Molecular modeling of human complement component C4 and its fragments by X-ray and neutron solution scattering

The solution structures of human complement component C4 and five derived fragments, C4u, C4(a + b), C4b, C4c, and C4d, were analyzed by synchrotron X-ray and neutron scattering. The X-ray radii of gyration RG for C4, C4u, and C4(a + b) in H2O buffers are similar at 5.23-5.28 nm, and likewise the cross-sectional radii of gyration RXS are similar at 2.48-2.52 nm. Molecular mass calculations using X-rays and neutrons show unexpectedly that C4c is dimeric; however, all the other forms are monomeric. C4c2 has an X-ray RG of 5.18 nm and an RXS of 2.89 nm. Neutron contrast variation gives RG values at infinite contrast of 4.87-4.93 nm for C4 and C4u, 4.79 nm for C4b, 4.94 nm for C4c2, and 2.69 nm for C4d. The RXS values at infinite contrast are 2.23-2.25 nm for C4 and C4u, 1.89 nm for C4b, and 2.62 nm for C4c2. These data show that a large conformational change occurs on going from C4 to C4b, but not on going from C4 to C4u, and this is attributed to the presence of the C4a moiety in C4u. Comparisons of the C4 and C4u scattering curves show that these are very similar out to a nominal resolution of 4 nm. Scattering-curve models were developed to account for the neutron scattering curves of C4, C4c2, and C4d in 2H2O buffers. The C4c monomer could be represented by a lamellar ellipsoid of size 8 nm x 2 nm x 18 nm. C4d was found to be 4 nm x 2 nm x 9 nm. The combination of these structures gave good accounts of the neutron data for C4, C4b, and C4c2 to resolutions of 5-6 nm. The C4 model was obtained by placing the long axis of C4d parallel to that of C4c such that the cross section is extended. C4b was best modeled by repositioning C4d relative to C4c such that this cross section becomes more compact. The C4 and C4b models are compared with possible structures for the C1 component of complement to show the importance of the surface accessibility of the protease domains and short consensus repeat domains in C1 for C4 activation.     

Possibility of wide-angle neutron scattering in the study of proteins and nucleic acids in solutions

A method is proposed for calculating wide-angle neutron scattering curves of biopolymers at any fraction of heavy water (D2O) in solution. The method permits to accurately take into account the phenomenon of deuteroexchange. By this method neutron scattering curves of proteins and DNA have been calculated. The calculations have shown that at optimal fractions of D2O in solution the profiles of neutron scattering curves and their sensitivity to conformational rearrangements in protein molecules turned out to differ very little from those of corresponding X-ray curves. Thus the neutron scattering curves do not contain any additional information (as compared with those contained in X-ray scattering curves) on the structure of proteins in solution. On the contrary, neutron and X-ray scattering curves of DNA differ significantly at all fractions of D2O in solution and therefore the methods of wide-angle neutron and X-ray scattering could become mutually complementary in studying the structure of nucleic acids in solution.     

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.     

Small-angle X-ray scattering on malate synthase from baker's yeast. The native substrate-free enzyme and enzyme-substrate complexes.

