Anomalous x-ray scattering from terbium-labeled parvalbumin in solution

We have used anomalous small-angle x-ray scattering as a structural probe for solutions of rabbit parvalbumin labeled with terbium. This technique makes use of the large changes in the terbium scattering factor that occur when the x-ray energy is tuned around an L3 absorption edge of this heavy-atom label. These changes in scattering result in changes in the small-angle scattering curve of the labeled protein as a whole, which can then be analyzed to derive structural information concerning the distribution of labels in the protein. Based on a Gaussian model for the protein electron density, the mean distance from the terbiums to the protein center of mass is determined to be 13.2 A and is consistent with crystallographic results. Our results demonstrate the usefulness of terbium as an anomalous scattering label and provide criteria to help establish anomalous scattering as a reliable structural technique for proteins in solution.     

X-ray solution scattering studies of the structural diversity intrinsic to protein ensembles

It is becoming increasingly clear that characterization of the protein ensemble-the collection of all conformations of which the protein is capable-will be a critical step in developing a full understanding of the linkage between structure, dynamics, and function. X-ray solution scattering in the small angle (SAXS) and wide-angle (WAXS) regimes represents an important new window to exploring the behavior of ensembles. The characteristics of the ensemble express themselves in X-ray solution scattering data in predictable ways. Here we present an overview of the effect that structural diversity intrinsic to protein ensembles has on scattering data. We then demonstrate the observation of these effects in scattering from four molecular systems; myoglobin; ubiquitin; alcohol dehydrogenase; and HIV protease; and demonstrate the modulation of these ensembles by ligand binding, mutation, and environmental factors. The observations are analyzed quantitatively in terms of the average spatial extent of structural fluctuations occurring within these proteins under different experimental conditions. The insights which these analyses support are discussed in terms of the function of the various proteins.     

A study of protein structure changes during hydration by diffuse X-ray scattering. I. The intensity and the shape of "10-angstrom" maximum

The angle dependencies of diffuse x-ray scattering intensities were studied in a wide range of angles from 3 to 80 degrees for water-soluble and membrane proteins with a different structural organization: alpha-helical protein myoglobin, alpha-helical protein serum albumen, alpha + beta protein lysozyme, and transmembrane proteins of photosynthetic reaction centers (RC) from purple bacteria Rhodobacter sphaeroides, and Blastochlorii (Rhodopseudomonas) viridis containing cytocrome c, situated out side the membrane, and for H and L+M subunits of membrane protein of reaction center from Rb. sphaeroides for various hydration degrees. The hydration/dehydration process was studied for water-soluble proteins (within hydration range from h = 0.05 to h = 1). The hydration/dehydration process appears to be reversible. All water-soluble proteins show a 10 angstroms peak, and proteins of reaction center do not show this peak. A quantitative comparable study of the behaviour for of the 10 angstroms peak different proteins the degree of lysozyme hydration increases from h = 0.05 to h = 0.45, the protein structure slightly changes (most probably the motifoffolding), the structure of myoglobin in solution is slightly different from the structure in crystal. By taking into account the changes in the shape and intensity of the 10 angstroms peak only, it is impossible to make the conclusion about structural changes in other proteins studied. A correlation between the structural changes observed and dynamic and functional properties of proteins is discussed.     

A study of protein structure changes during hydration by means of diffuse X-ray scattering. II. Fourier transform analysis of X-ray scattering data

