Small-Angle Solution Scattering of Proteins

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Ruth Prassl, Michal Hammel and Peter LaggnerInstitute of Biophysics and X-Ray Structure Research, Austrian Academy of Sciences, Schmiedlstr. 6, A-8042 Graz, Austria

Correspondence : Univ.Doz. Dr. Ruth Prassl, Institute of Biophysics and X-Ray Structure Research, Austrian Academy of Sciences, Schmiedlstr.6A-8042 Graz, Austria. E-mail : [email protected]

Small-Angle Solution Scattering of Proteins

ABSTRACTIn structural proteomics it has become increasingly evident

that the structure of proteins in solution and under the influenceof different, specific or non-specific additives, is often differentfrom the crystal structure. This feature might exert fundamentalfunctional influence on e.g. membrane association, ligand orreceptor binding, enzyme activity or immune response.

At the same time, the methods of small angle scattering havereached a state of development, both in hard- and software,where the routine application in the proteomics laboratory hasbecome feasible. Hence, questions such as oligomerization orconformational changes induced by different environmentalconditions can be answered. The impact of small molecules,ligands, lipids or drugs on the overall conformation of the proteincan be visualized. Moreover, a rational combination of smallangle scattering data with X-ray crystallographic data, molecular

dynamics and bioinformatics offers to structural biologists theopportunity to assemble individual protein modules in a bottom-up strategy to get information on complex macromolecularstructures. Thus, most recent advances in instrumentation andmethodology of small angle scattering have opened importantnew ways for molecular structure determination.

In this minireview we report on the rapid increasing progressachieved in the field of small angle scattering techniques andoutline some problems which can be addressed by solutionscattering. We briefly review the strengths and limitations of themethodology and finally discuss some of our recent resultsshowing how small angle scattering can be used to obtainessential information on protein structure in solution and howconclusions on the functional behavior of a protein can be drawnbased on solution structural data.

INTRODUCTION

Proteins are central elements of life and key players inhuman physiology and cell machinery. Their functionalityis often triggered by subtle changes in their structure, thus,knowledge of structure is fundamental to understandprotein specificity and metabolic pathways. In the lastdecades major efforts have been devoted to the elucidationof the three-dimensional structure of proteins by X-raycrystallography or by NMR. Due to rapid technicaldevelopments including high-throughput automation thereis an exponential increase in 3D- protein structures depositedin the Protein Data Bank (PDB) over the last few years. Atpresent more than 32000 structures are available in the PDB

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but only 79 unique membrane protein structures have beensolved although more than 30% genes of all genomes codefor membrane proteins. Likewise, just a somewhat highernumber of three-dimensional structures on nativeglycosylated or membrane-associated proteins are availablein the PDB. The reason for such a limited number of complexprotein structures solved at atomic resolution is that proteincrystallisation is a tedious task and the bottleneck in X-raycrystallography is still the growth of single crystals of highquality, whereas NMR techniques although steadilyimproving are limited to smaller proteins. However, valuableinformation on the three-dimensional structure of proteinsat the nanoscale can also be obtained by imagereconstruction techniques from e.g. cryo-electronmicroscopy images, and recently more and more by lowresolution modelling from small angle scattering (SAS)patterns. As solution scattering is very sensitive to changesin the tertiary and quarternary structure of proteins, thelatter methodology is excellently suitable for the analysis ofmulti-modular macromolecular assemblies which areotherwise difficult to be crystallized. However, even ifcrystallographic data of a protein are known, it wasdemonstrated that the conformation of the protein insolution might be different as compared to the crystalstructure.1-4 In this regard, it has to be considered that thecrystal structure represents a “frozen” snap-shot of the proteinstructure. In case of proteins with compact rigidconformation composed of well defined structural elementsthis static picture is perfectly realistic. However, in case ofmulti-domain proteins composed of distinct rigid domainsconnected by flexible linkers or hinge regions some inherentprotein motion has to be taken into account. This intrinsicdynamics takes place in linker regions or in disorderedrandom coil regions connecting protein modules. In view ofthis internal motional freedom the protein might adopt morethan one conformation in solution. This conformationalheterogeneity is directly reflected in an increased mobilityof atoms leading to a low intensity in the electron densitymap or high B-factors which makes a determination ofatomic coordinates for these regions extremely difficult oreven impossible. Besides, such complex proteins often refusecrystallisation setups or never yield suitable crystals. However,it has to be stressed that this internal flexibility might beimportant for proper function of the protein as such segmentsoften contain important regulatory sequences which arecritical for biological activity. Moreover, dynamicfluctuations between structural domains may play anintriguing role for ligand binding or receptor recognition. Inthis context, small angle solution scattering with X-rays(SAXS) or neutrons (SANS) is an excellent tool to provideessential information complementary to that obtained byX-ray crystallography or NMR. The informations obtainedby a rational combination of various techniques will produce

a more comprehensive structural picture on multi-modularmacromolecular assemblies with respect to differentenvironmental conditions.

