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PubMed1981

Neutron small angle scattering on selectively deuterated human plasma low density lipoproteins. The location of polar phospholipid headgroups.

Human plasma low density lipoproteins (LDL) were deuterated in the phospholipid headgroup region by exchange with phosphatidylcholine-N(CD3)3-apolipoprotein A complexes. The exchange was associated with a net transfer of phosphatidylcholine to LDL leading to an increase in total phospholipid content by 27%. Practically all of the endogenous phosphatidylcholine including lysophosphatidylcholine, and about one-third of the sphingomyelin pool was found to be exchangeable. Immunochemically, deuterated LDL was identical with native LDL. The hydrodynamic and ultrastructural properties were closely similar for the two particle species apart from a slight increase in overall particle size by about 2%. Both native and deuterated LDL were investigated by neutron small angle scattering at several representative contrasts in H2O/D2O buffers. Subtraction of the scattering amplitudes of native from deuterated LDL resulted in a radius of gyration of 103 +/- 5 A for the N(CD3)3 groups, and in a structure factor resembling that of a thin, spherical shell. Evaluation of the contrast variation experiments in combination with previous data from x-ray small angle scattering (Laggner, P., and Mueller, K. (1978) Q. Rev. Biophys. 11, 371-425) indicates that the phospholipids form a spherical monolayer shell in the radial range between 75 A and 103 A around the core of cholesteryl esters and triglycerides. For the protein moiety, a radius of gyration of 110 A was calculated, indicating that it is located, on average, 5 to 10 A from the polar phospholipid headgroups toward the aqueous environment.

Cholesterol↗

PubMed2004

Radiation damage to a protein solution, detected by synchrotron X-ray small-angle scattering: dose-related considerations and suppression by cryoprotectants.

In small-angle X-ray scattering experiments at high-brilliant synchrotron sources, protein aggregation results from radiation damage. The radiation-induced aggregation of lysozyme in solution was qualitatively evaluated based on forward scattering and radii of gyration. The scattering did not change below 400 Gy and increased exponentially above this dose. The aggregation is only seen beyond the critical dose rate, and the 'dilution effect' known in radiology was also observed. Mass spectroscopy of the lysozyme solution exposed to a monochromatic X-ray beam did not show any cleavage of the polypeptide chain. Small-angle X-ray scattering patterns suggested that the radiation-induced aggregation should be a non-specific association of intact lysozyme, without substantial alterations of the folding topologies. It was found that the addition of small amounts of cryoprotectants, such as glycerol, ethylene glycol and sucrose, effectively reduced the radiation damage. Glycerol and ethylene glycol were identically effective in the 100 mM concentration range. A similar effective concentration was observed for sucrose. The damage reduction by the cryoprotectants was mainly ascribed to changes in the protein-protein interactions, and rarely to decreases in the diffusion rates of activated species.

Cryoprotective Agents↗

PubMed1984

Structure of the cholesteryl ester core of human plasma low density lipoproteins: selective deuteration and neutron small-angle scattering.

The structural arrangement of cholesteryl esters in human plasma low density lipoproteins (LDL) has been studied by selective deuteration and neutron small-angle scattering. LDL were labeled by in vitro exchange with two different kinds of deuterated cholesteryl esters, one labeled in the fatty acyl chain (cholesteryl myristate-d27) and the other in the branched side chain of cholesterol (cholesteryl-25,26,27-d7 oleate). Neutron scattering data from deuterated and protonated LDL were compared to identify the locations of the fatty acyl and cholesterol side chain moieties. Below the thermotropic transition, radii of gyration of 60 A and 70 A were obtained for these two domains, respectively, indicating that the cholesteryl nuclei are situated more distantly from the center than the fatty acyl chains. At 37 degrees C, above the thermotropic transition of the cholesteryl esters in LDL, both parts have similar radii of gyration of approximately 56 A. This information is used in a discussion of possible structural models for the apolar lipid core of LDL.

Cholesterol Esters↗

PubMed2004

Influence of multiple well defined conformations on small-angle scattering of proteins in solution.

