of Michigan, Ann Arbor, MI, USA.Circadian clocks are entrained by zeitgebers, environmental cues such aslight and temperature that adapt living organisms to the physical rhythmsof the earth. Although temperature has been shown to be a major zeitgeberand can entrain the circadian clock of Drosophila, the neural and molecularmechanisms by which circadian clocks respond to temperature remainpoorly understood. In our work, we use in vivo calcium imaging to charac-

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terize the temperature response of clock neurons in Drosophila to tem-perature modulation. We show that a selective group of clock neuronsresponds to temperature changes and that dorsal neurons (DNs) areexcited by cooling and inhibited by warming. We further investigatedthe physiological input pathway of temperature sensing into the circadianclock. We find that arista and chordotonal organs are both criticalfactors that contribute to the response of circadian neurons to tempe-rature modulation. Our work reveals that clock neurons respond totemperature changes through multiple temperature input pathways, suggest-ing a complex network similar to the entrainment of circadian clocks bylight input.

667-Pos Board B432TRP Channel Function in iPSC-Derived Sensory NeuronsLaura Vangeel.KULeuven, Leuven, Belgium.Using somatic cells to generate induced pluripotent stem cells (iPSC) is anestablished method in research and has multiple applications and advan-tages. An increasing amount of cell types have been successfully differenti-ated from iPSCs, including hematopoietic cells, cardiomyocytes, smoothmuscle cells, pancreas, liver and renal tissue. Directing differentiation intoneuronal cells has the great benefit of bypassing the problematic isolationof human neuronal cells. Recently, a protocol using dual SMAD inhibitionwas shown to drive differentiation into sensory neuron-like cells. Whileexpression of canonical markers of sensory neurons has already been vali-dated, an in-depth characterization of sensory TRP channels in theseinduced neurons is still lacking. In this study, we use qPCR, Fura-2-basedmicrofluorimetry and patch-clamp experiments to evaluate the expressionand function of the sensory TRP channels at different time points duringthe differentiation toward a sensory neuron phenotype. We not only confirmexpression of the sensory channels TRPV1, TRPM8 and TRPA1, but alsodemonstrate for the first time strong molecular and functional expressionof TRPM3 in iPSC-derived sensory neurons. Interestingly, we found atemporarily increase of TRPM3 responses at an early time point in differen-tiation, which might indicate a role for this channel in the development ofsensory neurons. To conclude, we were able to produce sensory neurons us-ing iPSCs, and validated functional expression of TRP channels important insomatosensation. This approach has the potential to investigate the develop-ment of sensory neurons in vitro, and to explore the cellular physiology andpharmacology of TRPM3 and other sensory TRP channels in a humancontext. Moreover, it may open the door to generate patient-derived neuronsfor disease modeling and target validation.

668-Pos Board B433Effect of Spatial Complexity on Dopaminergic Signaling Revealed fromMultiscale SimulationsCihan Kaya1, Mary H. Cheng1, Ethan R. Block2, Alexander Sorkin2,James R. Faeder1, Ivet Bahar1.1Computational and Systems Biology, University of Pittsburgh, Pittsburgh,PA, USA, 2Cell Biology, University of Pittsburgh, Pittsburgh, PA, USA.Efficient clearance of neurotransmitters from the synapse by dopaminetransporters (DATs) is critical to regulating dopamine (DA) signalingin the central nervous system. Despite significant advances in the field,we still lack a complete mechanistic understanding of DA transport events.First it is still unclear how molecular structure and dynamics affectcellular neurosignaling events. Second, the structural and stochastic proper-ties of the cellular environment, including the morphology of the synapticregions, and the heterogeneous distribution of transporters on the cellmembrane may affect the efficiency of neurotransmitter transport, and norealistic simulations of the dopaminergic signaling has been carried out todate. We adopted a multiscale methodology to examine the effects ofspatial complexity and firing patterns on DA reuptake by DATs. We useda kinetic scheme derived from our earlier molecular, together with highresolution images dopaminergic neurons from fluorescence spectroscopyand electron microscopy, to reconstruct in silico the simulation environmentmediating DA signaling. Overall, our model provides a framework toinvestigate the effect of variations in different neuronal properties togain a better understanding of the modulation of DA signaling in the centralnervous system. Our results highlight the significance of consideringthe realistic geometry as well as the spatial heterogeneities from ex-periments as opposed to adopting well-mixed assumptions. Thecomputing platform also permits us for the first time to gain a quantitativeunderstanding of the effect of psychostimulants and antidepressants on DAsignaling.

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669-Pos Board B434Sensitivity and Robustness in an Axon Guidance Signaling SystemBrendan A. Bicknell1, Peter Dayan2, Geoffrey J. Goodhill1.1Queensland Brain Institute, University of Queensland, Brisbane, Australia,2Gatsby Computational Neuroscience Unit, University College London,London, United Kingdom.Correct functioning of the brain requires the orderly wiring of billions of cellsduring development. A major mechanism that mediates this is the guidance ofaxons to their targets by extracellular chemical gradients. Although well stud-ied, the computational rules by which even simple guidance decisions aremade are not yet understood at a biophysical level. The task of a growingaxon to estimate a direction signal is immense, as unavoidable biologicalnoise corrupts the measurement at all levels of processing. Here, we showhow the modulation of growth by a balance of positive and negative feedbackcan explain the remarkable chemotactic sensitivity observed in vitro. Weperform a detailed analysis of the experimental data of ref. [1] which charac-terizes the neurite growth of ~2500 rat dorsal root ganglia explants in veryshallow gradients of nerve growth factor. Constrained by these data, weconstruct a model chemical signaling system for growth and guidance inthe developing brain. In the model, amplification of the gradient signal occursvia paracrine signalling between cell bodies within the ganglion, while robust-ness is conferred by the dynamics of receptor trafficking. The model gives aunified and quantitative account of experimentally observed behavior, andyields testable predictions with implications for understanding brain develop-ment and repair after injury.[1] Mortimer, D. et al (2009) A Bayesian model predicts the response of axonsto molecular gradients. Proc. Natl. Acad. Sci. U.S.A, 106(25), 10296-10301.

670-Pos Board B435Mathematical Modeling and Analyses of Interspike-Intervals of Sponta-neous Activity in Afferent Neurons of the Zebrafish Lateral-LineSangmin Song1, Ji Ah Lee2, Ilya Kiselev1, Varun Iyengar1, Josef G. Trapani1,Nessy Tania2.1Biology, Amherst College, Amherst, MA, USA, 2Mathematics andStatistics, Smith College, Northampton, MA, USA.Temporal patterns of spontaneous activity may vary between sensory systemssuch as the auditory, vestibular, and lateral line systems due to differences inphysiology at the level of hair cells. In the absence of stimuli, hair cellsdisplay spontaneous synaptic vesicle fusion and neurotransmitter release,which lead to action potential (spike) generation in innervating afferent neu-rons. As a result, features of synaptic transmission and the innervation of haircells will affect the timing of spontaneous spike trains. We analyzed sponta-neous spiking recorded from the lateral line of zebrafish, and found that thedistribution of interspike-intervals (ISI) had an ‘‘L-shape’’ that decayed fasterand had a wider tail than a typical exponential distribution commonlyobserved in other models of spike timing. Additionally, successive ISI’s inthe lateral line recordings tended to have positive serial correlation, i.e., suc-cessive ISI pairs were either short/short or long/long. This pattern contrastsprevious findings from the auditory system where ISI’s tended to have nega-tive serial correlation presumably due to the effects of synaptic depletion.Computational models of spike trains that included the calcium-dependencyof neurotransmitter release at the ribbon synapse of hair cells were able togenerate ISI distributions consistent with those we acquired experimentally.These simulations suggested that fluctuations in total calcium channel activity,including both the number and cooperativity of channels in the population, area primary contributor to serial correlations in hair-cell evoked spike trains.Given the difference in innervation pattern between auditory and vestibular/lateral line hair cells, we further modeled the effects of single versus multiplesynapses on the temporal patterns of spontaneous spike trains. Altogether, ourfindings provide evidence for how physiological similarities and differencesbetween the auditory, vestibular, and lateral line systems can account fordifferences in spontaneous activity. Furthermore, our computational methodsallow for future characterization of mechanisms that underlie spontaneous ac-tivity in these different sensory systems.

671-Pos Board B436A Model for Assessing ATP Demands of Sustained High Frequency FiringBela Joos1, Michael R. Markham2, John E. Lewis3, Catherine E. Morris4.1Physics, University of Ottawa, Ottawa, ON, Canada, 2Biology, University ofOklahoma, Norman, OK, USA, 3Biology, University of Ottawa, Ottawa, ON,Canada, 4OHRI, Ottawa, ON, Canada.The continuous electric organ discharge (EOD) of the weakly electric fish,Eigenmannia, reflects action potentials (APs) fired by the EO’s muscle-derived electrocytes. EODs enable electrosensing and communication. AP fre-quency is neurally controlled, with acetylcholine-gated channels (AChRs)

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mediating synaptic transmission. According to EOD-linked whole-fish O2

consumption (for fish with EODs between ~300-500 Hz) ATP demand perAP grows exponentially with frequency. The unimodal task, continual firing,and simple homogeneous structure of the EO render it especially suitable forprobing excitable system energetics in relation to molecular, cellular andtissue features. We develop a model, Epm, to depict currents at the electro-cyte’s energy-dissipating Excitable posterior membrane. In situ, 3Na/2K-ATPases (pumps) counteract the dissipation of electrocyte ENa (and EK).Using Epm we calculate AP frequency-dependent ‘‘Naþ-entry budgets’’ forsynaptic activation (pulsatile and/or steady-state, with/without noise). Com-parison of Epm-calculated ATP consumption (inferred from total Naþ-entry)against published EOD-linked whole-fish O2 consumption suggests thatEOD-linked energy demands external to electrocytes (neuronal, circulatoryetc) exceed, several-fold, those of electrocyte excitability per se.Well-understood conductance processes (as modeled by Epm) proved fullyadequate for generating sustained APs (including during jamming avoidanceresponses) from 200-600 Hz. By contrast, although we computationallyimpose the equivalent of fast stimulatory variations in [ACh], even at thebottom of this frequency range the means by which synaptic transmissionmachinery so reliably achieves the requisite fast variations is a mystery.The simple Eigenmannia EO therefore continues to emerge as a fascinatingmodel system for studying not only the energetics but the subcellular andbroader-level dynamics of high frequency excitability.

672-Pos Board B437Analyzing and Modeling the Dysfunction of Inhibitory Neurons inAlzheimer’s DiseaseCarlos M. Perez1, Ghanim Ullah1, Jokubas Ziburkus2.1Physics, University of South Florida, Tampa, FL, USA, 2Biologyand Biochemistry, University of Houston, Houston, TX, USA.Alzheimer’s disease (AD) is characterized by the abnormal proteolytic pro-cessing of amyloid precursor protein, resulting in increased production of aself-aggregating form of beta amyloid (Ab). Several lines of work on AD pa-tients and transgenic mice with high Ab levels exhibit altered rhythmicity,aberrant neuronal network activity, reflected in clusters of hyperactive neu-rons, network hyperexcitability, and spontaneous epileptic activity. Recentstudies highlight that abnormal accumulation of Ab changes intrinsic proper-ties of inhibitory neurons, including their inability to reliably produce actionpotentials, one of the main reasons underlying the impaired network activity.Specific cellular mechanisms leading to the interneuron dysfunction are notcompletely understood. Using extended Hodgkin-Huxley (HH) formalismmodel in conjunction with patch-clamp experiments, we investigated themechanisms leading to the impaired activity of interneurons. Our detailedanalysis indicates that increased conductance of Naþ leak channels recreatesseveral observed inhibitory neuron behaviors in models of AD, includingfailure to reliably produce action potentials, smaller action potential ampli-tude, increased resting membrane potential, and higher membrane depolariza-tion in response to a range of stimuli in a model of APP-swe/PSEN1DeltaE9(APdE9) AD mice. Increasing the conductance of hyper-polarizationactivatedcyclic nucleotide-gated (HCN) ion channels (Ih) could account for most of thereported dysfunctions. However, the extent of changes required to reproducethese observations render such changes unphysiological. We investigate anddiscuss other pathways that would lead to some but not all observations withinHH formalism.

673-Pos Board B438Denied Access of Ions and Molecules to Axons in the Development ofAlzheimer’s DiseaseWade Dauberman, Samuel Breit, Shaohua Xu.Biology, Florida Institute of Technology, Melbourne, FL, USA.Self-assembly of amyloid-beta peptide and the formation of fibers andplaques in extracellular space of the brain is a characteristic of Alzheimer’sdisease. Whether and how fibers and plaques cause neurodegenerationremains unclear. Recently, we reported that amyloid fibers can furtheraggregate and form gels. Gels are known to eliminate bulk flow which isrequired for the circulation of ions and molecules essential for neuronalfunction. To understand how restriction of ions or molecules affectsneurons, we examined the gels’ role in the propagation of nerve fibers’action potential. Unlike amyloid fibers, fibrin, which forms gels fast, withina couple of minutes, was used to simulate amyloid fiber gels. We found a40% reduction of the compound action potential when fibrin gel wasformed outside frog sciatic nerve fiber epineuriums, a porous membranewrapping around axon bundles. A 70% reduction of the action potentialwas recorded when fibrin was formed inside the epineurium. Fibrin encap-sulating individual axons was verified under a confocal microscope when

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fluorescein-labeled fibrinogen was injected. Gelation also changes viscosity,which affects the diffusion of ions and molecules. An approximate 1.42-foldincrease of microviscosity during fibrin gelation was revealed using amolecular rotor. Glycerol caused a concentration dependent reduction ofthe compound action potential. These results imply that the formation ofamyloid plaques surrounding neuronal processes initially disrupt the propa-gation of action potential and then trigger the cascade of events leading toneuronal death seen in Alzheimer’s disease. Denied access of the neuritesto nutrients, signaling molecules, and/or waste drainage pathways mightbe responsible for the plaque’s effect on the action potential and underliethe pathogenesis of Alzheimer’s disease. The methods introduced in the pa-per for the study of gelation’s effect on action potential may have broadapplications.

674-Pos Board B439Biodistribution of Insulin-Nanogels in Mouse: A Preliminary Study for theTreatment of Alzheimer’s DiseaseDaniela Giacomazza1, Pasquale Picone2, Laura Ditta3,M. Antonietta Sabatino3, Valeria Militello4, P. Luigi San Biagio1,Laura Cristaldi2, Domenico Nuzzo2, Antonella Amato5, Flavia Mule5,Clelia Dispenza3, Marta Di Carlo2.1IBF, CNR, Palermo, Italy, 2IBIM, CNR, Palermo, Italy, 3DICGM,University of Palermo, Palermo, Italy, 4DCF, University of Palermo,Palermo, Italy, 5STEBICEF, University of Palermo, Palermo, Italy.A growing body of evidence shows that Insulin, Insulin Receptor (IR) and IRsignaling are involved in brain cognitive functions and their dysfunction isimplicated in Alzheimer’s disease (AD) neurodegeneration. Thus, adminis-tration of insulin could be a strategy for AD treatment. For this aim wehave designed, synthesized and characterized a nanogel system (NG) thathas been conjugated to insulin molecules (NG-In) to deliver the proteininto the brain, as a tool for the development of a new therapy against AD.In our preclinical study in mice, intraperitoneal injection of fluorescent-labeled NG has allowed to determine the biodistribution of NG vs time inthe whole body and its clearance through the kidneys and bladder. Further-more, we have observed that mice injected with nanogel did not experiencestress, discomfort, nor mortalities have been recorded during the observationtime. Thus, we may conclude that, under our experimental conditions, nano-gels did not cause any toxic effects and they are eliminated in urine. Theadministration of NG-In through the intranasal route to study its brain distri-bution has been done by fluorescence analysis and Western blot. Data haveshown that insulin signaling is improved in the different brain areas whenthe protein is conjugated with nanogels with respect to the free insulin. Inaddition, the histopathological analysis of the nasal cavity shows no signifi-cant change in epithelial cell lining indicating that the nano-solution is welltolerated in mouse. These results indicate that the synthesized NG-In was asuitable carrier for insulin delivery in the brain having a higher efficiencythan free-insulin.

675-Pos Board B440Studies of the Interaction between Ab Peptides and Carbon Nano-MaterialsDongdong Lin, Ruxi Qi, Luogang Xie, Shujie Li, Guanghong Wei,Xinju Yang.State Key Laboratory of Surface Physics and Physics Department, FudanUniversity, Shanghai, China.The aggregation of amyloid-b peptides (Ab) is considered as one of themainly possible causes of the Alzheimer’s diseases (AD). How to suppressthe formation of toxic Ab aggregates has been intensively concerned overthe past several decades. Increasing evidence shows that whether carbonnano-materials could suppress or promote the aggregation depend on theirphysicochemical properties. However, as amyloid fibrillization is a complexprocess, their interaction dynamics remains elusive. In this presentation, wehave investigated the interaction between Ab(16-22) peptides/full-lengthAb peptides and three kinds of carbon nanomaterials (carbon nanotube,fullerene, and graphene) by utilizing atomic force microscopy (AFM), elec-trostatic force microscopy (EFM), Thioflavin T (ThT) fluorescence, com-bined with molecular dynamic (MD) simulations. Our experimental resultsdemonstrate that all of the three carbon nano-materials could suppress theprocess of Ab fibrillation. EFM and ThT fluorescence results furtherconfirmed that the Ab peptides are strongly absorbed on the surface of carbonnano-materials by forming non-fibrillar aggregates. At the molecular scale, itis found that carbon nano-materials dramatically inhibit the formation ofordered b-sheet-rich structures as the strong hydrophobic and aromatic stack-ing interactions between carbon nano-materials and Ab peptides. In compar-ison, the fullerenes exhibit the most excellent inhibitory ability by the strong

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aromatic stacking of the fullerene hexagonal rings with the Phe rings, as wellas the outstanding biocompatibility. Furthermore, we found that the barecarbon nanotube could dissociate the preformed fibrils to some extent.Our results provide novel clues for studying interaction in amyloid/carbon-material system and seeking amyloidosis inhibitors with carbon nanomateri-als, which may have important potentials in the development of drugcandidates against Alzheimer’s disease.

676-Pos Board B441Ultrasound-Enhanced Molecular Therapy for Axon NeurogenesisAsis Lopez, Damir Khismatullin.Tulane University, New Orleans, LA, USA.Between 28M - 55M individuals experience spinal cord injuries (SCIs). In theU.S. alone, this amounts to an estimated 180k-230k individuals. In the centralnervous system (CNS) after an injury to a nerve, the environment prevents thehealing/regrowth of neurons and myelin, because myelin inhibitors arepresent, growth factor is not re-expressed, and glial tissue scars very quickly.Knowing that nerves need to be regenerated quickly, research has been doneto discover an optimal treatment for nerve regeneration. Unfortunately, noeffective method currently exists to stimulate nerve fiber regeneration (i.e.,neurogenesis). To address this gap, we propose a non-invasive method tostimulate neurogenesis through low-intensity focused or unfocused ultra-sound irradiation. This method is new and has high potential as a transforma-tive rehabilitation approach for people with disabilities due to SCIs. Acombination of therapeutic methods that mitigate the inhibitory microenvi-ronment at the site of the injury and restore the regeneration potential ofcorticospinal axons are required for achieving effective functional recoveryafter SCI. We developed an in vitro system integrating focused ultrasound(FUS) with neuron axon dynamics for neurogenesis and an approach usinga 3D microenvironment. Dorsal root ganglion (DRG) neurons are used forour studies and microsurgically removed at day 15 and E18 Sprague Dawleycortical neurons from BrainBits, LLC. To determine if stimulation of axongrowth is occuring, we measure density and growth (distance) using phasecontrast imaging and a custom MATLAB code for image analysis. We testeda variety of operating parameters to identify optimal conditions that induceaxon neurogenesis by ultrasound. We demonstrated that ultrasound can stim-ulate DRGs.

Molecular Dynamics I

677-Pos Board B442Asymmetric Membrane Models for the PM And TGN of Yeast, AnAll-Atom Molecular Dynamics StudyViviana Monje-Galvan, Jeffery B. Klauda.Chemical and Biomolecular Engineering, University of Maryland, CollegePark, MD, USA.We previously examined symmetric membrane models for the plasma (PM)and trans-Golgi-network (TGN) membranes of Saccharomyces cerevisiae(yeast) (Biochem 54:6852-6861). Although diverse in lipid nature and re-flecting sterol compositions characteristic to those organelles, our modelswere not an accurate enough representation as they lacked sphingolipids(SM), relevant for membrane structure and dynamics. Once SM wereparametrized for the CHARMM 36 lipid force field (BJ, 107:134-145),we included them in the new models and added lipid composition asymme-try to better reflect the complexity of these bilayers. The new modelsinclude inositol phosphoceramide (IPC) and mannose-(inositol-P)2-ceramideM(IP)2C lipids, both present in the non-cytosolic leaflets of the PM andTGN. We present a comparison study between the previous PM modeland the more complex ones using common membrane properties such assurface area per lipid, bilayer thickness, deuterium order parameters, lipiddiffusion constants, and lipid cluster formation. The last three propertieswill serve to investigate lipid phase coexistence and leaflet coupling inmembrane order and diffusion. Our simulation trajectories were at least2ms long for each of the three models (old-PM, PM, and TGN), for a totalof 15 ms of simulation data run in the Anton machine at the PittsburghSupercomputing Center.

678-Pos Board B443PI(4,5)P2 Binds to Phospholipase C Delta 1 in a Cholesterol ConcentrationDependent Manner: Perspective on Implications to PI(4,5)P2-BindingProteinsSami Rissanen1, Lauri Salmela1, Ilpo Vattulainen1,2, Tomasz Rog1,2.1Tampere University of Technology, Tampere, Finland, 2University ofHelsinki, Helsinki, Finland.

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Phosphoinositide, a phosphorylated form of phosphatidylinositol (PI), plays animportant role in, e.g., cell signaling and membrane trafficking. Phosphatidyli-nositol 4,5-bisphosphate (PI(4,5)P2) found in, for instance, the plasma mem-brane and the Golgi apparatus is known to bind to a number of proteins.How the PI(4,5)P2-binding proteins recognize their substrate and how the bind-ing is regulated and fine-tuned remains unclear. In this work, we hypothesizethat the increasing cholesterol concentration along a path from the nucleus to-wards the plasma membrane may be one of the key regulators in this process.To consider this hypothesis, we employed both atomistic and coarse-grained(CG) molecular dynamics simulations to study the binding of PI(4,5)P2 withphospholipase C delta 1 (PLC) that is an important signaling enzyme knownto bind PI(4,5)P2. Unbiased CG MARTINI simulations in POPC/PI(4,5)P2and POPC/PI(4,5)P2/cholesterol mixtures were complemented by umbrellasampling simulations to measure the free energy of PI(4,5)P2 binding toPLC. The final structures of the PLC-PI(4,5)P2 complexes were fine-grainedto atomistic detail and simulated over microseconds to explore the details ofthe binding process.In essence, we observed deeper free energy minima for the binding ofPI(4,5)P2 to PLC in the cholesterol-rich membrane system, compared tocholesterol-free membranes, thus supporting the underlying hypothesisthat PI(4,5)P2 binding to PLC and possibly also other PI(4,5)P2-bindingproteins is strengthened by increasing cholesterol concentration. Theatom-scale features such as hydrogen bonding patterns and lipid-specific in-teractions that dictate the binding preference are discussed in detail in thepresented work.

