Structure of Trypanosoma Brucei Flagellum Accounts for its Bihelical Motion
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1 INSERM / Institut Curie, Paris, France, 2 Universite ´ Aix-Marseille, Marseille, France, 3 C-CINA, Biozentrum, Basel, Switzerland, 4 Institut Curie, Paris, France. Membrane-mediated protein-protein and protein-lipid interactions, membrane protein localization, and related dynamics, modulate membrane protein func- tion. So far membrane structure and dynamics could not be studied altogether lacking the technique that analyzes unlabelled proteins at submolecular lateral and high temporal resolution. Here we used high-speed atomic force micros- copy (HS-AFM) to characterize the lateral and angular movements and interac- tions of unlabelled OmpF. OmpF distribution emerges from diffusion-limited aggregation, maximizing surface coverage and providing a multitude of local environments and contacts. Protein motion scales roughly with membrane crowding. However molecules display individuality of diffusion behavior rang- ing from fast moving (~400nm2/s) to immobile molecules trapped by favorable protein-protein associations. We derive the complete molecular interaction probability landscape that we compare with coarse-grained molecular dynam- ics simulation of the membrane protein interaction. HS-AFM may open a novel research avenue that bridges structure of individual membrane proteins and su- pramolecular membrane architecture. HS-AFM movie frames (frame rate 477 ms) showing the motion of OmpF trimers in the membrane. Right: Experimental interaction potential map 2103-Plat Probing Dielectric and Hydrogen Bonding Gradients in Biological Membranes Tatyana I. Smirnova 1 , Maxim A. Voynov 1 , Oleg G. Poluektov 2 , Alex I. Smirnov 1 . 1 North Carolina State University, Raleigh, NC, USA, 2 Argonne National Laboratory, Argonne, IL, USA. Nitroxide spin-labeling in combination with EPR spectroscopy has found many applications in studying structure and dynamics of proteins and biological membranes. Recently, there has been a substantial interest in utilizing EPR to characterize local effects of polarity and hydrogen bonding in proteins and biological membrane systems. Here we report on employing an arsenal of ad- vanced spin-labeling EPR methods to profile heterogeneous dielectric and hy- drogen bonding environment along the a-helical chain of an alanine-rich WALP peptide that is anchored in a lipid bilayer in a transmembrane orienta- tion. A series of WALP cysteine mutants was labeled with a pH-sensitive nitro- xide IMSTL (S-(1-oxyl-2,2,3,5,5-pentamethylimidazolidin-4-ylmethyl) ester) that is similar in molecular volume to phenylalanine. The protonation state of this nitroxide could be directly observed by EPR allowing us to follow pro- ton gradient across the membrane in the vicinity of the WALP a-helix, and, thus, to reconstruct the gradient in the effective dielectric constant. These ex- periments were complemented by assessing local polarity from characteristic changes in EPR spectra that were enhanced by the use of perdeuterated and 15 N-substituted nitroxides and high field EPR at 130 GHz (D-band). Formation of hydrogen bonds between the nitroxides and membrane-penetrating water molecules was observed directly in HYSCORE X-band experiments. Such measurements allowed us to derive experimental profiles of heterogeneous di- electric and hydrogen bonding environment along a typical transmembrane a-helix. Supported by: NSF-0843632 to TIS and NIH 1R01GM072897 to AIS. 2104-Plat Pulmonary Surfactant Reduces Surface Tension to Low Values near Zero through a Modified Squeeze-Out Mechanism Fred Possmayer 1 , Nora Keating 1 , Yi Y. Zuo 2 , Nils O. Petersen 3 , Ruud A. Veldhuizen 1 . 1 University of Western Ontario, London, ON, Canada, 2 University of Hawaii, Honolulu, HI, USA, 3 University of Alberta, Edmonton, AB, Canada. Pulmonary surfactant stabilizes the lung by reducing surface tension (ST) to low values near zero at end expiration. Atomic force microscopy studies re- vealed that, as surface pressure increases, spread surfactant films initially form solid micro- and nanodomains and then generate 3-5 multilayers which apparently remain associated with the surface monolayer. Time-of-flight Sec- ondary Ion Mass Spectroscopy analyses revealed selective squeeze-out must occur because the multilayers are enriched in unsaturated fluid phospholipids (PL) while the remaining monolayer becomes enriched in disaturated PL. Taken together, these results are consistent with a modified squeeze-out model where surfactant vesicles interact with the air-water interface through surfac- tant proteins B- and C-containing adsorption/fusion pores. PL migration onto the surface results in vesicle instability generating a monolayer at equilibrium surface pressure, which remains