A common structural motif for many proteins comprises rigid domains connected by a flexible hinge or linker. The flexibility afforded by these domains is important for proper function and such proteins may be able to adopt more than one conformation in solution under equilibrium conditions. Small-angle scattering of proteins in solution samples all conformations that exist in the sampled volume during the time of the measurement, providing an ensemble-averaged intensity. In this paper, the influence of sampling an ensemble of well defined protein structures on the small-angle solution scattering intensity profile is examined through common analysis methods. Two tests were performed using simulated data: one with the extended and collapsed states of the bilobal calcium-binding protein calmodulin and the second with the catalytic subunit of protein kinase A, which has two globular domains connected by a glycine hinge. In addition to analyzing the simulated data for the radii of gyration Rg, distance distribution function P(r) and particle volume, shape restoration was applied to the simulated data. Rg and P(r) of the ensemble profiles could be easily mistaken for a single intermediate state. The particle volumes and models of the ensemble intensity profiles show that some indication of multiple conformations exists in the case of calmodulin, which manifests an enlarged volume and shapes that are clear superpositions of the conformations used. The effect on the structural parameters and models is much more subtle in the case of the catalytic subunit of protein kinase A. Examples of how noise influences the data and analyses are also presented. These examples demonstrate the loss of the indications of multiple conformations in cases where even broad distributions of structures exist. While the tests using calmodulin show that the ensemble states remain discernible from the other ensembles tested or a single partially collapsed state, the tests performed using the simulated catalytic subunit of protein kinase A with noise added demonstrate that it can mask out the ensemble-dependent effects observed for the noiseless profiles.

Amino Acid Motifs↗

PubMed1993

Organization of turnip yellow mosaic virus investigated by neutron small angle scattering at 80 K: an intermediate state preceding decapsidation of the virion?

The organization of turnip yellow mosaic virus has been investigated by neutron small angle scattering at 300 K and 80 K in buffers containing various amounts of D2O. We confirm that in native virions, no substantial part of the RNA is located at a radius larger than ca. 100-110 A, i.e., that there is very little interpretation of the RNA into the capsid. At 80 K, scattering curves do not depend much upon contrast, from 40% D2O to 100% D2O buffers, but are strongly affected by interparticle interference. We could, however, show that it is not the case for the subsidiary intensity maximum at q approximately 0.06 A-1. From the position of this maximum, we conclude that upon freezing, the radius of the capsid expands by c.a. 3.5% and the RNA penetrates deeply into the protein shell. Biological implications of this conformational change immediately preceding decapsidation are discussed.

Capsid↗

PubMed1980

X-ray small-angle scattering study of mononucleosomes and of the close packing of nucleosomes in polynucleosomes.

The radius of gyration of mononucleosomes determined by X-ray small-angle scattering is 4.35 nm. The maximum dimension determined from the distance distribution function and the volume amount to 12.9 nm and 370 nm3, respectively. For a particular fraction of polynucleosomes a mean radius of gyration 16 nm, a maximum dimension 65 nm, and a mean volume 25,240 nm3 is obtained. The shape is approximated by an elongated cylinder having a diameter of 28 nm. A polynucleosome is built up from 69 nucleosomes, on the average. The distance of neighbouring nucleosomes in the polynucleosome amounts to 5.2 nm. Moreover, this distance shows that the nucleosomes in the polynucleosome are very closely packed.

Animals↗

PubMed1977

The lipid bilayer structure of the abnormal human plasma lipoprotein X. An X-ray small-angle-scattering study.

The structure of the abnormal lipoprotein X occurring in the plasma of patients with obstructive jaundice was investigated by X-ray small-angle scattering. The data were analyzed by discussing the distance distribution functions obtained directly from the experimental data by Fourier transformation, involving no a priori assumptions. The results provide evidence for lipoprotein X being essentially a random distribution of lamellae with a thickness of 5.1 nm and are consistent with hollow spherical (vesicular) structures of outer diameters greater than 30 nm with some overall size heterogeneity. Under the experimental conditions chosen, lipoprotein concentrations between 0.01 and 0.18 g/ml in in buffers of low ionic strength, lateral stacking as observed in negative-stain electron microscopy does not occur. The electron density profile perpendicular to the lamellar plane indicates that a lipid bilayer is the underlying structural principle, with the protein moieties partly bound within the polar head-group regions and partly occluded in soluble form in the vesicle interior.

Cholestasis↗

PubMed1998

Measurement of smoothed Wigner phase-space distributions for small-angle scattering in a turbid medium.