679-Pos Board B444STRD MARTINI: Simulating Quasi-2D Hydrodynamics with ChemicallyDetailed Lipid ModelsAndrew Zgorski, Edward Lyman.Physics and Astronomy, University of Delaware, Newark, DE, USA.Hydrodynamic interactions are an important component of lipid membranedynamics. A proper molecular dynamics simulation of lateral diffusion in amembrane requires coupling to hydrodynamic flows in the surrounding sol-vent. In the low Reynolds number limit, the interactions mediated by theseflows are long-ranged. Camley, et al recently generalized the Saffman-Delbruck result to periodic boundary conditions, and showed that hydrody-namic interactions introduce significant error into lateral diffusion of lipidsand proteins in typical molecular dynamics simulations. The system sizesrequired to overcome the finite size effect are computationally prohibitivewhen using explicit solvent models in both all-atom and coarse-grainedsimulation. Implicit solvent models offer a potential solution, but entirelyneglect hydrodynamic momentum transport. To remedy this, we have sup-plemented the Dry MARTINI implicit solvent model with an efficient meso-scopic particle-based hydrodynamic model called Stochastic RotationDynamics (SRD). Our implementation allows for fine control over fluidproperties of the solvent, such as viscosity and Reynolds number, and in-cludes a thermostat for the solvent that produces canonical energy fluctua-tions without interfering with the mesoscopic hydrodynamic flows. Theapproach, dubbed ‘‘STRD Martini’’, has been implemented in an in-houseversion of Gromacs v 5.1.

680-Pos Board B445Molecular Views of a Eukaryotic Plasma Membrane ModelKarelia H. Delgado-Magnero, Gurpreet Singh, Valentina Corradi,D. Peter Tieleman.Biological Science, University of Calgary, Calgary, AB, Canada.Plasma membranes are composed of hundreds of lipid species and differenttypes of membrane proteins. Lipid-lipid and lipid-protein interactions play acrucial role in regulating cell membranes functions and are involved inmany diseases when altered. Despite advances in experimental methods,the interplay between lipids and proteins is not completely understood. Inthis context, computer simulations have become a powerful tool to investi-gate the dynamic behavior of plasma membrane components and their inter-actions at a near atomic resolution. The goal of our research is to worktowards large-scale modeling of plasma membranes using computer simula-tions. In this study, we placed ten different types of eukaryotic membraneproteins in a membrane model containing various lipid species found inthe plasma membrane of eukaryotic cells. In our simulation setup, mem-brane proteins are present in different ratios, for a total of ca. 150 proteinmolecules, embedded in more than 60,600 lipids. The protein-membranesystem was prepared using the Martini force field, with in house software.Using molecular dynamic simulations, we investigate the local lipid envi-ronment around proteins, and provide a molecular view of a realistic mem-brane model.

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681-Pos Board B446Beyond Lateral Pressure Profiles: Local Stress and the Traction Vector inMD SimulationsJuan M. Vanegas1, Alejandro Torres-Sanchez2, Marino Arroyo2.1Dept. of Physics, University of Vermont, Burlington, VT, USA, 2Dept. ofApplied Mathematics III, Universitat Politecnica de Catalunya-BarcelonaTech, Barcelona, Spain.Lateral pressure or stress profiles are routinely used to understand the me-chanical behavior of lipid membranes from molecular dynamics (MD) sim-ulations. However, the 1-dimensional stress profiles are not adequate tounderstand the multidimensional mechanical state of complex systemswhere there are membrane-protein and protein-protein interactions. Further-more, the fact that the microscopic stress from MD simulations is notuniquely defined is a theoretical consideration that is most often ignored,which has acute practical consequences when atomistic models are consid-ered. I will present our recent work (Phys. Rev. Lett. 114, 258102, 2015 andJ. Chem. Theory Comput. 10, 691, 2014) on the development of objective3D local stress calculations and its applications to lipid bilayers, mechano-sensitive channels, and fibrous proteins. I will show how popular definitionsof the microscopic stress violate mechanical equilibrium and present anunambiguous and physically sound definition based on the central decompo-sition of forces from multi-body potentials. I will also demonstrate the useof the traction vector as a powerful tool to visualize the local stress tensor oninterfacial surfaces (such as the surface of a protein embedded in amembrane).

682-Pos Board B447Molecular Dynamics Study on Lipid Wrapped Carbon Nanotube as anArtificial Membrane ChannelMoon-Ki Choi, Hyunki Kim, Youngjin Kim, Kyunghoon Kim, Moonki Kim.Mechanical engineering, SungKyunKwan University, Suwon, Korea,Republic of.Carbon nanotube (CNT) is an ideal membrane channel because of its hydropho-bic inner pore and prominent transport properties like typical biologicalchannels. These CNT porins can transport water, small ions, and DNA.CNT-membrane interaction simulation shows that CNTs having a lengthsimilar to the thickness of lipid bilayer can be inserted into membrane andsustain angle almost perpendicular to membrane. Much longer ones can alsobe inserted into membrane but they are buried inside hydrophobic region ofmembrane (1). However, recent experimental research elucidated that longerCNTs wrapped by lipids can be inserted into lipid-bilayer and maintain theirangle within 15 degrees (2).In this study, we performed molecular dynamics simulation for investigatinginteraction between lipids wrapped CNT and lipid-bilayer. Martini coarse-grained force field was used to reduce computational cost significantly.Also, wrapping process was analyzed by using the coarse-grained carbonnanotube (CG-CNT) and the 1,2-dioleoyl-sn-glycero-3-phosphocholine(DOPC) in various CNT length and surface adsorption density, respectively.Furthermore, we performed insertion simulation to see how these propertiesaffect insertion process and finally compared CG-CNT’s insertion angleswith experimental data. As a result, CNT insertion takes longer time in gen-eral as the density of wrapped lipid increases. Also, we realized that CNT canmaintain its insertion angle almost perpendicular to lipid-bilayer at a certaincritical density.Finally, its insertion mechanism is fully discussed with respect to interactionenergy between the wrapped CG-CNT and lipid membrane.(1) Lelimousin, M., and M. S. P. Sansom. 2013. Small 9:3639-3646.(2) Geng, J., K. Kim, J. F. et al. 2014. Nature 514:612-þ.

683-Pos Board B448Molecular Dynamics Studies Support Elevator Type Transport Mecha-nisms in the Glucose EIIC Superfamily TransportersJumin Lee1, Zhenning Ren2, Ming Zhou2, Wonpil Im1.1Lehigh University, Bethlehem, PA, USA, 2Baylor College of Medicine,Houston, TX, USA.The phosphoenolpyruvate-dependent phosphotransferase system (PTS) pro-vides a distinct sugar transportation pathway in bacteria. Because PTS doesnot exist in eukaryotes, it is considered a novel target for antibiotics develop-ment. Among the components of PTS, the enzyme IIC (EIIC), a membranetransporter protein, is particularly important since it transports a sugar throughthe bacterial cell membrane. In 2011, Cao et al. first determined the crystalstructure of chitobiose-specific EIIC (bcChbC) in an inward-facing occludedstate. In addition, we have recently determined the crystal structure ofmaltose-specific EIIC (bcMalT) in an outward-facing occluded state. Thecrystal structures suggest an ‘‘Elevator’’ type transportation mechanism, but

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the accurate mechanism of how EIIC transports a sugar has not been clearlydemonstrated yet. To investigate more detailed and accurate transportationmechanisms, we performed collective variable-based steered moleculardynamics (CVSMD) simulations. Our simulation shows the spontaneoustransportation of the sugar toward the opposite side of the membrane withthe preserved H-bonding interactions, supporting the ‘‘Elevator’’ type trans-port mechanism. A set of cross-linking experiments was carried out on thebasis of the CVSMD model structures, and we were able to acquire a fewof stable cross-linked structures.

684-Pos Board B449Molecular Basis of GLUT4 in Glucose Transport: Atomistic MolecularDynamics StudyChetan S. Poojari1, Job Roodhuizen2, Fabio Lolicato3, Tomasz Rog1,3,Ilpo Vattulainen1,3.1Department of Physics, Tampere University of Technology, Tampere,Finland, 2Biomedical Engineering department, Eindhoven University ofTechnology, Eindhoven, Netherlands, 3Department of Physics, University ofHelsinki, Helsinki, Finland.The glucose transporter 4 (GLUT4) is one of the most important glucosetransporter proteins for the absorption of glucose from the plasma circula-tion after a meal or during exercise. It is present in skeletal muscle, adiposetissue cells, cardiac muscle, and it has also been found in brain cells. It playsa significant role in the development of various diseases such as type 2diabetes, cancer, and cardiac diseases. Inside a cell, GLUT4 is transportedtowards the cell membrane upon an insulin stimulus, leading to a 10- to40-fold increase in the glucose uptake. In spite of its importance, the molec-ular mechanism of glucose transport by GLUT4 is still not clear. There is nocrystal structure available for the GLUT4 protein either. However, usingexisting structural information of the other solved glucose transporterstructures, we have modeled and validated the GLUT4 structure inthree conformations: the outward-open, outward-occluded, and inward-open conformation. The GLUT4 models with glucose bound were subse-quently embedded in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine(POPC) membrane and simulated over microsecond time scales usingunbiased atomistic molecular dynamics simulations. In addition, we alsocarried out random acceleration molecular dynamics simulations to exploreall possible pathways for glucose transport across the membrane. Oursimulation studies revealed specific GLUT4 residues that captured theconformational changes in GLUT4 structure on glucose binding. Overall,the study provides atomistic level structural information on glucosetransport and also provides one with ideas for the development of therapeu-tic agents blocking the function of GLUT4. This development work isimportant given that cancer cells express elevated levels of glucosetransporter proteins and depend on increased glucose uptake forproliferation.

685-Pos Board B450Ions in Action - Studying Ion Channels by Computational Electrophysi-ology in GROMACSCarsten Kutzner1, R. Thomas Ullmann1, Bert L. de Groot2,Ulrich Zachariae3, Helmut Grubmueller1.1Theoretical and Computational Biophysics, Max Planck Institute forBiophysical Chemistry, Goettingen, Germany, 2Computational BiomolecularDynamics, Max Planck Institute for Biophysical Chemistry, Goettingen,Germany, 3Physics, University of Dundee, School of Science andEngineering, Dundee, United Kingdom.Ion channels play a fundamental role in maintaining vital electrochemicalgradients across the cell membrane and in enabling electrical signaling incells. Understanding their functional mechanism is crucial for facilitatingdrug design on this important class of membrane proteins. Key characteris-tics of ion channel function that are commonly quantified experimentallyare ionic permeation rates and selectivities. The Computational Electro-physiology (CompEL) protocol allows the investigation of ion channels inGROMACS all-atom molecular dynamics simulations. By employing anion/water exchange protocol in a double-membrane simulation setup, asteady state with a continuous flow of ions through the channels isachieved. The recorded ion permeation events directly reveal conductance,selectivity, and rectification behavior, which all allow a straightforwardcomparison to experimental quantities. In addition, the ionic pathwaysthrough the channels are readily revealed. CompEL is available inGROMACS and has a negligible impact on the simulation performanceeven in parallel.Recent application to the bacterial channel PorB resulted in conductance andselectivity values and revealed the pathways of traversing ions. For a

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mutant, pathway disruption was observed, thereby explaining its resistanceto certain antibiotics. The conductance calculated from the simulationsagreed very well with measured conductances. Other successful examplesof CompEL include the discovery of an unexpected ionic pathway in theanti-microbial peptide dermcidin as well as the identification of a novel con-duction mechanism in the potassium channel KcsA. We finally investigatedthe conductance and the gating mechanism of pentameric ligand-gated ionchannels such as GLIC. We constructed a reaction coordinate from twocrystal structures, which have been proposed to represent the open andclosed states of the channel, respectively. Constraining the pore to specificpositions on that reaction coordinate allowed us to sample the conductivityat these positions. CompEL confirms that the assumedly open structure isindeed conducting and cation-selective (with conductances in the pS range,comparable to published experimental values), whereas the closed structureis indeed non-conducting. Moreover, conductance values steadily increasealong the closed-to-open transition and are correlated with the amount ofwater in the pore.

686-Pos Board B451Modeling Membrane Associated Proteins through Neutron ReflectivityAugmented Molecular DynamicsBradley W. Treece1, Mathias Loesche1, Frank Heinrich1,Arvind Ramanathan2.1Physics, Carnegie Mellon University, Pittsburgh, PA, USA, 2ComputationalScience, Oak Ridge National Laboratory, Oak Ridge, TN, USA.The structural characterization of membrane-associated proteins on fluidlipid bilayers remains a challenge to most modern biophysical techniques.Neutron reflectivity (NR) has emerged as a method that provides sub-nanometer resolution of proteins in a functional lipid environment. Interpre-tation of NR data gives an envelope structure related to the distribution ofprotein density along the membrane normal direction and further refinementusing structural information may yield a full atomistic description of theprotein-membrane complex. However, for flexible or intrinsically disor-dered protein domains, such information is often not available or multipleconformational states may contribute to the average density profile asresolved by NR. Thus, characterization of such systems requires more elab-orate approaches. We previously demonstrated that molecular dynamics(MD) simulations can provide a full atomistic interpretation of NR resultsin cases where only partial internal protein structure is available, but suchsimulations are often plagued by long equilibration times. Here we presenta procedure to steer MD simulations toward configurations that reproduceexperimental NR results. Biasing potentials are calculated through a com-parison of the one-dimensional densities from NR data with the evolvingdensity profile derived from the MD trajectory at each time step. This resultsin steering forces that direct molecular conformations of the protein on thebilayer toward the experimental results. Steering becomes weaker as thedensity profiles match more closely, disappearing entirely for matched den-sities. The structure is guided toward the desired configuration, rather thanrigidly confined to the experimental density. Here we show the applicationof our method to model peptide and small protein systems, also discussingthe efficiency of the procedure and potential merits and pitfalls in itsapplication.

687-Pos Board B452Characterization of Apolipoprotein Mimetic Peptides on Nascent HighDensity LipoproteinsMohsen Pourmousa, Rafique Islam, Denis Sviridov, Scott Gordon,B. Scott Perrin, Jr., John Stonik, Alan T. Remaley, Richard W. Pastor.National Institutes of Health, Bethesda, MD, USA.A nascent high-density lipoprotein (HDL) is a discoidal bilayer composed ofphospholipids, cholesterols, and two apolipoproteins that form a scaffoldthat stabilizes the assembly. Apolipoprotein mimetic peptides are shortsynthetic peptides that share features of apolipoproteins and have potentialtherapeutic value based on their ability to form and stabilize nascent HDL.A key question in designing mimetic peptides is why some are moreefficient than others. We characterize the properties of four mimetic pep-tides rich in the amino acids E, L, and K (called ELK peptides), using acombination of Molecular Dynamics simulations and experimental tech-niques. Experiments show that the hydrophobic and neutral ELKs have asignificantly higher ability to form nascent HDLs than the positive or nega-tive peptides. An in silico model of a discoidal bilayer was developed byintroducing a water slab perpendicular to the bilayer head group surface,leading to acyl chains of edges exposed to water. Simulations on this modeldiscoidal bilayer with peptides indicate that hydrophobic and neutral ELKsstabilize the assembly by forming scaffolds at the edges in a picket fence

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arrangement; the adjacent peptides are held together by strong salt-bridges.In contrast, the positive and negative ELKs diffuse to the head groupsurface, do not form effective scaffolds, and fail to stabilize the discoidalassembly. Hence, the simulations provide a structural rational for the exper-imental observations, and provide an avenue for computer based design ofapolipoprotein mimetic peptides.

688-Pos Board B453Computational and Experimental Studies of Gold Nanoparticle TemplatedHDL-Like Nanoparticles for Cholesterol MetabolismCheng-Tsung Lai1, Wangqiang Sun2, Rohun U. Palekar2, C. Shad Thaxton2,George C. Schatz1.1Department of Chemistry, Northwestern University, Evanston, IL, USA,2Department of Urology, Northwestern University, Chicago, IL, USA.High-density lipoprotein (HDL) is involved in the transport and metabolismof phospholipids, triglycerides, and cholesterol. Mimics of HDL are beingexplored as potential therapeutic agents for removing excess cholesterolfrom arterial plaques. Gold nanoparticles (AuNPs) functionalizedwith apolipoprotein A-I (apoA-I), and with the lipids 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (PDP PE) havebeen demonstrated to be acceptors of cellular cholesterol. However, detailedstructural properties of this functionalized HDL AuNP are not well under-stood. We combined coarse-grained and all-atom MD simulations to simu-late the self-assembly of lipids and cholesteryl ester on the AuNP/apoA-Iconstruct to gain insights into its structural properties, and further makecomparisons with experimental results. We find that lipids are orienteddifferently in regions with and without apoA-I. We also find that in thisfunctionalized HDL AuNP, the distribution of cholesteryl ester maintainsa reverse concentration gradient that is similar to the gradient found innative HDL. Incubating the HDL AuNP with cholesterol and lecithin:cholesterol acyltransferase (LCAT) demonstrate the HDL AuNP is able toefficiently activate LCAT and form esterified cholesterol from free choles-terol and phospholipid.

689-Pos Board B454High-Throughput Thermodynamics of Drug-Membrane Interactions fromMultiscale SimulationsRoberto Menichetti, Kiran Kanekal, Kurt Kremer, Tristan Bereau.Theory Group, Max Planck Institute for Polymer Research, Mainz, Germany.The number of small organic molecules is overwhelmingly large–so large,that most of it remains unexplored. Computer simulations offer an appealingframework to probe many of these compounds without the need to synthe-size them in the laboratory. The main hurdles preventing a high-throughputcharacterization of many small molecules relies on the time investment toparametrize the force field—a process that typically requires significant hu-man intervention—and extensive sampling requirements. We address theseissues by first sampling from the coarse-grained Martini model, for whichwe developed an automated parametrization protocol for small molecules.The resulting potential-of-mean-force (PMF) curves for the insertion ofsmall molecules in lipid membranes show excellent agreement for a numberof benchmark cases. Our framework allows us to run high-throughput mo-lecular dynamics simulations and estimate tens of thousands of relativefree energies of different small molecules in a lipid bilayer. They provideuseful data to explore structure-property relationships from computersimulations.

Optical Microscopy and Super-resolutionImaging: Novel Approaches and Analysis I

690-Pos Board B455Combining Expansion Microscopy and STED Nanoscopy for the Study ofCellular OrganizationIsotta Cainero1,2, Michele Oneto1,2, Luca Pesce1,2, Giulia Zanini1,2,Luca Lanzano1,2, Alberto Diaspro1,2, Paolo Bianchini1,2.1Nanophysics, Istituto Italiano di Tecnologia, Genoa, Italy, 2University ofGenoa, Genoa, Italy.Expansion microscopy (ExM) is a novel method that allows super-resolutionimaging with conventional microscopes(1, 2). It consists in soaking the cellswith a polymer, inducing the polymerization to form a dense meshworkthroughout the cell, cross-linking the fluorophores to the polymer and, afterdigestion of cellular protein, rehydrating of the sample. The swelling ofthe polymer gel led to a fourfold isotropic stretching of the sample. Therefore,it increases the distance between two objects that otherwise couldnot be seen as

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two different things with an ordinary microscope. One of the drawback of sucha technique is the long preparation made of several stages, i.e. immunostaining,gelation, digestion and expansion. They are really crucial steps for a good im-aging post-expansion.In our work we present a comparison between ExM and stimulated emissiondepletion (STED) nanoscopy(3). Our aim is to study the e possible combinationof STED and ExM as a method to further enhance the final resolution achiev-able. We will in particularly take advantage of the use of separation of photonsby lifetime tuning (SPLIT) STED (4).We show application of these methods from single fixed cells to slices of fixedmouse retina tissue.We are also interested in applying the approach to high-density compartmentslike the cell nucleus to decipher the high-order structure organization of chro-matin-DNA.1. Chen, F., P.W. Tillberg, and E.S. Boyden. 2015. Optical imaging. Expansionmicroscopy. Science. 347: 543-548.2. Chozinski, T.J., A.R. Halpern, H. Okawa, H.-J. Kim, G.J. Tremel, R.O.L.Wong, and J.C. Vaughan. 2016. Expansion microscopy with conventionalantibodies and fluorescent proteins. Nat Meth. 13: 485-488.3. Bianchini, P., C. Peres, M. Oneto, S. Galiani, G. Vicidomini, and A. Diaspro.2015. STED nanoscopy: a glimpse into the future. Cell Tissue Res. 360:143-150.4. Lanzano, L., I. Coto Hernandez, M. Castello, E. Gratton, A. Diaspro, andG. Vicidomini. 2015. Encoding and decoding spatio-temporal informationfor super-resolution microscopy. Nature Communications. 6: 6701.

691-Pos Board B456Quantitative Microstructure Analysis of Casein Network Dynamics usingSTED Microscopy with Relation to Macroscopic Gel PropertiesZachary J. Glover, Adam Cohen Simonsen, Jonathan Brewer.MEMPHYS, FKF, University of Southern Denmark, Odense, Denmark.Casein proteins are the main structural element in many fermented dairyproducts. Understanding how protein functionality is affected during mod-ern food processing, such as filtration and spray drying, is key to productand process optimization. Advances in Super Resolution Microscopy haveprovided novel tools to investigate protein microstructures. Herein, Stimu-lated Emission Depletion (STED) Microscopy has been used to imagecasein micelle aggregation, gel formation and network coarsening overtime. Protein structures have been resolved to > 100 nm. Native skimmilk and reconstituted skim milk powder were used to produce gelled sys-tems. Gelation was induced either through rennet addition and acidificationwith GDL. Images have quantitatively analyzed to monitor the developmentof casein network over time, and the fractal dimensions of the different sys-tems have been determined. Label free Coherent Anti-Stokes Raman Spec-troscopy (CARS) Microscopy was used to verify that the presence offluorophore did not interfere with the gelation process during STED micro-scopy. Image data has been directly correlated with dynamic textureanalysis.We demonstrate the ability to resolve casein micelle structures on thescale of a single micelle, and extract valuable information that directlyrelates to the macroscopic properties of a gelled system. This work pro-vides the basis for the use of STED microscopy to analyze other colloidalstructures. We present a framework to extract meaningful informationthat directly relates to the macroscopic properties and functionality ofthe system.

692-Pos Board B457A Novel STED Microscope with Nanometer Axial SectioningIvan Coto Hernandez1, Siddharth Sivankutty2, Nicolas Bourg3,Sandrine Lecart3, Guillaume Dupuis3, Sandrine Leveque-Fort1.1Univ. Paris-Sud, Universite Paris-Saclay, CNRS, Institut des SciencesMoleculaires d’Orsay (ISMO), Paris, France, 2Aix-Marseille Universite,CNRS, Institut Fresnel, Marseille, France, 3Universite Paris-Sud, Centre dePhotonique BioMedicale (CPBM), Paris, France.The axial resolution of conventional Stimulated emission depletion (STED)microscopy is limited by diffraction to about 500 nm. Overcoming this lim-itation usually comes at the cost of complex instrumentation, as 3D confine-ment usually implies to combine two STED beams. By trying to combinewith TIRF excitation this decrease the lateral performances. In this workwe reported a straightforward approach to restrict the axial extension ofthe STED microscope by only modifying the detection path, thus keepingoptimal lateral resolution. This new membrane imaging takes advantageof the supercritical angle (SAF) emission of fluorophore that allows oneto discriminate their position regarding the glass coverslip. Indeed only

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when fluorophore are closed to the interface, their evanescent near field canbecome propagative and appears above the critical angle. This SAF emis-sion represents up to 50% of the emission collected by the objective for afluorophore at the interface. By filtering out of the undercritical emissionin a conjugated plane of the back focal plane of the objective lens, onlythe SAF emission is detected. It allows nanometric axial sectioning of fluo-rescent emitters close to the water-glass interface; simply by adding a SAFmodule in the detection path of all STED microscopes. This STED-SAF im-plementation reduces the complexity and cost of its early implementationand as side effect, the photobleaching and light dose sent to the sample isreduced since not extra STED beam is needed for isotropic resolution. Inthis work we show for the first time the coupling of STED and SAF micro-scopy and we highlight the benefits of this implementation by imagingfluorescent beads and sub-cellular structures. The image quality of rawdata is improved by using deconvolution techniques. Finally the specificimportance of the method towards the implementation of Tomography-STED microscopy is discussed.