METHODOLOGICAL DEVELOPMENT

Since early reports in the seventies on the application ofsmall angle scattering for biological macromolecules (forreviews see5,6) SAS techniques have developed rapidly overthe years. First results were limited to characteristicmolecular parameters like overall shape and size of themacromolecule and were used for molecular massdeterminations.7 These parameters are still fundamentalfor the methodology, however, with the developement offast computer and software algorithms, the evaluation ofSAS patterns has become more sophisticated, reliable andconvenient and has soon enabled the construction of simplemolecular models.8 The first low resolution models restoredfrom SAS curves were assembled using simple spherical,cylindrical or disk-like models for comparison.9 Subsequently,the first ab initio approaches have been started to determinemolecular envelopes. Hitherto, fundamental work in thedevelopment of modelling theorems was performed bySvergun.10,11 With the implementation of the so-called “beadmodel” reliable ab initio shape reconstructions could beperformed.12,13 Briefly, ab initio modelling is done byarranging a number of beads within a confined volume,which is given by the maximal particle dimension derivedfrom the scattering pattern. Distinct bead arrangementswhich yield reasonable fits to the experimental scatteringcurve are taken as results. Thus, bead modelling yieldsmultiple models regarding spatial distribution. In this vein,the final most reliable low resolution model is obtained byalignment, superposition and averaging of numerousdifferent models14 Another promising ab initio approach isto take dummy residues for all amino acids and to arrangethem to find the spatial distribution that best fits theexperimental scattering curve.15 Often, a direct comparisonof several ab initio approaches is performed and certainlyterminates in the most probable three dimensional model ofthe protein. For excellent detailed reviews on modellingapproaches the reader is directed to Svergun and Koch16

and Koch et al.17

FROM THE CRYSTAL STRUCTURE TOTHE SOLUTION SCATTERINGPATTERN

If the crystal structure of the protein is known, a theoreticalsolution scattering pattern can be calculated from thecrystallographic atomic coordinates of the protein by specificprogrammes.18 If the scattering profile computed from thecrystallographic coordinates displays significant deviations

Ruth Prassl


from the experimental curve, molecular design andmanipulation programmes can be applied to performinteractive modifications of the atomic structures. By thismeans, sequences or loops missing in the crystal structurecan be supplemented and modelled.19 The theoreticalscattering curves are thus computed from these artificiallymodified structures, which are automatically compared tothe experimental scattering curve.18 By integration ofmolecular dynamics simulations the conformation of thesetentative residues can be optimized to fit the experimentalcurves. Thereby, large segments of the structure remainstatic but their relative positions to each other are varied.These procedures are repeated iteratively to yield the bestmatch.20

SAS is also an excellent method to obtain structuralinformation on multimeric enzymes or multidomain proteins.To facilitate crystallisation parts of the protein are oftenremoved during cloning leading to truncated fragmentswith cut-off hinge regions or single protein domains areused for crystallogaphy. All these single module structuresare deposited in the PDB and can be used to build wholeentities from distinct parts followed by reconstruction of theentire protein by e.g. rigid body refinement. Thus, singleprotein domains, which have been studied and depositedseparately, can be taken and built together like lego bricks,interconnected by dummy residues or randomly orientedamino acid sequences. Their relative conformations can beoptimized till the best match to experimental SAS data isobtained. Similarly, different protein modules or specificligands can be combined .21 Even missing modules in thehigh resolution structure can be added by homologymodelling22 combined with sequence homology search andsecondary structure alignment. The spatial orientation ofthese parts can be optimized by molecular dynamicssimulations. Interactively, the theoretical scattering curveis calculated and compared to the experimental one. Bythis way, thousands of different conformations can bemodelled to obtain the best fit to the experimentalcurves.2,23,24 However, one has to bear in mind that thesolution is not unique, and different distinct proteinconformations might exhibit the same structural probability.Again, the reliability of the model has to be confirmed byalignment and superposition of different models.