Malate synthase from baker's yeast has been investigated in solution by the small-angle X-ray scattering technique. Size, shape and structure of the native substrate-free enzyme and of various enzyme-substrate complexes have been determined. As the enzyme was found to be rather unstable against X-rays, several precautions as well as sophisticated evaluation procedures had to be adopted to make sure that the results were not influenced by radiation damage. These included use of low primary intensity, short time of measurement, the presence of high concentrations of dithiothreitol, combined use of the conventional slit-collimation system and the new cone-collimation system. 1. For the native substrate-free enzyme the following molecular parameters could be established: radius of gyration R = 3.96 +/- 0.02 nm, maximum particle diameter D = 11.2 +/- 0.6 nm, radius of gyration of the thickness Rt = 1.04 +/- 0.04 nm, molecular weight Mr = 187000 +/- 3000, correlation volume Vc = 338 +/- 5 nm3, hydration x = 0.35 +/- 0.02 g/g, mean intersection length - l = 5.0 +/- 0.2 nm. Comparison of the experimental scattering curve with theoretical curves for various models showed that the enzyme is equivalent in scattering to an oblate ellipsoid of revolution rather than to a circular cylinder. The semiaxes of this ellipsoid are a = b = 6.06 nm and c = 2.21 nm. Thus with an axial ratio of about 1:0.36 the enzyme is of very anisometric shape. 2. Binding of the substrates (acetyl-CoA, glyoxylate) or the substrate analogue pyruvate causes slight structural changes of the enzyme. These changes are reflected mainly by a slight decrease of the radius of gyration (0.3--1.3%, as established both with the slit-smeared and the desmeared curves). Concomitantly there occurs a decrease of the maximum particle diameter and an increase of the radius of gyration of the thickness. These changes imply an increase of the axial ratio by 2.2--6.9%, i.e. substrate binding induces a decrease of anisometry. While the particle volume appears to be unchanged on binding glyoxylate or its analogue pyruvate, binding of acetyl-CoA causes slight changes of this parameter. In a similar manner the binding of acetyl-CoA leads to a slight enhancement of the molecular weight; this increase corresponds to the binding of 2.7 +/- 1 molecules of acetyl-CoA.     

A small angle X-ray study of the elongation factor complex Tu . GDP from Escherichia coli.

The protein elongation factor complex Tu. GDP from Escherichia coli was investigated in the presence of 0.01 mM GDP using the small-angle X-ray method. The overall shape and the molecular parameters of the Tu . GDP complex were determined using a least-squares method where the experimental data were used directly without desmearing. The best fit to the experimental data was obtained assuming the molecule to be an ellipsoid of revolution with the semiaxes A = B = 4.08 nm, and C = 1.18nm. Determination of the molecular weight gave the result Mr = 46 000, which corresponds to a water content equal to 26% (by weight).     

Low-angle neutron scattering analysis of Na/K-ATPase in detergent solution

Purified Na/K-ATPase from guinea pig renal outer medulla has been delipidated and solubilized in Brij 58 (polyoxyethylene ether; C-16, E-20). At a concentration of 2 mg of Brij 58/mg of protein, about one-half the enzyme complement was solubilized and almost 50% of Na/K-ATPase activity was retained by the enzyme-micelle complex. Guinier plots of the neutron scattering profiles yielded no evidence of heterogeneity with respect to subunit composition or the state of aggregation in the solubilized oligomers. Contrast matching with D2O used to obtain estimates of the molecular weight of the micellar form of Na/K-ATPase gave a mean value of 310,000 +/- 42,700, which corresponds to an alpha 2 beta 2 tetramer. A Stuhrmann plot of the neutron scattering data yielded an estimated radius of gyration of 67 A. The Stuhrmann plot also indicated an asymmetrical distribution of neutron scattering density. On the basis of the Stuhrmann plot parameters, the estimated molecular weight, and the radius of gyration, a low-resolution model was formulated of the oligomeric unit of Na/K-ATPase.     

Yeast tRNA(Asp)-aspartyl-tRNA synthetase complex: low resolution crystal structure

Yeast aspartyl-tRNA synthetase, a dimer of molecular weight 125,000, and two molecules of its cognate tRNA (Mr = 24160) cocrystallize in the cubic space group I432 (a = 354 A). The crystal structure was solved to low resolution using neutron and X-ray diffraction data. Neutron single crystal diffraction data were collected in five solvents differing by their D2O content in order to use the contrast variation method to distinguish between the protein and tRNA. The synthetase was first located at 40 A resolution using the 65% D2O neutron data (tRNA matched) tRNA molecules were found at 20 A resolution using both neutron and X-ray data. The resulting model was refined against 10 A resolution X-ray data, using density modification and least-squares refinement of the tRNA positions. The crystal structure solved without a priori phase knowledge, was confirmed later by isomorphous replacement. The molecular model of the complex is in good agreement with results obtained in solution by probing the protected part of the tRNA by chemical reagents.     

