Jill Trewhella- Biological Neutron Scattering: Now and the Future

8/3/2019 Jill Trewhella- Biological Neutron Scattering: Now and the Future

1/8

LA-UR-96- 1 4 3 3Title:

A ufhor(s):

Submitted to:

Los AlamosN A T I O N A L L A B O R A T O R Y

cl

Biological Neutron Scattering: Now and the Future

Jill Trewhella, CST-4

International Workshop on Science in Neutron-Arena ofJHPJXEK, Tsukuba, JapanMarch 26-27, 1996

LLos Aiamos National Laboratory, an affirmative action/equal opportunity employer, is ope rated by the University of Ca lifornia for th e U S . Departm ent of Energy ucontrac t W-7405-ENG-36. By acc ept anc e of this article, the publisher recognizes that the U.S. Government retains a nonexclusive, royalty-free lice nse to publireproduce the published form of this contribution, or to allow others to do so , for U S . Government purposes. The Los Alamos National Laboratory requests thaDublisher identify this article a s work Deriormed under the aus pic es of the U.S. Departm ent of Energy.

Form No. 8Ex2629DISTRIBUTION OF THIS DOCUMENT IS MMmeD p~


2/8

International Workshop on Science in Neutron-Arena of JHPKEK, Tsukuba, JapanMarch 26-27,1996BIOLOGICAL NEUTRON SCATTERING: NOW AND THE FUTURE

Jill TrewhellaChemical Science and Technology Division, Los Alamos National Laboratory,Mail Stop G758, os Alamos NM 87545ABSTRACT

Neutrons have an important role to play in st ruc ixk l biology. Neutron crystallography, small-angle neutron scattering and inelastic neutron scattering techniques can all contribute uniqueinformation on biomolecular structures. In particular, solution scattering techniques can givecritical information on the conformations and dispositions of the com ponents of com plexassemblies under a wide variety of relevant conditions. The power of these methods aredemonstrated by examples Pro teinD NA complexes, and Ca*"-binding proteins complexed withtheir regulatory targets. In addition, we demonstrate the utility of a new structural approachusing neutron resonance scattering. The impact of biological neutron scattering to date has beenconstrained principally by the available fluxes at neutron sources and the true potential of theseapproaches will only be realized with the development of new more powerful neutron sources.1. IntroductionThe 20th century has seen great advances in medicine and biotechnology that have been foundedon detailed knowledge of the structures and interactions of biological polymers: thepolynucleotides, DNA and RNA, which contain coded information that determines all themolecular structures needed to assemble a living system; and the polypeptides, or proteins, thatcarry out almost a ll the " ~ ~ r k "f living systems and are the ultimate expression of the codedinformation carried in the DNA. It has been the miracle of nature to design these polymersusing unique linear sequences of a relatively small number of chemical subunits (4 nucleotideacids for DNA, 20 am ino acids for proteins) such that they form highly specialized three-dimensional structures that fold reliably into their functional forms. U nderstanding biologicalfunction has become a study of these molecular forms, in sufficient detail to be ab le to describeand predict the physics and chemistry they perform in the microenvironments they create bybringing together specific chemical subunits. The latter half of the 20th century has seen rapidadvances in technologies that have led to an impressive structural data base on biologicalpolymers. Importantly, molecular biology and cloning techniques has enabled structuralbiologists to routinely obtain large amounts of pure samples for physical studies. X-raycrystallography has provided a wealth of high resolution structural information on protein s andpolynucleotides that can be coaxed to form crystals. NMR pectroscopy with isotope labelinghas opened the window to high resolution structure determination of small proteins, and evensmall polynucleotides in solution. Optical and laser spectroscopy techniques are used to probethe active sites of proteins at high resolution, and provide critical information on proteinstructural dynamics over the many orders of magnitude relevant to biological function(femtoseconds to hours). Electron microscopy and a number of novel new microscopytechniques are giving clearer images of molecular assemblies.In thisworld of high technology structural tools, neutron techniques have a special place. Asnon-ionizing radiation, neutrons are benign probes of biomolecular structures, that can locate


