Contrast variation studies of clathrin coated vesicles by small-angle neutron scattering

Structural information on clathrin coated vesicles has been obtained by small angle neutron scattering using contrast variation. A characteristic peak in the neutron scattering profile, which is apparent in 75 % D₂O, as well as in H₂O, disappears when contrast matching the protein component of the coated vesicles in 42% D₂O. Neutron, as well as dynamic, light scattering give a coated vesicle size of about 900 Å in H₂O and D₂O, but for neutron scattering the diameter decreases when matching out the protein coat of the clathrin coated vesicles. From the match point for the clathrin coated vesicles it is demonstrated that the clathrin cages do contain internal membrane. The mass of 34 MD and composition of 75% protein and 25% lipid found from the analysis of the small-angle scattering data are both in good agreement with the values reported in the literature. Electron microscopy gives an average outer diameter of 880 Å for the coated vesicles and an average diameter of 460 Å for the vesicle itself. Offprint requests to: Correspondence to: R. Bauer     

Joint use of small-angle X-ray and neutron scattering to study biological macromolecules in solution

Novel techniques for simultaneous analysis of X-ray and neutron scattering patterns from macromolecular complexes in solution are presented. They include ab initio shape and internal structure determination of multicomponent particles and more detailed rigid body modeling of complexes using high resolution structures of subunits. The methods fit simultaneously X-ray and neutron scattering curves including contrast variation data sets from selectively deuterated complexes. Biochemically sound interconnected models without steric clashes between the components displaying a pre-defined symmetry are generated. For rigid body modeling, distance restraints between specified residues/nucleotides or their ranges are taken into account. The efficiency of the methods is demonstrated in model examples, and potential sources of ambiguity are discussed.     

A review of multi-domain and flexible molecular chaperones studies by small-angle X-ray scattering

Intrinsic flexibility is closely related to protein function, and a plethora of important regulatory proteins have been found to be flexible, multi-domain or even intrinsically disordered. On the one hand, understanding such systems depends on how these proteins behave in solution. On the other, small-angle X-ray scattering (SAXS) is a technique that fulfills the requirements to study protein structure and dynamics relatively quickly with few experimental limitations. Molecular chaperones from Hsp70 and Hsp90 families are multi-domain proteins containing flexible and/or disordered regions that play central roles in cellular proteostasis. Here, we review the structure and function of these proteins by SAXS. Our general approach includes the use of SAXS data to determine size and shape parameters, as well as protein shape reconstruction and their validation by using accessory biophysical tools. Some remarkable examples are presented that exemplify the potential of the SAXS technique. Protein structure can be determined in solution even at limiting protein concentrations (for example, human mortalin, a mitochondrial Hsp70 chaperone). The protein organization, flexibility and function (for example, the J-protein co-chaperones), oligomeric status, domain organization, and flexibility (for the Hsp90 chaperone and the Hip and Hep1 co-chaperones) may also be determined. Lastly, the shape, structural conservation, and protein dynamics (for the Hsp90 chaperone and both p23 and Aha1 co-chaperones) may be studied by SAXS. We believe this review will enhance the application of the SAXS technique to the study of the molecular chaperones.     

Nonuniformity in natural rubber as revealed by small-angle neutron scattering, small-angle X-ray scattering, and atomic force microscopy

The microscopic structures of natural rubber (NR) and deproteinized NR (DPNR) were investigated by means of small-angle neutron scattering (SANS), small-angle X-ray scattering (SAXS), and atomic force microscopy (AFM). They were compared to those of isoprene rubber (IR), which is a synthetic analogue of NR in terms of chemical structure without any non-rubber components like proteins. Comparisons of the structure and mechanical properties of NR, DPNR, and IR lead to the following conclusions. (i) The well-known facts, for example, the outstanding green strength of NR and strain-induced crystallization, are due not much to the presence of proteins but to other components such as the presence of phospholipids and/or the higher stereoregularity of NR. It also became clear the naturally residing proteins accelerate the upturn of stress at low strain. The protein phases work as cross-linking sites and reinforcing fillers in the rubbery matrix. (ii) The microscopic structures of NR were successfully reproduced by SANS intensity functions consisting of squared-Lorentz and Lorentz functions, indicating the presence of inhomogeneities in bulk and thermal concentration fluctuations in swollen state, respectively. On the other hand, IR rubbers were homogeneous in bulk. (iii) The inhomogeneities in NR are assigned to protein aggregates of the order of 200 A or larger. Although these aggregates are larger in size as well as in volume fraction than those of cross-link inhomogeneities introduced by cross-linking, they are removed by deproteinization. (iv) Swelling of both NR and IR networks introduces gel-like concentration fluctuations whose mesh size is of the order of 20 A.     