ADVANTAGES OF SAS METHODS

The great advantage of SAS technique lies in thepossibility to investigate protein structures in solution atdistinct environmental conditions without any specialsample preparation. Most likely, at mild conditions, i.e. atdefined, low ionic strengths or at physiological temperatureand pH-value. SAS offers the opportunity to detect changes

in structure that might occur just by adding salts, smalladditives, ligands or drugs. All these options can hardly beachieved during crystallisation, in which high salt orprecipitant concentrations are required to induce crystalgrowth. Thus, crystallisation has strong restrictions to fixedexternal conditions and might also be prone toconformational restrictions induced by crystal latticeformations.

SAS can be applied for small protein species, however, thespecial strength of this methodology consists in itsapplicability for very complex systems, as proven for examplefor ribosomes and factor H of human complement.25

Nowadays, performing time resolved measurements atsynchrotron radiation sources SAS can be used to visualizedomain motion, to view conformational changes in real timeor to follow up agglomeration behavior upon ligandassociation.26 With the high intensity and brilliance ofsynchrotron radiation resolvable data can be obtained in amilllisecond time scale, thus, processes like protein foldingand unfolding and intermediate states can be followed insitu.27,28

Taken together, SAS techniques have reached a state ofdevelopment in which they become more and moreinteresting not only to scattering experts but also to structuralbiologists in general.

LIMITATIONS OF SAS METHODS

One has to be aware of the fact that SAS is a low resolutionmethod and experimental data are limited to a resolution of1 to 1.5 nm although some additional information on proteinfolding is buried in the outer parts of the scattering curves.In contrast, by X-ray crystallography resolution limits of about0.1 nm can be achieved. Thus, SAS is not the appropriatemethod to determine the secondary structure of a proteinand most significantly, SAS does not present a uniquesolution as the small angle scattering profile provides anaverage of all conformations that the protein adopts withinthe sample volume during the time course of measurement.Accordingly, SAS strongly needs the support of additionalbiochemical or biophysical data to be able to draw explicitconclusions.

LOW RESOLUTION PROTEIN MODELS

As already outlined above, a variety of problems can beaddressed by solution scattering serving as complementarysource of information. In the following two examples ofmultidomain mammalian proteins which exhibit a distinctdifferent spatial orientation in solution as compared to their



crystal structure will be briefly outlined.In both proteins,the domains are interconnected by disordered linkerregions, which bear some intrinsic flexibility. In both casesthe differences in conformation are of physiologicalrelevance.

β2 -Glycoprotein-I (β2GPI) is a highly glycosilated plasmaprotein comprised of four conserved complement controlprotein (CCP) domains and a domain V, which wasidentified as phospholipid-binding domain. 29,30

Functionally, β2GPI appears to have several roles. The mostprominent role is as cofactor in autoimmune diseases suchas antiphospholipid syndrome or systemic lupuserythematosus.31-33 However, β2GPI seems also to beinvolved in platelet aggregation, in blood coagulationcascade and in thrombotic diseases, moreover, through itsbinding affinity to acidic phospholipids or macrophagesβ2GPI seems to play a role in apoptotic processes.34 Thecrystal structure of β2GPI was solved in 1999 and showedan extended rod-like arrangement of the CCP domainsfollowed by the lipid-binding domain V.35,36 Although thelow resolution model of the protein reconstructed fromsolution scattering data also revealed an elongated particle,the overall shape of the protein in solution was more bulkyand bent.4 Consequently, large deviations were observedby comparing the experimental to the theoreticalscattering curve, which was calculated from the crystalcoordinates. To overcome this gap, the crystal structurewas modified based on the knowledge that mostcarbohydrate residues are missing in the crystal structuredue to a lack of interpretable electron density. Thus, themissing carbohydrate chains were modelled and theirconformation interactively optimized for the best fit to theexperimental data. Further assuming a rigid internal CCPdomain structure, the observed flexibility in structure hasto be buried within the interdomain short linker regions.In this respect, a simple rotation between CCP-domain 2and 3 resulted in a conversion of the overall shape yieldingthe best fit to the experimental data (Figure 1). Hence,we could show that in solution at nearly physiologicalconditions the protein adopts a bent spatial conformationthrough interdomain movement.4 This conformationalflexibility is probably an essential structural feature for theproper function of the protein as it has been reported thatafter immobilisation or cross-reaction of β2GPI domainswith ligands the protein adopts a conformation that revealsdistinct differences in autoantibody recognition andimmunological activity.37,38

Mammalian lipoxygenases are lipid peroxidizing enzymes,which have been implicated in the biosynthesis ofinflammatory mediators, in cell differentiation and in thepathogenesis of atherosclerosis and osteoporosis.