We study Wigner phase-space distributions W (x, p) in position (x) and momentum (p) for light undergoing multiple small-angle scattering in a turbid medium. Smoothed Wigner phase-space distributions are measured by using a heterodyne technique that achieves position and momentum resolution determined by the width and the diffraction angle of the local oscillator beam. The sample consists of 5.7-micron-radius polystyrene spheres suspended in a water-glycerol mixture. The momentum distribution of the transmitted light is found to contain a ballistic peak, a narrow diffractive pedestal, and a broad background. The narrow diffractive pedestal is found to decay more slowly than the ballistic peak as the concentration of scatterers is increased. The data are in excellent agreement with a simple theoretical model that explains the behavior of the narrow pedestal by including multiple diffractive scattering and treating large-angle scattering as a loss.

Light↗

PubMed1980

The core subunit structure in RNA polymerase holoenzyme determined by neutron small-angle scattering.

The core subunit arrangement of alpha 2-beta-beta' within DNA-dependent RNA polymerase holoenzyme alpha 2 beta beta' sigma from Escherichia coli was investigated by neutron small-angle scattering using label triangulation. The quaternary structure of multisubunit biomolecules can be studied by this new method if total reconstitution works in a quantitative way and if extensive replacement of C-bound hydrogen (H) by deuterium (2H) is possible. A substitution of the selected subunits by their fully deuterated analogues was used for the analysis of the overall shapes of the core subunits, alpha 2, beta and beta' in situ and for the determination of the intersubunit centre-to-centre distances. The contrast between the buffer and the remaining 'hydrogenated' enzyme vanishes if the buffer contains 42% 2H2O (matching of scattering length densities). The isotopic hybridization of the enzyme fulfils the conditions of isomorphous replacement as required: molecular functions, like enzyme activity, were completely preserved. The orientations of the core subunits within the holoenzyme were derived by comparing theoretical and experimental pair distance distribution functions, P(r), obtained from the scattering intensity differences of the pair-labelled (e.g. both beta and beta' labelled) and both mono-labelled molecules by direct Fourier transformations. Additional, the subunit shapes were refined by P(r) analyses. The arrangement of the stable core structure within the holoenzyme, which contains sigma as a dissociable factor, is presented in a three-dimensional model.

DNA-Directed RNA Polymerases↗

PubMed1990

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.

Hydrogen-Ion Concentration↗

PubMed2005

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.

Algorithms↗

PubMed1991

Correlation between the human and porcine complement system: a small-angle scattering study of cross immunity and methylamine-induced conformational changes of porcine C3 and C4 proteins.

The porcine complement proteins C3 and C4 have been isolated and then characterized using small-angle scattering methods. Within the limits of experimental errors, the porcine proteins are virtually identical with the corresponding human proteins as measured in terms of mol. wt, Mr and radius of gyration, R,: Mr(C3) = 198,000, Mr(C4) = 207,000, and R(C3) = 4.4 nm, R(C4) = 4.5 nm. The C3 and C4 proteins from pigs show cross-immunity with monoclonal antibodies (mAbs) raised against human C3 and C4, respectively. Using the Fab fragments of these mAbs as markers, it is indicated that porcine C3 and C4 undergo a conformational change after reaction with methylamine. The relatively large increase in the radius of gyration observed, delta R = 1.0-1.2 nm, going from the Fab complexes to the Fab complexes of the methylamine derivatives, is similar to that observed for human C3 under similar conditions. This may indicate that methylamine cleaves a labile thiol ester bond supposed to be present within the porcine proteins and that the epitopes interacting with the Fab fragments are very similar to those of the human proteins. Porcine C3 also resembles the human analogue by forming dimers after being subjected to methylamine and dilute lauryl sulphate: Mr = 404,000 and R = 7.9 nm.

Animals↗

PubMed2003

Analytical model for determination of parameters of helical structures in solution by small angle scattering: comparison of RecA structures by SANS.

The filament structures of the self-polymers of RecA proteins from Escherichia coli and Pseudomonas aeruginosa, their complexes with ATPgammaS, phage M13 single-stranded DNA (ssDNA) and the tertiary complexes RecA::ATPgammaS::ssDNA were compared by small angle neutron scattering. A model was developed that allowed for an analytical solution for small angle scattering on a long helical filament, making it possible to obtain the helical pitch and the mean diameter of the protein filament from the scattering curves. The results suggest that the structure of the filaments formed by these two RecA proteins, and particularly their complexes with ATPgammaS, is conservative.

Adenosine Triphosphate↗

PubMed1998

X-ray small angle scattering study of chromatin as a function of fiber length.