3/8

hydrogen atoms the very high precision in crystals, give information on the shapes anddispositions of the components of biomolecular complexes in solution, as well as probe themolecular dynamics of biological molecules over very broad range of timescales. My own workusing neutrons has focused on studies of biomolecular complexes in solution using small-anglescattering.2. Small-Angle Neutron ScatteringSmall-angle scattering from monodisperse particles in solution gives information on the particleshape. With neutrons, one can capitalize on the large difference in neutron scattering amplitudebetween the isotopes of hydrogen('H nd *H>. Selective deuteration of one componen t of acomplex of proteins in solution provides a way of altering the mean neutron scattering density ofthat component. Further, by changing the deuterium level in the solvent., the neutron scatteringcontrast of each component is varied. If internal scattering density fluctuations are negligible,then "solvent matching" can be achieved by ad justing the deuteration level in the so lvent so thatthe m ean solvent and particle (or component) scattering densities are the same, i.e. there is zerocontrast and hence no small-angle scattering fiom the particle rendering it "invisible" in theneutron experiment. "Contrast variation" techniques provide methods for extracting structuralinformation on the individual components of a complex and their relative dispositions [1,2]. Inthe case of DNN protein complexes one can take advantage of the natural difference in neutronscattering density between DNA and protein, thus making contrast variation experimentsfeasible without labeling either component. This ability to study interacting components in acom plex or assembly is extremely important if you wish to understand biomolecular function,since healthy function depends upon highly coordinated interactions between b iomolecules thatallow for signals to be transmitted in order to obtain the appropriate molecular responses tophysiological stimuli. Highlighted here are a few examples from my ownwork thatdemonstrate the pow er of neutron small-angle scattering fiom biomolecules in solution.3. Studies of Gene 5 proteinDNA ComplexesEssential proteins that bind nonspecifically, cooperatively, and preferentially to single strandedDNA (ssDNA) are required in the life cycles of viruses, prokaryots, and eukaryots. The ssDNAbinding protein (the gene 5 protein, or g5p) of filamentous bacterial viruses is among thesmallest of such proteins, having a molecular weight jus t under 10,000daltons, and there is awealth of detailed information about the physical properties of g5p which iswidely regarded asa 9 nodel" ssDNA binding protein whose further study will elucidate fundam ental mechanismsgoverning this class of proteinDN A interactions. The g5p forms a dimer (Fig. l) , and electronmicroscopy of fd g5p complexed with fd-ssDNA shows left-handed nucleoprotein superheliceswith variable pitch.W e undertook small-angle scattering experiments in combination with electron microscopy toprovide definitive experimental data to test m odels for gSp/fd-ssDNA superhelices, as well as toevaluate how unique the models might be [3]. In addition, we obtained new information on thedependence of nucleotide type and proteidnucleotide (PN)atio on the structure of thecomplexes. Reconstituted complexes were made with single-stranded fd viralDNA (fdssDNA), poly [d(A)] and poly [r(A)]. AU complexes formed similar left-handed, flexiblesuperhelices (Fig. 2) having approximately the sam e diameter, but the pitch d iffers among thesecomplexes. The g5p dimers associate to form a superhelical framework to which thepolynucleotide is attached. A combination of X-ray and neutron small-angle scattering data wasneeded to prove the nucleic acid is inside the protein superhelix. The neutron and X-rayexperiments provided two se ts of scattering data in w hich the relative contributions of theprotein and DNA to the scattering were significantly different ("contrast variation"). These data,


4/8

along with known structural parameters, provided sufficient constraints to define the relativelocations of the DNA and protein. A Monte Carlo integration modeling procedure applied to thescattering data was used to systematically test large numbers of possible superhelical models foreach complex, and previously proposed models based on param eters obtained from electronmicroscopy were found to be essentially correct and unique. The data on the complexes withdifferent P/N ratios showed that mass per un it length values decreased while the rise per dimerand pitch of the superhelix increased for gSp/fd-ssDNA complexes with decreasingP/N ratios.

Fig.1: g5p dimer with the its twossDNA-binding sites depicted bycrosses. The prote in structure isdepicted as a trace of its a-carbonbackbone. solid lines.

Fig. 2: Schematic drawing of the superhelixconstruction or the g5p DN A complex.Ellipses represent the g5p dimers, the 2 SSDNAsinterior to the protein envelop are indicated as4. Protein Complexes in Biochemical RegulationThe divalent calcium ion is the simplest of natures "messengers" used to regulate a broad rangeof cellular processes. Ca2+messages are generally "delivered" when Ca2+ inds to a protein andtriggers a cascade of molecular interactions leading to a physiological response. Small-angleneutron scattering has proven extr exely useful for studying the evolutionarily related,structurally similar dumbbell-shaped Ca2+-binding roteins calmodulin and troponin C and theirinteractions with the target proteins whose activity they regulate.4 . 1 Calmodulin Target Enzyme InteractionsCalmodulin is a multifunctional protein that acts as the major intra-cellular receptor for Ca2+, ndregulates the activity of a diverse array of target enzymes including a large number of kinases.The crystal structure of calmodulin shows it has two globular domains connected by anextended, solvent exposed a-helix of 7-8 turns [4] (Fig. 32. Each globular domain is made upof two pairs of helices that form a "cup-shape", with 2 Ca +-binding sites at the base of the cup(Fig. 5). Ca2+ inding to calmodulin causes the pairs of helices to move away from each otherexposing hydrophobic residues lining the inner surface of the cup. Small-angle neutron (and X-ray) scattering experiments showed calmodulin contracts about target enzym e binding domainswith the common characteristic of having a high propensity for forming a basic, amphipathic a-helix (for review see [5]). Fig. 3 shows the structure of calmodulin complexed with itshelicaltarget binding domain from myosin light chain kinase, determined at high resolution by NMR[6] and crystallography [7] subsequent to our neutron scattering experiments [SI. Thecontraction of calmodulin is achieved via flexibility in the interconnecting helix region of the


5/8

binding to different arrangements of hydrophobic and charged residues important in formingthese complexes.

p.