functionally associated with excess bilayer material. Initially, the monolayer and associated reservoir have similar compo- sition, but film compression causes fluid PL to migrate through the fusion pores into the multilayers. Gel phase PL are restricted because they are sequestered in micro- or nanodomains. This process results in monolayers highly enriched in disaturated PL which reduce ST to near zero. Film expansion allows fluid PL to regain the surface through the fusion pores. This mechanism explains both the rapid reincorporation of surfactant PL into the monolayer during adsorption and during film expansion and the progressive improvement in surface activity dur- ing repeated compression. Platform: Cell & Bacterial Mechanics & Motility II 2105-Plat Flexural Rigidity and Shear Stiffness of Flagella Gang Xu, Kate S. Wilson, Ruth J. Okamoto, Jin-Yu Shao, Susan K. Dutcher, Philip V. Bayly. Washington University, Saint Louis, MO, USA. Cilia are thin subcellular organelles that line airways and other passages and bend actively to propel fluid and foreign materials. The ciliary cytoskeleton (the axoneme) consists of nine outer microtubule doublets surrounding a central pair of singlet microtubules. Large bending deformations of the axoneme in- volve relative sliding of the outer doublets driven by the motor protein dyneins. The genetics and cell biology of the ciliary structure and function have been studied extensively, but the mechanics of the axoneme remain unclear. In this study, we used the unicellular alga Chlamydomonas reinhardtii as the model system for their flagellum replicates the highly conserved molecular structure of the ciliary axoneme. Piconewton forces were applied perpendicu- larly on the tip of a single flagellum (length L) through a microsphere trapped in optical tweezers. Dividing the force (P) by the corresponding deflection of the flagellar tip (d) yields the flexural stiffness of the flagellum (K = P/d), which was then used to calculate the apparent flexural rigidity (EI = KL 3 /3). The con- tributions of major structural components to passive mechanical properties were quantified by testing on flagella of specific mutations. The average appar- ent flexural rigidity of wild-type, pf-3 (without nexin links), and pf-13 flagella (without outer dynein arms) was about 2700 5 1100, 1300 5 550, and 650 5 140 pN$mm 2 , respectively. In addition, the ratio of elastic shear stiffness (resis- tance to interdoublet sliding) to true flexural rigidity was estimated by the coun- terbend response in bent flagella manipulated with a glass microneedle. The quantitative understanding of axonemal mechanics will help illuminate the roles of certain genes and molecular structures in the normal and abnormal axoneme. 2106-Plat Structure of Trypanosoma Brucei Flagellum Accounts for its Bihelical Motion Michael F. Schmid 1 , Alexey Y. Koyfman 1 , Ladan Gheiratmand 2 , Caroline J. Fu 1 , Dandan Huang 2 , Htet A. Khant 1 , Cynthia Y. He 2 , Wah Chiu 1 . 1 Baylor College of Medicine, Houston, TX, USA, 2 National University of Singapore, Singapore, Singapore. Trypanosoma brucei is a parasitic protozoan that causes African sleeping sick- ness. It contains a flagellum required for locomotion and viability. In addition to a microtubular axoneme, the flagellum contains a crystalline paraflagellar rod (PFR) and connecting proteins. We show here, by cryoelectron tomogra- phy, the structure of the flagellum in three bending states. The PFR lattice in straight flagella repeats every 56 nm along the length of the axoneme, matching the spacing of the connecting proteins. During flagellar bending, the PFR crys- tallographic unit cell lengths remain constant while the interaxial angles vary, similar to a jackscrew. The axoneme drives the expansion and compression of the PFR lattice. We propose that the PFR modifies the in-plane axoneme mo- tion to produce the characteristic trypanosome bihelical motility as captured by high-speed light microscope videography. 2107-Plat Hydrodynamics of the Double-Wave Structure of Insect Spermatozoa On Shun Pak 1 , Saverio Eric Spagnolie 2 , Eric Lauga 1 . 1 University of California San Diego, La Jolla, CA, USA, 2 Brown University, Providence, RI, USA. 414a Tuesday, February 28, 2012
Transcript of Structure of Trypanosoma Brucei Flagellum Accounts for its Bihelical Motion
414a Tuesday, February 28, 2012
