Statistical conformation of human plasma fibronectin

Fibronectin is a multifunctional glycoprotein (molecular mass, M = 530 kg/mol) of the extra cellular matrix (ECM) having a major role in cell adhesion. In physiological conditions, the conformation of this protein still remains debated and controversial. Here, we present a set of results obtained by scattering experiments. In "native" conditions, the radius of gyration (R(g) = 15.3 +/- 0.3 nm) was determined by static light scattering as well as small-angle neutron scattering. The hydrodynamic radius (R(H) = 11.5 +/- 0.1 nm) was deduced from quasi-elastic light scattering measurements. These results imply a low internal concentration compared to that of usual globular proteins. This is also confirmed by the ratio R(H)/R(g) = 0. 75 +/- 0.02 consistent with a Gaussian chain, whereas R(H)/R(g) = 1. 3 for spherical shaped molecules. However, adding a denaturing agent (urea 8 M) increases R(g) by a factor 2. This means that fibronectin "native" chain is not either completely unfolded. The average shape of fibronectin conformation was also probed by small-angle neutron scattering performed for reverse scattering vector q(-)(1) smaller than R(g) (0.2 < q(-)(1) < 15 nm). The measured form factor is in complete agreement with the form factor of a random string of 56 beads of 5 nm diameter. It rules out the possibility of unfolded chain as well as globular structures. These results have structural and biological implications as far as ECM organization is concerned.     

Dynamic Properties of Human α-Synuclein Related to Propensity to Amyloid Fibril Formation

α-Synuclein (αSyn) is an intrinsically disordered protein that can form amyloid fibrils. Fibrils of αSyn are implicated with the pathogenesis of Parkinson's disease and other synucleinopathies. Elucidating the mechanism of fibril formation of αSyn is therefore important for understanding the mechanism of the pathogenesis of these diseases. Fibril formation of αSyn is sensitive to solution conditions, suggesting that fibril formation of αSyn arises from the changes in its inherent physico-chemical properties, particularly its dynamic properties because intrinsically disordered proteins such as αSyn utilize their inherent flexibility to function. Characterizing these properties under various conditions should provide insights into the mechanism of fibril formation. Here, using the quasielastic neutron scattering and small-angle x-ray scattering techniques, we investigated the dynamic and structural properties of αSyn under the conditions, where mature fibrils are formed (pH 7.4 with a high salt concentration), where clumping of short fibrils occurs (pH 4.0), and where fibril formation is not completed (pH 7.4). The small-angle x-ray scattering measurements showed that the extended structures at pH 7.4 with a high salt concentration become compact at pH 4.0 and 7.4. The quasielastic neutron scattering measurements showed that both intra-molecular segmental motions and local motions such as side-chain motions are enhanced at pH 7.4 with a high salt concentration, compared to those at pH 7.4 without salt, whereas only the local motions are enhanced at pH 4.0. These results imply that fibril formation of αSyn requires not only the enhanced local motions but also the segmental motions such that proper inter-molecular interactions are possible.     

Physical characteristics of ribosomal protein S4 from Escherichia coli

A hydrodynamic study of protein S4 from Escherichia coli 30 S ribosomal subunits indicates that this protein is moderately asymmetric. A sedimentation coefficient of 1.69 S and a diffusion coefficient of 7.58 X 10(-7) cm2/s suggest that S4 has an axial ratio of about 5:1 using a prolate ellipsoidal model. This structure should give a radius of gyration of about 29-30 A from small-angle neutron or small-angle x-ray scattering studies. This study has utilized quasi-elastic light scattering as an analytical tool to obtain a diffusion coefficient as well as a method to monitor sample quality. Using quasi-elastic light scattering in this manner allows an assessment of problems associated with protein purity which may be responsible for the many disparate results reported for ribosomal proteins and especially protein S4.     