Radial distribution functions were deduced by Fourier transform analysis of angular dependences of diffuse x-ray scattering intensities for the following proteins with different hydration degree: water-soluble a-protein myoglobin, water-soluble alpha+beta protein lysozyme, and transmembrane proteins of photosynthetic reaction centers from purple bacteria Rhodobacter sphaeroides and Blastochlorii viridis. The results of Fourier analysis of x-ray scattering intensities give the quantitative characteristics of the mechanisms underlying the influence of water on the formation of biomacromolecules. Water, on the one hand, weakens the intraglobular hydrogen bond net, loosens the protein structure, and increases the internal conformational dynamics. Concurrently water arranges the stability and ordering of the macromolecule. A sharp correlation is observed between the shift of the "first" peak of radial distribution functions, the weakening of the intraglobular hydrogen bond net, the increase in intraglobular mobility, and the appearance of functional activity in macromolecules. The behavior of the "first" peak is similar to that observed in transmembrane protein of reaction center and water-soluble proteins. The "first" peak for transmembrane protein of reaction center reaches its maximum value much faster (at smaller hydration degrees) than for water-soluble proteins. The fast transfer of reaction center protein to its native state during hydration is due to the fact that the dehydrated conformation of reaction center protein is very close to the native one. From a comparison of the radial distribution functions for water, water-soluble proteins and transmembrane proteins, one may conclude that water has the lowest packing density and the lowest order; water-soluble proteins have a larger packing density and are more ordered than water, and transmembrane proteins have the highest degree of packing density and ordering.     

Protein conformations explored by difference high-angle solution X-ray scattering: oxidation state and temperature dependent changes in cytochrome C

We demonstrate the use of high-angle X-ray scattering to explore protein conformational states in solution by resolving oxidation state- and temperature-dependent changes in the conformation of horse heart cytochrome c. Several detailed models exist for oxidation-dependent changes in mitochondrial class I c cytochromes determined by X-ray crystallography and solution NMR techniques. These models differ in the magnitude and locations of structural change. Our scattering measurements show that high-angle X-ray scattering can discriminate between these models, and that the experimental scattering data for horse cytochrome c can be best reconciled with selected NMR models for the same protein. These results demonstrate the ability to use high-angle X-ray scattering to resolve conformational states of proteins in solution, and to relate these measurements to detailed structural models. Furthermore, temperature-dependent changes are found in the high angle scattering patterns for horse cytochrome c, illustrating the sensitivity of these measurements to dynamic aspects of protein structure. These results demonstrate the ability to use difference high angle scattering as a quantitative monitor of reaction-linked changes in protein conformation and structural dynamics. Synchrotron-based high-angle scattering holds promise as a widely applicable, high throughput technique for exploring conformational states linked to physiological protein function, for resolving configurational differences between protein structures in solution and crystalline states, and for bridging the gap between solution NMR and crystallographic structure techniques.     

Anomalous diffusion of proteins due to molecular crowding

We have studied the diffusion of tracer proteins in highly concentrated random-coil polymer and globular protein solutions imitating the crowded conditions encountered in cellular environments. Using fluorescence correlation spectroscopy, we measured the anomalous diffusion exponent alpha characterizing the dependence of the mean-square displacement of the tracer proteins on time, r(2)(t) approximately t(alpha). We observed that the diffusion of proteins in dextran solutions with concentrations up to 400 g/l is subdiffusive (alpha < 1) even at low obstacle concentration. The anomalous diffusion exponent alpha decreases continuously with increasing obstacle concentration and molecular weight, but does not depend on buffer ionic strength, and neither does it depend strongly on solution temperature. At very high random-coil polymer concentrations, alpha reaches a limit value of alpha(l) approximately 3/4, which we take to be the signature of a coupling between the motions of the tracer proteins and the segments of the dextran chains. A similar, although less pronounced, subdiffusive behavior is observed for the diffusion of streptavidin in concentrated globular protein solutions. These observations indicate that protein diffusion in the cell cytoplasm and nucleus should be anomalous as well, with consequences for measurements of solute diffusion coefficients in cells and for the modeling of cellular processes relying on diffusion.     