1INSERM / Institut Curie, Paris, France, 2Universite Aix-Marseille,Marseille, France, 3C-CINA, Biozentrum, Basel, Switzerland, 4Institut Curie,Paris, France.Membrane-mediated protein-protein and protein-lipid interactions, membraneprotein localization, and related dynamics, modulate membrane protein func-tion. So far membrane structure and dynamics could not be studied altogetherlacking the technique that analyzes unlabelled proteins at submolecular lateraland high temporal resolution. Here we used high-speed atomic force micros-copy (HS-AFM) to characterize the lateral and angular movements and interac-tions of unlabelled OmpF. OmpF distribution emerges from diffusion-limitedaggregation, maximizing surface coverage and providing a multitude of localenvironments and contacts. Protein motion scales roughly with membranecrowding. However molecules display individuality of diffusion behavior rang-ing from fast moving (~400nm2/s) to immobile molecules trapped by favorableprotein-protein associations. We derive the complete molecular interactionprobability landscape that we compare with coarse-grained molecular dynam-ics simulation of the membrane protein interaction. HS-AFMmay open a novelresearch avenue that bridges structure of individual membrane proteins and su-pramolecular membrane architecture.HS-AFMmovie frames (frame rate 477ms) showing themotion ofOmpF trimers
in the membrane.Right: Experimentalinteraction potentialmap2103-PlatProbing Dielectric and Hydrogen Bonding Gradients in BiologicalMembranesTatyana I. Smirnova1, Maxim A. Voynov1, Oleg G. Poluektov2,Alex I. Smirnov1.1North Carolina State University, Raleigh, NC, USA, 2Argonne NationalLaboratory, Argonne, IL, USA.Nitroxide spin-labeling in combination with EPR spectroscopy has found manyapplications in studying structure and dynamics of proteins and biologicalmembranes. Recently, there has been a substantial interest in utilizing EPRto characterize local effects of polarity and hydrogen bonding in proteins andbiological membrane systems. Here we report on employing an arsenal of ad-vanced spin-labeling EPR methods to profile heterogeneous dielectric and hy-drogen bonding environment along the a-helical chain of an alanine-richWALP peptide that is anchored in a lipid bilayer in a transmembrane orienta-tion. A series of WALP cysteine mutants was labeled with a pH-sensitive nitro-xide IMSTL (S-(1-oxyl-2,2,3,5,5-pentamethylimidazolidin-4-ylmethyl) ester)that is similar in molecular volume to phenylalanine. The protonation stateof this nitroxide could be directly observed by EPR allowing us to follow pro-ton gradient across the membrane in the vicinity of the WALP a-helix, and,thus, to reconstruct the gradient in the effective dielectric constant. These ex-periments were complemented by assessing local polarity from characteristicchanges in EPR spectra that were enhanced by the use of perdeuterated and15N-substituted nitroxides and high field EPR at 130 GHz (D-band). Formationof hydrogen bonds between the nitroxides and membrane-penetrating watermolecules was observed directly in HYSCORE X-band experiments. Suchmeasurements allowed us to derive experimental profiles of heterogeneous di-electric and hydrogen bonding environment along a typical transmembranea-helix. Supported by: NSF-0843632 to TIS and NIH 1R01GM072897 to AIS.
2104-PlatPulmonary Surfactant Reduces Surface Tension to Low Values near Zerothrough a Modified Squeeze-Out MechanismFred Possmayer1, Nora Keating1, Yi Y. Zuo2, Nils O. Petersen3,Ruud A. Veldhuizen1.1University ofWestern Ontario, London, ON, Canada, 2University of Hawaii,Honolulu, HI, USA, 3University of Alberta, Edmonton, AB, Canada.Pulmonary surfactant stabilizes the lung by reducing surface tension (ST) tolow values near zero at end expiration. Atomic force microscopy studies re-vealed that, as surface pressure increases, spread surfactant films initiallyform solid micro- and nanodomains and then generate 3-5 multilayers whichapparently remain associated with the surface monolayer. Time-of-flight Sec-ondary Ion Mass Spectroscopy analyses revealed selective squeeze-out mustoccur because the multilayers are enriched in unsaturated fluid phospholipids(PL) while the remaining monolayer becomes enriched in disaturated PL.Taken together, these results are consistent with a modified squeeze-out modelwhere surfactant vesicles interact with the air-water interface through surfac-
tant proteins B- and C-containing adsorption/fusion pores. PL migration ontothe surface results in vesicle instability generating a monolayer at equilibriumsurface pressure, which remains functionally associated with excess bilayermaterial. Initially, the monolayer and associated reservoir have similar compo-sition, but film compression causes fluid PL to migrate through the fusion poresinto the multilayers. Gel phase PL are restricted because they are sequestered inmicro- or nanodomains. This process results in monolayers highly enriched indisaturated PL which reduce ST to near zero. Film expansion allows fluid PL toregain the surface through the fusion pores. This mechanism explains both therapid reincorporation of surfactant PL into the monolayer during adsorption andduring film expansion and the progressive improvement in surface activity dur-ing repeated compression.