Protein structure and hydration probed by SANS and osmotic stress

Interactions governing protein folding, stability, recognition, and activity are mediated by hydration. Here, we use small-angle neutron scattering coupled with osmotic stress to investigate the hydration of two proteins, lysozyme and guanylate kinase (GK), in the presence of solutes. By taking advantage of the neutron contrast variation that occurs upon addition of these solutes, the number of protein-associated (solute-excluded) water molecules can be estimated from changes in both the zero-angle scattering intensity and the radius of gyration. Poly(ethylene glycol) exclusion varies with molecular weight. This sensitivity can be exploited to probe structural features such as the large internal GK cavity. For GK, small-angle neutron scattering is complemented by isothermal titration calorimetry with osmotic stress to also measure hydration changes accompanying ligand binding. These results provide a framework for studying other biomolecular systems and assemblies using neutron scattering together with osmotic stress.     

Effects of Macromolecular Crowding on the Structure of a Protein Complex: A Small-Angle Scattering Study of Superoxide Dismutase

Macromolecular crowding can alter the structure and function of biological macromolecules. We used small-angle scattering to measure the effects of macromolecular crowding on the size of a protein complex, SOD (superoxide dismutase). Crowding was induced using 400 MW PEG (polyethylene glycol),TEG (triethylene glycol), α-MG (methyl-α-glucoside), and TMAO (trimethylamine n-oxide). Parallel small-angle neutron scattering and small-angle x-ray scattering allowed us to unambiguously attribute apparent changes in radius of gyration to changes in the structure of SOD. For a 40% PEG solution, we find that the volume of SOD was reduced by 9%. Considering the osmotic pressure due to PEG, this deformation corresponds to a highly compressible structure. Small-angle x-ray scattering done in the presence of TEG suggests that for further deformation—beyond a 9% decrease in volume—the resistance to deformation may increase dramatically.     

Structural characterization and pH-induced conformational transition of full-length KcsA

The bacterial K+ channel KcsA from Streptomyces lividans was analyzed by neutron and x-ray small-angle solution scattering. The C-terminally truncated version of KcsA, amenable to crystallographic studies, was compared with the full-length channel. Analyzing the scattering data in terms of radius of gyration reveals differences between both KcsA species of up to 13.2 A. Equally, the real-space distance distribution identifies a 40 to 50 A extension of full-length KcsA compared to its C-terminally truncated counterpart. We show that the x-ray and neutron scattering data are amenable for molecular shape reconstruction of full-length KcsA. The molecular envelopes calculated display an hourglass-shaped structure within the C-terminal intracellular domain. The C-terminus extends the membrane spanning region of KcsA by 54-70 A, with a central constriction 10-30 A wide. Solution scattering techniques were further employed to characterize the KcsA channel under acidic conditions favoring its open conformation. The full-length KcsA at pH 5.0 shows the characteristics of a dumbbell-shaped macromolecular structure, originating from dimerization of the tetrameric K+ channel. Since C-terminally truncated KcsA measured under the same low pH conditions remains tetrameric, oligomerization of full-length KcsA seems to proceed via structurally changed C-terminal domains. The determined maximum dimensions of the newly formed complex increase by 50-60%. Shape reconstruction of the pseudooctameric complex indicates the pH-induced conformational reorganization of the intracellular C-terminal domain.     

The denaturation transition of DNA in mixed solvents

The helix-to-coil denaturation transition in DNA has been investigated in mixed solvents at high concentration using ultraviolet light absorption spectroscopy and small-angle neutron scattering. Two solvents have been used: water and ethylene glycol. The "melting" transition temperature was found to be 94 degrees C for 4% mass fraction DNA/d-water and 38 degrees C for 4% mass fraction DNA/d-ethylene glycol. The DNA melting transition temperature was found to vary linearly with the solvent fraction in the mixed solvents case. Deuterated solvents (d-water and d-ethylene glycol) were used to enhance the small-angle neutron scattering signal and 0.1M NaCl (or 0.0058 g/g mass fraction) salt concentration was added to screen charge interactions in all cases. DNA structural information was obtained by small-angle neutron scattering, including a correlation length characteristic of the inter-distance between the hydrogen-containing (desoxyribose sugar-amine base) groups. This correlation length was found to increase from 8.5 to 12.3 A across the melting transition. Ethylene glycol and water mixed solvents were found to mix randomly in the solvation region in the helix phase, but nonideal solvent mixing was found in the melted coil phase. In the coil phase, solvent mixtures are more effective solvating agents than either of the individual solvents. Once melted, DNA coils behave like swollen water-soluble synthetic polymer chains.     