Molecular modelling of human complement component C3 and its fragments by solution scattering

Solution scattering experiments using both X-rays and neutrons are reported for human complement component C3 and up to six other glycoprotein fragments that are derived from C3. The X-ray and neutron molecular masses and neutron matchpoints are in agreement with the known primary sequence of C3. The X-ray radius of gyration RG of C3 is 5.2 nm and is similar for the related forms C3u, C3(a + b) and C3b. The X-ray cross-sectional radius of gyration RXS of C3b is however less than that of C3, C3u and C3(a + b). The major fragments of C3b, namely C3c and C3dg, were studied. The RG of C3c is 4.7 nm and for C3dg is 2.9 nm. C3c and C3dg do not interact when they coexist in solution in equimolar amounts. When C3u is cleaved into iC3u, the RG of iC3u increases to 5.9 nm and its RXS decreases, showing that C3c and C3dg behave as independent entities within the parent glycoprotein. Analyses of the neutron RG and RXS values by contrast variation techniques confirm the X-ray analyses, and show no evidence for significant hydrophobic or hydrophilic domains within C3 or any of its fragments. Shape analyses show that C3, C3c and C3dg are elongated particles. Debye models were developed using the scattering curve out to Q = 1.6 nm-1. These show that C3 and C3c resemble oblate ellipsoids while C3dg resembles a prolate ellipsoid. C3dg lies on the long edge of C3c within C3. The dimensions of the models are 18 nm X 2 nm X 10 nm for C3, 18 nm X 2 nm X 7 nm for C3c and 10 nm X 2 nm X 3 nm for C3dg. These models are compatible with analyses of the scattering curve RG and RXS values, data from sedimentation coefficients, and images of C3 and C3c seen by electron microscopy.     

Divalent ion-dependent swelling of tomato bushy stunt virus: a multi-approach study

Time-resolved small-angle X-ray and neutron scattering (SAXS and SANS) in solution were used to study the swelling reaction of TBSV upon chelation of its constituent calcium at mildly basic pH. SAXS intensities comprise contribution from the protein capsid and the RNA moiety, while neutron scattering, recorded in 72% D2O, is essentially due to the protein capsid. Cryo-electron micrographs of compact and swollen virus were used to produce 3D reconstructions of the initial and final conformations of the virus at a resolution of 13 A and 19 A, respectively. While compact particles appear to be very homogeneous in size, solutions of swollen particles exhibit some size heterogeneity. A procedure has been developed to compute the SAXS pattern from the 3D reconstruction for comparison with experimental data. Cryo-electron microscopy thereby provides an invaluable starting (and ending) point for the analysis of the time-resolved swelling process using the scattering data.     

Protein S1 from Escherichia coli ribosomes: an improved isolation procedure and shape determination by small-angle X-ray scattering.

Ribosomal protein S1 from Escherichia coli was studied in solution by small-angle X-ray scattering and the following parameters were obtained. The radius of gyration R = 8.0 +/- 0.2 nm; largest diameter D = 28 nm; molecular weight = (8--9) x 10(4). The data also yielded (with the assumption of a rigid particle with almost constant electron density) two radii of gyration of cross-section Rq1 = 2.5 +/- 0.1 nm and Rq2 = 1.05 +/- 0.05 nm and molecular volume = 140 nm3. The experimental scattering curve of S1 was compared with the theoretical scattering curves for several rigid triaxial homogeneous bodies and the closest fit was given by that of a flat elliptical cylinder with the dimensions of 4.5 nm and 0.88 nm for the two semiaxes and 26.5 nm for height. The results from the present X-ray scattering studies and those from limited proteolytic digestion of protein S1 [J. Mol. Biol. 127, 41--54, (1979)] support the notion that the structure of protein S1 is organized into two distinct subdomains within its elongated overall shape. Protein S1 was purified for this study by an efficient procedure which yielded 12 mg S1/g ribosomes. The isolated protein was fully active in functional tests both before and after X-ray irradiation.     