Figure1. Discrepancies between experimental SAS data andthose calculated from the crystal structure are seen directly inthe scattering curve as well as in the distance distributionfunction p(r). In (A) the experimental SAXS pattern of humanβ2GPI (dotted line) is shown in comparison to the theoreticalSAXS profile calculated from the crystal structure PDB-code :1C1Z (solid line)35 by the program CRYSOL.18 Thecorresponding distance distribution functions p(r) calculatedfrom the scattering patterns by the program GNOM45 aredisplayed in (B). Both profiles, theoretical and experimental,are typical for elongated particles, however, the shape of thetheoretical p(r) function for the crystal structure shows a higheranisotropy, while the pronounced first maximum at 1.9 nmcorresponds to the cross section thickness of the complementcontrol protein domains. The low resolution model wasreconstructed from the solution scattering data by the programDAMMIN13 and shows an “S”-like shape (C). In contrast, inthe crystal structure shown in (D), the CCP domains arearranged like beads on a string followed by domain V formingan elongated “J”-like overall structure. By modelling ofcarbohydrate residues to the crystal structure and by takinginto account some inherent flexibility of the interdomainlinkers a modified crystal structure was designed whichoptimally fits the low resolution model as shown bysuperposition in (C). Reproduced fromHammel et al.4

Ruth Prassl


Lipoxygenases are composed of two compact domains andthe crystal structures of the rabbit enzyme and of two plantisomers are already known.39-41 The availability of the crystalstructure allows for a direct comparison to the solutionscattering data. Superposition of the crystal structure of rabbit15-lipoxygenase-1 and the low resolution model of thesolution structure lead to a perfect superposition in the regionof the catalytic domain, which is spherical as confirmed bySAXS studies on the isolated N-terminal deletion mutant.However, the low resolution model showed a stretched-outmolecular shape, which poorly fits the N-terminal domain.

Thus, the data could only be explained as motional flexibilityof the N-terminal domain relative to the catalytic domain.42

This hypothesis of a relaxed surface structure in solutionwith the N-terminal β-barrel domain dislocated from thecatalytic domain as shown in Figure 2, seems biologicallyreasonable, as Trp100 which was shown to be involved inmembrane binding becomes surface exposed during swing-out and thus accessible to lipids. In contrast, in the crystalstructure Trp100 is deeply buried in the contact plane of thedomains unaccessible to lipids. The validity of the modelwas further supported by membrane binding and site-directed mutagenesis studies.

Another example shows how single protein modules canbe combined to yield a comprehensive picture of a complexprotein assembly. Cellulosomes are multienzymatic,extracellular complexes in a molecular range of 0.7 to 2MDa produced by anaerobic bacteria for the degradationof plant cell walls. Through assembling cellusosomes start toact highly synergistic as compared to the free enzymes. Thestructural arrangement, which finally leads to the synergisticproperties constitutes the basis for the design of cellulosomesand is therefore of special industrial interest. Clostridialcellulosomes are composed of a cellulose binding module,several cohesin modules, which bind to the catalytic subunitsvia a module named dockerin (Figure 3). So far, onlytruncated forms of cellulosomal enzymes lacking theirdockerin domain have been crystallized. SAXS experimentson full length cellulase containing the native dockerin aswell as their assemblies with the cognate cohesin have beenperformed. The models obtained for the full length cellulasesindicate that the catalytic as well as the dockerin domainare folded and that the dockerin domain adopts an identicalconformation as the isolated dockerin domain, whosestructure was solved by X-ray crystallography and NMR.43,44

The models inferred from the SAXS data and the resultsfrom normal mode analysis (NMA) also show that the linkerconnecting the catalytic module to the dockerin is extendedand flexible. On the other hand the results obtained for thecellulase – cohesin complex clearly define a more compactcharacter for the complex, with a lower flexibility (Figure3). This fact implies that docking of the full length cellulaseto the cohesin leads to a pleating of the cellulase linker.21 Bythis means, the synergistic interplay of single componentscould be investigated and has yielded a more comprehensivepicture of the spatial orientations of molecular assemblies insolution.