Charge centers and formation of the protein folding core

Electrostatic interactions are important for protein folding. At low resolution, the electrostatic field of the whole molecule can be described in terms of charge center(s). To study electrostatic effects, the centers of positive and negative charge were calculated for 20 small proteins of known structure, for which hydrogen exchange cores had been determined experimentally. Two observations seem to be important. First, in all 20 proteins studied 30-100% of the residues forming hydrogen exchange core(s) were clustered around the charge centers. Moreover, in each protein more than half of the core sequences are located near the centers of charge. Second, the general architecture of all-alpha proteins from the set seems to be stabilized by interactions of residues surrounding the charge centers. In most of the alpha-beta proteins, either or both of the centers are located near a pair of consecutive strands, and this is even more characteristic for alpha/Beta and all-beta structures. Consecutive strands are very probable sites of early folding events. These two points lead to the conclusion that charge centers, defined solely from the structure of the folded protein may indicate the location of a protein's hydrogen exchange/folding core. In addition, almost all the proteins contain well-conserved continuous hydrophobic sequences of three or more residues located in the vicinity of the charge centers. These hydrophobic sequences may be primary nucleation sites for protein folding. The results suggest the following scheme for the order of events in folding: local hydrophobic nucleation, electrostatic collapse of the core, global hydrophobic collapse, and slow annealing to the native state. This analysis emphasizes the importance of treating electrostatics during protein-folding simulations.     

Small-angle scattering: a view on the properties, structures and structural changes of biological macromolecules in solution

A self-contained presentation of the main concepts and methods for interpretation of X-ray and neutron-scattering patterns of biological macromolecules in solution, including a reminder of the basics of X-ray and neutron scattering and a brief overview of relevant aspects of modern instrumentation, is given. For monodisperse solutions the experimental data yield the scattering intensity of the macromolecules, which depends on the contrast between the solvent and the particles as well as on their shape and internal scattering density fluctuations, and the structure factor, which is related to the interactions between macromolecules. After a brief analysis of the information content of the scattering intensity, the two main approaches for modelling the shape and/or structure of macromolecules and the global minimization schemes used in the calculations are presented. The first approach is based, in its more advanced version, on the spherical harmonics approximation and relies on few parameters, whereas the second one uses bead models with thousands of parameters. Extensions of bead modelling can be used to model domain structure and missing parts in high-resolution structures. Methods for computing the scattering patterns from atomic models including the contribution of the hydration shell are discussed and examples are given, which also illustrate that significant differences sometimes exist between crystal and solution structures. These differences are in some cases explainable in terms of rigid-body motions of parts of the structures. Results of two extensive studies--on ribosomes and on the allosteric protein aspartate transcarbamoylase--illustrate the application of the various methods. The unique bridge between equilibrium structures and thermodynamic or kinetic aspects provided by scattering techniques is illustrated by modelling of intermolecular interactions, including crystallization, based on an analysis of the structure factor and recent time-resolved work on assembly and protein folding.     

BCL::SAXS: GPU accelerated Debye method for computation of small angle X‐ray scattering profiles

Small angle X‐ray scattering (SAXS) is an experimental technique used for structural characterization of macromolecules in solution. Here, we introduce BCL::SAXS—an algorithm designed to replicate SAXS profiles from rigid protein models at different levels of detail. We first show our derivation of BCL::SAXS and compare our results with the experimental scattering profile of hen egg white lysozyme. Using this protein we show how to generate SAXS profiles representing: (1) complete models, (2) models with approximated side chain coordinates, and (3) models with approximated side chain and loop region coordinates. We evaluated the ability of SAXS profiles to identify a correct protein topology from a non‐redundant benchmark set of proteins. We find that complete SAXS profiles can be used to identify the correct protein by receiver operating characteristic (ROC) analysis with an area under the curve (AUC) > 99%. We show how our approximation of loop coordinates between secondary structure elements improves protein recognition by SAχS for protein models without loop regions and side chains. Agreement with SAXS data is a necessary but not sufficient condition for structure determination. We conclude that experimental SAXS data can be used as a filter to exclude protein models with large structural differences from the native. Proteins 2015; 83:1500–1512. © 2015 Wiley Periodicals, Inc.     

Structural genomics plucks high-hanging membrane proteins

Recent years have seen the establishment of structural genomics centers that explicitly target integral membrane proteins. Here, we review the advances in targeting these extremely high-hanging fruits of structural biology in high-throughput mode. We observe that the experimental determination of high-resolution structures of integral membrane proteins is increasingly successful both in terms of getting structures and of covering important protein families, for example, from Pfam. Structural genomics has begun to contribute significantly toward this progress. An important component of this contribution is the set up of robotic pipelines that generate a wealth of experimental data for membrane proteins. We argue that prediction methods for the identification of membrane regions and for the comparison of membrane proteins largely suffice to meet the challenges of target selection for structural genomics of membrane proteins. In contrast, we need better methods to prioritize the most promising members in a family of closely related proteins and to annotate protein function from sequence and structure in absence of homology.     