Solution structure of a short DNA fragment studied by neutron scattering

The solution structure of a DNA fragment of 130 base pairs and known sequence has been investigated by neutron small-angle scattering. In 0.1 M NaCl, the overall structure of the DNA fragment which contains the strong promoter A1 of the Escherichia coli phage T7 agrees with that expected for B-DNA. The neutron scattering curve is well fitted by that of a rigid rod with a length of 44 nm and a diameter of 2 nm. The results were confirmed by quasi-elastic light scattering and analytical centrifugation. The neutron measurements in H2O and D2O buffer reveal a cross-sectional inhomogeneity not detected by X-ray small-angle scattering. This inhomogeneity is caused by the hydration layer around the DNA core and not by the helical structure. The primary solvent shell has a density increased by at least 4-9% compared to bulk water.     

The radius of gyration of native and reductively methylated myosin subfragment-1 from neutron scattering.

Reductive methylation of nearly all lysine groups of myosin subfragment-1 (S1) was required for crystallization and solution of its structure at atomic resolution. Possible effects of such methylation on the radius of gyration of chicken skeletal muscle myosin S1 have been investigated by using small-angle neutron scattering. In addition, we have investigated the effect of MgADP.Vi, which is thought to produce an analog of the S1.ADP.Pi state, on the S1 radius of gyration. We find that although methylation of S1, with or without SO42- ion addition, does not significantly alter the structure, addition of ADP plus vanadate does decrease the radius of gyration significantly. The S1 crystal structure predicts a radius of gyration close to that measured here by neutron scattering. These results suggest that the overall shape by crystallography resembles nucleotide-free S1 in solution. In order to estimate the effect of residues missing from the crystal structure, the structure of missing loops was estimated by secondary-structure prediction methods. Calculations using the complete crystal structure show that a simple closure of the nucleotide cleft by a rigid-body torsional rotation of residues (172-180 to 670) around an axis running along the base of the cleft alone does not produce changes as large as seen here and in x-ray scattering results. On the other hand, a rigid body rotation of either the light-chain binding domain (767 to 843 plus light chains) or of a portion of 20-kDa peptide plus this domain (706 to 843 plus light chains) is more readily capable of producing such changes.     

Behavior of Tn3 resolvase in solution and its interaction with res

The solution properties of Tn3 resolvase (Tn3R) were studied by sedimentation equilibrium, sedimentation velocity analytical ultracentrifugation, and small-angle neutron scattering. Tn3R was found to be in a monomer-dimer self-association equilibrium, with a dissociation constant of K(D)(1-2)=50 microM. Sedimentation velocity and small-angle neutron scattering data are consistent with a solution structure of dimeric Tn3R similar to that of gammadelta resolvase in a co-crystal structure, but with the DNA-binding domains in a more extended conformation. The solution conformations of sites I, II, and III were studied with small angle x-ray scattering and modeled using rigid-body and ab initio techniques. The structures of these sites do not show any distortion, at low resolution, from B-DNA. The equilibrium binding properties of Tn3R to the individual binding sites in res were investigated by employing fluorescence anisotropy measurements. It was found that site II and site III have the highest affinity for Tn3R, followed by site I. Finally, the affinity of Tn3R for nonspecific DNA was assayed by competition experiments.     

Complexes of RecA protein in solution. A study by small angle neutron scattering

RecA complexes on DNA and self-polymers were analysed by small-angle neutron scattering in solution. By Guinier analysis at small angles and by model analysis of a subsidiary peak at wider angles, we find that the filaments fall into two groups: the DNA complex in the presence of ATP gamma S, an open helix with pitch 95 A, a cross-sectional radius of gyration of 33 A and a mass per length of about six RecA units per turn, which corresponds to the state of active enzyme; and the compact form (bound to single-stranded DNA in the absence of ATP, or binding ATP gamma S in the absence of DNA, or just the protein on its own), a helical structure with pitch 70 A, cross-sectional radius of gyration 40 A and mass per length about five RecA units per turn, which corresponds to the conditions of inactive enzyme. The results are discussed in the perspective of unifying previous conflicting structural results obtained by electron microscopy.     