Platform: Cell & Bacterial Mechanics& Motility II
2105-PlatFlexural Rigidity and Shear Stiffness of FlagellaGang Xu, Kate S. Wilson, Ruth J. Okamoto, Jin-Yu Shao, Susan K. Dutcher,Philip V. Bayly.Washington University, Saint Louis, MO, USA.Cilia are thin subcellular organelles that line airways and other passages andbend actively to propel fluid and foreign materials. The ciliary cytoskeleton(the axoneme) consists of nine outer microtubule doublets surrounding a centralpair of singlet microtubules. Large bending deformations of the axoneme in-volve relative sliding of the outer doublets driven by the motor protein dyneins.The genetics and cell biology of the ciliary structure and function have beenstudied extensively, but the mechanics of the axoneme remain unclear. Inthis study, we used the unicellular alga Chlamydomonas reinhardtii as themodel system for their flagellum replicates the highly conserved molecularstructure of the ciliary axoneme. Piconewton forces were applied perpendicu-larly on the tip of a single flagellum (length L) through a microsphere trapped inoptical tweezers. Dividing the force (P) by the corresponding deflection of theflagellar tip (d) yields the flexural stiffness of the flagellum (K = P/d), whichwas then used to calculate the apparent flexural rigidity (EI = KL3/3). The con-tributions of major structural components to passive mechanical propertieswere quantified by testing on flagella of specific mutations. The average appar-ent flexural rigidity of wild-type, pf-3 (without nexin links), and pf-13 flagella(without outer dynein arms) was about 27005 1100, 13005 550, and 6505140 pN$mm2, respectively. In addition, the ratio of elastic shear stiffness (resis-tance to interdoublet sliding) to true flexural rigidity was estimated by the coun-terbend response in bent flagella manipulated with a glass microneedle. Thequantitative understanding of axonemal mechanics will help illuminate theroles of certain genes and molecular structures in the normal and abnormalaxoneme.
2106-PlatStructure of Trypanosoma Brucei Flagellum Accounts for its BihelicalMotionMichael F. Schmid1, Alexey Y. Koyfman1, Ladan Gheiratmand2,Caroline J. Fu1, Dandan Huang2, Htet A. Khant1, Cynthia Y. He2, Wah Chiu1.1Baylor College of Medicine, Houston, TX, USA, 2National University ofSingapore, Singapore, Singapore.Trypanosoma brucei is a parasitic protozoan that causes African sleeping sick-ness. It contains a flagellum required for locomotion and viability. In additionto a microtubular axoneme, the flagellum contains a crystalline paraflagellarrod (PFR) and connecting proteins. We show here, by cryoelectron tomogra-phy, the structure of the flagellum in three bending states. The PFR lattice instraight flagella repeats every 56 nm along the length of the axoneme, matchingthe spacing of the connecting proteins. During flagellar bending, the PFR crys-tallographic unit cell lengths remain constant while the interaxial angles vary,similar to a jackscrew. The axoneme drives the expansion and compression ofthe PFR lattice. We propose that the PFR modifies the in-plane axoneme mo-tion to produce the characteristic trypanosome bihelical motility as captured byhigh-speed light microscope videography.
2107-PlatHydrodynamics of the Double-Wave Structure of Insect SpermatozoaOn Shun Pak1, Saverio Eric Spagnolie2, Eric Lauga1.1University of California San Diego, La Jolla, CA, USA, 2Brown University,Providence, RI, USA.