693-Pos Board B458Well-Characterised Time-Gated Detector Photon Flux Resolves theUltrastructure of DNA-Damage Nuclear Bodies with G-STED NanoscopyKok-Lung Chan1, Esther Garcia Gonzalez2, Sergi Padilla-Parra3,Jorge Bernardino de la Serna2.1Genome Damage and Stability Centre, University of Sussex, Brighton,United Kingdom, 2Central Laser Facility, Science and Technology FacilitiesCouncil, Harwell-Oxford, Didcot, United Kingdom, 3The Wellcome TrustCentre for Human Genetics, University of Oxford, Oxford, United Kingdom.Widely spread commercial STED nanoscopes, and low-cost custom-builtsystems are generally equipped with continuous-wave STED lasers(cwSTED) combined with pulsed excitation lasers (peLaser), and time-gated detection. This modality, termed gated-STED (gSTED), permits usinglower cwSTED powers reducing background noise, at the expense of lowintensity images. gSTED critically depends on the photon-counting detec-tors sensitivity, and photon-flux optimization to the temporal gate. The com-mercial system employed in this work utilise an electronic trigger-box tosynchronise the peLaser with time-gate hybrid detectors (gHyD). Unfortu-nately, commercial gSTED set-ups do not provide an accurate detectiontemporal gate characterization. Hence, users blindly select the time-gatedwindow with none a priori knowledge of neither the shape, nor the delayrelative to the peWLL. As a result, often the acquisition of sharp, highly-resolved images turns into seemingly random events. Here, we report howto measure the gate shape in our gSTED microscope. Our gate starts at2.5ns and last 4.5ns when exciting at 488nm, depleting at 592nm, andwith both notch filters. The effective gate shape is highly affected by thefluorescence emission and detection, as well as the optical elements in thepathway. For instance, neither a detection window from 500nm and above560nm, nor short lifetime dyes yield sharp, highly-resolved images. Further-more, a thorough study on how different peLaser and cwSTED power ratios,and different time-gates impact the lateral resolution and image signal-to-noise allowed generating resolution maps showing a priori knowledge tobest gSTED performance. Our method allowed resolving the nuclear bodies(NB) ultrastructure, a previously unresolved sub-nuclear DNA assembly inhuman cells. They spontaneously appear in ~10% of G1/early S phase cells,and it is suggested to be DNA damage sheltering centre for transgenera-tional DNA lesions originated in the previous S phase. Our method revealedthe NB organisation in high detail, showing that NB contains multiplechromatin looped structures in the periphery. To gain further details ofthe NB inner core, we extended the chromatin by pre-extracting cells priorto staining. Compared to the previous condition, we observe chromatin fibrebundles of approximate 15-40nm diameter, and can now resolve a higher-order chromatin organisation reminiscent of a molecular plait-like sub-struc-ture. In conclusion, a well-characterised time-gate, and cwSTED/peWLLpower ratio in commercial gSTED nanoscopes allows resolving the NBultra-structure with a consistent resolution (within ~15nm) and possiblyother biologically-relevant structures.

694-Pos Board B459Localization Microscopy Theory and Practice for Data Analysis onSparsely Labeled SamplesBrian T. DeVree, Sarah L. Veatch.Biophysics, University of Michigan, Ann Arbor, MI, USA.In the last several years, the development of myriad localization-basedfluorescence microscopy techniques has enabled the study of cellular

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structures at spatial resolutions up to an order of magnitude better than pre-vious optical techniques allowed. These localization microscopy techniqueswork by acquiring the positions of single fluorophore molecules withdiverse methods of varying complexity, but they all share the requirementthat in order to create quality images, the cellular structures being studiedthey must be labeled at quite high densities. Such dense labeling is notalways experimentally achievable nor desirable, most especially for livecell imaging studies. However, it is still possible to learn much about theunderlying structures in more sparsely labeled samples using correlativeanalysis. In this study, we use an information theory framework to representthe process of building a localization microscopy image and develop a‘‘completeness index’’ for the resulting images that aids in imagequality assessment. We develop a test with human observers to identifythe baseline range of completeness index values required for identificationof features commonly found in fluorescence images of cellular structures.We then use simulated and real localization microscopy datasets to deter-mine how to maximize the information gained from correlation analysisof datasets with low completeness and determine guidelines to avoidintroducing data processing artifacts into the data. We also confirm theapplication of bootstrapping methods to estimate the uncertainty of themeasurements taken from correlations with these sparsely labeled datasets.Finally, we apply the analysis an example of TIRF-based localizationimaging; the distribution of actin-modifying proteins around the site ofHIV-entry into a T-cell.

695-Pos Board B460Super-Resolution Imaging of Organelle Membrane Contact Sites viaFar-Red Hyperspectral Localization MicroscopyAdriano Vissa1, Maximiliano Giuliani2, Peter K. Kim3, Christopher M. Yip2.1BIochemistry, University of Toronto, Toronto, ON, Canada, 2IBBME,University of Toronto, Toronto, ON, Canada, 3Biochemistry, University ofToronto, Toronto, ON, Canada.The communication and exchange of material between subcellular organ-elles is critical to many cellular processes. Areas of close appositionbetween organelles, termed membrane contact sites (MCS), have recentlybecome a focus of study for intracellular communication. Through enrich-ment of specific proteins and protein complexes, these sites are implicatedin the exchange of lipids and metabolites such as calcium. As MCScharacteristically display inter-membrane distances of 10-30 nm, electronmicroscopy is often used to visualize these phenomena. However, themolecular specificity of the proteins involved in MCS is of great interest.In order to address these questions more closely, we have developed ahyperspectral localization microscopy technique to spectrally distinguishfar-red fluorophores using a Versachrome thin film tunable filter (Sem-rock) integrated into the emission path of an Olympus IX-83 dual deck in-verted TIRF microscope. Using this hyperspectral 3D Direct StochasticOptical Reconstruction Microscopy (dSTORM) imaging approach, wewere able to spectrally and spatially distinguish between Alexa 647 andAlexa 680 labels in our studies of the nanoscale spatial distribution ofER and peroxisome membranes under WT and MCS knockdown condi-tions and the clustering of MCS proteins at key sites of membraneapposition.

696-Pos Board B461Towards Quantitative High-Throughput 3D Localization MicroscopyJoran Deschamps, Markus Mund, Jonas Ries.Cell biology and biophysics, EMBL, Heidelberg, Germany.Single-molecule localization microscopy (SMLM) has become a widelyused technique to observe cellular structures at the scale of tens of nanome-ters. However, major bottlenecks are still faced and limit the scope of appli-cations. Here we present new technologies to overcome three of the majorlimitations of SMLM in throughput, quantitative counting of proteins and3D resolution. Indeed, SMLM is an intrinsically slow method with acquisi-tion and data analysis times in the minute to hour range. This severely limitsthe possibility to acquire a sufficient number of experiments in quantitativestudies. To nevertheless achieve high throughput in SMLM, we automatedthe entire workflow of data acquisition and analysis. This enabled us tocontinuously acquire new experiments over the course of days and analyzethem in real time. Another limitation concerns the inhomogeneous photo-dynamics of molecules across the field of view, which is a consequenceof the Gaussian intensity profile in microscopes using lasers for excitation.Such inhomogeneity impedes accurate protein counting. To tackle this issue,we developed a homogeneous illumination scheme with the help of a

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multimode fiber and a speckle reducer. This simple illumination achieves avery high uniformity and results in homogeneous measured localization pre-cisions across the field of view, removing statistical bias from quantitativestudies. Finally, we present the use of adaptive optics to improve the 3D res-olution of supercritical angle localization microscopy (SALM), with the aimto reach isotropic localization precision in the first few hundreds of nanome-ters above the coverslip. Altogether, these improvements open the way tobetter sampled and more precise structural studies in superresolutionmicroscopy.

697-Pos Board B462Versatile Super-Resolution Calibration Standard for Quantifying ProteinCopy NumberFrancesca Cella Zanacchi1, Carlo Manzo2, Angel Sandoval Alvarez3,Nathan D. Derr4, Maria Garcia Parajo2, Melike Lakadamyali1.1AFIB group, ICFO-Institut de Ciencies Fotoniques, Barcelona, Spain,2SMB group, ICFO-Institut de Ciencies Fotoniques, Barcelona, Spain,3ICFO-Institut de Ciencies Fotoniques, Barcelona, Spain, 4Smith College,Northampton, MD, USA.Single molecule based super-resolution microscopy offers a unique opportu-nity for quantifying protein copy numbers with nanoscale resolution [1,2].While fluorescent proteins have been extensively characterized for quantita-tive imaging using calibration standards, similar calibration tools for smallorganic fluorophores used in conjunction with immunofluorescence arelacking.Within this framework, the development of methods able to access a precisemolecular counting of protein copy numbers is essential, clearing the way toaddress several biological questions using super-resolution techniques suchas stochastic optical reconstruction microscopy (STORM).The development of a suitable calibration method represents the bestway to address the challenges of molecular counting using super-resolution [3,4]. Within this project, we demonstrate that DNA origamiin combination with GFP antibodies is a versatile platform for quantifyingprotein copy number in immunofluorescence based super-resolution micro-scopy. We show that this calibration method, besides quantifying theaverage protein copy number in a cell, allows determining the abundanceof various oligomeric states. Furthermore, we apply this calibration methodto quantify nucleoporins (NUP107) [5] and molecular motors (dynein inter-mediate chain) [6] in vivo. Overall, we provide a versatile strategy forquantifying a large number of proteins of interest using various labelingapproaches.1. Durisic, N., et al., Single-molecule evaluation of fluorescent protein photo-activation efficiency using an in vivo nanotemplate. Nat Methods, 2014. 11(2):p. 156-62.2. Ulbrich, M.H. and E.Y. Isacoff, Subunit counting in membrane-bound pro-teins. Nat Methods, 2007. 4(4): p. 319-21.3. Schmied, J.J., et al., DNA origami-based standards for quantitative fluores-cence microscopy. Nat Protoc, 2014. 9(6): p. 1367-91.4. Jungmann R. et al. Quantitative superresolution imaging with qPAINTNature methods doi:10.1038/nmeth.3804 (2016)5. Szymborska, A., et al., Nuclear Pore Scaffold Structure Analyzed by Super-Resolution Microscopy and Particle Averaging. Science, 2013. 341(6146):p. 655-658.6. Derr, N.D., et al., Tug-of-war in motor protein ensembles revealedwith a programmable DNA origami scaffold. Science, 2012. 338(6107):p. 662-5.

698-Pos Board B463Heterogeneity of the Nuclear Environment Investigated by Superresolu-tion Microscopy and Fluorescence Correlation SpectroscopyLuca Lanzano01, Melody Di Bona1,2, Lorenzo Scipioni1,3,Maria J. Sarmento1, Enrico Gratton4, Giuseppe Vicidomini5,Alberto Diaspro1,2.1Nanoscopy, Nanophysics, Istituto Italiano di Tecnologia, Genoa, Italy,2Department of Physics, University of Genoa, Genoa, Italy, 3Department ofComputer Science, Bioengineering, Robotics and Systems Engineering,University of Genoa, Genoa, Italy, 4Biomedical Engineering, University ofCalifornia, Irvine, CA, USA, 5Molecular Spectroscopy and Microscopy,Nanophysics, Istituto Italiano di Tecnologia, Genoa, Italy.Genomes are more than one-dimensional entities purely defined by theirlinear DNA sequences [Misteli T, Cell (2007)]. A long standing challenge

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in biology is to decipher the principles of organization of what can be con-sidered, by analogy with human-created libraries, the cell’s primary unit ofinformation storage and retrieval. In this respect, the development of super-resolved fluorescence microscopy has provided a new toolbox to peer intothe nucleus. For instance, super-resolution microscopy has been recentlyapplied to the investigation of nanoscale chromatin architecture, revealingnucleosome higher-order organization into heterogeneous ‘clutches’ andepigenetically dependent folding motives [Ricci M et al, Cell, (2015);Boettiger A et al, Nature (2016)].However, dynamic properties of proteins in the nucleus are also critical fortheir function [Misteli T, Cell (2007)]. Fluorescence Correlation Spectroscopy(FCS) has been used to map the dynamics of several proteins in the nucleuswith diffraction-limited spatial resolution. Here we combine a novel STED-based super-resolution method [Lanzano’ L et al, Nat Commun (2015)]with FCS to measure protein dynamics in the nucleus with an improvedspatial resolution of about 100 nm. We sample several positions within thenucleus by performing line scanning. The measured spatial and temporalheterogeneity of the dynamics, quantified by a recently introduced algorithm[Scipioni L et al, Biophys J (2016)], is discussed in relation to nuclearorganization.

699-Pos Board B464Analysis of Fibrous Spatial Point Patterns from Single-Molecule Super-Resolution Microscopy DataRuby Peters, Dylan Owen, Juliette Griffie.King’s College London, London, United Kingdom.Unlike conventional microscopy methods which generate pixelatedimages, SMLM techniques produce images comprising of a list of molec-ular coordinates - a spatial point pattern (SPP). SMLM methods thereforenecessitate a statistical approach to data analysis. SPPs are routinelyanalyzed using robust cluster analysis algorithms to quantify molecularclustering across diverse biological systems. The analysis of fibrousSPPs however, such as those derived from components of the cyto-skeleton, remains relatively understudied. Here, we present statisticalmethodology based upon a variant of Ripley’s K function to quanti-tatively assess fibrous patterns generated by SMLM. We demonstratethe technique using various simulated fibrous spatial arrangementsand extract spatial descriptors of the pointillist input. We further demon-strate the technique on experimental data acquired using the image recon-struction by integrating exchangeable single-molecule localization (IRIS)approach to SMLM. We quantitatively assess the fibrous distribution offilamentous actin at the T cell immunological synapse, whose structurehas been shown to be important for cell morphology, polarization andactivation.

700-Pos Board B465Expanding the Spectral Resolution of Single-Molecule LocalizationMicroscopy with Bodipy-Based Photoswitchable FluorophoresAmy M. Bittel, Ashley Davis, Tao Huang, Xiaolin Nan, Summer L. Gibbs.Biomedical Engineering, Oregon Health & Science University, Portland,OR, USA.Single-molecule localization microscopy (SMLM) has become an impor-tant tool for studying molecular biology. Through the use of photoswitch-able fluorophores, SMLM can accurately localize individual moleculeswith 10-20 nm resolution, an order of magnitude better than conventionalfluorescence microscopy. While SMLM succeeds in locating individualmolecules, it is limited to 4-color emission based imaging due to thestandard bandpass filter technology used to generate multicolor images,restricting the number of molecular entities that can be simultaneouslylocalized in a single sample. The development of multi-spectral super-resolution microscopy (MSSRM) improves the spectral resolution ofSMLM enabling up to 20 color imaging in a single sample. However,MSSRM is currently restricted by the spectrally limited conventionalfluorophores with adequate photoswitching for SMLM. Herein, we de-signed, synthesized and validated a series of novel BODIPY-based fluoro-phores with appropriate photoswitching for SMLM that span the visiblespectrum permitting high-resolution, multi-color images using our MSSRM.The BODIPY-based probes were selected from a 110 member libraryof compounds synthesized through modification of a core BODIPY FLscaffold with diverse aromatic rings using a solid phase synthetic plat-form. All BODIPY-based probes were characterized for absorption andemission properties as well as key photoswitching properties to facilitate

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selection of the ideal probes for SMLM and MSSRM. Through labelingknown cellular structures with selected BODIPY-based probes, wedemonstrated that multiple fluorophores could be accurately resolvedwhen excited by the same laser line due to varied length Stokes shifts.The developed BODIPY-based probes for SMLM will be beneficial inadvancing understanding of cellular spatial organization by enablingthe localization of an increased number of molecular compoundssimultaneously.

701-Pos Board B466DNA-Paint Imaging of DNA-Origami Rings Mimicking BiologicallyRelevant StructuresArunima Chaudhuri1, Yang Yang1, Kenny Kwok Hin Chung1,Zhao Zhang1, Fredric Pincet2,3, Shyam Krishnakumar1, Chenxiang Lin1,James E. Rothman1, David Baddeley1.1Department of Cell Biology, School of Medicine, Yale University;Nanobiology Institute, School of Medicine, Yale University, New haven, CT,USA, 2Department of Cell Biology, School of Medicine, Yale University,New Haven, CT 06520; Nanobiology Institute, School of Medicine, YaleUniversity, New Haven, CT 06520, New haven, CT, USA, 3Laboratoire dePhysique Statistique, Ecole Normale Superieure, Paris Sciences et LettresResearch University; Laboratoire de Physique Statistique, Universite ParisDiderot Sorbonne Paris Cite; Laboratoire de Physique Statistique, SorbonneUniversites, Paris, France.Single-molecule localization microscopy methods such as DNA-PAINT(point accumulation for imaging in nanoscale topography), utilize successivelocalizations of single molecules over time to reveal of biological structureswith photon-limited resolution1,2. However, visualization of sub-diffracteddiscrete bimolecular assemblies still remains a challenge. ‘‘DNA origami’’has emerged as powerful nano-templating technique capable of generating sta-ble self-assembled scaffold for positioning of molecular species of interestwith nanometer precision3,4. Recent studies of DNA-PAINT have focusedon imaging different molecular species, such as fluorophores and proteins,positioned on synthetic grid-like DNA nanostructures1,4. These grids arewell suited for determining the resolution limit of sparsely labelled points(e.g. one fluorophore per 5x5 nm2). However, biological structures oftenhave a much higher local density and complex structure, making imagingmore challenging. In this study, as a proof-of-concept, we explored imagingof circular ‘‘ring-like’’ DNA-origami nanostructures of dimension mimickingsmall bio-molecular structures (25-50 nm inner diameter), like membrane re-ceptors, ion channels etc. Using a TIRF-based imaging technique, we haveoptimized the fluorophores density and its positioning on DNA-origami ringsto reconstruct ‘‘doughnut’’ shaped images and have been able to resolve innerdiameter of ~25 to 50 nm. We have carried out these analyses on glass sur-faces and also on supported bilayers. Our study utilizing DNA rings provideschallenging targets for DNA-PAINT technology and underscores the use ofsize-templated nanostructures, as approximates for bio-molecular complexes,to optimize imaging and analysis protocols independent of biological samplepreparation.1. Dai et al. (2016) Nat. Nanotechnol. 11:798-807.2. Baddeley, D. (2015) Nat. Methods 12:1019-1020.3. Yang et al. (2016) Nat. Chem. 8:476-483.4. Schlichthaerle et al. (2016) Curr. Opin. Biotechnol. 39: 41-47.

702-Pos Board B467Characterization of PS-CFP2 for Reliable Super-Resolution MicroscopyBenedikt Rossboth1, Rene Platzer2, Johannes Huppa2, Gerhard Sch€utz1,Mario Brameshuber1.1Institute of Applied Physics, TU Wien, Vienna, Austria, 2Institute forHygiene and Applied Immunology, Medical University of Vienna, Vienna,Austria.Single molecule localization microscopy (SMLM) methods constitute anelegant way to infer information about cellular organization at high resolution,e.g. distinguished structures such as the cytoskeleton or the nuclear pore com-plex. Hence, their application to identify (nano-)clustering on the plasmamembrane, which is widely acknowledged to be a common feature of manyproteins, is well reasoned. Identifying those, however, ultimately convergesto quantitation of SMLM images. Overcounting artefacts caused by multipleobservations of the same fluorophore molecules due to blinking or incompletebleaching can be easily introduced. In this study we aimed at characterizingthe blinking behaviour of the commonly used fluorescent protein, PS-CFP2.Recombinant, his-tagged PS-CFP2 was anchored to supported lipid bilayers

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via DGS-NTA(Ni), representing a system with adjustable surface densitiesand diffusion characteristics. Our data suggests the existence of severalprolonged bright and dark states resulting in off times up to several seconds.Only about 66% of PS-CFP2 molecules were identified in a single image.Merging localisations from consecutive images increased this fraction to77%. Although the maximum number of localisations per PS-CFP2 wasgreater than 10 in only ~3% of the cases, the influence of these moleculeson the resulting image is rigorous. Including this blinking statistics in simula-tions and evaluation using popular clustering algorithms, such as Ripley’s Kfunction, showed no differences between randomly distributed data containingblinks versus clustered non-blinking data. We conclude, that SMLM methodspossess limitations for investigating protein clusters. Merging can be used forthe correction of suboptimal bleaching, however, for considering blinking offluorophores, this correction is - by the nature of the imaging method itself -inherently limited. Appropriate ways to tackle the investigation of possibleclustering include varying the labelling density and multi-color colocalizationtechniques.

703-Pos Board B468A Quantitative Platform for Super-Resolution Microscopy ImagingOttavia Golfetto1, Devin L. Wakefield1, Eliedonna E. Cacao1,Kendra N. Avery1, Raphael Jorand1, Steven J. Tobin1, Ronald M. Clinton1,Jennifer Gutierrez1, Yuelong Ma1, Daniel Ganjali2, Athanasios Sideris2,David A. Horne1, John C. Williams1, Tijana Jovanovic-Talisman1.1Molecular Medicine, Beckman Research Institute-City of Hope, Duarte, CA,USA, 2Mechanical and Aerospace Engineering, University of California,Irvine, Irvine, CA, USA.Pointillistic super-resolution microscopy techniques provide nanometerscale spatial resolution and single molecule sensitivity. Thus, they are excel-lent tools for probing the lateral organization of molecules, especially inbiological environments. Additional information on such organization canbe obtained by subjecting super-resolution data to quantitative analyses.These powerful approaches are able to quantify the extent to which mole-cules localize into clusters and the size/occupancy of clusters. However,quantitative super-resolution approaches typically encounter two majorchallenges. First, it is difficult to determine optimum imaging parametersand characterize, under these conditions, the fluorescent probe’s biophysicalproperties. Second, it is challenging to identify efficient labeling strategiesfor detecting endogenous proteins, particularly those that yield stoichio-metric and site specific labeling. To efficiently characterize fluorescentprobes, we have developed an easily implementable nano-biology assay.Using chemistry compatible with super-resolution imaging (i.e. producingminimal background signal), proteins were covalently immobilized toglass surfaces with specific orientations. Interrogating such surfaces re-vealed important features of both optical highlighter proteins (paGFPand pamCherry1) and proteins labeled with organic fluorophores. Toefficiently label endogenous receptors, we leveraged a unique cyclicpeptide called meditope, which binds within the Fab framework of engi-neered monoclonal antibodies. A fluorescent meditope was complexedwith the engineered trastuzumab Fab. This complex was used to imageboth SK-BR-3 and BT-474 cells, where we delineated the distribution ofepidermal growth factor receptor 2 (HER2). According to our results, themajority of HER2 receptors were monomers as expected. However, afraction of receptors were organized as oligomers, and more significantlyin BT-474 cells that are oncogene-addicted. This demonstrates that ourmethod is capable of detecting subtle differences in endogenous receptororganization and thus offers a powerful platform for quantitative super-resolution imaging.

704-Pos Board B469Estimating the PSF from Single Molecule DataKenny K.H. Chung, David Baddeley.Cell Biology Department/Nanobiology Institute, Yale University, NewHaven, CT, USA.A common method of extracting 3D information in localization microscopy isto use PSF engineering techniques where depth information is encoded in thePSF shape. These techniques are very sensitive to aberrations and poor estima-tion of the experimental PSF will not only reduce localization precision but alsodistort the reconstructed structure (e.g. by introducing a z-dependent lateralshift). Sub-resolution beads are typically used as a PSF reference, but are aninexact match for a single fluorophore in terms of size and spectral properties,and fail to capture sample-induced aberrations.

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We have established a method which eliminates the need for an externalPSF reference and directly estimates the experimental PSF from singlemolecule data collected during a localization trial. Although individual sin-gle molecule data have poor signal-to-noise, we can employ a PSF modelparameterized in terms of Zernike polynomials and ensemble fitting to esti-mate the PSF. For each choice of PSF parameters, we perform a 3D fit to allthe single molecules in our training set and use the summed residuals as afigure of merit. As Zernike modes are nearly independent, we were ableto perform fitting of each Zernike mode sequentially, drastically reducingthe computation required.We have validated this method on both simulated data and bead-derived PSFdata, with either an astigmatic or biplane 3D model. On experimental STORMdata, a small subset of single molecule images was sufficient for PSF extrac-tion. The use of a PSF extracted directly from the single molecule data led toan overall improvement in z precision for the complete data set. Althoughhere we have demonstrated a post-acquisition approach, further speed improve-ments may allow for on-the-fly extraction to provide feedback for adaptiveoptics.