The 15 SCR flexible extracellular domains of human complement receptor type 2 can mediate multiple ligand and antigen interactions

Complement receptor type 2 (CR2, CD21) is a cell surface protein that links the innate and adaptive immune response during the activation of B cells. The extracellular portion of CR2 comprises 15 or 16 short complement regulator (SCR) domains, for which the overall arrangement in solution is unknown. This was determined by constrained scattering and ultracentrifugation modelling. The radius of gyration of CR2 SCR 1-15 was determined to be 11.5 nm by both X-ray and neutron scattering, and that of its cross-section was 1.8 nm. The distance distribution function P(r) showed that the overall length of CR2 SCR 1-15 was 38 nm. Sedimentation equilibrium curve fits gave a mean molecular weight of 135,000 (+/- 13,000) Da, in agreement with a fully glycosylated structure. Velocity experiments using the g*(s) derivative method gave a sedimentation coefficient of 4.2 (+/- 0.1) S. In order to construct a model of CR2 SCR 1-15 for constrained fitting, homology models for the 15 SCR domains were combined with randomised linker peptides generated by molecular dynamics simulations. Using an automated procedure, the analysis of 15,000 possible CR2 SCR 1-15 models showed that only those models in which the 15 SCR domains were flexible but partially folded back accounted for the scattering and sedimentation data. The best-fit CR2 models provided a visual explanation for the versatile interaction of CR2 with four ligands C3d, CD23, gp350 and IFN-alpha. The flexible location of CR2 SCR 1-2 is likely to facilitate interactions of C3d-antigen complexes with the B cell receptor.     

Structural comparisons of the native and reactive-centre-cleaved forms of alpha 1-antitrypsin by neutron- and X-ray-scattering in solution.

alpha 1-Antitrypsin is the best-characterized member of the serpin (serine-proteinase inhibitor) superfamily. Its solution structure was studied by high-flux neutron-scattering and synchrotron X-ray-scattering. Neutron data show that its absorption coefficient A1% 280,1cm is 5.4. The neutron radius of gyration RG at infinite contrast for native alpha 1-antitrypsin is 2.61 nm, characteristic of a moderately elongated structure, and its cross-sectional RG is 1.34 nm. The internal inhomogeneity of scattering densities within alpha 1-antitrypsin is high at 29 x 10(-5). The X-ray RG is 2.91 nm, in good agreement with the neutron RG of 2.82 nm in 1H2O. This RG is unchanged in reactive-centre-cleaved alpha 1-antitrypsin. These parameters are also unchanged at pH 8 in sodium/potassium phosphate buffers up to 0.6 M. The neutron and X-ray curves for native alpha 1-antitrypsin were compared with Debye simulation based on the crystal structure of reactive-centre-cleaved (papain) alpha 1-antitrypsin. After allowance for residues not visible in the crystallographic electron-density map, and rejoining the proteolysed site between Met-358 and Ser-359 by means of a relatively minor conformational re-arrangement, good agreement to a structural resolution of 4 nm is obtained with the neutron data in two contrasts and with the X-ray data. The structures of the native and cleaved forms of alpha 1-antitrypsin are thus similar within the resolution of solution scattering. This places an upper limit on the magnitude of the presumed conformational changes that occur in alpha 1-antitrypsin on reactive-centre cleavage, as indicated in earlier spectroscopic investigations of the Met-358-Ser-359 peptide-bond cleavage. Methods for scattering-curve simulations from crystal structures are critically assessed. The RG data lead to dimensions of 7.8 nm x 4.9 nm x 2.2 nm for native alpha 1-antitrypsin. The high internal inhomogeneity and the asymmetric shorter semi-axes of 4.9 nm and 2.2 nm suggest that the three oligosaccharide chains of alpha 1-antitrypsin are essentially freely extended into solvent in physiological conditions. This conclusion is also supported by the Debye simulations, and by modelling based on hydrodynamic parameters.     