Refining conformational ensembles of flexible proteins against small-angle x-ray scattering data

Intrinsically disordered proteins and flexible regions in multidomain proteins display substantial conformational heterogeneity. Characterizing the conformational ensembles of these proteins in solution typically requires combining one or more biophysical techniques with computational modeling or simulations. Experimental data can either be used to assess the accuracy of a computational model or to refine the computational model to get a better agreement with the experimental data. In both cases, one generally needs a so-called forward model (i.e., an algorithm to calculate experimental observables from individual conformations or ensembles). In many cases, this involves one or more parameters that need to be set, and it is not always trivial to determine the optimal values or to understand the impact on the choice of parameters. For example, in the case of small-angle x-ray scattering (SAXS) experiments, many forward models include parameters that describe the contribution of the hydration layer and displaced solvent to the background-subtracted experimental data. Often, one also needs to fit a scale factor and a constant background for the SAXS data but across the entire ensemble. Here, we present a protocol to dissect the effect of the free parameters on the calculated SAXS intensities and to identify a reliable set of values. We have implemented this procedure in our Bayesian/maximum entropy framework for ensemble refinement and demonstrate the results on four intrinsically disordered proteins and a protein with three domains connected by flexible linkers. Our results show that the resulting ensembles can depend on the parameters used for solvent effects and suggest that these should be chosen carefully. We also find a set of parameters that work robustly across all proteins.     

Addition of missing loops and domains to protein models by x-ray solution scattering

Inherent flexibility and conformational heterogeneity in proteins can often result in the absence of loops and even entire domains in structures determined by x-ray crystallographic or NMR methods. X-ray solution scattering offers the possibility of obtaining complementary information regarding the structures of these disordered protein regions. Methods are presented for adding missing loops or domains by fixing a known structure and building the unknown regions to fit the experimental scattering data obtained from the entire particle. Simulated annealing was used to minimize a scoring function containing the discrepancy between the experimental and calculated patterns and the relevant penalty terms. In low-resolution models where interface location between known and unknown parts is not available, a gas of dummy residues represents the missing domain. In high-resolution models where the interface is known, loops or domains are represented as interconnected chains (or ensembles of residues with spring forces between the C(alpha) atoms), attached to known position(s) in the available structure. Native-like folds of missing fragments can be obtained by imposing residue-specific constraints. After validation in simulated examples, the methods have been applied to add missing loops or domains to several proteins where partial structures were available.     

Protein structures in solution by nuclear magnetic resonance and distance geometry. The polypeptide fold of the basic pancreatic trypsin inhibitor determined using two different algorithms, DISGEO and DISMAN

A set of conformational restraints derived from nuclear magnetic resonance (n.m.r.) measurements on solutions of the basic pancreatic trypsin inhibitor (BPTI) was used as input for distance geometry calculations with the programs DISGEO and DISMAN. Five structures obtained with each of these algorithms were systematically compared among themselves and with the crystal structure of BPTI. It is clear that the protein architecture observed in single crystals of BPTI is largely preserved in aqueous solution, with local structural differences mainly confined to the protein surface. The results confirm that protein conformations determined in solution by combined use of n.m.r. and distance geometry are a consequence of the experimental data and do not depend significantly on the algorithm used for the structure determination. The data obtained further provide an illustration that long intramolecular distances in proteins, which are comparable with the radius of gyration, are defined with high precision by relatively imprecise nuclear Overhauser enhancement measurements of a large number of much shorter distances.     