705-Pos Board B470Robust Nonparametric Descriptors for Clustering Quantification inSingle-Molecule Localization MicroscopyShenghang Jiang1, Sai Divya Challapalli2, Yong Wang1.1Department of Physics, University of Arkansas, Fayetteville, AR, USA,2Microelectronics and Photonics Graduate Program, University of Arkansas,Fayetteville, AR, USA.Single-molecule localization microscopy (SMLM) has been utilized broadlyin imaging biological molecules in various biological systems, allowingquantitative analyses on the spatial organizations and patterns of these mol-ecules. However, parameters are needed in many of the currently availablemethods or algorithms, likely introducing subjective bias in the analyses.Here, we report a robust nonparametric descriptor, J’(r) , for quantifyingthe spatial organization of molecules in single-molecule localization micro-scopy. J’(r) , based on nearest neighbor distribution functions, does notrequire any parameter as an input for analyzing point patterns. We showthat it displays a valley shape in the presence of clusters of molecules,and the characteristics of the valley reliably report the clustering featuresin the data. More importantly, the position of the J’(r) valley (rJ’m) dependsexclusively on the density of clustering molecules. Therefore, it is ideal fordirect measurements of clustering density of molecules in single-moleculelocalization microscopy.

706-Pos Board B471Perturbation Upon Observation: User Defined, Nanoscale Labeling ofSuper-Resolution ImagesNinning Liu, Mingjie Dai, Peng Yin.Wyss Institute, Boston, MA, USA.A basic process to investigate biomolecular systems is observing and manipu-lating single molecules within their native context. While many recent ad-vances in super-resolution microscopy methods (e.g. STORM, PALM,PAINT) have allowed observation of molecular architecture down to the scaleindividual components (~5 nm), there has not been a parallel development ofmanipulation methods at a comparable scale. Current light-based manipulationtechniques, including laser capture micro-dissection or focused laser crosslink-ing, are still diffraction-limited, and therefore incapable of selective targetingbelow a ~200 nm sized area.Here we present a nanoscale, targeted manipulation method based on thePAINT (point accumulation for imaging in nanoscale topography) principle.Briefly, PAINT utilizes a fluorescent probe that transiently and repetitivelybinds to imaging targets. Under TIRF illumination, these transient bindingevents will appear as apparent blinks that can be localized and reconstructedinto a super-resolution image. In contrast with localization-based methodssuch as STORM or PALM, the fluorescent probe in PAINT is freely diffusing,with a reasonable expectation of only one probe bound to an imaging targetwithin a diffraction-limited area at any given time. This makes a light-induced targeting technique possible, whereby a laser pulse will be introducedat the precise moment when a probe binds to a pre-determined region ofinterest.This active management of the PAINT process will utilize our previouslydeveloped DNA-PAINT super-resolution imaging technique combined with afast, photo-activated crosslinking chemistry. Currently, we have demonstratedmulti-target, single-molecule labeling of points on a DNA nanostructure test-board with 30 nm spatial discrimination.

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707-Pos Board B472Methods to Eliminate Localization Bias and Reduce Localization Error inLocalization Microscopy Datasets with Non-Uniform Background Fluo-rescenceThomas Shaw1, Sarah L. Veatch2.1Applied Physics, University of Michigan, Ann Arbor, MI, USA,2Biophysics, University of Michigan, Ann Arbor, MI, USA.Single molecule localization microscopy has become a standard tool fornanoscale imaging of biological systems. However, improper treatment ofnon-uniform background fluorescence can degrade image accuracy andprecision. In particular this can be problematic in cases where the structurebeing imaged is correlated in space with high background fluorescence.Various methods have already been implemented for correction ofbackground fluorescence. Here, we demonstrate that these backgroundestimation and correction procedures lead to both systematic and random er-ror in emitter localization, under experimentally relevant background condi-tions. Part of these errors result from background estimation procedures, andpart from fitting procedures that incorrectly model (or fail to model) thepresence of background. We find that background estimation proceduresfrom the literature may overestimate local background in the presence ofa high density of foreground fluorophores, and show implications of thisbias for systematic localization errors when these estimates are used forbackground correction before or during emitter fitting. We have developeda novel estimator of background fluorescence that uses selective temporalaveraging to reduce this bias. We further show that standard backgroundcorrection procedures for emitter fitting produce experimentally relevantsystematic and random localization errors, even when the background isexactly known. We implement two new background-corrected fitting algo-rithms that have no systematic error and less random error than standardprocedures. We use simulations to compare the statistical performance ofthese methods to that of earlier methods, and demonstrate improvementsin images of biological systems.

708-Pos Board B473The Characterization of Cellulose Nanostructure using Super-ResolutionFluorescence MicroscopyMouhanad Babi.McMaster University, Hamilton, ON, Canada.Cellulose - a major and critical component of plant cell walls - constitutesthe largest component of Earth’s biomass and is an attractive raw materialto exploit in the production of biodegradable and renewable products,such as biocomposites, biofuels and other biomaterials. Manufacturing theseproducts often entails the chemical or biochemical depolymerization ofcellulose, a process that is limited by its crystalline structure. To better un-derstand these manufacturing processes and improve their efficiency, werequire insight into the nanoscale structure of cellulose and the mechanismof its depolymerization. In this work, direct stochastic optical reconstructionmicroscopy (dSTORM) was used to study the structure of fluorescently-labelled bacterial microcrystalline cellulose (BMCC) at the nanoscale.Super-resolution imaging unveiled regular repeating patterns of high andlow fluorophore density regions on BMCC microfibrils that are hypothesizedto represent disordered and crystalline regions of cellulose. Grafting cellu-lose using different dyes or labelling reactions produced similar patterns,evidencing that the fluorescent patterns are labeling chemistry-independentand are instead encoded within the native cellulose structure. The lengthof the dark regions was measured and their distribution was found to corre-late with the length of cellulose nanocrystals produced by a short hydrolysistreatments, as observed using transmitted electron microscopy. The lengthof the microfibril dark regions were also determined to be dependent onthe concentration of dye during the grafting procedure, strongly suggestingthat the observed labelling patterns are due to intervening crystalline anddisordered regions of cellulose microfibrils. A fluorescently-labeled cellu-lase - CBHI-Cy5 - bound to labelled BMCC was imaged in the same wayand the results show that the enzyme does not exhibit any preferentialbinding to either the crystalline or disordered cellulose regions. Understand-ing the nanostructure of cellulose will allow us to improve cellulosemanufacturing processes and give insight into how plant cells assemble,restructure and degrade their cell wall.

709-Pos Board B474Quantitative Analysis of Membrane Protein Clustering from Live-Cell,Single-Molecule Super-Resolution Microscopy DataJuliette Griffie1, Dylan Owen1, Patrick Rubin-Delanchy2, Garth Burn1.1King’s college London, London, United Kingdom, 2University of Oxford,Oxford, United Kingdom.

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To address the increasing need for a precise characterisation of the molecularorganisation in cells, a new family of fluorescence microscopy techniques,super-resolution, has arisen. Single molecule localisation microscopy(SMLM) in particular, allows the localisation of fluorophores with a precisionof 10-30 nm, hence revealing the cells’ nanoscale architecture at a molecularlevel. Over recent years, 2D SMLM has been extended to 3D and live-cellacquisitions, unravelling cellular machinery at the nano-scale. Using suchtools, it has been confirmed that modification of molecular spatio-temporalorganisation at the plasma membrane, particularly molecular clustering, isan important component of cell signalling. Despite the advances in imagingmethodology, analysis tools which are able to accurately and objectivelyquantify these phenomena remain limited, especially for 3D and live-cellSMLM data. Here, we present novel model-based Bayesian cluster analysistechnique suitable for 2D SMLM as well as its extension to 3D and live-cell analysis. We demonstrate the validity of these approaches on simulateddata sets. We further show that the transition from limited visual inspectionof the data to the extraction of quantitative descriptors provides a uniqueinsight into key biological processes. Using the presented analysis tools, wedemonstrate the importance of clustering for T cell activation and the trig-gering of an immune response. Analysis of live-cell PALM data demonstratesthe importance of molecular clustering and cluster dynamics at the membraneduring T cell activation. Our analysis, based on live-cell single-moleculesuper-resolution data is the first to provide real time descriptors of the clus-tering, as well as a detailed tracking of cluster dynamics and interactions atthe membrane.

710-Pos Board B475Selective Plane Illumination Microscopy in the Conventional InvertedMicroscope GeometryPer Niklas Hedde, Leonel Malacrida, Enrico Gratton.Biomedical Engineering, University of California Irvine, Irvine, CA, USA.During the last decade, selective plane illumination microscopy (SPIM)has proven to be one of the most suitable techniques for three-dimensional time lapse imaging. By confining the excitation light to a sheet,SPIM combines axial sectioning capability with minimal light exposure andfast, camera-based image acquisition [1]. However, the typical arrangementof two objective lenses perpendicular to each other provides a number ofchallenges in terms of instrument design and sample geometry, especiallyif the use of high numerical aperture (NA) lenses is desired. A popularapproach is to dip into the sample container from the top, both lenses at a45 degree angle with respect to the sample plane [2,3]. Instead, our newdesign is based on a regular inverted microscope where the sample is illu-minated from the side via an accessory. A custom designed chamber isused to allow side illumination. This way, all microscope ports remainavailable for other purposes and there is unrestricted access from the top.Without the need of dipping into the sample container, smaller sample vol-umes (< 1 ml) can be realized and the use of high NA lenses is facilitated.Also, isolation of optics and sample allows imaging of sealed sample con-tainers when demanded, e.g., for samples treated with potent toxins. Further,in this design, the orientation of the imaging plane is parallel to the surfaceof the sample container which is desirable for flat samples where it maxi-mizes the field of view.Work supported in part by NIH grants P50 GM076516 and P41 GM103540.[1] Huisken, J. et al., Optical Sectioning Deep Inside Live Embryos by Selec-tive Plane Illumination Microscopy. Science 305, 1007–1009 (2004).[2] Hedde, P.N. et al., Rapid Measurement of Molecular Transport and Inter-action inside Living Cells Using Single Plane Illumination. Sci. Rep. 4, 7048(2014).[3] Hedde, P.N. et al., 3D fluorescence anisotropy imaging using selectiveplane illumination microscopy. Opt. Express 23, 22308–22317 (2015).

711-Pos Board B476Light-Sheet Microscopy using Attenuation-Compensating Airy BeamCong Liu, Yen-Liang Liu, Tim Yeh.Biomedical Engineering, University of Texas at Austin, Austin, TX, USA.Diffraction-free light fields such as Airy beam have attracted significantattention due to their ability to retain their transverse intensity profile duringpropagation. This ability is highly desirable for light-sheet microscopy whosefield of view (FOV) is usually limited by the Rayleigh range of the Gaussianbeam used to create the light sheet. However, the extended FOV of Airy

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beam light-sheet microscopy can be comprised by the non-uniformity of illumi-nation, especially in thick, highly scattering and absorbing tissues. To provide amore uniform illumination, light-sheet excitation from both sides of the sampleis adopted, which results in higher system complexity and optical alignmentdifficulties. Here we introduce the concept of a digital micromirror device(DMD)-based Airy beam light-sheet microscope (DMD-ALS) that cancompensate the intensity loss caused by scattering and absorption in biologicaltissues. In our modelling, DMD-ALS can provide thin and uniform illuminationover a large range, thus eliminating the necessity of dual excitation. Theattenuation-compensating Airy beam is generated by phase-modulating aGaussian laser beam with a DMD displaying the Lee hologram. The fluores-cence intensity profile along the optical axis is measured and used for the calcu-lation of DMD spatial pattern in real time. This feedback adaptive proceduresensures optimal attenuation compensation for a variety of biological tissueswith different optical properties, without human intervention. Taking advan-tage of the fast frame-rate (~32kHz) of DMD, we expect that the adaptivecontrol algorithm converges within 50 ms.

712-Pos Board B477Light Sheet Microscopy by Dual Line Scanning of Two Bessel BeamsJames Werner, Pengfei Zhang, Elizabeth Phipps, Peter Goodwin.Los Alamos National Laboratory, Los Alamos, NM, USA.We have developed a light-sheet microscope that uses confocal scanning ofdual-Bessel beams for illumination. A digital micro-mirror device (DMD) isplaced in the intermediate image plane of the objective used to collect fluores-cence and is programmed with two lines of pixels in ‘on’ state such that theDMD functions as a spatial filter to reject the out-of-focus background gener-ated by the side-lobes of the Bessel beams. The optical sectioning and out-of-focus background rejection capabilities of this microscope were demonstratedby imaging of human A431 cells whose actin was fluorescently stained. Thedual-Bessel beam system enables twice as many photons to be detected perimaging scan, which is useful for low light applications (e.g. single moleculelocalization) or imaging at high speed with superior signal to noise. Whiledemonstrated for two Bessel beams, this approach is scalable to a larger numberof beams.

713-Pos Board B478Single Molecule Fluroescence Approaches to Plasma MembraneBiophysicsPhilip R. Nicovich.University of New South Wales, EMBL Australia Node in Single MoleculeScience þ ARC Centre of Excellence in Advanced Molecular Imaging,Sydney, Australia.The plasma membrane is the fundamental component of a cell. Not only is theplasma membrane the outer barrier of the cell, it also regulates many cellularfunctions including endocytosis, exocytosis, receptor signalling and cell migra-tion. As essential as the plasma membrane is, methods to capture the structureof and the proteins within the membrane on biologically-relevant length scalesremain lacking. A particularly vexing issue is whether membrane rafts - regionsof locally-ordered lipids and associated proteins observed in model membranesystems - are present in intact cells. Much of the previous evidence for theserafts relies on invasive biochemical techniques and the little imaging evidenceavailable shows that these regions are small (< 100 nanometres) and short-lived (< 100 milliseconds).While still limited by the diffraction limit of visible light, single-molecule fluo-rescence imaging allows such small and transient features to be detected. Wewill present three new approaches to investigating these structures in intactmembranes. The first is a spectral fluorescence correlation spectroscopyapproach demonstrating the presence of regions of local membrane order anddisorder within live cell membranes. These regions have lifetimes of 5 to25 ms and through simulations we can show that both probe diffusion anddomain diffusion give rise to the observed fluctuations in emission intensity.The second technique is a self-calibrating image analysis method that iscapable of capturing the coordination of local differences in membrane orderwith membrane protein distribution within intact cells. This approach demon-strates the presence of protein sorting within intact cells as well as the effect ofexpression level on effective protein sorting. The final technique is a 3D super-resolution method to map the nanometer-scale topography of the plasmamembrane. We will show initial results demonstrating our ability to map thesetopographical features and their correlation with protein sorting.

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Optical Microscopy and Super-resolutionImaging: Applications to Cellular Molecules

714-Pos Board B479A Fast and Reliable Online-System for Platelet Viability StudiesMichael D. Brodesser1, Sandra Mayr1, Fabian Hauser1, Johannes Breuss2,Michael Aspetsberger3, Andreas Hangler3, Lukas Bindreiter3,Daniela Borgmann4, Stephan Winkler4, Christian Gabriel5,Eleni Priglinger5,6, Jaroslaw Jacak1, Birgit Plochberger1.1Medical Engineering, University of Applied Sciences Upper Austria, Linz,Austria, 2Medical University of Vienna, Vienna, Austria, 3Catalysts GmbH,Linz, Austria, 4School of Informatics, University of Applied Sciences UpperAustria, Hagenberg, Austria, 5AUVA Research Center, Ludwig BoltzmannInstiute for Experimental and Clinical Traumatology, Vienna, Austria,6Austrian Cluster for Tissue Regeneration, Vienna, Austria.Platelets are blood cells with the capability to adhere to injured tissues and bloodvessels after trauma. Self-adherence is also known in thrombosis and inflamma-tion. Platelets release factors triggering the activation of other platelets, leuco-cytes and endothelial cells near the site of adherence. Here we show a fast andreliable detection system for the activation status of platelets, even in earlystages. In particular, the method allows the classification of the cell state, theviability and its reversibility under selected environmental parameters. An opti-cal microscope in combination with a contrast enhancing technique like Differ-ential Interference Contrast and single molecule fluorescence microscopy isutilized for grading unique features like e.g. cell morphology, cytoskeletalarrangement, protein diffusion and clustering. The online-systemobserves adhe-sion of platelets at controlled temperatures and buffer/surface conditions.Focusing on platelet apheresis concentrates labeled with a fluorescent antibodyto the activation receptor CD62p, we observed a distinct change in protein diffu-sion and clustering depending on activation state and associated morphologicalchanges. A systematic correlation between microtubule/actin cytoskeleton- andmorphological change was made. Activation steps were triggered either by tem-perature or buffer conditions on cleaned glass and epoxysilane-coated surfaces.In order to allow a classification of activation states of platelets in real-time, fea-tures of diffusion, cluster properties and morphologywere cross-correlated. Thefeature analysis system, useful for the classification of platelets in a clinicalcontext, might prove applicable also to other specimen (e.g. pathology samples).

715-Pos Board B480Visualizing Signaling Complexes in Filamentous FungiAlexander W.A.F. Reismann1, Lea Atanasova2, Alexander Lichius2,Sabine G. Gruber3, Susanne Zeilinger2, Gerhard J. Schuetz1.1Institute of Applied Physics, TU Wien, Vienna, Austria, 2Institute ofMicrobiology, University of Innsbruck, Innsbruck, Austria, 3Institute of FoodScience, University of Natural Resources and Life Science Vienna, Vienna,Austria.Due to itsmycoparasitic lifestyle, thefilamentous fungusTrichodermaatrovirideis a widely used biofungicide for the control of fungal plant pathogens in today’sagriculture. However, the exact signaling mechanisms regulating the mycopara-sitic attack of fungal preys are not yet fully understood. The sensing and locationof its prey fungi, such as Botrytis cinerea, is mediated by membrane receptorswhich react to small chemical substances secreted by the prey. Single moleculetechniques have hardly been used in filamentous fungi and can open up efficientpossibilities to observe these molecular processes in great detail. These methodscan be used under different physiological conditions by adding specific stimuli orby co-cultivating Trichoderma with a prey fungus and detect behavioral differ-ences. In this study, we examine Gpr1, a G-protein coupled receptor, and its pu-tative interaction partner Sur7, which are known to contribute to the molecularsignaling process, using single molecule techniques. By using strains expressingSur7-mEOS3.2 fusion proteins, we performed PALMmeasurements and gener-ated detailed localization maps in growing hyphae. Analyzing single moleculetrajectories of GFP tagged Sur7, we furthermore provide insights into themobility of the protein in vivo. In the future, we will apply AF647-labeled nano-bodies to perform STORM within fixed hyphae expressing Sur7-GFP fusions.The obtained insights into the properties of the involved proteins will lead to adeeper understanding of the molecular organization in mycoparasitic fungi.

716-Pos Board B481Flim-FRET Imaging of Plitidepsin-eEF1A2 Complexes in Live CancerCells using the Phasor ApproachCarolina Garcia1, Alejandro Losada2, Miguel A. Sacristan1,Jose M. Molina-Guijarro2, Juan F. Martinez-Leal2, Carlos M. Galmarini2,M. Pilar Lillo1.1Quimica Fisica Biologica, IQFR-CSIC, Madrid, Spain, 2Biologia Celular yFarmacogenomica, PharmaMar S.A., Colmenar Viejo. Madrid, Spain.

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Eukaryotic Elongation Factor 1A2 (eEF1A2) is an isoform of the alpha subunitof eEF1A complex. Differently from the A1 isoform, the expression of eEF1A2is restricted to brain, heart and skeletal muscle. eEF1A2 is overexpressed in tu-mors, including multiple myeloma (MM), prostate, pancreas, and ovarian, andhas also an oncogenic behavior favoring tumor cell proliferation while inhibit-ing apoptosis. Thus, eEF1A2 is an interesting target for cancer treatment. Pli-tidepsin (Aplidin�, APL) is an antitumor agent, originally isolated from themarine tunicate Aplidium albicans, which is being tested in MM patients in aphase III pivotal trial in combination with dexamethasone and a phase I trialin combination with bortezomib and dexamethasone. HeLa-WT and HeLa-APLR (resistant to Aplidin�) cell lines have been previously described.HeLa-APLR present lower eEF1A2 levels than HeLa-WT cells. Herein wereveal the interaction of Plitidepsin with eEF1A2 using a fluorescent Aplidin�derivative, APL-DMAC as a FRET donor.We stably transfected HeLa-WT andHeLA-APLR cells with plasmids encoding GFP-tagged eEF1A2 (FRETacceptor), and selected clones expressing physiological levels of the fusion pro-teins. We applied the FLIM-phasor approach [Redford and Clegg 2005; Dig-man et al. 2008], to localize and quantify APL-DMAC molecular speciesand to measure FRET efficiencies, DMAC - GFP, thus identifying differenteEF1A2-APL complexes [Losada et. al. 2016].

717-Pos Board B482Image Correlation Spectroscopy based Assay to Investigate G-ProteinCoupled ReceptorsNader Danaf.Chemie und Pharmazie, Ludwig Maximilian University, Munich, Germany.G-Protein coupled receptors (GPCRs) coordinate and regulate several cellularmechanisms and vital human activities such as behavior and sensing. Hence,GPCRs constitute a superfamily of membrane proteins that encode approxi-mately 4 - 5 % of the entire human genome.1 Interestingly, few GPCRs repre-sent a huge pharmacological interest making them the target of ~ 50% of theprescribed drugs on the market. GPCRs, being ubiquitos and diverse, madethese membrane proteins an interesting system to study and understand.Most current assays lack the ability to provide the essential physiologicalexperimental conditions, which enables in vivo studies addressing the GPCR-ligand interactions. Here, we provide an image correlation spectroscopy basedassay that monitors and investigates GPCRs in live cells on a single moleculebasis. The presently developed assay utilizes fluorescence and image correla-tion methods2,3 to investigate several aspects of the GPCR membrane proteins.Raster image correlation spectroscopy, allows to scan and determine diffusioncoefficients of the receptors, was implemented to study the behavior ofdifferent GPCRs, like the b2-adrenergic and neurotensin receptors, beforeand after the binding of a certain ligand. Other image correlation spectroscopytechniques would be implemented to investigate the behaviour of these recep-tors at the membrane. For example, to study the associaton of GPCRs in themembrane, number and brightness would be a feasible analysis to perform inorder to question the degree of oligomerization the GPCRs would show inthe membrane.1 a) Rosenbaum, D. M; Søren, S. G. F.; Kobilka, B. K. Nature 2009, 459, 356-363; b) Bjarndottir, T. K; Gloriam, D. E.;Hellstrand, S. H.; Kristiansson, H.; Fredriksson, R.; Schioth, H. B Genomics2006, 88, 263-273.2 Foo, Y. H.; Naredi-Rainer, N; Lamb, D. C.; Ahmed, S.; Wohland, T. Biophys.J. 2012, 102, 1174-1183.3 a) M€uller, B. K.; Zaychikov, E.; Br€auchle, C.; Lamb, D. C. Biophys. J. 2005,89, 3508-3522; b) Hendrix, J.; Schrimpf,W.; Holler, M.; Lamb, D. C. Biophys. J. 2013, 105, 848-861.