DETERGENT SOLUBILIZED PROTEINS

Detergents are indispensable for the isolation, expressionand finally for the handling of membrane associated proteinswhich are poorly water-soluble. For solubilization purposes


Figure 2. In the surface presentation based on the SAXS model(A), Trp100 of rabbit 15-lipoxygenase-1 becomes solventexposed and accessible for membrane binding. In contrast, asseen in the surface presentation of the crystallized enzyme (B),Trp100 is shielded by the catalytic domain. In bothpresentations, the amino acid residues, which have been ruledout as determinants for membrane binding by mutation studies,are highlighted in red, whereas Trp 100 is shown in yellow. Theflexibility of the interdomain linker region, shown in pink,allows for the movement of the N-terminal domain, whichmost likely adopts different conformations in solution. Thus,the low resolution model displays an average of all motionswhich occur during the measuring period.Reproduced from Hammel et al.42


the protein has to be depleted from lipids by the aid ofdetergents, which shield the hydrophobic parts of the proteinfrom water contact. After the purification the membraneprotein solution contains a protein-detergent complex,which often exhibits a very complex geometry. As alreadymentioned, crystallisation of such protein-detergentcomplexes is a tedious and time-consuming task and highresolution X-ray crystallography is often hampered by thelack of suitable, well-ordered crystals. Accordingly, it is ofprincipal interest to obtain knowledge on the morphologyand the oligomeric state of the solubilized protein. To thisend, SAS can be applied to obtain information on thearchitecture of the protein in solution in terms of overall sizeand shape. Furthermore, additional information onmolecular asymmetry, dimerisation, domain assembly anddomain orientation can be derived from the reconstructedlow resolution models. Moreover, an estimation of rigid andflexible regions within the molecule can be made. However,for detergent-protein complexes the use of small angle X-ray scattering (SAXS) is limited, because of the additionalscattering contribution of the detergent to the scatteringintensity yielding a composite detergent-protein scatteringcurve. More benefical is the application of neutron scattering(SANS), as neutron technique offers the possibility ofselective contrast matching by exploiting the differences inscattering length density of H2O and D2O. H2O/D2Osubstitution hence allows to completely match thecontributions of the detergent to the neutron scatteringprofile. Consequently, with SANS the shape and size of theprotein without contribution of the detergent can be

determined. In principle, similar equations and programmesas for SAXS can be applied to determine the characteristicscattering data and to restore the low resolution modelsfrom experimental curves. By this way, valuable informationon the morphology of membrane proteins or proteins of poorwater solubility which are only stable in the presence ofdetergents can be obtained (for an example, see Figure 4).

CONCLUDING REMARKS

The increasing number of SAS low resolution paperspublished in high ranking scientific journals, sometimes inparallel or comparative to crystal structures, strongly arguesfor the advances achieved in structure determination fromsmall-angle scattering data within the last few years.Questions such as oligomerization or conformationalchanges of a given protein in solution at differentenvironmental conditions can now be answered easily andquickly, thus enabling the researchers to make structure-based decisions in a time commensurate to the rate ofproduction of new proteins. Reconstitution of high molecularassemblies starting with single fragments, as well as thedescription of complexes with ligands, antibodies or lipidsbecome possible. Alternatively, important informations onthe morphology and folding of solubilized membraneproteins in solution prior to crystallisation can be derived. Inconclusion, this review represents a short excerpthighlighting some recent promising results that underlinethe emerging potential of SAS for molecular structuredetermination.

Figure 3. Atomic model of the entire cellulase based on the SAS model. The restored low resolution model of a cellulase complexed with cohesin is shown in surface presentation. The secondary structural elements of the crystal structure of the catalyticmodule of Cel48F (PDB-code: 1G9G) and of the cohesin-dockerin module from C.celluloyticum (PDB-code: 1G1K) aresuperimposed. A typical conformation for the constructed linker and the missing loops is shown. For clarity, the modules used forthe cellusosome assembly are schematically depicted in the left panel. Reproduced from Hammel et al. 21.

Ruth Prassl


ACKNOWLEDGMENTS

The authors would like to thank all colleagues, who havecontributed to the studies outlined in this article. The studiespresented herein were in part supported by the Fonds zurFörderung der wissenschaftlichen Forschung (P16479-N11).

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Ruth Prassl

Small-Angle Solution Scattering of Proteins

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