Structure of casein micelles studied by small-angle neutron scattering

The structure of casein micelles has been studied by small-angle neutron scattering and static light scattering. Alterations in structure upon variation of pH and scattering contrast, as well as after addition of chymosin, were investigated. The experimental data were analyzed by a model in which the casein micelle consists of spherical submicelles. This model gave good agreement with the data and gave an average micellar radius of about 100–120 nm and a submicellar radius of about 7 nm both with a polydispersity of about 40–50%. The contrast variation indicated that the scattering length density of the submicelles was largest at the center of the submicelles. The submicelles were found to be closely packed, the volume fraction varying slightly with pH. Upon addition of chymosin the submicellar structure remained unchanged within the experimental accuracy. Correspondence to: S. Hansen     

Use of small-angle neutron scattering to study tubulin polymers

Small-angle neutron scattering has been used to examine taxol-stabilized microtubules and other tubulin samples in both H(2)O and D(2)O buffers. Measurements were made at pH/pD values between 6.0 and 7.8, and observed scattered intensities, I(Q), have been interpreted in terms of multicomponent models of microtubules and related tubulin polymers. A semiquantitative curve fitting procedure has been used to estimate the relative amounts of the supramolecular components of the samples. At both pH and pD 7.0 and above, the tubulin polymers are seen to be predominantly microtubules. Although in H(2)O buffer the polymer distribution is little changed as the pH varies, when pD is lowered the samples appear to contain an appreciable amount of sheetlike structures and the average microtubule protofilament number increases from ca. 12.5 at pD > or = approximately 7.0 to ca. 14 at pD approximately 6.0. Such structural change indicates that analysis of microtubule solutions based on H(2)O/D(2)O contrast variation must be performed with caution, especially at lower pH/pD.     

beta-lactoglobulin under high pressure studied by small-angle neutron scattering

We used small-angle neutron scattering to study the effects of the high hydrostatic pressure on the structure of beta-lactoglobulin. Experiments were carried out at pH 7 on the dimeric form of the protein in a pressure range going from 50 MPa to 300 MPa. These measurements allow the protein size and the interactions between macromolecules to be studied during the application of pressure. Increasing pressure up to 150 MPa leads to a swollen state of the protein that gives rise to an increase of the radius of gyration by about 7%. Within this pressure range, we also show that the interaction between macromolecules weakens although it remains repulsive. The measurements show an aggregation process occurring above 150 MPa. From the spectra analysis, it appears that the aggregation occurs mainly by association of the dimeric units.     

A neutron small-angle scattering study of bovine fibrinogen.

A number of glycyl-tRNA synthetase (glyS) mutants have been isolated as glycine auxotrophs in Salmonella typhimurium. One of the mutants, glyS141, has a glycyl-tRNA synthetase with a K_m for glycine that is 700 times higher than the wild-typeK_m. Prototrophic revertants glyS141 occur at high spontaneous frequencies (>5 × 10^?5). The majority of these revertants contain large tandem duplications including the mutant glyS gene. Some of the duplications cover at least 22% of the chromosome. The duplications overlap with a large duplication isolated previously by a different selection procedure (Straus &; Hoffmann, 1975). Evidence has been obtained which suggests that formation of the duplications may occur by recA-dependent recombination. The Gly⁺ phenotype of revertants carrying the duplications does not appear to be explainable simply by the increased gene dosage of glyS.     

Structural study of rhodopsin in detergent micelles by small-angle neutron scattering

Monodisperse solutions of bovine rhodopsin monomers, devoid of lipid, associated with a linear polyoxyethylene alcohol detergent have been prepared. The composition and homogeneity of these complexes have been determined by hydrodynamic characterisation. Each rhodopsin molecule is associated with about 110 monomers of the detergent. These rhodopsin-detergent complexes have been studied by small-angle neutron scattering. Partial or total deuteration of the detergent, as well as variation of the ²H₂O/H₂O ratio in the solvent, were used to eliminate the detergent—solvent contrast at various protein—solvent contrasts. The size and shape of the detergent micelle and of the rhodopsin-detergent complexes were shown to be independent of solvent or detergent deuteration. Mixture of selectively deuterated detergent molecules allowed us to obtain an homogeneous scattering density for the detergent part of the micelles and therefore to eliminate totally its contribution to the scattering when it is contrast matched. Neutron scattering from rhodopsin alone was then measured even in highly deuterated solvents, with low incoherent background, as for a water-soluble protein. Supplementary neutron scattering measurements on rhodopsin-dodecyl dimethylamine oxide micelles confirmed essentially the results reported by Yeager (1975). Analysis of the neutron scattering data indicates that most of the hydrophobic residues of rhodopsin form a compact region which has zero hydration, this probably being the part which is embedded in the disc membrane, and that the unhydrated rhodopsin molecule is asymmetrically arranged with respect to the membrane. Comparison with the results of a small-angle X-ray scattering study (Sardet et al., 1976) implies that the peripheral regions on both sides of the membrane are highly hydrated. Several schematic models are discussed.     