718-Pos Board B483Single Molecule Imaging Reveals Dysferlin-Mediated Recruitment ofPhosphatidylserine in Cell Membrane RepairLu Zhou1, Volker Middel1, Uwe Str€ahle1, G. Ulrich Nienhaus1,2.1Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany, 2Universityof Illinois at Urbana-Champaign, Urbana, IL, USA.Dysferlin (Dysf) has been implicated as a key protein for membrane repair inmuscle cells, although its precise biological function is still unknown. Dysfis a transmembrane protein, and its rapid recruitment of Dysf to membrane le-sions has been observed; direct observation of Dysf transport has so far re-mained elusive [1]. Moreover, interaction of Dysf with various other proteinshas been investigated for many years to understand its role in cell membranerepair; however, studies addressing the role of phospholipids, the main compo-nents of the plasma membrane, are still scarce.We have observed fast transport of plasma membrane-derived Dysf in livingzebrafish by using fluorescence loss in photobleaching (FLIP) and single

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molecule localization imaging [2]. In addition, fluorescence imaging of humancells and zebrafish has revealed rapid phosphatidylserine (PS) accumulation inthe repair patch in a Dysf-mediated fashion. Single molecule trajectory analysisprovides evidence that Dysf facilitates PS translocation from adjacent mem-brane regions to the site of lesion.[1] A. Lek et al., ‘‘Calpains, cleaved mini-dysferlinC72, and L-type channelsunderpin calcium-dependent muscle membrane repair’’, J. Neurosc., 2013,33, 5085-94.[2] Middel, V., Zhou, L., Takamiya, M., Beil, T., Shahid, M., Roostalu, U.,Grabher, C., Rastegar, S., Reischl, M., Nienhaus, G. U., & Str€ahle, U., ‘‘Dys-ferlin-Mediated Phosphatidylserine Sorting Engages Macrophages in Sarco-lemma Repair’’, Nat. Commun., 2016, 7, 12875.

719-Pos Board B484Rotation of Single Cell Surface Molecules Examined via Polarized FCSMeasurements using Quantum Dot ProbesDomgmei Zhang1, Peter W. Winter1, Deborah A. Roess2,B. George Barisas1.1Department of Chemistry, Colorado State University, Fort Collins, CO,USA, 2Department of Biomedical Sciences, Colorado State University, FortCollins, CO, USA.Rotational motions of cell surface molecules are of particular interest giventhe sensitivity of such motions to molecular size and aggregation state. Thetime-autocorrelation function (TACF) of fluctuations in fluorescence polariza-tion from asymmetric quantum dots (QD) such as Molecular Probes’ Qdot655labeling cell surface molecules indicates the rotational correlation times(RCT) of the QD-containing complexes. Type I Fcε receptors (FcεRI) and in-sulin receptors (IR) on 2H3 cells, subjected to various treatments potentiallyaffecting receptor rotation, including polyvalent DNP-BSA, methyl-b-cyclo-dextrin, cytochalasin D or paraformaldehyde, were labeled by Qdot655s.Over 700 individual QD were examined. Side-by-side vertically(v)- andhorizontally(h)-polarized fluorescence images of QD-labeled cells were ob-tained simultaneously as image stacks at 10 ms per frame by means of aPrinceton Instrument Dual-View. To minimize apparent polarization TACFarising from QD lateral diffusion, v- and h-substacks were aligned to sub-pixel accuracy. From fluorescence in identical regions around each QD ineach stack, intensity and polarization fluctuation TACF were calculated.Given the blinking of QD, experimental parameters such as g-factor, camerabackground, etc. were carefully optimized for each QD to minimizecross-correlation between polarization and intensity fluctuations. This couldotherwise appear as intensity fluctuation TACF contributing to the apparentpolarization fluctuation TACF. Using these techniques, the initial polarizationfluctuation TACF for FcεRI averages about 0.0015 and the geometrically-averaged RCT is about 55-85 ms, both independent of cell treatment. Morelimited results on IR appear similar. Absence of treatment effects on magni-tudes or decay rates of polarization TACF is puzzling. Previoustime-resolved phosphorescence anisotropy studies on FcεRI suggest limitedpresence of orientational relaxations slower than 1 ms. However, the absenceof treatment effects here suggests such slow reorientation may be a property ofthe membrane itself, perhaps reflecting large-scale fluctuations of mesoscalemembrane regions. Supported by NSF grant MCB-1024668 and NIH grantCA175937 to BGB.

720-Pos Board B485Super-Resolution Imaging Reveals Protein-Templated Patterns for Bio-silica FormationPhilip Groger, Nicole Poulsen, Jennifer Klemm, Nils Kroger,Michael Schlierf.B CUBE – Center for Molecular Bioengineering, TU Dresden, Dresden,Germany.The intricate, genetically controlled biosilica nano- and micropatterns pro-duced by diatoms are a testimony for biology’s ability to control mineral for-mation (biomineralization) at the nanoscale and regarded as paradigm fornanotechnology. Several recently discovered protein families involved indiatom biosilica formation remain tightly associated with the final biosilicastructure. Determining the locations of biosilica-associated proteins with highprecision is therefore expected to provide clues to their roles in biosilicamorphogenesis. To achieve this, we introduce single-molecule localization mi-croscopy to diatoms based on photo-activated light microscopy (PALM) toovercome the diffraction limit. We identified six photo-convertible fluorescentproteins (FPs) that can be utilized for PALM in the cytoplasm of Thalassiosirapseudonana. However, only three FPs that share a common molecularconversion-mechanism were also functional when embedded in diatom bio-

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silica and localized with a mean precision of 25 nm to resolve structural fea-tures. Further co-localization studies on proteins of the Cingulin family whenextracted from the biosilica using a combined two-color PALMþSTORMapproach revealed characteristic protein filaments with distinct protein specificpatterns. The enhanced microscopy techniques introduced here for diatoms willaid in elucidating the molecular mechanism of silica biomineralization as wellas other aspects of diatom cell biology.

721-Pos Board B486Quantitative Super-Resolution Microscopy Detects HER2 ReorganizationFollowing Meditope-Antibody TreatmentDevin Wakefield, Raphael Jorand, Cindy Zer, John Williams,Tijana Jovanovic-Talisman.Beckman Research Institute at the City of Hope, Duarte, CA, USA.We combine quantitative super-resolution microscopy and meditope technol-ogy to investigate the effects of cross-linked antibodies on the organization ofcancer receptor targets. A cyclic peptide, known as a meditope, has beenrecently discovered to bind within the Fab framework of Cetuximab andvarious meditope enabled monoclonal antibodies (memAbs). In this work,several divalent, Fc-based meditope ligands were generated with differentlinkers (linear sequences with 10, 20, or 30 amino acids). BT-474 cellswere incubated with these meditopes, together with memAb Trastuzumab,and subsequently fixed for dSTORM imaging of human epidermal growthfactor receptor 2 (HER2). The spatial organization of HER2 was character-ized through pair-correlation (PC) analysis, using streamlined Matlab codewith minimal user input and methods to reduce bias in selecting regions foranalysis. According to our results, HER2 organization strongly depends onmeditope linker length. Importantly, an increase in linker length was foundto correlate with an increase in the proportion of HER2 oligomers. PC anal-ysis further revealed that these HER2 oligomers organize within clustersranging from approximately 14 to 25 nm in radius. Ultimately, exploringthe molecular organization of receptors with our approach serves as an impor-tant step toward optimizing a diversity of ligands with potential therapeuticpurposes.

722-Pos Board B4873D Orbital Tracking of Single Gold Nanoparticles: A New Approach toStudy Vesicle Trafficking in Chromaffin CellsManuela Gabriel1, Jose Moya-Diaz2, Fernando D. Marengo2,Laura C. Estrada1.1Quantum Electronics Laboratory, Physics Department, University ofBuenos Aires, Buenos Aires, Argentina, 2Institute of Physiology, MolecularBiology and Neuroscience, University of Buenos Aires, Buenos Aires,Argentina.Endocytosis and subsequent vesicle recycling serves to keep constant thepools of transmitter-containing vesicles ready for release in neurons andendocrine cells. The study of these processes has been carried out by usingdifferent experimental approaches, like electrophysiological measurementsand single photon fluorescence microscopy. However, the diverse experi-mental limitations of these techniques restricted a detailed and high resolvedstudy of the dynamics of vesicle trafficking after endocytosis in the wholecellular volume. Multiphoton microscopy provides optical sectioning forhigh-resolution imaging. In biological systems, most multiphoton micro-scopy studies have relied on two-photon excited fluorescence (TPEF) to pro-duce images. Because of their strong brightness and imaging durability,metallic NPs have been recently introduced as labels in fluorescence micro-scopy. The use of TPEF and metallic nanoparticles in combination provides anoninvasive, spatially localized, in vivo characterization for biological sam-ples. In this work, we tracked single gold nanoparticles after endocytoticinternalization in mouse chromaffin cells stimulated with high potassium.We use an orbital-scanning tracking method in a two-photon absorption mi-croscope. In the first place, we compare constitutive and active internalizationof gold nanoparticles (AuNPs), assessing the number of internalized AuNPsand evaluating its dynamics in terms of velocity and displacement after stim-ulation. In the second place, we evaluate these parameters pharmacologicallyblocking proteins classically involved in the development of endocytotic pro-cess. Our results show that endocytosis of AuNPs was much more efficientwhen exocytosis was induced with high K in comparison with constitutivecycling. In addition, the dynamics of AuNPs had a strong dependence on cla-thrin dependent endocytosis as well as on cortical actin polymerization. Thisstudy shows that the combination of 3D orbital tracking and AuNPs is aninteresting tool for the study of vesicle trafficking after endocytosis in livecells.

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723-Pos Board B488Super-Resolution Imaging of DNA Replisome Dynamics in Live BacillusSubtilisYilai Li1, Jeremy W. Schroeder2, Yi Liao3, Lyle A. Simmons1,Julie S. Biteen1.1University of Michigan, Ann Arbor, MI, USA, 2University of Wisconsin,Madison, WI, USA, 3University of Chicago, Chicago, IL, USA.DNA replication happens in all living organisms, and assures that the genome isaccurately copied and maintained. The replisome is the molecular machine incells that replicates DNA, and it is composed of several different proteins,including DNA polymerases, which directly synthesize DNA by adding nucle-otides. Although the bacterial replisome has been studied extensively in vitro,little is known about the dynamics and architecture of replisome componentsin vivo. Here we use Bacillus subtilis, a Gram-positive bacterium commonlyfound in soil, as a model organism in which to study the architecture anddynamics of several replisome components in vivo. Photoactivated localizationmicroscopy (PALM) and single-molecule tracking give a resolution of 20-40 nm, far below the diffraction limit of conventional microscopy, enablingus to localize and track every single protein molecule. In our study, we inves-tigated the dynamics of a number of replisome components under different con-ditions, including the DNA polymerases PolC and DnaE, and the b-clamploader DnaX. We can watch the real time behavior of different replisome com-ponents during the DNA synthesis process, and study them quantitatively. Sur-prisingly, our investigations have revealed that all these replisome componentsare highly dynamic and exchange more rapidly than previously expected, andwe characterize the molecular scale distribution of each replisome componentwithin the dynamic replication machinery.

724-Pos Board B489Single-Molecule Tracking Reveals Altered Dynamics of a TranscriptionRegulator Expressed at Similar Levels from Different Gene ExpressionSystemsChanrith Siv1, David J. Rowland1, Victor J. DiRita2, Julie S. Biteen3.1Biophysics, University of Michigan, Ann Arbor, Ann Arbor, MI, USA,2Microbiology & Molecular Genetics, University of Michigan, Ann Arbor,East Lansing, MI, USA, 3Chemistry, University of Michigan, Ann Arbor,Ann Arbor, MI, USA.The development of fluorescent proteins and their application in live-cell imag-ing have not only validated previously known molecular processes but havealso unraveled new biological mechanisms. Though the functionality and sta-bility of fusions to fluorescent proteins are routinely characterized, there isno strict agreement in the community on how to express these fusion proteinsin cells. Here, we measure differences in the single-molecule trajectories of atranscription regulator protein, TcpP, depending on its expression method inVibrio cholerae. TcpP was fluorescently labeled with the photoactivatable fluo-rescent protein PAmCherry at its C-terminus and was either expressed off of thenative TcpP promoter in the chromosome or from an arabinose-inducible pro-moter PBAD. Even though we detected similar levels of tcpP-pamcherrymRNAtranscripts and TcpP-PAmCherry protein concentrations in vitro with bothexpression methods, single-molecule tracking of the protein in live V. choleraereveals differences which can obfuscate biological interpretation of the role ofTcpP in toxin production. In the plasmid-induced strain, we observed (1) moreslowly diffusing TcpP-PAmCherry molecules, (2) an at least a five-fold in-crease in copies of TcpP-PAmCherry, contrary to the in vitro western results,and (3) the absence of a very rapidly diffusing population that is observedupon endogenous expression. In addition, we compared the dynamics ofTcpP-PAmCherry in V. cholerae to TcpP-PAmCherry in wildtype Escherichiacoli, in which background TcpP should not have any relevant biological activ-ity. Our findings suggest that single-molecule tracking of proteins provides avery sensitive assay to detect subtle differences in protein dynamics—andthus activities—that are hidden in vitro.

725-Pos Board B490Fluorescence Diffusion Tensor Maps of p53 Activation Acquired withSPIMMichelle A. Digman, Lukas Rottsch€afer, Enrico Gratton, Per Niklas Hedde.Biomedical Engineering, University of California, Irvine, Irvine, CA, USA.The transcription factor, p53, can activate the DNA repair or apoptoticpathway in response to cellular stress. Specific binding sites must be acces-sible to active either pathway. Thus, specific chromatin location as well asaccessibility are required for stable tetramers of p53 to activate eitherresponse pathway. To gain information regarding p53 fast dynamic bindinginteraction and diffusion to localized nuclear regions, we used single planeillumination microscopy (SPIM) to acquire high speed frame rate imageswith a time resolution below 1 ms. Cells were treated with either anisomyocin

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or cisplatin to induce cellular stress. To obtain maps of protein diffusion anddirectional local motion the fluorescence Diffusion Tensor Imaging (fDTI)method, inspired by the Diffusion Tensor imaging in the MRI field, wasused. This powerful technique provides maps of molecular diffusivity andprotein interactions in the context of their endogenous environments. ThefDTI is based on spatial pair correlations between adjacent pixels in a givennumber of radial angles that are explored in a given point to determine theanisotropic diffusion. The time points for the pCF correlation function arecalculated in a log scale using by dividing the tau axis in 32 segments equallyspaced in the log scale. Thus diffusion and connectivity maps are obtained bycalculating pair correlation functions of each voxel with all the neighbor vox-els. For this work we show changes in molecular diffusion and alterations ofprotein connectivity post activation of the cell stress response mechanism andare able to construct molecular connectivity maps of p53. This type of anal-ysis can provide a well-informed picture of spatial temporal p53 regulationthroughout the living cell. This work is supported in part by NIH grantsP50 GM076516 and P41 GM103540.

726-Pos Board B491Association of Endophilin B1 with Cytoplasmic VesiclesJinhui Li1, Barbara Barylko2, Joachim D. Mueller1, Joseph Albanesi2,Yan Chen1.1Physics, University of Minnesota, Minneapolis, MN, USA,2UT Southwestern Medical Center, Dallas, TX, USA.Studying protein association with cytoplasmic vesicles within the live cell re-mains a general challenge for optical microscopy techniques. Here, we showthat fluorescence fluctuation microscopy offers an effective approach to tacklethis problem by using endophilins as a model system. Endophilins are SH3-and BAR domain-containing proteins implicated in membrane remodelingand vesicle formation. Endophilins A1 and A2 promote the budding ofendocytic vesicles from the plasma membrane, whereas endophilin B1 hasbeen implicated in vesicle budding from intracellular organelles, includingthe trans-Golgi network and late endosomes. We previously reported thatendophilins A1 and A2 exist almost exclusively as soluble dimers in thecytosol. Here we present results of fluorescence fluctuation spectroscopy an-alyses indicating that, in contrast, the majority of endophilin B1 is presentin multiple copies on small, highly mobile cytoplasmic vesicles. Formationof these vesicles was enhanced by overexpression of wild-type dynamin 2,but suppressed by expression of a catalytically inactive dynamin 2 mutant.Using dual-color heterospecies partition analysis, we identified the epidermalgrowth factor receptor (EGFR) on EndoB1 vesicles. Moreover, a proportionof EndoB1 vesicles also contained caveolin, whereas clathrin was almost un-detectable on those vesicles. These results raise the possibility that endophilinB1 participates in dynamin 2-dependent formation of a population of transportvesicles distinct from those generated by A-type endophilins. This work hasbeen supported by a grant from the National Institutes of Health (R01GM64589).

727-Pos Board B492Characterization of Ire1 Interactions and Dynamics with QuantitativeSuper-Resolution MicroscopyElizabeth M. Smith, Ragnar Stefansson, Maria Paz Ramirez Lopez,Elias M. Puchner.Physics, University of Minnesota, Minneapolis, MN, USA.Quantitative Super-Resolution Microscopy is a powerful technique that can beused to study biological processes below the diffraction limit. In this work, weemploy our intracellular calibrated Photoactivated Localization Microscopy(PALM) technique to perform quantitative molecular counting of proteinsinvolved in the unfolded protein response (UPR). The UPR is a signalingpathway which dynamically regulates endoplasmic reticulum (ER) proteinfolding capacity in response to cellular stress. As is true with many signalingpathways, the spatiotemporal organization of the UPR-specific biomoleculesis an inherent feature of the pathway activation and downstream response. Spe-cifically, in response to stress, Ire1 (a bifunctional transmembrane kinase/endoribonuclease) oligomerizes and forms discrete signaling clusters whichrecruit and splice an mRNA encoding a transcription activator. Using PALMin conjunction with traditional fluorescence microscopy we characterize the in-teractions and dynamics of Ire-1 at wild type expression levels in yeast cells.Specifically, we quantify the oligomeric state, of Ire1 under stressed andunstressed conditions and track the motion of Ire1 during signaling activityto study the kinetics of cluster formation. Finally we perform colocalization ex-periments with downstream UPR biomolecules to further characterize the roleof Ire1 signaling centers in control of gene expression. This study providesinsight into the spatiotemporal organization of Ire1 and its downstream partnersin the signaling response of the UPR.

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728-Pos Board B493Diffusion of DNA-Binding Species in Thenucleus: A Transient AnomalousSubdiffusion ModelMichael J. Saxton.Biochemistry & Molec Med, University of California, Davis, CA, USA.Recent single-particle tracking experiments have measured the distribution ofdwell times of DNA-binding species diffusing in living cells: CRISPR-Cas9,TetR, and LacI [Knight, Science 2015; Normanno, Nat Commun 2015; Caccia-nini, Faraday Disc 2015]. The observed distribution, a truncated power law, im-plies transient anomalous subdiffusion, in which diffusion is anomalous at shorttimes (mean-square displacement proportional to t^a, a< 1) and normal at longtimes (MSD proportional to t) [Saxton, Biophys J 66 (1994), 70 (1996), 92(2007)]. Monte Carlo simulations are used to characterize the time-dependentdiffusion coefficient D(t) in terms of the exponent a, the crossover time, D(0),and D(N), and these quantities in terms of the dwell time distribution. Thesimplest interpretation is that the dwell time is an actual binding time toDNA. One alternative interpretation is that the dwell time is the period of 1Ddiffusion on DNA in the standard model combining 1D and 3D search. Themodel has several implications for cell biophysics. (1) The initial anomalousregime represents the search of the DNA-binding species for its target DNAsequence. (2) Non-target DNA sites have a significant effect on search kinetics.False positives in bioinformatic searches are potentially rate-determiningin vivo. For simple binding, the search would be speeded if false-positive se-quences were eliminated from the genome. (3) Both binding and obstructionaffect diffusion. The proper controls for obstruction are GFP as a calibrationstandard among laboratories and cell types, and the DNA-binding specieswith the binding site inactivated. (4)Overexpression of theDNAbinding speciesreduces anomalous subdiffusion because the deepest binding sites are occupiedand unavailable. (5) The model provides a coarse-grained phenomenologicaldescription of diffusion of a DNA-binding species, useful in larger-scalemodeling of kinetics and FRAP. (Supported in part by NIH grant GM038133).

729-Pos Board B494Spatial Dynamics of SIRT1 Relates to Metabolic Transitions in the CellNucleusSuman Ranjit1, Lorena Aguilar-Arnal2,3, Chiara Stringari1,4,Paolo Sassone-Corsi2, Enrico Gratton1.1Biomedical Engineering, University of California Irvine, Irvine, CA, USA,2Center for Epigenetics and Metabolism, University of California Irvine,Irvine, CA, USA, 3Institute for Biomedical Research, UNAM, Mexico,Mexico, 4Laboratory for Optics and Biosciences, Ecole Polytechnique, Paris,France.SIRT1 is a NADþ -dependent deacetylase functioning as metabolic sensor ofcellular energy and it adapts different biochemical pathways to the changesin the environment. SIRT1 substrates include histones and proteins related toenhancement of mitochondrial and antioxidant protection. Fluctuations in intra-cellular NADþ levels regulate SIRT1 activity, yet the exact pathway SIRT1enzymatic activity impacts NADþ levels and its intracellular distribution re-mains unclear. Here, we demonstrate that SIRT1 determines the nuclear orga-nization of protein bound NADH. Using multiphoton microscopy in live cells,we show that free and bound NADH are compartmentalized inside of the nu-cleus, and its subnuclear distribution depends on SIRT1. Importantly, SIRT6,a chromatin-bound deacetylase of the same class does not influence NADH nu-clear localization. In addition, using fluorescence fluctuation spectroscopy,especially phasorFCS in single living cells, we reveal that NADþ metabolismin the nucleus is linked to subnuclear dynamics of active SIRT1. SIRT1 dif-fuses faster on the periphery of nucleus and the diffusion is slower in the center.Comparison of results from phasorFCS and autofluorescence FLIM divulge arelationship between NADþ metabolism, NADH distribution and SIRT1 activ-ity in the nucleus of live cells, and leads off to decipher links between nuclearorganization and metabolism.

730-Pos Board B495Spatiotemporal Fluctuation Analysis: A Powerful Tool for the FutureNanoscopy of Dynamic Molecular ProcessesFrancesco Cardarelli1, Enrico Gratton2, Fabio Beltram3,Carmine Di Rienzo3.1Nanomedicine, Center for Nanotechnology Innovation at NEST, IstitutoItaliano di Tecnologia, Pisa, Italy, 2Department of Biomedical Engineering,Laboratory for Fluorescence Dynamics, University of California at Irvine,California, US, Irvine, CA, USA, 3Center for Nanotechnology Innovation atNEST, Istituto Italiano di Tecnologia; NEST, Scuola Normale Superiore andIstituto Nanoscienze-CNR, Pisa, Italy.A major challenge of present and future biophysics is to quantitatively studyhow biomolecules dynamically fulfill their physiological role in living cells, tis-

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sues, or entire organisms. In the last few decades, new experimental methodol-ogies were introduced that are able to unveil details on a length scale that is atiny fraction of the wavelength of light, thus moving spatial resolution farbeyond the diffraction limit set by Ernst Abbe’s equation. Still, the enormouswealth of information available today from optical microscopy measurementson living samples is often underexploited. We argue that spatiotemporal fluo-rescence correlation spectroscopy (spFCS) can enhance the performances ofcurrent nanoscopy methods and provide further insight into dynamic molecularprocesses of high biological relevance (Di Rienzo, C. et al. Biophys J, 111, 679-685, 2016). We present exemplary biological applications of spFCS to measuredynamic molecular parameters well below the diffraction limit in a standardoptical setup, including the measurement of the nanoscale displacement ofGFP in the cell cytoplasm (Di Rienzo, C. et al. Nat comm 5, n.5891, 2014),and of Transferrin Receptor-GFP (TfR-GFP) sub-diffraction confinement onthe plasma membrane of live cells (Di Rienzo, C. et al. PNAS, 110 (30),12307-12312, 2013). Also we discuss how standard super-resolution methods,which are intrinsically endowed with high static spatial resolution properties,can take advantage of the resolution improvements that are accessible throughspFCS to describe dynamical molecular processes. Finally, we argue that by us-ing spFCS we can definitely integrate the arsenal of methods at our disposal toinvestigate living matter at the nanoscale.