Fig. 3: a-carbon backbone trace showing the structures of 4Cd+*ca lmodulin lej?) and4Ca2+*cabnodulinomplexed with its binding domain in myosin light chain kinase (ri ht).& & -A second type of calmodulin binding domain has been identified in the catalytic subunit ofphosphorylase kinase. This system is distinctive because calmodulin is part of a multisubunitenzyme and its binding domain encompasses approximately70 residues (compared with themore usual 17-25residues). Using deuterium labeling and neutron scattering we showedcalmodulin remains extended in its interaction with the cataly tic subunit of phosphorylase kinaseand the nature of the interaction is fundamentally different to the previously characterizedcontracted structures [9]. he ability of calmodulin to modulate its conformation via flexibilityin its interconnecting helix region in order to accommodate different target binding domains is aremarkable exam ple nature building functional diversity aswell as specificity into a compact,unusual shape.4.2 Troponin C and Troponin ILike calmodulin in phosphorylase kinase, troponin C (TnC) is an integral component of amultisubunit complex: troponin. Muscle contractiodrelaxation is achieved when thick and thinfilaments made up of com plex assemblies of proteins slide past each other. The slidingmechanism is regulated by Ca" binding to TnC, resulting in a conformational change thattransmits a signal to troponin I (TnI), which in turn eleases its inhibition of the interactionbetween the thick and thin filaments which drives the sliding action. Our neutron scatteringexperiments show that, similar to calmodulin in phosphorylase kinase, TnC rem ains extended inits complex with its regulatory target TnI. The neutron data further show that Tn I is even moreextended than TnC and the centers-of-mass of the two components are approximately coincident[101. W e have modeled the complex [1 1 with TnC having a structure very similar o its crystalstructure [12,13] and TnI forming a spiral structure that encompasses the TnC (Fig. 4). TheTnI spiralhas he approximate dimensions of an a-helix and its central portion w inds throughthe two hydrophobic cup-shaped regions in each of Tnc 's two globular domains.This solution structure of 4Caz+*TnC*TnI ives important insights into the m olecular basis forthe Ca2+-dependent egulation of muscle contraction. In the Ca2+-bound omplex, both ends ofthe TnI central spiral region are anchored by interactions with the hydrophobic cup regions ofTnC, and th e TnI inhibitory sequence is constrained to be associated with TnC and theinhibitory function is switched off. The loss of Ca" from the N-terminal low-affinity Ca2'-specific binding sites results in a closing of that domain, lowering its affinity for Tn I at the N-


6/8

terminal end, and allowing the T nI inhibitory sequence (96-115)the flexibility to shift from itsbinding site on TnC to that on actin, switching the inhibition on. The regulatory signal is thustransmitted via the central spiral region of TnI, which contains the inhibitory sequence.

Fig. 4: Right Stereo drawing of the model of 4Ca2-1-eTnCeTnI. The white crosses depict TnI,which ollows a spiral path winding around the TnC represented as an a-carbon backboneribbon drawing. The N- nd C-terminal domains of TnC are labeled asare helices C,E, and Gwhich were know n to interact with Tnl. Left The same model with TnC represented as a CPKmodel.5. Neutron Resonance Scattering from 240Pu-labeledCalmodulinNuclei with strong resonances in their coherent neutron scattering amplitudes include the softmetals 'I3Cd, '"In, rare earths '49Sm,1 5 1 E ~ ,S7Gd,'63Dy,68Yby77Hfyhe noble metal 1931r,and actinides U"Th .mPu [14,15]. Of these nuclei, *?Pu has the strongest and sharpestresonance (at 0.281, ig. 6 ) hat lies within the wavelength regime accessible using the Low-Q

A530,A1 I ' I I ' 1 I I ----3---

E -'E0 -10t: -h e l i x p - 2 0 = l I I I I I 1 I 1 1 $ 9

0.24 0.26 0.28 0.30 0.32(4IIFig. 5: Backbone structuresof the'structurallyhomologousN- nd C-terminal domains ofca lm du lin showing the Ca'+-bindingsitesthat were labeled with 240Pu.

Fig 6: Coherent scattering length or240Pun the vicinity of the resonance.


7/8


8/8

DISCLAIMERThis report was prepared as an account of work sponsored by an agency of theUnited States Government, Neither the United St ates Government nor any agencythereof, nor any of their employees, makes any warranty, express or implied, orassumes any legal liability or responsibility for the accuracy, completeness, or use-fulness of any information, apparatus, product, or process disclosed, or representsthat its use would not infringe privately owned rights. Reference herein to any spe-cific commercial product, process, or service by trade name, trademark, manufac-turer, or otherwise does not necessarily constitute or imply its endorsement, m m -mendation, or favoring by the United States Government or any agency thereof.The views and opinions of authors expressed herein do not necessarily state orreflect those of the United State s Government or any agency thereof.

.

Jill Trewhella- Biological Neutron Scattering: Now and the Future

Documents

Transcript of Jill Trewhella- Biological Neutron Scattering: Now and the Future