A study of protein structure changes during hydration by diffuse X-ray scattering: II. Fourier transform analysis of diffuse X-ray scattering data

Radial distribution functions were deduced by Fourier transform analysis of the angular dependences of diffuse X-ray scattering intensities for the following proteins with different hydration degrees: water-soluble α-protein myoglobin, water-soluble (α + β) protein lysozyme, and transmembrane proteins from the photosynthetic reaction centers of purple bacteria Rhodobacter sphaeroides and Blastochlorii (Rhodopseudomonas) viridis. The results of Fourier transform analysis of X-ray scattering intensities give quantitative characteristics of the mechanism underlying the influence of water on the formation of biological macromolecules. On the one hand, water loosens the network of hydrogen bonds, which results in a considerable conformational mobility in the molecules of lysozyme and myoglobin and the reaction centers. On the other hand, water stabilizes and orders the protein globule. A strict correlation was found between the shift of the “first” maximum of the radial distribution function, loosening of the intraglobular hydrogen bonds, increase in the intramolecular mobility, and appearance of pronounced functional activity in macromolecules. The pattern of behavior of the first maximum in the transmembrane proteins of the reaction center was similar to that observed for the water-soluble proteins. However, the first maximum reached the limiting value of 2.9 Å at a considerably lower hydration degree compared with the water-soluble proteins. A quick transition of the protein complex of the reaction center to its native state is due to the fact that the dehydrated conformation of this complex is very close to the native conformation. Comparison of the radial distribution function for water, water-soluble proteins, and transmembrane proteins suggests a quantitative conclusion that water is the least densely packed and ordered system, the water-soluble proteins are more densely packed than water, and the transmembrane proteins are the most densely packed and ordered system.     

Effect of the fluctuations in electron density within a globular protein on the excess small-angle X-ray scattering

Excess small angle X-ray scattering in solvents of differing electron density has been calculated from the crystal structures obtained for rubredoxin, trypsin inhibitor, myogen, ferricytochrome c₂, ribonuclease S, lysozyme, nuclease, myoglobin, α-chymotrypsin, elastase, subtilisin, carboxypeptidase A, thermolysin, methemoglobin, deoxyhemoglobin, and a single polypeptide chain of M₄ lactate dehydrogenase. The scattering curves for each protein can be reproduced by the sum of three curves, with the weighting of the three curves depending on the electron density of the solvent. The radius of gyration obtained from the small angle X-ray scattering by globular proteins in aqueous solution will usually exceed the values defined by the shape of the macromolecule. Deviations for certain of the proteins cited are calculated to be as large as 6%. These deviations arise from the tendency for the amino acid residues with low electron density to be situated closer to the center of the protein than the amino acid residues of high electron density. An upper limit of 19% is obtained for the discrepancy between the radius of gyration defined by the shape of a spherical globular protein of typical amino acid composition and the apparent radius of gyration measured for that protein in water by small angle X-ray scattering.     

Magnetic interaction between the tyrosyl free radical and the antiferromagnetically coupled iron center in ribonucleotide reductase

Ribonucleotide reductases from Escherichia coli and from mammalian cells are heterodimeric enzymes. One of the subunits, in the bacterial enzyme protein B2 and in the mammalian enzyme protein M2, contains iron and a tyrosyl free radical that both are essential for enzyme activity. The iron center in protein B2 is an antiferromagnetically coupled pair of high-spin ferric ions. This study concerns magnetic interaction between the tyrosyl radical and the iron center in the two proteins. Studies of the temperature dependence of electron paramagnetic resonance (EPR) relaxation and line shape reveal significant differences between the free radicals in proteins B2 and M2. The observed temperature-dependent enhanced EPR relaxation and line broadening of the enzyme radicals are furthermore completely different from those of a model UV-induced free radical in tyrosine. The results are discussed in terms of magnetic dipolar as well as exchange interactions between the free radical and the iron center in both proteins. The free radical and the iron center are thus close enough in space to exhibit magnetic interaction. For protein M2 the effects are more pronounced than for protein B2, indicating a stronger magnetic interaction.     