Neutron scattering studies of nucleosome structure at low ionic strength

Ionic strength studies using homogeneous preparations of chicken erythrocyte nucleosomes containing either 146 or 175 base pairs of DNA show a single unfolding transition at about 1.5 mM ionic strength as determined by small-angle neutron scattering. The transition seen by some investigators at between 2.9 and 7.5 mM ionic strength is not observed by small-angle neutron scattering in either type of nucleosome particle. The two contrasts measured (H2O and D2O) indicate that only small conformational changes occur in the protein core, but the DNA is partially unfolded below the transition point. Patterson inversion of the data and analysis of models indicate that the DNA in both types of particle is unwinding from the ends, leaving about one turn of supercoiled DNA bound to the histone core in approximately its normal (compact) conformation. The mechanism of unfolding appears to be similar for both types of particles and in both cases occurs at the same ionic strength. The unfolding observed for nucleosomes in this study is in definite disagreement with extended superhelical models for the DNA and also disagrees with models incorporating an unfolded histone core.     

The location of DNA in complexes of recA protein with double-stranded DNA. A neutron scattering study

Purified recA protein is found as rodlike homopolymers, and it forms filamentous complexes with double-stranded DNA that are stable in the presence of ATP gamma S, a nonhydrolyzable analogue of ATP. The structure of these filaments has been described in some detail by electron microscopy. Here we confirm the mass per length of 6.5 recA/100 A in solution by small-angle neutron scattering and extend the analysis to homopolymers of recA protein, finding a mass per length of about 7 recA/100 A and a radial mass distribution (cross-sectional radius of gyration) significantly different for the two filaments. The models proposed so far for the structure of the complex have placed the DNA in the center of the filament. Here we verify this assumption using small-angle neutron scattering to locate the DNA in the complexes, exploiting the contrast variation method in D2O/H2O mixtures. Model calculations show that the natural contrast difference between DNA and protein is not sufficient to locate the DNA (which accounts for only 4.7% of the mass in the complex). When deuterated DNA is used, the contrast difference is enhanced, and model calculations and experiment then converge, indicating that the DNA is indeed near the axis of the complex.     

Assembly of bacteriophage T7. Dimensions of the bacteriophage and its capsids.

The dimensions of bacteriophage T7 and T7 capsids have been investigated by small-angle x-ray scattering. Phage T7 behaves like a sphere of uniform density with an outer radius of 301 +/- 2 A (excluding the phage tail) and a calculated volume for protein plus nucleic acid of 1.14 +/- 0.05 x 10(-16) ml. The outer radius determined for T7 phage in solution is approximately 30% greater than the radius measured from electron micrographs, which indicates that considerable shrinkage occurs during preparation for electron microscopy. Capsids that have a phagelike envelope and do not contain DNA were obtained from lysates of T7-infected Escherichia coli (capsid II) and by separating the capsid component of T7 phage from the phage DNA by means of temperature shock (capsid IV). In both cases the peak protein density is at a radius of 275 A; the outer radius is 286 +/- 4 A, approximately 5% smaller than the envelope of T7 phage. The thickness of the envelope of capsid II is 22 +/- 4 A, consistent with the thickness of protein estimated to be 23 +/- 5 A in whole T7 phage, as seen on electron micrographs in which the internal DNA is positively stained. The volume in T7 phage available to package DNA is estimated to be 9.2 +/- 0.4 x 10(-17) ml. The packaged DNA adopts a regular packing with 23.6 A interplanar spacing between, DNA strands. The angular width of the 23.6 A reflection shows that the mean DNA-DNA spacing throughout the phage head is 27.5 +/- less than 2.2 A. A T7 precursor capsid (capsid I) expands when pelleted for x-ray scattering in the ultracentrifuge to essentially the same outer dimensions as for capsids II and IV. This expansion of capsid I can be prevented by fixing with glutaraldehyde; fixed capsid I has peak density at a radius of 247 A, 10% less than capsid II or IV.     