731-Pos Board B496Bacterial Type 3 Secretion Systems: High-Throughput 3D Single-MoleculeTracking of Sorting Platform Proteins in Live CellsJulian Rocha1, Andreas Diepold2, Judith P. Armitage2,Andreas Gahlmann3.1Department of Chemistry, University of Virginia, Charlottesville, VA, USA,2Department of Biochemistry, University of Oxford, Oxford, UnitedKingdom, 3Departments of Chemistry and Molecular Physiology &Biological Physics, University of Virginia, Charlottesville, VA, USA.Bacterial secretion systems are large biomolecular assemblies that rely on staticand transient interactions between individual molecular subunits. A centralexample is the Type 3 Secretion System (T3SS) which consists of both thestatic membrane-embedded needle complex and the much more dynamic cyto-plasmic sorting platform. Single-subunit turnover in the sorting platform andthe resulting structural heterogeneity have made it challenging to decipherthe molecular-level mechanism of Type 3 secretion. Live-cell single-moleculesuper-resolution microscopy is ideally suited to measure spatial locations andtrajectories of individual molecular subunits with nanoscale precision. Extract-ing meaningful biological results, however, requires characterizing the entiredistribution of molecular behaviors, which in turn, necessitates a large numberof individual measurements. Here, we apply high-throughput aberration-cor-rected 3D single-molecule localization microscopy to quantitatively measurethe diffusion behaviors of over 100,000 individual T3SS sorting platform pro-teins. The single-molecule trajectories reveal multiple diffusive populations inthe bacterial cytoplasm suggesting the pre-formation of functionally importanthigher-order molecular complexes. By providing information on the spatiotem-poral regulation of protein function in living cells, our results complementrecent structural and biochemical findings that the cytoplasmic T3SS sortingplatforms contain large pod-like structures and that cytoplasmic C-ring proteinsmay pre-assemble into oligomeric complexes prior to binding to the T3SS sort-ing platforms.

732-Pos Board B497Molecular Tattoo: Subcellular Confinement of Drug Effects In Vivo withTwo-Photon MicroscopyBoglarka Varkuti1, Miklos Kepiro1, Anna A. Rauscher2, Laszlo Vegner1,Aron Zsigmond1, Vanda Imrich1, Szilvia Rati1, Adam I. Horvath1,Mate Varga3, Miklos S. Kellermayer4, Malnasi-Csizmadia Andras1.1Department of Biochemistry, MTA-ELTE Molecular Biophysics ResearchGroup, Budapest, Hungary, 2Printnet Ltd., Budapest, Hungary, 3Departmentof Genetics, Eotvos University, Budapest, Hungary, 4Department ofBiophysics and Radiation Biology, MTA-SEMolecular Biophysics ResearchGroup, Budapest, Hungary.Technological resources for sustained local control of molecular effects withinorgans, cells, or subcellular regions are currently unavailable, even though suchtechnologies would be pivotal for unveiling the molecular actions underlyingcollective mechanisms of neuronal networks, signaling systems, complexmachineries, and organism development. We present a novel optopharmacolog-ical technology namedmolecular tattooing, which combines photoaffinity label-ing with two-photon microscopy.Moleculartattooing covalently attaches aphotoreactive bioactive compound to its target by two-photon irradiationwithout any systemic effects outside the targeted area, thereby achievingsubfemtoliter,long-term confinement of target-specific effects in vivo. As wedemonstrated in melanoma cells and zebrafish embryos, molecular tattooing is

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suitable for dissecting collective activities by the separation of autonomous andnon-autonomous molecular processesin vivo ranging from subcellular to organ-ism level. Since a series of drugs are available for molecular tattoo, the technol-ogy can be implemented by a wide range of fields in the life sciences. Supportedby the Hungarian Research and Innovation Fund (VKSZ_14-1-2015-0052).

733-Pos Board B498Human Subcutaneous Adipose Tissue Adipocytes Demonstrate Two Phys-iological States: Insulin Responsive or Insulin RefractoryChad D. McCormick, Hang Waters, Ludmila Bezrukov, Brad Busse,Andrew Demidowich, Paul S. Blank, Jack A. Yanovski,Joshua J. Zimmerberg.NICHD, NIH, Bethesda, MD, USA.Insulin resistance is a precursor to Type II Diabetes. Peripheral insulin resis-tance, as exemplified by the response of tissues like adipose to an insulin chal-lenge, is a tissue average traditionally monitored by glucose uptake assays. Wehypothesize that these assays may not reflect cellular level heterogeneousbehavior and instead report a weighted response of insulin responsive and re-fractory adipocytes. We developed new assays to monitor the adipocyte insulinresponse in the context of ex vivo tissue samples. Within one hour of biopsyfrom subjects recruited to the NIH Clinical Center, we tested if AKT phosphor-ylation, one of the major signaling nodes of the canonical insulin signalingpathway, is sufficient to monitor the number of insulin responsive or refractorycells in fixed human tissue. Immunostaining revealed two adipocyte popula-tions: low pAKT cells, primarily seen in the absence of insulin stimulation,and high pAKT cells, primarily seen in tissue from healthy subjects after insulinstimulation. The fraction of tissue with a large pAKT response to insulin cor-relates well with the fraction of tissue with GLUT4, the insulin-stimulatedglucose transporter, localized in the adipocyte plasma membrane. The pAKTfraction also supports a two-component model: insulin responsive versus insu-lin refractory adipocytes, rather than a graded continuum of insulin responses,when the pAKT fraction is matched with each subject’s insulin sensitivity in-dex (SI), calculated using the insulin-modified frequently sampled intravenousglucose tolerance test (FSIVGTT). These results agree with previous work fromour lab using isolated cells in which the healthier SI value a subject has, thegreater fraction of their subcutaneous adipocytes respond to insulin.

734-Pos Board B499Single Cell Examination of Membrane Fluidity and Cellular RespirationKrishna Ojha, John Ertle, Michael C. Konopka.Chemistry, The University of Akron, Akron, OH, USA.Bulk measurements of oxygen consumption rates by bacteria have previouslybeen shown to be related to the cells’ average membrane fluidity (as measuredby the diffusion of fluorescently-labeled molecules). Within a bacterial popula-tion, there is also significant cell-to-cell variation in both the diffusion coeffi-cient of membrane probes and oxygen consumption rates of individual cells.One possibility is the diffusion of ubiquinone, an electron carrier in the electrontransport chain (ETC), is being limited by the fluidity of the membrane which iscausing the heterogeneity.We describe Fluorescence Recovery After Photobleaching (FRAP) measure-ments to monitor cell-to-cell variation in membrane diffusion coefficient.These results are compared to two approaches to look at what influence it mighthave on respiration at the single cell level. The first uses a fluorescent indicatorof the activity of the ETC. The second directly measures the consumption ofoxygen by individual cells using a phosphorescent Pt-porphyrin dye.

735-Pos Board B500An Improved Single Molecule Imaging Vivo Method for In Vivo Stoichio-metric and Functional Analysis of Protein ComplexesWarren R. Zipfel1, Avtar Singh2, Maria Sirenko3, Alexander Song4,Paul Kammermeier5.1Biomedical Engineering, Cornell University, Ithaca, NY, USA,2Applied Physics, Cornell University, Ithaca, NY, USA, 3Cornell University,Ithaca, NY, USA, 4Princeton University, Princeton, NJ, USA, 5University ofRochester Medical Center, Rochester, NY, USA.Elucidating the composition and stoichiometry of membrane bound and cyto-solic protein complexes is critical to understand their biological function andthe underlying molecular mechanisms involved. Existing techniques used todetermine subunit stoichiometry in single molecule experiments may signifi-cantly bias experimental results due to the need for either extremely lowexpression levels required to obtain concentrations suitable for single moleculeimaging, or in the case of protein complex isolation for in vitro characteriza-tion, perturbation of the normal stoichiometric relationships by the isolationmethod used. Here we present an alternative approach in which protein com-plexes are assembled at physiological concentrations and subsequently dilutedby conditionally controlled cell fusion to obtain protein levels suitable for

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single-molecule observation while preserving the complexes in a near-nativelive cell environment. Methods such as stepwise photobleaching can thenbe used to study the subunit stoichiometry of membrane receptors, or for cyto-solic complexes, this strategy greatly facilities fluorescence correlation spec-troscopy based methodologies which can provide a quantitative assessmentof cytoplasmic oligomerization state. We present examples that illustratethe advantages of the method for both membrane and intracellular cytosolic in-vestigations. The method, called Single Protein Recovery After Dilution(SPReAD), is a simple and versatile means of extending the concentrationrange of single molecule measurements to what are often more normal cellularlevels with minimal perturbation of protein complex stoichiometry.

736-Pos Board B501Visualizing Heterogeneous Single-Molecule Dynamics of MolecularAssemblies in Live CellsMichael Lacy1, David Baddeley2, Julien Berro1.1Molecular Biophysics and Biochemistry, Yale University, West Haven, CT,USA, 2Cell Biology, Yale University, West Haven, CT, USA.Molecular assemblies can have highly heterogeneous dynamics within the cell.Classic fluorescence microscopy methods, such as FRAP (Fluorescence Recov-ery After Photobleaching), have been invaluable to characterize these dynamicsat the micrometer scale. However, it has been particularly difficult to charac-terize molecular heterogeneities inside diffraction limited zones within multi-molecular assemblies in live cells. We have developed a novel fluorescentlabeling and imaging protocol, called Single Molecule Recovery After Photo-bleaching (SMRAP), which has allowed us to reveal the heterogeneous dy-namics of the eisosome, a multi-protein structure on the cytoplasmic face ofthe plasma membrane in fungi. By fluorescently labeling only a small fractionof cellular Pil1p, the core eisosome protein in fission yeast, we were able tovisualize whole eisosomes before photobleaching and, after photobleaching,the recovery of individual Pil1p molecules that bound to the structure with~30 nm precision. Further analysis of these sparsely labeled, dynamic struc-tures allowed us to show that Pil1p turnover is spatially heterogeneous. Weobserved that Pil1p molecules from the cytoplasm bind and unbind at theends of eisosomes, but not along the interior, supporting a new model of theeisosome as a dynamic filament. We expect our new SMRAP method will beeasily and broadly applicable to any molecular assembly in the cell, since itonly requires sparse labelling of proteins of interest and a standard TIRF setupwith single molecule detection capabilities.

Single-Molecule Spectroscopy I

737-Pos Board B502Single-Molecule Counting Applied to ImmunoassaysPatrick J. Macdonald, Qiaoqiao Ruan, Kerry M. Swift, Sergey Y. Tetin.Abbott Laboratories, Libertyville, IL, USA.We investigated the sensitivity of single-molecule TIRF counting in diagnosticimmunoassay applications. This work focused on using single-molecule tech-niques purely for detection, with the assay itself taking place on a separate plat-form with the detectable label being eluted for single-molecule measurement.Such an approach both limits the amount of background in the final measure-ment and sets up a universal detection procedure for a potentially wide varietyof immunoassays. We took advantage of the low volume required for single-molecule measurements and demonstrated a sample reloading approach tofurther concentrate the sample on the single-molecule surface. SM reload-ing—for a biotin-streptavidin surface capture reaction—is a remarkably robustprocedure, independent of starting concentration, and so can be used for assay-ing unknown samples. We tested model assays with single-molecule detectionto substantiate this approach and demonstrate the potential of single-moleculecounting for diagnostic applications.

738-Pos Board B503Comparing Antibody-Antigen Binding in Serum Versus Buffer with Fluo-rescence Correlation SpectroscopyDavid Ortiz, Isabel Yannatos, Abhinav Nath.Medicinal Chemistry, University of Washington, Seattle, WA, USA.Therapeutic proteins or ‘biologics’ such as monoclonal antibodies (mAbs),antibody-drug conjugates (ADCs) or Fc-fusions comprise a growing percent-age of drugs in the development pipeline. A majority of techniques used tocharacterize these drugs require dilution into standard buffers, conditions thatdo not reflect the complexity of crowded biological environments such asserum. Here, we use fluorescence correlation spectroscopy (FCS) to directlyassess the effect of biological solutions on the binding affinity of mAbs toantigen. FCS is a single-molecule technique that measures the mean time alabeled particle takes to diffuse across a small (~1fL) confocal volume. Diffu-sion time is directly proportional to hydrodynamic radius, thus binding can be

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tracked with changes in diffusion time of the larger mAb-antigen complex.With this approach, we measured the binding affinities of Alexa-488 labeledstreptavidin and anti-streptavidin immunoglobulin G2 (IgG2) in buffer andneat serum, and found that binding is ~3-fold tighter in serum versus buffer.Through control experiments in viscosity-matched sucrose solution, we foundthat differences in viscosity may account for some but not all of this effect.Serum contains high concentrations of co-solutes of various sizes includingalbumin and g-globulins. We tested the effects of macromolecular crowdingon binding using physiological concentrations of bovine serum albumin aswell as polymeric crowders, and found that crowding also partially accountsfor the tighter binding of the streptavidin-IgG2 pair in serum. Conversely,serum does not affect the binding affinity of anti-streptavidin IgG1 to strepta-vidin. Accurate characterization of therapeutic proteins in relevant conditions isintegral to assessing their safety and efficacy. The implications of these resultswith respect to drug development are discussed.

739-Pos Board B504In Vitro Binding of 6S RNA Mango to RNA Polymerase by Two PhotonFluorescence Cross Correlation SpectroscopyS. Shyam Sundar Panchapakesan1, Eric J. Hayden2, Peter Unrau1,Matthew L. Ferguson3.1Department of Molecular Biology and Biochemistry, Simon FraserUniversity, Burnaby, BC, Canada, 2Department of Biological Science, BoiseState University, Boise, ID, USA, 3Department of Physics, Boise StateUniversity, Boise, ID, USA.The assembly of RNA and protein complexes is a fundamental process for lifebut has been difficult to study both in vitro and in vivo. Recently a novelaptamer based method to fluorescently label RNA molecules has beenreported(Dolgosheina et al. 2014). Here we report the successful utilizationof the RNAMangoþ thiazole orange derivative to measure the in vitro bindingof 6S RNA to RNA Polymerase from E. coli. By two photon FluorescenceCross Correlation Spectroscopy, we are able to independently measure the mo-lecular brightness, diffusion coefficient and concentration of 6S RNA Mango,RNA Polymerase GFP and complex as we increase RNA Polymerase concen-tration giving us an estimate for the equilibrium binding constant of the com-plex in vitro. This experiment demonstrates the utility of RNA Mango for bothin vitro and in vivo single molecule experiments.Dolgosheina, Elena V., Sunny C. Y. Jeng, Shanker Shyam S. Panchapakesan,Razvan Cojocaru, Patrick S. K. Chen, Peter D. Wilson, Nancy Hawkins, PaulA. Wiggins, and Peter J. Unrau. 2014. ‘‘RNA Mango Aptamer-Fluorophore:A Bright, High-Affinity Complex for RNA Labeling and Tracking.’’ ACSChemical Biology 9 (10): 2412-20.

740-Pos Board B505Single-Molecule Fluorescence Study of RNA Recognition by Viral RNAiSuppressorsMohamed Fareh1, Jasper van Lopik1, Iason Katechis1, Ronald van Rij2,Chirlmin Joo1.1Kavli Institute of Nanoscience, Bionanoscience Department, DelftUniversity of Technology, Delft, Netherlands, 2Department of MedicalMicrobiology, Radboud University Nijmegen Medical Centre, RadboudInstitute for Molecular Life Sciences, Nijmegen, Netherlands.RNA interference (RNAi) is an indispensable antiviral defensemechanism in in-sects including mosquitoes that transmit human diseases such as dengue or Zikafever. To escape the cellular defense, viruses employ various proteins called viralsuppressors of RNAi (VSRs). VSRs are thought to suppress the RNAi pathway atseveral different levels. This interaction precludes the recognition and elimina-tion of viral RNA, leading to viral survival and proliferation in detriment of thehost organism. Despite a decade of research on VSRs, how VSRs antagonizeRNAi remains incompletely understood. Here we employed single-moleculefluorescence techniques to investigate how VSRs inhibit viral RNA recognitionby Dicer-2, a key enzyme in RNAi antiviral defense. Our single-molecule datashowed that the majority of VSRs tested directly interact with double-strandedRNA (dsRNA), a viral replication intermediate. The stable binding inhibitsrecognition of viral dsRNA by Dicer-2 and consequently suppresses the RNAiantiviral pathway. The length of the double-stranded stem region is a crucialfeature in forming a stable interaction between viral dsRNA and VSRs.

741-Pos Board B506Dual Role of Munc13 in Regulating SNARE Assembly for Fast Neuro-transmitter ReleaseUcheor Brandon Choi.Molecular and Cellular Physiology, Stanford University, Stanford, CA, USA.

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Neuronal SNAREs (synaptobrevin, syntaxin, SNAP-25) are the minimal fusionmachinery, but require synaptotagmin, complexin, Munc13, and Munc18 forfast Ca2þ-triggered neurotransmitter release. Munc13s have a central role insynaptic vesicle priming through their MUN domain. Using single moleculeFRET we identified two distinct roles of the MUN domain in SNARE complexassembly for efficient Ca2þ-triggered fusion events. Prior to SNARE complexassembly syntaxin adopts a closed conformation tightly bound to Munc18. Wefound that the MUN domain changes the conformation of the linker region ofsyntaxin when bound to Munc18 providing a nucleation site for ternarySNARE complex formation. Interestingly we found an additional role of theMUN domain where it cooperates with Munc18 to ensure properly assembledSNARE complex, which resulted in a dramatic increase in Ca2þ-triggeredfusion efficiency and Ca2þ-sensitivity.

742-Pos Board B507Monitoring Small Molecule and G-Quadruplex Interactions and Kineticsusing Single Molecule FRETParastoo Maleki.Kent State University, Kent, OH, USA.Telomestatin and oxazole telomestatin derivatives (OTD) are small moleculesstabilizing G-quadruplex (GQ) structures and are prominent due to their anti-cancer drug potential. Despite the observation of enhanced thermodynamic sta-bility imparted by such small molecules on GQ, the underlying dynamics ofsmall molecule-GQ interactions are not known. To have a better understandingof these interactions we employed single molecule Forster resonance energytransfer (smFRET) to study the system, where we utilized a Cy5-labeledOTD (L1Cy5-7OTD). These studies demonstrate that interactions of this smallmolecule with GQ are dynamic in terms of binding kinetics and possibly interms of rotational freedom. The Cy5 fluorophore has enabled monitoringand quantifying binding, dwell, and dissociation of a single L1Cy5-7OTDmolecule as it interacted with GQ, which to our knowledge has not beendemonstrated for any GQ stabilizing small molecule before. We show thatL1Cy5-7OTD remains bound to GQ for tens of seconds, with significantlylonger dwell times and higher binding frequencies for more stable GQ. In addi-tion, we propose L1Cy5-7OTD to have at least two preferred primary bindingorientations and is able to transition between these orientations while it remainsstacked on a G-tetrad.

743-Pos Board B508Engineering ClpXP for Single-Molecule Protein SequencingMike Filius, Jetty van Ginkel, Chirlmin Joo.BioNanoScience, Delft University of Technology, Delft, Netherlands.Proteins are vital in all biological systems as they are involved in a largenumber of structural and functional pathways. Mass sprectometric tech-niques have been proposed to provide valuable tools for complete proteomicstudies of the human proteome. However, current mass spectrometers lackthe sensitivity for determining the least abundant proteins within a cell. Asingle-molecule method that analyses the proteins molecule by moleculemay have the sensitivity to shed light of the least abundant proteins withina single cell. The major challenge of protein sequencing lies in the factthat protein sequences consist of 20 different amino acids. Here, we aimto develop a novel protein sequencing technique that could identify proteinsbased on their fingerprint at the single-molecule level. In the novel method,proteins will be identified based on two types of amino acids only instead ofall 20 types. In the present study we demonstrate since-molecule FRETdetection of an acceptor labeled protein substrate by a donor labeled proteinanalyzer ClpXP. This highly sensitive approach holds promise to detect theleast abundant proteins of a cell, that can have high value for biomedicalsciences.

744-Pos Board B509Investigating the Mechanism of Ultra-Fast Energy Transfer betweenVenus Oligomers using Time-Resolved Anisotropy, Fluorescence Correla-tion Spectroscopy, and Photon AntibunchingYoungchan Kim1, Grace H. Taumoefolau1, Tuan A. Nguyen1,Henry L. Puhl1, Paul S. Blank2, Steven S. Vogel1.1NIH/NIAAA, Rockville, MD, USA, 2NIH/NICHD, Bethesda, MD, USA.FRET experiments using Fluorescent proteins (FPs) typically assume that indi-vidual FPs act independently (i.e. weak coupling limit). However, severalrecent studies have observed that assemblies of FPs can act as a single photonsource, indicating that FPs in these complexes interact in the strong couplinglimit and may explain our observation of ultra-fast (<6 ps) energy transfer be-tween FPs in Venus oligomers. To investigate the mechanism of this ultra-fast

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energy transfer, time-resolved anisotropy was used to measure the dynamics ofenergy transfer between fluorescent molecules, fluorescence correlation spec-troscopy (FCS) to measure the number of fluorescent molecules diffusing inthe focal volume, and photon antibunching (AB) to determine the number ofindependent fluorescent emitters in the focal volume. Because the number offluorescent molecules measured by FCS is not necessarily equal to the numberof independent emitters, a comparison of these values can provide insight intothe coupling strength required for ultra-fast energy transfer. We studied threeFP constructs; Venus monomers (V1), Venus dimers separated by a 5 flexibleamino acid linker (V2, ~2 nm separation), and Venus dimers separated by asemi-rigid linker containing 4 fibronectin type III domains (VFNV, ~14 nmseparation). Ultra-fast energy transfer and homo-FRET was only observed inV2. Thus, close proximity, even via a flexible linker, is correlated with ultra-fast energy transfer. FCS and ABmeasurements of systematic dilutions of thesethree constructs revealed that while VFNV has two independent emitters permolecule, V2 has less than two and because V1 is a monomer, each V1 mole-cule detected must have one independent emitter. These results suggest thatultra-fast energy transfer requires strong coupling between FPs, and implicatesproximity as an essential factor in the mechanism.

745-Pos Board B510Plasma Membrane Organization and Dynamics is Probe and Cell LineDependent: An Imaging FCS StudyThorsten Wohland1, Shuangru Huang2, Shi Ying Lim3, Anjali Gupta2,Nirmalya Bag2.1Biological Sciences and Chemistry, National University of Singapore,Singapore, Singapore, 2Biological Sciences, National Universityof Singapore, Singapore, Singapore, 3Chemistry, National University ofSingapore, Singapore, Singapore.The plasma membrane is a complex assembly comprising hundreds of differentlipids and proteins. Its properties are temperature dependent and change fromcell line to cell line. This raises the question whether measurements at differenttemperatures and within different cell lines are comparable even when usingthe same probe. Here, we investigate the dynamics and organization of fivedifferent cell lines - Chinese hamster ovary (CHO-K1), Hela, neuroblastoma(SH-SY5Y), fibroblast (WI-38) and rat basophilic leukemia (RBL-2H3) - usingup to four different lipid probes. For the outer membrane leaflet we use DiI-C18,a common liquid disordered marker and GFP-GPI, a GFP tagged glycosylphos-phatidylinositol anchored protein as a marker for the liquid ordered phase. Forthe inner membrane leaflet we use PH-PLCd-RFP, a PIP2 binding domain, andPMT-GFP, a GFP tagged plasma membrane targeting domain that had beenshown to be sensitive to the cytoskeleton. We applied Imaging FluorescenceCorrelation Spectroscopy (imaging FCS) on a Total Internal Reflection Micro-scope (TIRFM) which provided diffusion coefficients (D), the Arrhenius acti-vation energy for diffusion (EArr), and the FCS diffusion law intercept (t0),which report on membrane fluidity, molecular packing, and diffusion modeof the probes (free, domain, or hop diffusion), respectively. The combinationof these parameters is unique for each of the probes. Our results showed,that each of the labels is characterized by a unique set of parameters (D,EArr, t0). Furthermore, the parameters do not stay constant for a particularprobe or change in a concerted manner, but can change individually fromcell to cell line, indicating that the organization and mode of diffusion of thesemolecules depends strongly on the environment. This variability has importantimplications for membrane measurements in general and the comparability ofmeasurements is only given in a particular cell line and at a given temperature.