Salt-dependent compaction of di- and trinucleosomes studied by small-angle neutron scattering

Using small-angle neutron scattering (SANS), we have measured the salt-dependent static structure factor of di- and trinucleosomes from chicken erythrocytes and from COS-7 cells. We also determined the sedimentation coefficients of these dinucleosomes and dinucleosomes reconstituted on a 416-bp DNA containing two nucleosome positioning sequences of the 5S rDNA of Lytechinus variegatus at low and high salt concentrations. The internucleosomal distance d was calculated by simulation as well as Fourier back-transformation of the SANS curves and by hydrodynamic simulation of sedimentation coefficients. Nucleosome dimers from chicken erythrocyte chromatin show a decrease in d from approximately 220 A at 5 mM NaCl to 150 A at 100 mM NaCl. For dinucleosomes from COS-7 chromatin, d decreases from 180 A at 5 mM to 140 A at 100 mM NaCl concentration. Our measurements on trinucleosomes are compatible with a compaction through two different mechanisms, depending on the salt concentration. Between 0 and 20 mM NaCl, the internucleosomal distance between adjacent nucleosomes remains constant, whereas the angle of the DNA strands entering and leaving the central nucleosome decreases. Above 20 mM NaCl, the adjacent nucleosomes approach each other, similar to the compaction of dinucleosomes. The internucleosomal distance of 140-150 A at 100 mM NaCl is in agreement with distances measured by scanning force microscopy and electron microscopy on long chromatin filaments.     

Denaturation of a halophilic enzyme monitored by small-angle neutron scattering

Malate dehydrogenase from Halobacterium maris mortui exists in 4 M-NaCl as a stable protein dimer, with which are associated unusually large amounts of salt and water. In 1 M-NaCl at 25 degrees C, it denatures with a time-constant of about 0.5 h-1. Small-angle neutron scattering data from the protein under these conditions were monitored regularly over more than 12 hours during denaturation. They are quantitatively consistent with a model in which the protein dimer loses its exceptional salt and water-binding properties, and dissociates into monomers that partially unfold and have the interactions with solvent expected from their relatively charged amino acid composition. The exceptional salt and water-binding by the native enzyme, therefore, is associated with the native structure of the dimer.     

Global rigid body modeling of macromolecular complexes against small-angle scattering data

New methods to automatically build models of macromolecular complexes from high-resolution structures or homology models of their subunits or domains against x-ray or neutron small-angle scattering data are presented. Depending on the complexity of the object, different approaches are employed for the global search of the optimum configuration of subunits fitting the experimental data. An exhaustive grid search is used for hetero- and homodimeric particles and for symmetric oligomers formed by identical subunits. For the assemblies or multidomain proteins containing more then one subunit/domain per asymmetric unit, heuristic algorithms based on simulated annealing are used. Fast computational algorithms based on spherical harmonics representation of scattering amplitudes are employed. The methods allow one to construct interconnected models without steric clashes, to account for the particle symmetry and to incorporate information from other methods, on distances between specific residues or nucleotides. For multidomain proteins, addition of missing linkers between the domains is possible. Simultaneous fitting of multiple scattering patterns from subcomplexes or deletion mutants is incorporated. The efficiency of the methods is illustrated by their application to complexes of different types in several simulated and practical examples. Limitations and possible ambiguity of rigid body modeling are discussed and simplified docking criteria are provided to rank multiple models. The methods described are implemented in publicly available computer programs running on major hardware platforms.     

Small-angle neutron scattering contrast variation studies of biological complexes: Challenges and triumphs

Small-angle neutron scattering (SANS) has been a beneficial tool for studying the structure of biological macromolecules in solution for several decades. Continued improvements in sample preparation techniques, including deuterium labeling, neutron instrumentation and complementary techniques such as small-angle x-ray scattering (SAXS), cryo-EM, NMR and x-ray crystallography, along with the availability of more powerful structure prediction algorithms and computational resources has made SANS more important than ever as a means to obtain unique information on the structure of biological complexes in solution. In particular, the contrast variation (CV) technique, which requires a large commitment in both sample preparation and measurement time, has become more practical with the advent of these improved resources. Here, challenges and recent triumphs as well as future prospects are discussed.     