The Fab and Fc fragments of IgA1 exhibit a different arrangement from that in IgG: a study by X-ray and neutron solution scattering and homology modelling

Human immunoglobulin A (IgA) is an abundant antibody that mediates immune protection at mucosal surfaces as well as in plasma. The IgA1 isotype contains two four-domain Fab fragments and a four-domain Fc fragment analogous to that in immunoglobulin G (IgG), linked by a glycosylated hinge region made up of 23 amino acid residues from each of the heavy chains. IgA1 also has two 18 residue tailpieces at the C terminus of each heavy chain in the Fc fragment. X-ray scattering using H2O buffers and neutron scattering using 100 % 2H2O buffers were performed on monomeric IgA1 and a recombinant IgA1 that lacks the tailpiece (PTerm455). The radii of gyration RG from Guinier analyses were similar at 6.11-6.20 nm for IgA1 and 5.84-6.16 nm for PTerm455, and their cross-sectional radii of gyration RXS were also similar. The similarity of the RG and RXS values suggests that the tailpiece of IgA1 is not extended outwards in solution. The IgA1 RG values are higher than those for IgG, and the distance distribution function P(r) showed two distinct peaks, whereas a single peak was observed for IgG. Both results show that the hinge of IgA1 results in an extended Fab and Fc arrangement that is different from that in IgG. Automated curve-fit searches constrained by homology models for the Fab and Fc fragments were used to model the experimental IgA1 scattering curves. A translational search to optimise the relative arrangement of the Fab and Fc fragments held in a fixed orientation resembling that in IgG was not successful in fitting the scattering data. A new molecular dynamics curve-fit search method generated IgA1 hinge structures to which the Fab and Fc fragments could be connected in any orientation. A search based on these identified a limited family of IgA1 structures that gave good curve fits to the experimental data. These contained extended hinges of length about 7 nm that positioned the Fab-to-Fab centre-to-centre separation 17 nm apart while keeping the corresponding Fab-to-Fc separation at 9 nm. The resulting extended T-shaped IgA1 structures are distinct from IgG structures previously determined by scattering and crystallography which have Fab-to-Fab and Fab-to-Fc centre-to-centre separations of 7-9 nm and 6-8 nm, respectively. It was concluded that the IgA1 hinge is structurally distinct from that in IgG, and this results in a markedly different antibody structure that may account for a unique immune role of monomeric IgA1 in plasma and mucosa.     

An analysis by low-angle neutron scattering of the structure of the acetylcholine receptor from Torpedo californica in detergent solution.

The acetylcholine receptor from the electric tissue of Torpedo californica is a large, integral membrane protein containing four different types of polypeptide chains. The structure of the purified receptor in detergent solution has previously been investigated by sedimentation analysis and gel filtration. Sedimentation analysis yielded a molecular weight of 250,000 for the protein moiety of the receptor monomer-detergent complex; hydrodynamic characteristics such as the Stokes radius, however, refer to the receptor-detergent complex. In this paper we report the results of our use of low-angle neutron scattering to investigate the shape of the receptor-detergent (Triton X-100 from Rohm & Haas Co., Philadelphia, Pa.) complex and separately of its protein and detergent moieties. By adjustment of the neutron-scattering density of the solvent with D2O to match that of one or the other of the moieties, its contribution to the scattering can be nearly, if not completely, eliminated. Neutron scattering from Triton X-100 micelles established that this detergent is contrast matched in approximately 18% D2O. Scattering measurements on the receptor-detergent complex in this solvent yielded a radius of gyration of the acetylcholine receptor monomer of 46 +/- 1A. The radius of gyration and molecular volume (305,000 A3) of the receptor are inconsistent with a compact spherical shape. These parameters are consistent with, for example, a prolate cylinder of dimensions (length x diameter) approximately 150 x approximately 50 A or an oblate cylinder, approximately 25 x approximately 130 A. More complex shapes are possible and in fact seem to be required to reconcile the present results with previous electron microscopic and x-ray analyses of receptor in membrane and with considerations of the function of the receptor in controlling ion permeability. The neutron-scattering data yield, in addition, an independent determination of the molecular weight of the receptor protein (240,000 +/- 40,000), the extent of Triton X-100 binding in the complex (approximately 0.4 g/g protein), and from the extended scattering curve, an approximation to the shape of the receptor-Triton X-100 complex, namely an oblate ellipsoid of axial ratio 1:4.     