Analytical ultracentrifugation combined with X-ray and neutron scattering: Experiment and modelling

Analytical ultracentrifugation and solution scattering provide different multi-parameter structural and compositional information on proteins. The joint application of the two methods supplements high resolution structural studies by crystallography and NMR. We summarise the procedures required to obtain equivalent ultracentrifugation and X-ray and neutron scattering data. The constrained modelling of ultracentrifugation and scattering data is important to confirm the experimental data analysis and yields families of best-fit molecular models for comparison with crystallography and NMR structures. This modelling of ultracentrifugation and scattering data is described in terms of starting models, their conformational randomisation in trial-and-error fits, and the identification of the final best-fit models. Seven applications of these methods are described to illustrate the current state-of-the-art. These include the determination of antibody solution structures (the human IgG4 subclass, and oligomeric forms of human IgA and its secretory component), the solution structures of the complement proteins of innate immunity (Factor H and C3/C3u) and their interactions with macromolecular ligands (C-reactive protein), and anionic polysaccharides (heparin). Complementary features of joint ultracentrifugation and scattering experiments facilitate an improved understanding of crystal structures (illustrated for C3/C3u, C-reactive protein and heparin). If a large protein or its complex cannot be crystallised, the joint ultracentrifugation-scattering approach provides a means to obtain an overall macromolecular structure.     

Prediction of protein conformational freedom from distance constraints

A method is presented that generates random protein structures that fulfil a set of upper and lower interatomic distance limits. These limits depend on distances measured in experimental structures and the strength of the interatomic interaction. Structural differences between generated structures are similar to those obtained from experiment and from MD simulation. Although detailed aspects of dynamical mechanisms are not covered and the extent of variations are only estimated in a relative sense, applications to an IgG-binding domain, an SH3 binding domain, HPr, calmodulin, and lysozyme are presented which illustrate the use of the method as a fast and simple way to predict structural variability in proteins. The method may be used to support the design of mutants, when structural fluctuations for a large number of mutants are to be screened. The results suggest that motional freedom in proteins is ruled largely by a set of simple geometric constraints. Proteins 29:240–251, 1997. © 1997 Wiley-Liss, Inc.     

Hydrodynamic radii of native and denatured proteins measured by pulse field gradient NMR techniques

Pulse field gradient NMR methods have been used to determine the effective hydrodynamic radii of a range of native and nonnative protein conformations. From these experimental data, empirical relationships between the measured hydrodynamic radius (R(h)) and the number of residues in the polypeptide chain (N) have been established; for native folded proteins R(h) = 4.75N (0.29)A and for highly denatured states R(h) = 2.21N (0.57)A. Predictions from these equations agree well with experimental data from dynamic light scattering and small-angle X-ray or neutron scattering studies reported in the literature for proteins ranging in size from 58 to 760 amino acid residues. The predicted values of the hydrodynamic radii provide a framework that can be used to analyze the conformational properties of a range of nonnative states of proteins. Several examples are given here to illustrate this approach including data for partially structured molten globule states and for proteins that are unfolded but biologically active under physiological conditions. These reveal evidence for significant coupling between local and global features of the conformational ensembles adopted in such states. In particular, the effective dimensions of the polypeptide chain are found to depend significantly on the level of persistence of regions of secondary structure or features such as hydrophobic clusters within a conformational ensemble.     

Structural features that govern enzymatic activity in carbonic anhydrase from a low-temperature adapted fish, Chionodraco hamatus

The carbonic anhydrase (CA) family of zinc metalloenzymes includes many known isozymes that have different subcellular distributions. The study described here focuses on identification of the structural features that define low-temperature adaptation in a Chionodraco hamatus protein, both for the reaction center, at an atomic level, and for the tertiary structure of the protein. To this aim, an x-ray absorption near-edge spectroscopy/Minuit x-ray absorption near-edge spectroscopy analysis of the reaction center was undertaken for both a structurally characterized human CAII and CA of C. hamatus. Higher structural levels were analyzed by sequence comparison and homology modeling. To establish whether the structural insights acquired in fish CAs are general, theoretical models were generated by homology modeling for three temperate-climate-adapted fish CAs. The measured structural differences between the two proteins are discussed in terms of the differences in the electrostatic potential between human CAII and CA of C. hamatus. We conclude that modulation of the interaction between the catalytic water molecule and the zinc ion could depend on the effect of the electrostatic potential distribution.