Solution scattering approaches to dynamical ordering in biomolecular systems

Clarification of solution structure and its modulation in proteins and protein complexes is crucially important to understand dynamical ordering in macromolecular systems. Small-angle x-ray scattering (SAXS) and small-angle neutron scattering (SANS) are among the most powerful techniques to derive structural information. Recent progress in sample preparation, instruments and software analysis is opening up a new era for small-angle scattering. In this review, recent progress and trends of SAXS and SANS are introduced from the point of view of instrumentation and analysis, touching on general features and standard methods of small-angle scattering. This article is part of a Special Issue entitled “Biophysical Exploration of Dynamical Ordering of Biomolecular Systems” edited by Dr. Koichi Kato.     

Neutron and x-ray scatter studies of the histone octamer and amino and carboxyl domain trimmed octamers

The structure of the nucleosome has been under intense investigation using neutron crystallography, x-ray crystallography, and neutron solution scattering. However the dimension of the histone octamer inside the nucleosome is still a subject of controversy. The radius of gyration (Rg) of the octamer obtained from solution neutron scattering of core particles at 63% 2H2O, 37% 1H2O is 33 A, and x-ray crystallography study of isolated histone octamer gives a Rg of 32.5 A, while the reported values using x-ray crystallography of core particles from two individual studies are 29.7 and 30.4 A, respectively. We report here studies of isolated histone octamer and trypsin-limited digested octamer using both neutron solution scattering and small angle x-ray scattering. The Rg of the octamer obtained is 33 A, whereas that of the trimmed octamer is 29.8 A, similar to the structure obtained from the crystals of the core particles. The N-terminal domains of the core histones in the octamer have been shown by high resolution nuclear magnetic resonance (Schroth, G.P., Yau, P., Imai, B.S., Gatewood, J.M., and Bradbury, E.M. (1990) FEBS Lett. 268, 117-120) to be mobile and flexible; it is likely that these regions are disordered and "not seen" by x-ray crystallography.     

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     

Salt-dependent DNA superhelix diameter studied by small angle neutron scattering measurements and Monte Carlo simulations.

Using small angle neutron scattering we have measured the static form factor of two different superhelical DNAs, p1868 (1868 bp) and pUC18 (2686 bp), in dilute aqueous solution at salt concentrations between 0 and 1.5 M Na+ in 10 mM Tris at 0% and 100% D2O. For both DNA molecules, the theoretical static form factor was also calculated from an ensemble of Monte Carlo configurations generated by a previously described model. Simulated and measured form factors of both DNAs showed the same behavior between 10 and 100 mM salt concentration: An undulation in the scattering curve at a momentum transfer q = 0.5 nm-1 present at lower concentration disappears above 100 mM. The position of the undulation corresponds to a distance of approximately 10-20 nm. This indicated a change in the DNA superhelix diameter, as the undulation is not present in the scattering curve of the relaxed DNA. From the measured scattering curves of superhelical DNA we estimated the superhelix diameter as a function of Na+ concentration by a quantitative comparison with the scattering curve of relaxed DNA. The ratio of the scattering curves of superhelical and relaxed DNA is very similar to the form factor of a pair of point scatterers. We concluded that the distance of this pair corresponds to the interstrand separation in the superhelix. The computed superhelix diameter of 16.0 +/- 0.9 nm at 10 mM decreased to 9.0 +/- 0.7 nm at 100 mM salt concentration. Measured and simulated scattering curves agreed almost quantitatively, therefore we also calculated the superhelix diameter from the simulated conformations. It decreased from 18.0 +/- 1.5 nm at 10 mM to 9.4 +/- 1.5 nm at 100 mM salt concentration. This value did not significantly change to lower values at higher Na+ concentration, in agreement with results obtained by electron microscopy, scanning force microscopy imaging in aqueous solution, and recent MC simulations, but in contrast to the observation of a lateral collapse of the DNA superhelix as indicated by cryo-electron microscopy studies.     

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.