Characterization of a half-molecular fragment obtained by reduction of human α2-macroglobulin with dithiothreitol

A half-molecular fragment of ₂-macroglobulin has been prepared by reducing and alkylating the inter-subunit disulfide bonds in the tetrameric ₂-macroglobulin molecule with 1 mM dithiothreitol (40 min) and 3 mM iodoacetamide (40 min). Further purification was made by gel chromatography and the homogeneous population of halfmolecules has been characterized by the techniques of small-angle X-ray and neutron scattering. The radii of gyration found by the two methods are 57.0 and 58.0 Å, respectively. The match point, obtained by neutron scattering from solutions with different H₂O/D₂O rations, is at 43% D₂O; the data are consistent with a particle having a higher scattering density at large distances from the particle centre. From the X-ray and neutron intensities scattered at zero angle, the specific volume was determined to be 0.73 cm³/g at+5°C and the molecular weight to be 390,000; the latter value is associated with a relatively large error due to the uncertainty in the concentration determination. Shape analysis indicates that the best-fitting scattering-equivalent threeaxial bodies are oblate shaped, with two of their axial dimensions about three to four times larger than the third one. From the volume of the best-fitting scattering-equivalent three-axial bodies, 0.72×10⁶ ų, we obtain a water content equal to 0.38 g H₂O/g protein (dry weight).Abbreviations SANS small-angle neutron scattering - SAXS small-angle X-ray scattering - ₂ M ₂-macroglobulin - DTT dithiothreitol - Tris tris(hydroxymethyl)aminomethane     

Interactions in human casein systems: self-association of nonphosphorylated human beta-casein

Since caseins were originally defined as phosphoproteins, nonphosphorylated beta-casein, comprising nearly 5% of the total beta-casein in the isoelectric precipitate from human milk, appears to be unique. Despite the relatively small amount present, its properties suggest that it may play an important role in micelle formation and structure. It has a partial specific volume, v, of 0.749 +/- 0.008 and an absorbance, E1% 1 cm,280 nm of 6.2 +/- 0.2. Sedimentation and viscosity data yield a solvation of 3 g H2O/g protein and an axial ratio of about 5 for the monomer. This would be consistent with a prolate ellipsoid of 10 nm length and 2 nm width. Equilibrium in the system is attained quite slowly and the temperature-dependent polymerization was found to be reversible. With calcium, the solubility behavior reflects an increased hydrophobicity and lower electrostatic repulsion in the molecule. There is essentially no strong calcium binding to this protein but there is evidence which strongly suggests that calcium binds to nonphosphate groups at higher concentrations. Increasing the temperature from 4 to 37 degrees C causes an apparent conformational change and an increase in protein aggregation which is further increased by addition of NaCl at 37 degrees C until a limiting size is reached at about 0.1 M NaCl. This limiting size polymer contains about 75 monomers and is nearly spherical with a radius of about 12 nm and a solvation of 1.5 g H2O/g protein. Laser light scattering measurements on the solution in 0.25 M NaCl revealed a relatively homogeneous particle size with a corrected diffusion coefficient, D20,w, of 2.8 X 10(-7) cm2/s.     