746-Pos Board B511Single Molecule Imaging of Transcription Factor-DNA Interactions inZebrafish DevelopmentMatthias Reisser1, Shai R. Joseph2, Nadine L. Vastenhouw2,J. Christof M. Gebhardt1.1Department of Physics, Ulm University, Ulm, Germany, 2Max PlanckInstitute of Molecular Cell Biology and Genetics, Dresden, Germany.Cellular tasks such as transcription rely on stochastic interactions of biomole-cules. Thus, single molecule methods are beneficial in revealing the kinetic andstructural underpinnings of these tasks, and consequently single moleculetracking has been developed for live cells and organisms. Yet, following indi-vidual fluorescently labeled biomolecules throughout the development of anorganisms is still challenging.Here, we establish single molecule tracking of genetically encoded fluorescentproteins in live developing Zebrafish embryos using reflected light-sheet micro-scopy. In addition, we design a novel time-lapse acquisition scheme allowingfor fast quantification of biomolecular interaction kinetics. We use our methodsto monitor and quantify the interactions between the general transcription fac-tor TBP and DNA during embryo development. We find that TBP interacts with

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DNA at all developmental stages, but binding to DNA changes from transientto stable within the first twelve cell division cycles. This kinetic transitiontemporally coincides with the delayed onset of transcription in the Zebrafishembryo. Our single molecule experiments reveal a potential mechanism ofan important step in Zebrafish embryo development.

747-Pos Board B512A Hidden Markov Model Approach to Measure Two-State Diffusion ofThermobifida Fusca CellulasesMarkus Rose1, Jose Moran-Mirabal2.1Department of Physics and Astronomy, McMaster University, Hamilton,ON, Canada, 2Department of Chemistry, McMaster University, Hamilton,ON, Canada.The hydrolysis of crystalline cellulose by cellulases is a bottleneck in the gen-eration of fermentable sugars, which is an important step in biofuel production.However, the detailed interaction between the enzymes, i.e. catalytic domain(CD) and carbohydrate binding module (CBM), and cellulose remains unclear.In this work, the motion of Thermobifida fusca cellulases Cel5A, Cel6B andCel9A on cellulose fibrils is observed through single-molecule fluorescence im-aging. A tracking algorithm provides trajectories, which display distinct sta-tionary states as well as states of fast motion. A hidden Markov model,implemented with a Markov chainMonte Carlo algorithm, separates the motioninto states of fast and slow diffusion and calculates the corresponding state-switching probabilities. The results show a slow diffusion coefficientD1=10

�4um2s�1 and a fast diffusion coefficient D2=10�1um2s�1, with calcu-

lated state-switching probabilities of p12=4% and p21=45% respectively. D1

gives an average diffusion speed of 20nm s�1 which corresponds to the local-ization uncertainty and therefore incorporates immobility as well as processivehydrolysis. Although different binding combinations of CBM, CD and linker tocellulose make for a complex system, we propose a simplified model of threeobservable states: A) a bound stationary state where the cellulase CD isengaged and directly interacts with the cellulose, resulting in limited motionand a slow diffusion coefficient, B) a bound state where the CD is disengaged,but the enzyme is otherwise loosely bound to the cellulose, e.g. via the CBM,leading to a mobile state with a faster diffusion, C) a completely disengagedenzyme that can freely diffuse - a state that is too fast for observation. Withthis approach we have developed a method to correlate the observed single-molecule motion with the underlying interaction between cellulases and thecellulose substrate.

Micro- and Nanotechnology I

748-Pos Board B513Biomolecule Transport across Droplet Interface Bilayer NetworksHeather E. Findlay, Grant Pellowe, Paula J. Booth.Chemistry, Kings College London, London, United Kingdom.An important aspect of biochemical reactions as they occur in vivo is thesequential and spatial control that arises from the compartmentalisation pro-vided by the membranes of cells and organelles within them. Integral mem-brane proteins control the traffic of ions and biomolecules across thesemembranes and the lipid composition of the bilayer in turn influences the struc-ture, function and stability of the proteins. Artificial systems such as DropletInterface Bilayers are being developed that have the potential to replicatethis feature, where sub-microlitre aqueous droplets are surrounded by a lipidmonolayer and assembled together to form bilayer regions at the connections.Here, we seek to functionalise a simple droplet network by the incorporation ofdifferent channels and transporters and by varying the selection of the compos-ite synthetic phospholipids, allowing for the selective transport of substrates be-tween compartments.

749-Pos Board B514Hierarchically Layered Platform for the Formation of Free-StandingLipid Bilayer MembraneHyunil Ryu1,2, Sangbaek Choi1,2, Sun Min Kim2,3, Tae-Joon Jeon1,2.1Biological Engineering, Inha University, Incheon, Korea, Republic of,2Biohybrid Systems Research Center (BSRC), Inha University, Incheon,Korea, Republic of, 3Department of Mechanical Engineering, InhaUniversity, Incheon, Korea, Republic of.Free-standing lipid membranes are commonly used in biophysical applicationsand they often require physically and chemically controlled conditions. Theyare traditionally formed over small apertures in Teflon thin film by manually‘‘painting’’ a droplet of organic solvent containing lipid molecules. However,as the conventional technique requires highly skilled expertise, its use hasbeen limited to only the research field. To resolve these issues, frozen mem-brane precursor with expedited self-assembly (MPES) that uses hydrophobic,porous material film to absorb solvent for expediting self-assembly was devised

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with a few limitations (Choi et al., Journal of Visualized Experiments, e54258-e54258, 2016). In this study, we have improved and optimized the MPES usinga hierarchically layered platform.

750-Pos Board B515Dynamics and Energetics of Phage T4 Injection MachineryAmeneh Maghsoodi1, Anupam Chatterjee2, Ioan Andricioaei2,Noel C. Perkins1.1Department of Mechanical Engineering, University of Michigan, AnnArbor, MI, USA, 2Department of Chemistry, University of California, Irvine,Irvine, CA, USA.Bacteriophage T4 is one of the most common and complex tailed viruses fromthe family Myoviridae, and it infects E. coli using a highly efficient contractilegenome delivery machine. Phage T4 possesses a multi-protein capsid contain-ing the genomic DNA and a long contractile tail assembly that consists of arigid tail tube surrounded by a six helical-stranded sheath. The tail assemblytransmits the genomic DNA from the capsid to the host during injection. Whilethe atomistic structure of phage T4 has been studied extensively using Cryo-EM and X-Ray crystallography, the dynamics of the injection process is notwell understood. Questions remain regarding the energetics of injection aswell as the pathway and time scale of the large conformational change of thesheath. In this study, we employ a dynamic multi-scale model to address thesefundamental questions. To this end, a two-stage solution process is employed;first, a molecular dynamics (MD) simulation is carried out to estimate theelastic properties of the sheath strands. Second, a continuum model is devel-oped for the entire-assembled T4 injection machinery using the elastic con-stants estimated from MD. The resulting multi-scale model simulates thedynamics of the sheath represented by six interacting helical strands that arecoupled to the capsid represented by a massive cylinder. Dynamic simulationsreveal the time scale, the pathway of sheath contraction, and the energeticsdriving the injection process. While these results are specific to T4 as anexample, the resulting model and methodology may also inform the futuredesign of nanotechnology injection devices.

751-Pos Board B516Using Magnetic Field to Purify Membrane Proteins in Supported CellPlasma MembranesKai-Hung Hsiao, Ling Chao.National Taiwan University, Taipei City, Taiwan.Processing and separating membrane proteins in their native bilayer environ-ment still remains challenging. The conventional methods to purify membraneproteins for characterize require detergents or harsh chemicals, which maycause the membrane protein to denature. Here, we developed a supportedplasma membrane platform and a magnetic tweezer to purify the target mem-brane proteins. We deposited giant plasma membrane vesicles (GPMVs)directly derived from Hela cells on a polymer cushioned support and filledthe region which was not covered by the GPMV patch with artificially preparedlipid membrane in order to form a continuous membrane platform. We addedmagnetic beads coated with antibodies to fish the target membrane proteins andused the developed magnetic tweezer to move the beads with the membraneproteins to the desired region for the collection and purification purpose. Weapplied a polymer cushion at the support to prevent some membrane proteinsfrom being stuck to the support. The continuous membrane allowed us tomove the membrane proteins in the 2-D bilayer platform so that the membraneproteins can be always kept in their native bilayer environment, which reducesthe possibility for them to denature or aggregate. Our preliminary result showedthat we can collect a native membrane protein in Hela Cells, aquaporin3, fromour supported plasma membrane platform.

752-Pos Board B517Targeting Specific Membranes with the Pore-Forming Peptide Cerato-toxin a using Click ChemistrySimon F. Mayer1, Aziz Fennouri1, Jerry Yang2, Michael Mayer1.1Adolphe Merkle Institute, Fribourg, Switzerland, 2Department of Chemistryand Biochemistry, University of California San Diego, San Diego, CA, USA.Targeting specific cellular membranes with selective pore-forming moleculesis a major component of the innate immune response of all mammals. Herewe explore the potential of a pore-forming peptide to specifically target lipidmembranes that were activated by a click reagent. With this idea in mind wemodified ceratotoxin A (CtxA), a 36-amino acid peptide found in the medflyCeratitis capitata, at its N-terminal region to bear an azide group. When intro-duced into a solution in contact with a lipid bilayer containing dibenzocyclooc-tyne (DBCO) moieties on the lipid headgroups, a spontaneous alkyne-azidecycloaddition covalently links the peptide to the bilayer. CtxA and its modifiedversion (CtxA-azide) form pores according to the barrel-stave model similar toalamethicin, displaying well-defined characteristic conductance states for

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transmembrane ion flux. Here, we show that the reaction between the azideof the peptide and the DBCO of the lipids increases the potency of the pore-forming peptide, resulting in pore-forming activity at peptide concentrationsapproximately ten times lower than the usual concentration required for activitywithout conjugation. We also show that the peptides and the lipids are indeedlinked covalently by observing channel activity for several hours after replac-ing the buffer solution in the aqueous compartments. This work suggests thatclick reactions could be used for targeting cellular membranes after theirsite-specific activation with a bio-orthogonal conjugation reagent.

753-Pos Board B518Identifying Peptide-Peptide Interactions with Lysenin NanoporesChristopher A. Thomas1, Nisha Shrestha1, Juliette Tinker1,Devon Richtsmeier1, Sheenah Bryant1, Xinzhu Pu2, Daniel Fologea1.1Department of Physics/Biomolecular Sciences Graduate Program, BoiseState University, Boise, ID, USA, 2Biomolecular Research Center, BoiseState University, Boise, ID, USA.Numerous peptides have been identified as biomarkers, providing valuable in-formation about diseases such as cancer, heart disease, rheumatoid arthritis,Alzheimer’s, and kidney disease. Similarly, the analysis of peptides hasemerged as a powerful tool for various diagnostic, therapeutic, and biomedicalapplications. In this context, there is a tremendous need for investigating andcharacterizing peptides beyond the established methods such as mass spectrom-etry, fluorescence spectroscopy, and immune staining. These methods can belabor intensive, time-consuming, require sophisticated instruments, and cansuffer from low sensitivity. As an alternative approach, we propose to usethe biological nanopore lysenin for peptide identification and characterizationat the single-molecule level. Lysenin is a nonameric pore-forming protein ex-tracted from the earthworm E. foetidawhich self-inserts large conducting poresinto natural and artificial lipid bilayer membranes containing sphingomyelin.Using the resistive-pulse technique, we investigated the translocation of theTrp-Trp-His-Pro-Cys peptide, which can exist either as a monomer or dimerdue to the cysteine group that can form a disulfide bond. The electronic signa-tures generated during the translocation of the peptide through single lyseninchannels indicate the ability of lysenin to differentiate between the monomerand dimer form. Similarly, translocation of another peptide, His-Met-Trp-Trp-Met, which lacks cysteine and hence is only present as a single monomer,presented the electronic signature of a single monomer. Mass spectrometryanalysis of the samples collected in the presence of large populations of lyseninchannels for an extended time period confirmed the translocation of peptidesacross the artificial bilayer. These results indicate that lysenin pores can beused for peptide detection, identification, characterization, and hence cancontribute to various biomedical and therapeutic applications.

754-Pos Board B519Sensing ssDNA Molecules with Single Lysenin ChannelsPhilip Belzeski, Nisha Shrestha, Sheenah Bryant, Juliette Tinker,Christopher Alex Thomas, Devon Richtsmeier, Daniel Fologea.Department of Physics/Biomolecular Sciences Graduate Program, BoiseState University, Boise, ID, USA.In this work we exploit the use of wild-type lysenin as a stochastic sensor forcharacterization of ssDNA molecules. Lysenin, a potent pore-forming toxin,self-oligomerizes into lipid membranes to form large conductance pores. Thelarge size of the pore (~3 nm) is deemed suitable for translocation of chargedmacromolecules driven by electric fields. In our experiments, we exploited thebiophysical properties of lysenin channels to build a sensing device based onthe long-revered resistive pulse technique, largely used for single moleculeanalysis by employing either synthetic or biological nanopores. We inserteda single lysenin channel into a planar bilayer lipid membrane composed ofneutral lipids (to avoid voltage-induced gating at positive voltages) and bathedby 1M KCl solutions. Addition of 69 nt DNA molecules into the reservoir sit-uated at the trans side of the protein channels did not induce any transients ofthe open current irrespective of the orientation of the transmembrane electricfield, indicative of absence of translocation. However, addition of ssDNA tothe reservoir bathing the cis side of the protein yielded multiple transients inthe open current, resembling the translocation pattern reported for syntheticand biological nanopores. In addition, the transients have been observed onlywhen the electric field orientation was such that the DNA molecules were elec-trophoretically driven toward the nanopore. The analysis of the translocationevents yielded a sharp distribution of the current blockages induced by the pass-ing molecules. In contrast, the dwell time distribution, although indicative oftranslocation speeds much lower than other nanopores, was relatively broad,suggesting strong interactions between the channel and ssDNA molecules.PCR performed on the translocated molecules and electrophoresis demon-strated that the DNA molecules may thread lysenin pores. This work may

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contribute to further development of single molecule biosensors, fast DNA andprotein sequencing, And single molecule characterization.

755-Pos Board B520Solid State Nanopore Kit for Real-Time Analysis of DNA and OtherAnalytesFederico Thei1, Michele Rossi1, James Yates2, Marco Bennati1.1Elements SRL, Cesena, Italy, 2Nanopore Solutions LDA, Oeiras, Portugal.Recent improvements in nanofabrication techniques have greatly increased thereproducibility and stability of nanopores fabricated in ultrathin dielectrics suchas silicon nitride or silicon oxide. These nanopores are now suitable for a widerange of accurate and reliable molecular analyses.Here we present a compact, portable and easy to use nanopore kit, intended forlaboratory experiments and development of prototype devices. Experimentsrange from simple nanopore conductance and stability determination, to themore complex analysis of translocations of DNA and other analytes (resistivepulse sensing/nanopore spectroscopy). The kit consists of:-A Teflon flow-cell with two 1.0 ml chambers, hosting a central cassette con-taining the silicon chip with the nanopore.-A miniaturized low-noise and high bandwidth amplifier.The entire kit is contained within a 16x10x5 cm Faraday cage and connects to alaptop via a USB cable.The real-time analyses are performed using the amplifier control software,enabling a fast characterization of nanopore conductance, production of I/Vcurves and production of histograms for blockade sizes and dwell times.The kit was tested under different conditions, using nanopores in the range from5 to 50nm. A 3,000bp fragment of DNA was successfully translocated on a6 nm nanopore. A 48,500bp phage lambda DNA was successfully translocatedon a 11 nm nanopore. Translocations were performed between potentials of200mV and 380mV in 1M KCl buffer, 10mM HEPES pH 8.0.The versatility of the nanopore kit combined with the real-time analysis featureallows for quick and easy characterizations of nanopores and their interactionwith analytes, for both educational and research purposes.

756-Pos Board B521Ionic Transport Property under an Extremely High Electrical Field in aThin PorePinyao He, Kun Li, Kabin Lin, Zhongwu Li, Haojie Yang, Yunfei Chen.Southeast University, Nanjing, China.Abnormal conductivity of electrolytic solutions is observed under anextremely high electrical field, this phenomenon is called the Wien effect.Generally, alternating voltage is applied on the electrodes because it isintractable to produce a direct voltage as high as 100,000 volts. However,extremely high electrical field which is akin to the high alternating electricalfield could be generated by several-volt direct voltage if DC voltage is appliedon such pore whose thickness is approaching nanoscale. Through our exper-iment increasing conductivity of electrolytic solutions is observed whenincreasing the DC voltage over 2 volts. Additionally, we propose some hy-pothesis to account for the result. We speculate the mode of ionic transportwill convert to a novel mode under very high electrical field. Our findingsdemonstrate that DC voltage could produce similar phenomenon to theWien effect, and could help to explore the property of ionic transport underextreme conditions.

757-Pos Board B522Defect-Guided Transport of BiomacromoleculesManish Shankla1, Aleksei Aksimentiev2.1Biophysics, University of Illinois at Urbana-Champaign, Champaign, IL,USA, 2Physics, University of Illinois at Urbana-Champaign, Champaign,IL, USA.Defects in the structure of solid-state materials often deteriorate their functionalproperties yet sometimes, they may grant the materials a new functionality.Graphene, one of the most versatile 2D nanomaterials does not lack imperfec-tions. In fact, one of the most common graphene fabrication procedures - me-chanical exfoliation - produces terraced islands of multi-layered carbon sheets.Using all-atom molecular dynamics simulations, here, we show that such de-fects in graphene can be used to direct the transport of adhered bio-macromolecules. Our key observation is that, subject to an external force, asingle-stranded DNA molecule moves much faster down a step-like defecton a graphene surface than against the defect, and even faster along the defect’sedge. We find the effect to be robust regardless of the nature of the force used todrive the motion of the biomolecules (mechanical or electrokinetic), the heightof the defects or its chemical termination. As a possible practical application ofthe effect, we demonstrate a system for capturing and transporting a DNAmolecule toward a graphene nanopore. Such defect-directed transport can beutilized for building two-dimensional nanoscale highways for precise delivery,

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concentration and storage of scarce biomolecular species, on-demand chemicalreactions and nanopore sensing.

758-Pos Board B523Anomalous Ionic Conductance in Carbon Nanotube NanochannelsSteven F. Buchsbaum1, Shirui Guo1, Eric Meshot1, Preston Hinkle2,Anh Pham1, Zuzanna Siwy2, Francesco Fornasiero1.1Physical and Life Sciences, Lawrence Livermore National Laboratory,Livermore, CA, USA, 2Physics, University of California, Irvine, Irvine,CA, USA.Simulations and experimental studies have reported an unusually high ionicconductance in carbon nanotube (CNT) nanochannels. The origin of this phe-nomenon is, however, poorly-understood: literature reports often disagree inthe magnitude of the different transport mode contributions to the measuredionic current and even in what ions are actually carrying the current; moreover,results obtained with single pore measurements differ frequently from thosewith membranes containing billions of open CNT channels, i.e. the averageCNT behavior. Toward shedding light on these phenomena, we fabricated anovel nanofluidic platform having a >30-nm wide, FIB-nanomachined siliconnitride nanopore (SiNx) in series with vertically-aligned sub-5 nm carbon nano-tubes, the number of which can be controlled from one to billions. By employ-ing this platform with only one or a few open CNTs, we observed giant ioniccurrents in CNT channels and a power-law increase of conductance withKCl concentration (G ~ cn, n=0.1-0.4), a dependence that seems to be uniqueof CNT pores. A few literature reports attributed this giant ionic current inCNTs to a strong electro-osmotic flow. To quantify electro-osmosis in CNTpores, we investigated translocation of neutral molecules in a single CNT nano-channel with the resistive pulse technique. Furthermore, we employed finiteelement analysis to elucidate the relationship between CNT pore characteristics(e.g., size, surface charge, slip length) and the resulting ionic transport andmagnitude of the electro-osmotic component. We confirmed computationallythat the unusually large conductance in our experimental platform resultsfrom the presence of a strong electro-osmotic coupling between the CNT chan-nel and the SiNx nanopore in series with it. Finally, we apply first-principle sim-ulations to show that cation-CNT interactions may help explain the origin ofthis electro-osmotic flow.

759-Pos Board B524Role of Solid State Nanopore Size and Charge on Molecular TransportKineticsMeni Wanunu.Physics, Northeastern University, Boston, MA, USA.Nanopore-based detection of macromolecules has gained interest in recentyears, because of the enormous potential of single-molecule analysis at ultra-low analyte concentrations. In this work, we describe how nanopores can beused to trap, manipulate, and study individual bimolecular complexes with con-trol over the applied force and other experimental parameters, such as temper-ature and pressure. Key to our results is the combination of ultrathin nanoporesand high-bandwidth electronics, which together enable high-resolution mea-surements. In addition, characterizing the pore’s surface charge enables morequantitative assessment of the role of electroosmosis on molecular captureand transport. We will discuss specific examples of molecular transport onwell-characterized nanopores in different materials. Our results represent akey advance towards more quantitative macromolecular characterization atthe single-molecule level.

760-Pos Board B525Structural Characterization of Vascular Endothelial Growth Factor bySolid-State NanoporesNitinun Varongchayakul1, Mark Grinstaff1, Amit Meller2.1Biomedical Engineering, Boston University, Boston, MA, USA,2Biomedical Engineering, Technion Israel Institute of Technology, Haifa,Israel.A novel solid-state nanopore sensing technique is described to study vascularendothelial growth factor (VEGF). VEGF is a cytokine that stimulates vascu-larization and is used as a cancer biomarker. Its biological activity dependson the isomerization state. Dimeric VEGF facilitates dimerization of theVEGF receptor which initiates the signal transduction pathway. MonomericVEGF consists of two domains; a VEGF receptor recognition domain and aheparin binding domain, separated by a peptide sequence cleavable by plasmin.Translocation of VEGF through a 4 to 6 nm-diameter pore, fabricated via trans-mission electron microscope sculpting on an thinned 10 nm free-standing SiNmembrane, produces a structural-dependent electronic signature. By fine-tuning the pore’s geometry and experimental conditions, VEGF translocationconsistently produces multi-step patterns, which are used to differentiate mono-meric and dimeric isoforms. The equilibrium dissociation constant depends on

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concentration and pH. Further study with TCEP and plasmin show that the steppattern corresponds to the translocation of individual protein domains. Theseresults demonstrate the potential for nanopores to detect small proteins andits capability to resolve the higher-order structure of proteins at the single-molecule level.

761-Pos Board B526Solid-State Nanopore Detection of Hydrophobic ProteinsAdam R. Hall, Dhruba Jyoti Basu Roy.Biomedical Engineering, Wake Forest School of Medicine, Winston-Salem,NC, USA.We report on a modified solid-state nanopore measurement scheme to probehydrophobic molecules that cannot be studied in conventional systems. Takingadvantage of the intrinsic alcohol solubility of LiCl as an electrolyte, we showthat the devices can be operated in alcohol-based azeotropic mixtures. We firstcharacterize nanopore conductivity as a function of ethanol content, nanoporediameter, and salt concentration, showing predictable ionic response. Then, as ademonstration of resistive-pulse sensing, we measure and interpret electricaltranslocations of zeins, a class of water-insoluble maize protein.