Structure and composition of influenza virus. A small-angle neutron scattering study

A detailed analysis is presented of the small-angle neutron scattering curves of homogeneous solutions of influenza B virus, both intact and after treatment with bromelain, which removes the external glycoprotein spikes. The two sets of data are consistent with the following low-resolution structure: the virus particles are spherical, about 1200 A in diameter and of Mr about 180 X 10(6). The lipid bilayer is centred at a radius of 425 A, is 40 A to 50 A thick and constitutes 25% to 28% of the virus mass. The surface glycoproteins, predominantly haemagglutinin, contribute 40% to 46% of the total mass. Surprisingly little protein is found in the interior of the virus. It is suggested that the reason for this is that many particles do not contain the full complement of ribonucleoprotein complexes. These results are in good agreement with recent scanning transmission electron microscopic measurements of molecular mass and cryo-electron microscopic observations of the same preparations. Appendix 1 describes a new method of deriving spherical shell models from contrast variation neutron scattering data on viruses, in which scattering curves from all measured contrasts are used simultaneously. There is also a discussion of the assumptions and limitations implicit in the structural interpretation of such models, with emphasis on viruses containing lipid bilayers. Appendix 2 examines the effect on the scattering curves of various arrangements of the surface glycoproteins.     

Integration of small-angle X-ray scattering data into structural modeling of proteins and their assemblies

A major challenge in structural biology is to determine the configuration of domains and proteins in multidomain proteins and assemblies, respectively. All available data should be considered to maximize the accuracy and precision of these models. Small-angle X-ray scattering (SAXS) efficiently provides low-resolution experimental data about the shapes of proteins and their assemblies. Thus, we integrated SAXS profiles into our software for modeling proteins and their assemblies by satisfaction of spatial restraints. Specifically, we modeled the quaternary structures of multidomain proteins with structurally defined rigid domains as well as quaternary structures of binary complexes of structurally defined rigid proteins. In addition to SAXS profiles and the component structures, we used stereochemical restraints and an atomic distance-dependent statistical potential. The scoring function is optimized by a biased Monte Carlo protocol, including quasi-Newton and simulated annealing schemes. The final prediction corresponds to the best scoring solution in the largest cluster of many independently calculated solutions. To quantify how well the quaternary structures are determined based on their SAXS profiles, we used a benchmark of 12 simulated examples as well as an experimental SAXS profile of the homotetramer d-xylose isomerase. Optimization of the SAXS-dependent scoring function generally results in accurate models if sufficiently precise approximations for the constituent rigid bodies are available; otherwise, the best scoring models can have significant errors. Thus, SAXS profiles can play a useful role in the structural characterization of proteins and assemblies if they are combined with additional data and used judiciously. Our integration of a SAXS profile into modeling by satisfaction of spatial restraints will facilitate further integration of different kinds of data for structure determination of proteins and their assemblies.     

The aggregation behavior of zinc-free insulin studied by small-angle neutron scattering

The aggregation behavior of zinc-free insulin has been studied by small-angle neutron scattering as a function of pH and ionic strength of the solution. The pair distance distribution functions for the 12 samples have been obtained by indirect Fourier transformation. The results show that the diameter of the aggregates is 40 Å at pH 11 and 10 mM NaCl, independent of the protein concentration. The largest diameter of about 120 Å is found for pH 8, 100 mM NaCl, and a protein concentration of 10 mg/ml. Estimates of the pair distance distribution functions, free of inter-particle correlation effects, were obtained by an indirect Fourier transformation, omitting the data at small scattering vectors, which are influenced by these effects. By this procedure the weight-averaged molecular mass and the average radius of gyration were determined. These parameters vary from 1.3 times the monomer mass and 14 Å, to 6.8 times the monomer mass and 31 Å, respectively. The mass distribution between the oligomers was determined by a model based on the crystal structure of zinc-free insulin. The results from this model and the Fourier transformations have been compared to an equilibrium model recently introduced by Kadima et al. (1993). The neutron scattering results agree well with the predictions of this model except that broader mass distributions are suggested by neutron scattering. Correspondence to: J. Skov Pedersen     

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.     

Architecture of bacterial lipid A in solution. A neutron small-angle scattering study

The phase structure of isolated bacterial lipid A, the lipid anchor of the lipopolysaccharides of the outer membrane of Gram-negative bacteria, has been investigated by neutron small-angle scattering. The shape of the scattering curves obtained at different H2O/2H2O ratios revealed a lamellar organisation of the lipid A at neutral pH both above and below its main phase temperature (approximately 40-45 degrees C). Analysis of the scattering curves and interpretation of the corresponding thickness distance distribution functions of the lamellar aggregates led to a model in which the lipid A molecules form a bilayer of about 5 nm in thickness. This value for the thickness of the bilayer, as well as the neutron-scattering density profile across the bilayer, can be explained by a molecular model which shows interdigitation of the fatty acid chains of the lipid A.