A neutron scattering study of the ternary complex EF-Tu.GTP-valyl-tRNAVal1A

The complex formation between elongation factor Tu (EF-Tu), GTP, and valyl-tRNAVal1A has been investigated in a hepes buffer of "pH" 7.4 and 0.2 M ionic strength using the small-angle neutron scattering method at concentrations of D2O where EF-Tu (42% D2O) and tRNA (71% D2O) are successively matched by the solvents. The results indicate that EF-Tu undergoes a conformational change and contracts as a result of the complex formation, since the radius of gyration decreases by 15% from 2.82 to 2.39 nm. tRNAVal1A, on the other hand, seems to mainly retain its conformation within the complex, since the radii of gyration for the free (after correction for interparticular scattering) and complexed form are essentially the same, 2.38 and 2.47 nm, respectively.     

Frozen spin targets in ribosomal structure research.

Polarized neutron scattering strongly depends on nuclear spin polarisation, particularly on proton spin polarisation. A single proton in a deuterated environment then is as efficient as 10 electrons in X-ray anomalous diffraction. Neutron scattering from the nuclear spin label is controlled by the polarisation of neutron spins and nuclear spins. Pure deuteron spin labels and proton spin labels are created by NMR saturation. We report on results obtained from the large subunit of E. coli ribosomes which have been obtained at the research reactor of GKSS using the polarized target facility developed by CERN. The nuclear spins were oriented with respect to an external field by dynamic nuclear polarisation. Proton spin polarisations of more than 80% were obtained in ribosomes at temperatures below 0.5 K. At T = 130 mK the relaxation time of the polarized target is one month (frozen spin target). Polarized small-angle neutron scattering of the in situ structure of rRNA and the total ribosomal protein (TP) has been determined from the frozen spin targets of the large ribosomal subunit, which has been deuterated in the TP and rRNA respectively. The results agree with those from neutron scattering in H2O/D2O mixtures obtained at room temperature. This is a necessary prerequisite for the planned determination of the in situ structure of individual ribosomal proteins and especially of that of ribosome bound mRNA and tRNAs.     

Reinvestigation of the shape and state of hydration of the skeletal myosin subfragment 1 monomer in solution

Hydrodynamic calculations lead to the conclusion that chymotryptic (or ethylenediaminetetraacetic acid) myosin S1 in solution (hydrated), at 1-5 degrees C, can be modeled as a prolate ellipsoid, with an axial ratio lying between p = 1.0 and 2.5 (major axis between 100.5 A, for p = 1.0, and 162.5 A, for p = 2.5). The degree of hydration is considerable (1.24 g/g for p = 2.5 and 2.02 g/g for p = 1.0). The dehydrated myosin head is pear-shaped under the electron microscope, and its narrowest part is located near the junction with the tail [Elliott, A., & Offer, G. (1978) J. Mol. Biol. 123, 505-519]. Mendelson & Kretzschmar [Mendelson, R. A., & Kretzschmar, K.M. (1980) Biochemistry 19, 4103-4108] have shown that the pear-shaped molecule does not predict the experimental X-ray scattering curve. Nor is this model able to predict the hydrodynamic values. The three-dimensional model for S1 used by Mendelson and Kretzschmar gives a rather good fit to the experimental X-ray scattering curve, but it does not predict the hydrodynamic values. In order to try to reconcile the three models and to fit the X-ray scattering curve and the hydrodynamic data, we suggest that, in solution, the S1 monomer has the shape of a prolate ellipsoid and that an inclusion of bound water exists at one extremity of the protein. The rest of bound water surrounds the protein. As first approximation, the dry protein and the hole are assumed to have the same shape as the hydrated molecule (prolate ellipsoid; p).(ABSTRACT TRUNCATED AT 250 WORDS)     

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.