762-Pos Board B527The Nanometric Golden Ratio: The Relation between Gold Volume andNanopore DiameterLennart J. de Vreede.Adolphe Merkle Institute, University of Fribourg, Fribourg, Switzerland.In this work we build further on a recently discovered nanopore manufacturingscheme which is based on the heating of nanometric gold particles on a siliconoxide surface. When heated to 1064�C the lithographically patterned and sput-tered gold particles dewet and create nanopores into the silicon oxide surface.The three leading phenomena which allow this nanopore creation are; a differ-ence in the surface energies of the gold and the silicon oxide, mobility of thesilicon oxide and evaporation of the gold. [1] This approach promises to berelevant in the field of proteomics, since a transfer of this technique to siliconoxide or silicon nitride membranes allows the creation of nanopore throughholes. [1] [2] Nanopores, either in singular or in array configuration, made insilicon oxide or silicon nitride membranes could form interesting tools, forexample, for the characterization of proteins.Currently however, it is not clear which volume of gold, combined with whichheat treatment, is needed to create which nanopore diameter. We conducted ex-periments in which a silicon oxide surface is coated with a photosensitive layerand patterned by using deep ultraviolet (DUV) lithography and sputtered with alayer of gold. The created nanometric gold volumes are measured and subse-quently heated to 1064�C. After cooling down the substrates are cleaved andthe nanopore diameters are measured. DUV lithography is used because ithas the ability to expose and develop features of 200 nm in diameter, thus al-lowing accurate volumetric variance when using different sputtering times.[1] Nanopore fabrication by Heating Au-Particles on Ceramic substrates, NanoLett. 15:727-731, 2015 [2] Nanopore fabrication in silicon oxynitride mem-branes by heating Au-Particles, J. Micromech. Microeng. 26:037001, 2016.

763-Pos Board B528Multiple-Nanopores Fabrication based on Dielectric BreakdownYunlong Wang, Cuifeng Ying, Wenyuan Zhou, Zhibo Liu, Jianguo Tian.NanKai University, TianJin, China.Single molecule detection based on nanopore technology is becoming more andmore established recently. Due to the Coulter counter and ion channels detec-tion principle, single nanopore suffers from limitation of low throughput.Thus high-throughput nanopore detection based is a definite direction of devel-opment. Dielectric breakdown, a novel method reported by Tabard-Cossarecently, shows advantages for low-cost and high fabrication efficiencycompared to previous transmission electro microscopy (TEM) and focus ionbeam (FIB) drilling. Here we show that the by means of dielectric breakdown,several nanopores can be formed in one SiN membrane. By controlling theexperiment conditions, we explore the number and diameters of nanoporesfabricated by our methods. Although it is still a challenge in making nanoporearrays due to the random formation, we show a simple and convenient methodto fabricate two or three nanopores at the same time.

764-Pos Board B529Artificial DNA-Based Channels for Controlled Membrane TransportJonathan R. Burns1, Astrid Seifert2, Niels Fertig2, Stefan Howorka1.1UCL Chemistry, London, United Kingdom, 2Nanion Technologies GmbH,Munich, Germany.We describe synthetic membrane channels that control transport of molecu-lar cargo across a lipid bilayer[1]. Unlike biological protein channels, the

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synthetic versions are made from DNA which is ideal for predictablede-novo design. The DNA channels are composed of six hexagonallyarranged, interlinked duplexes that enclose a 2 nm-wide lumen. Lipid an-chors hold the negatively charged channels in the membrane[1–3] to forman electrical seal required for electrical recordings[1, 4]. One DNA versionmimics the function of biological ligand-gated ion channels. The gate ofthe synthetic channel is a DNA strand that blocks the lumen, but a specificDNA ligand can re-open the channel[1] (see figure). The pore can also distin-guish with high selectivity the transport of small-molecule cargo that differsby the presence of a positive or negative charge. The synthetic analoguemay be used for controlled drug release and the building of cell-likenetworks.Related DNA channels show other hallmarks of the biological templates suchas voltage-gating at high transmembrane potentials[1, 4, 5]. The artificial porescan furthermore be programmed to function as cytotoxic agents by killing can-cer cells via membrane-rupturing[7]. The synthetic pores expand the range ofother DNA nanostructures that mimic biological functions of membrane pro-teins to control bilayer and cell shape[6].References:[1] J. R. Burns et al, Nat. Nanotechnol. 2016, 11, 152.[2] J. Burns et al, Nano Lett. 2013, 13, 2351.[3] J. R. Burns et al, Angew. Chem. Int. Ed. 2013, 52, 12069.[4] A. Seifert et al, ACS Nano 2015, 9, 1117.[5] V. Maingi et al, ACS Nano 2015, 9, 11209.[6] S. Howorka, Science 2016, 352, 890.[7] J. R. Burns et al, Angew. Chem. Int. Ed. 2014, 53, 12466.

765-Pos Board B530Slowing Down DNA Translocation using Integrated Nanopore and Nano-pillars Precisely Deposited by Helium Ion BeamYunsheng Deng, Qimeng Huang, Daming Zhou, Shuo Zhou, Liyuan Liang,Shaoxi Fang, Wanyi Xie, Shixuan He, Peng Tang, Deqiang Wang.Chongqing Institute of Green and Intelligent Technology, Chinese Academyof Sciences, Chongqing, China.Slowing down DNA translocation speed in a nanopore is essential to real-izing high resolution of individual bases. In this study, a uniform array ofPlatinum (Pt) and Silicon Oxide (SiOx) nanopillars created with Heliumion beam is introduced onto the trans side of Silicon Nitride layer toslow down DNA translocation. Both of Pt and SiOx nanopillars show acapability of great magnitude deceleration of dsDNA translocation. Andthe periodic space and diameter of nanopillars are precisely controlled,enabling further investigation of the interaction between pillars and thedsDNA. Pt and SiOx Plliars have different surface properties, hence wecould explore the different interaction mechanism between Pt/SiOx Plliarsand dsDNA.

766-Pos Board B531Single-Channel Measurements of Conductance through Sub-NanometerCarbon Nanotube PorinsYun-Chiao Yao1,2, Robert Henley3, Ramya Tunuguntla2, Meni Wanunu3,Aleksandr Noy1,2.1Chemistry and Chemical Biology, School of Natural Sciences, University ofCalifornia, Merced, CA, USA, 2Biology and Biotechnology Division,Physical and Life Sciences Directorate, Lawrence Livermore NationalLaboratory, Livermore, CA, USA, 3Department of Physics, NortheasternUniversity, Boston, MA, USA.Carbon nanotubes (CNTs) have narrow hydrophobic inner pores, highlyreminiscent of protein channels, that enable fast transport of water andions. Their rigid structure and chemical robustness allow for diverse ex vivoapplications. We have recently reported biomimetic membrane channelscased on carbon nanotubes—carbon nanotube porins (CNTPs). We haveshown that CNTPs self-insert into lipid membranes and form stable pores.In this study, we used electrophysiological single-channel measurementsto quantify ion conductance and selectivity of 0.8 nm diameter CNTPs. Wereport the conductance of single CNTP channels, as well as the scalingof the conductance with the ion concentration. We also used the reversalpotential measurements to characterize the selectivity of the ion transportand found that CNTPs are highly selective to cations over anions. We willdiscuss the mechanism of this selectivity, and demonstrate ways to controlit. These findings are important and relevant for developing CNTPs intoa potential candidate nanopore for water treatment applications. Ourstudies on intrinsic characterization of CNTP transport properties will alsoenable researchers to consider this new material for a variety of biophysicalapplications.

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767-Pos Board B532Tunable Ion Selectivity in Sub-Nanometer Diameter Carbon NanotubePorinsRobert Y. Henley1, Yun-Chiao Yao2, Ramya Tunuguntla2,Pradeep Waduge1, Meni Wanunu1, Aleksandr Noy2.1Physics, Northeastern University, Boston, MA, USA, 2Lawrence LivermoreNational Lab, Livermore, CA, USA.Carbon nanotube porins (CNTPs), ultra-short carbon nanotubes (5-20nm) thatcan self-insert into lipid bilayers, allow single-channel planar lipid bilayer mea-surements of ionic transport through carbon nanotube (CNT) pores. CNTs withsub-nanometer diameters have been predicted to display ultra-fast water trans-port and high levels of ionic exclusion resulting from one-dimensional waterwire transport through their hydrophobic cores. Here, we show that ultra-short0.8-nm-diameter CNTPs, can spontaneously insert into lipid bilayers to formhighly cation selective channels that exhibit low ionic conductance. Usingreversal potential in various asymmetric salt conditions, we observe that theseCNTPs exhibit large permselectivity values, corresponding to a high selectivityfor both potassium and sodium over chloride. We also show evidence thatneutralization of charges on the CNTs enhances ion selectivity. We also showthat by using a solid-state nanopore to act as a support and form a completelysolvent-free bilayer system, we can observe extremely stable ionic currentsthrough these nanotubes, in contrast to previous reports with solvent containingbilayers. These results establish CNTPs as a promising biomimetic platform fordeveloping cell interfaces, creating stochastic sensors, and water desalination.

768-Pos Board B533Sub-1-nm Carbon Nanotube Porins: Water Transport and Ion Selectivityin a Single-File Water Membrane NanoporeAleksandr Noy.Biology and Biotechnology Division, Lawrence Livermore NationalLaboratory, Livermore, CA, USA.Living systems control transport of ions or small molecules across biologicalmembranes using ion channels that form pores in lipid bilayers. Membranepores formed by ultra-short carbon nanotubes (CNTs) assembled in the lipidmembranes have transport properties that come remarkably close to replicatingthe transport properties of biological channels. The defining features of thesenanostructures are their inner pores that have atomically smooth hydrophobicwalls, which can confine water on a molecular level, and, in case of 0.8 nmdiameter CNTPs, down to a single-file configuration. We present experimentalresults that demonstrate efficient water transport in CNTPs and explore itsphysical origins. We also use single pore conductance measurements to demon-strate ion selectivity in these pores and show that they can be configured into aswitchable nanofluidic diode. CNTPs represent a simplified biomimetic systemthat is ideal for studying fundamentals of nanofluidic transport and transport inbiological channels, and for building complex engineered mesoscale structuresthat could be the foundation of next-generation water treatment technologies.

769-Pos Board B534Carbon Nanostructures of Different Spatial Geometry: Their Dispersionand Influence on Model Biological SystemsJustyna I _zykowska1,2, Michalina Skupin1, Weronika Andrzejewska1,Maria Dobies1,2, Stefan Jurga1,2, Maciej Kozak1,3.1Department of Macromolecular Physics, Adam Mickiewicz University,Pozna�n, Poland, 2NanoBioMedical Center, Adam Mickiewicz University,Pozna�n, Poland, 3Joint Laboratory for SAXS Studies, Adam MickiewiczUniversity, Pozna�n, Poland.The discovery of carbon nanostructures (CN) in the last century has revolution-ized science, opening new research opportunities in biophysics, materialsciences, biomedicine and pharmacology [1]. Unfortunately, carbon nanomate-rials show a strong tendency towards aggregation and poor stability in solutions.Such properties, especially the suspension stability, are critical for bio-applications of carbon nanostructures [2]. Surface functionalization of nanocar-bons improves the dispersing properties in water solutions but it may alsoinfluence their physicochemical properties [3]. For preparation of biocompatibleCN samples we used novel dicationic surfactant (gemini) with imidazole headgroups. Nanocarbon suspensions were investigated in the context of their anti-cancer activity, as well as their application as multimodal contrast/drug deliveryagents. Different spatial geometries of CN may be of key importance fordesigning of the efficient anti-cancer system. Such systems were subjected tothe cytotoxicity tests on HeLa cell cultures. The structural properties of selected,the most effective systems based on CN suspensions were characterized by theuse of SEM, AFM and NMRmethods. This study was supported by theMinistryof Science and Higher Education (Poland), within the project ‘‘Najlepsi z naj-lepszych!’’ (DEC - POWR.03.03.00-00-P001/15). [1] Krokosz, A. et al., J. Ra-diat. Phys. Chem. (2016), 128, 143–150. [2] Wang, L. et al., J. Biomed.

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Nanotechnol. (2015), 11 (9), 1653–1661. [3] Bogdanovic, G., Djordjevic, A.Srp. Arh. Celok. Lek. (2016), 144 (3–4), 222-231.

770-Pos Board B535The Influence of Surfactant Structure and Geometry on Nanotoxicity andDispersion of Carbon NanotubesJakub Zareba1, Justyna I _zykowska1, Michalina Skupin1,Augustyn Moli�nski1, Maria Dobies1,2, Stefan Jurga1,2, Maciej Kozak1,3.1Department of Macromolecular Physics, Adam Mickiewicz University,Pozna�n, Poland, 2NanoBioMedical Center, Adam Mickiewicz University,Pozna�n, Poland, 3Joint Laboratory for SAXS Studies, Adam MickiewiczUniversity, Pozna�n, Poland.Graphene, nanotubes and fullerenes, also known as carbon nanomaterials (CN),because of the their unique properties have potential applications not only inpreparation of new composite materials or in electronics, but they can be alsocomponents of drug delivery systems. However, the applications of CN in lifesciences or biomedicine require stable dispersions of these materials in water.The hydrophobic nature of CN causes serious problems with their separationin water solutions. Our study is aimed to find a way of dispersing such CN sys-tems in water, by the use of non-covalent surface modification via different sur-factants. In this work we focused on the use of trimeric, dimeric and monomericsurfactants fornon-covalent surface modification of selected carbon nanotubes.The toxicity of these systems (aqueous solutions of CNwith surfactants and sur-factants solutions) towards selected cell cultures (e. g. HeLa cells) was alsotested. The surfactants studied can interact with carbon nanotubes in a numberof different modes: hydrophobic interactions (surfactant chains with CN sidewalls) or p-p interactions (aromatic rings of surfactants with CN surface).Infrared spectroscopy and atomic force microscopy were used for characteriza-tion of the systems studied. Finally, we focused onmechanical properties of cellsexposed to surfactants and CN. This study was supported by the Ministry ofScience and Higher Education (Poland), within the project ‘‘Najlepsi z najleps-zych!’’ (DEC - POWR.03.03.00-00-P001/15).

771-Pos Board B536Designer Peptides Self-Assemble on Graphene to Create Remarkably Sta-ble, Precisely Organized SubstratesGina-Mirela Mustata1, Meni Wanunu2, Gevorg Gregoryan3,Yong Ho Kim4, Jian Zhang5, William F. DeGrado6.1Chemistry and Physics, Simmons College, Boston, MA, USA, 2Physics,Northeastern University, Boston, MA, USA, 3Dartmouth College, Hanover,NH, USA, 4Sungkyunkwan University, Seoul, Korea, Republic of,5Chemistry, University of Nebraska-Lincoln, Lincoln, NE, USA, 6Universityof California, San Franciso, San Francisco, CA, USA.We present a study of designed self-assembly of 2D peptide monolayer crystalson the surface of graphene and graphitic interfaces and their properties in variousbiologically significant conditions. Atomic force microscopy imaging of driedpeptides adsorbed on graphitic surfaces reveals an amorphous monolayer struc-ture that contains voids due to drying. After rehydration, the peptide monolayerreorganizes into highly ordered domains comprised by parallel arranged pep-tides that are oriented on the graphitic structure with C3 symmetry, in closeagreement with computational predictions. The monolayers are remarkably sta-ble in awide range of pH, ionic strengths, urea concentrations, and temperatures.Importantly, we find that alternating peptides that do not contain aromatic resi-dues organize similarly, and conclude that aromatic residues are not essential forthis organization. The monolayers are highly stable to proteolytic digestionwhen full coverage is acquired, while voids in the layer become seeds to slowdegradation from the void inwards. A striking quality of these substrates is thepreference to bind double stranded DNA imposing a preferred alignment tomatch their own molecular arrangement on the graphene surface. This systemof designed peptide-coated graphene surfaces, with its stability over a widerange of situations, presents new opportunities for the design of structures andsystems that are significant in the study of various biological entities and pro-cesses, such as specific binding or designed catalysis.

772-Pos Board B537Modulation of Graphene Oxide Probiotic and Antibiotic Activity by Crit-ical Coagulation ConcentrationMassimiliano Papi1, Valentina Palmieri2, Francesca Bugli3,Maria Carmela Lauriola1, Margherita Cacaci3, Claudio Conti4,Maurizio Sanguinetti3, Marco De Spirito1.1Physics, Universita Cattolica del Sacro Cuore, Rome, Italy, 2Physics,Universita Cattolica del Sacro Cuore, Roma, Italy, 3Microbiology Institute,Universita Cattolica del Sacro Cuore, Rome, Italy, 4Institute for ComplexSystems, National Research Council (ISC-CNR), Rome, Italy.In recent years, significant research related to antibacterial properties and ef-fects on eukaryotic cells of Graphene Oxide (GO) and GO-based materials

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has been conducted. Despite substantial efforts, results are controversial and aframework clarifying all effects reported in literature against bacteria still hasto be defined. In our work, we demonstrate that by modulating the stability ofGO in solution, the antibacterial or growth enhancement effect can beencontrolled both on S. aureus and E. coli bacteria. We expose these two speciesto concentrations of GO ranging from 3 to 200 ug/ml, in different incubationconditions (ultrapure water, PBS, NaCl, MgCl2 and CaCl2). We analyze thegrowth of microorganisms and characterize GO effects on cells with AtomicForce Microscopy and Colony Forming Units Assay. Our data indicate thatany buffer solution utilized during the GO-bacteria interaction alters specif-ically the GO surface zeta potential and the consequent GO clusters size andstructure. The GO stability influences antimicrobial activity and, while atlow concentration, the sheets cut microorganisms membranes and, at high con-centration, complexes between pathogens and aggregates inhibit or enhancebacteria growth in a surface potential-dependent manner. The main conse-quence of our results is the surprising possibility to finely modulate the GO ef-fects on bacteria and to produce versatile applications of GO materials in theenvironmental and medical sciences from treatments against multidrug resis-tant bacteria to water remediation systems and probiotic therapies.

773-Pos Board B538Real-Time Flow Deformability Cytometry via Resistive Pulse SensingPreston Hinkle.Physics and Astronomy, UC Irvine, Irvine, CA, USA.We present resistive pulse and optical data of cancer cells deforming whilepassing through microsized constrictions. Cell stiffness, the degree to whichcells deform under strain forces, is known to be an important physical param-eter that along with other physical and chemical biomarkers can be used todifferentiate cell types and enable clinical detection of cancerous cells. Manydesirable applications, such as detecting metastatic circulating tumor cells,rely on a high throughput of measurements, since the specimen of interestmay comprise only a total fraction of the total number of particles in a sample.Capable of measuring 1000s of cells/sec, real-time flow deformability cytom-etry is a high throughput method for determining a cell’s stiffness that works byinducing cell deformation under strain forces in the pressure-driven laminarflow through a microfluidic channel. Currently, real-time flow deformabilitycytometry relies on taking high-speed camera data of the sample, which isexpensive to acquire and analyze. We propose to measure cell stiffness usingresistive pulse sensing, whereby a particle’s physical properties are measuredfrom the change in the transchannel electrical current during the particle’stransit. By using a microfluidic channel with regions differing in diameter,the particle is deformed to different degrees, distorting the resistive pulse signalrelative to the signal expected of a hard sphere. We show resistive pulse datataken of hard spheres and cells with varying degrees of deformability, and usinghigh speed video, show cells deforming in the channel.

774-Pos Board B539Quantifying Extracellular ROS Levels in Individual Pancreatic Islets usingan Optical Sensor and Microfluidic DeviceRomario Regeenes1, Jonathan V. Rocheleau1,2.1IBBME, University of Toronto, Toronto, ON, Canada, 2Physiology,University of Toronto, Toronto, ON, Canada.The metabolism of pancreatic islet beta-cells is negatively regulated by reactiveoxygen species (ROS), which is a toxic byproduct of the electron transportchain and excessive aerobic respiration. During oxidative stress it is evidentthat ROS levels increase and result in apoptotic signaling. Chronic oxidativestress is associated with beta-cell failure and type 2 diabetes. Oxidative stressincreases expression of antioxidant enzymes as a protective mechanism thatparadoxically reduces glucose stimulated insulin secretion. To remove ROS,the cell uses superoxide dismutase (SOD) an enzyme that catalyzes the conver-sion of superoxide into hydrogen peroxide and oxygen. The hydrogen peroxideis then converted into water and oxygen via glutathione peroxidase or catalase.Therefore, both hydrogen peroxide (H2O2) release and molecular oxygen (O2)consumption of islets can serve as proxies to measure conversion rates in thetissue. We have designed a microfluidic device to hold individual pancreaticislets separately in chambers optimized for live cell confocal imaging. This de-vice puts islets very close to the planar slide and also collects islet effluent in a

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well-defined tail, thus offering us two positions to place a sensor: planar slideand solution. We will first modify the device by introducing a clear fluorescentsensor for oxygen directly beneath the islets. We are currently exploringRuII(bpy)3 [Tris(2,20-bipyridyl)dichlororuthenium(II) hexahydrate as an opti-cal sensor which has excitation and emission maxima at 450nm, and 600nm,respectively. The ruthenium compound will serve as a seed layer on the baseof the device that will increase in fluorescence intensity when oxygen levelsdecrease. Furthermore, we are exploring putting the dye in solution and usingother sensors such as palladium and platinum compounds that react to oxygen.This device will enable toxicity screening of living pancreatic islets and othertypes of cells.

775-Pos Board B540Microcantilever Investigation of Nanoconfinement Effects on WaterTransportMichael DeLay.Columbia University, Queens, NY, USA.Nanoconfinement of water influences myriad applications including environ-mental engineering, desalination/filtration, as well as biological activitiesincluding membrane transport, signaling/turgor-sensing, and protein folding.Understanding confinement effects in nature, outside of in situ SFM andAFM systems, requires consideration of heterogeneous, dynamic enclosures.The nanoporous and water-responsive, peptidoglycan matrix atop the genomiccore of Bacillus spores provides an opportunity to probe water confinement ef-fects when spores are layered atop an AFM cantilever. By measuring and inte-grating numerous transient nanomechanical responses as well as water massdisplacements after brief evaporative photothermal pulses, we are able toexamine transport kinetics of confined water and observe that spores behaveas a poroelastic material and display an effective viscosity higher than bulk.We then investigate the temperature dependence of these kinetics and findthat the activation energy of spore water is higher than bulk and close to ice.We suggest two possible models for this phenomenon based on spore geome-try: high viscosity water due to total confinement, and decelerated transport dueto frequent liquid-vapor-liquid transitions at negative pressure-driven nano-scale cavitations. We note that the slowed confinement kinetics observed couldbe involved in controlling reactions central to a range of biological processesincluding spore germination.

776-Pos Board B541Sub-Diffraction STED Lithography using Orthogonally FunctionalizedResinsThomas A. Klar1, Richard Wollhofen1, Johannes Kreutzer1,Bianca Buchegger1, Christine Eder2, Jaroslaw Jacak1,2.1Johannes Kepler University Linz, Linz, Austria, 2Upper Austria Universityof Applied Sciences, Linz, Austria.Stimulated emission depletion (STED) is not only apt to provide diffraction-unlimited imaging in fluorescence microscopy, but it also allows for sub-diffraction photo-polymerization. Nanoscale anchors to fix proteins on twoand three dimensional surfaces, even down to the single protein level, havebeen developed in the past. However, the proteins were bound electrostatically.Covalent bonds would be much stronger, insensitive to variations of buffer pHand they would allow for orthogonal functionalization of a structure exhibitingtwo different types of reactive groups.In this contribution, we present two acrylate-based photo-resins with chem-ically reactive surfaces. Chemically functional monomers are copolymer-ized with a highly crosslinking triacrylate monomer to yield mechanicallystable structures. Thereby, the polymer structure is equipped with chemicalfunctionality for post lithographical reactions. Two nanoscopic patterns,one functionalized with mercapto- and the other one with carboxylategroups, can be structured on top of each other with sub-diffractionprecision. In order to prove the surface reactivity, the bi-functionalizedstructure was labelled covalently with two different fluorophores. Theresins show excellent performance in stimulated emission depletion lithog-raphy and line widths below 60 nm can be achieved. Compound structures,which are fabricated of both resins, will allow fabrication of two andthree dimensional scaffolds for proteins, for instance inside microfluidicflow cells.

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