This work investigates the structure of native calf thymus chromatin as a function of fiber length and isolation procedures by using X-ray small angle scattering technique. Two methods of chromatin isolation have been compared in order to better understand the differences reported by various authors in terms of chromatin high order structure. In addition to these experimental results the effects of shearing have also been studied. In order to explain the differences among these chromatin preparations we built several models of chromatin fibers (represented as a chain of spherical subunits) assuming increasing level of condensation at increasing salt concentrations. For all these fiber models the corresponding theoretical X-ray scattering curves have been calculated and these results have been used to explain the influence of fiber length on the scattering profiles of chromatin. The comparison between experimental and theoretical curves confirms that the high molecular weight chromatin-DNA prepared by hypotonic swelling of nuclei (without enzymatic digestion) displays a partially folded structure even at low ionic strength, whereas the low molecular weight chromatin-DNA prepared by a brief nuclease digestion appears very weakly folded at the same ionic conditions.

Animals↗

PubMed1997

Insights into biomolecular function from small-angle scattering.

Recent advances in neutron and X-ray sources and instrumentation, new and improved scattering techniques, and molecular biology techniques, which have permitted facile preparation of samples, have each led to new opportunities in using small-angle scattering to study the conformations and interactions of biological macromolecules in solution as a function of their properties. For example, new instrumentation on synchrotron sources has facilitated time-resolved studies that yield insights into protein folding. More powerful neutron sources, combined with molecular biology tools that isotopically label samples, have facilitated studies of biomolecular interactions, including those involving active enzymes.

Aspartate Carbamoyltransferase↗

PubMed1990

Low-resolution structure of the tetrameric phenylalanyl-tRNA synthetase from Escherichia coli. A neutron small-angle scattering study of hybrids composed of protonated and deuterated protomers.

Escherichia coli phenylalanyl-tRNA synthetase is a tetrameric protein composed of two types of protomers. In order to resolve the subunit organization, neutron small-angle scattering experiments have been performed in different contrasts with all types of isotope hybrids that could be obtained by reconstituting the alpha 2 beta 2 enzyme from the protonated and deuterated forms of the alpha and beta subunits. Experiments have been also made with the isolated alpha promoter. A model for the alpha 2 beta 2 tetramer is deduced where the two alpha promoters are elongated ellipsoids (45 x 45 x 160 A3) lying side by side with an angle of about 40 degrees between their long axes and where the two beta subunits are also elongated ellipsoids (31 x 31 x 130 A3) with an angle of 30 degrees between their axes. This model was obtained by assuming that the two pairs of subunits are in contact in an orthogonal manner and by taking advantage of the measured distance between the centers of mass of the alpha 2 and beta 2 pairs (d = 23 +/- 2 A).

Amino Acyl-tRNA Synthetases↗

PubMed1994

Small-angle scattering study of alpha 1 inhibitor III from rat blood plasma.

The alpha 1 proteinase inhibitor III from rat blood plasma, homologous to the alpha 2-macroglobulin family of proteins, has been studied in solution using small-angle scattering of X-rays and of neutrons: the radius of gyration, Rg, was found to be 4.5 nm, and the largest distance within the molecule, Dmax = 14 nm. When the inhibitor reacts with chymotrypsin or methylamine, the resulting derivatives yield slightly higher Rg-values, 4.7 and 4.85 nm, respectively. The data of the native protein are consistent with a model, the projection of which resembles the letter V and which is formed by the two identical halves of an elliptic cylinder with semi-axes of 2.1 and 5.5 nm and a length of 11 nm. This elliptic cylinder model also explained the scattering from the monomeric complement proteins C3 and C4, as well as that from the monomers of the dimeric and tetrameric alpha 2-macroglobulin family of proteins (Osterberg, R., et al. (1991), Biochemistry 30, 7873-7878). Due to the conformational change occurring when the thiol ester bond is split, the cleft in the V-form seems to be closed; and as a result, the models of the chymotrypsin and methylamine derivatives are more compact than that of the native protein.

Acute-Phase Proteins↗

PubMed1991

C-terminal structure and mobility of rabbit skeletal muscle light meromyosin as studied by one- and two-dimensional 1H NMR spectroscopy and X-ray small-angle scattering.

Intact rabbit myosin and two different C-terminal fragments of rabbit muscle light meromyosin (LMM) expressed in Escherichia coli, LMM-30, and LMM-30C', were studied by 1H NMR spectroscopy. X-ray small-angle scattering shows that at high ionic strength two polypeptide chains of LMM-30 (which consists of the C-terminal 262 amino acids of myosin heavy chain) or LMM-30C' (which corresponds to LMM-30 but lacks the last 17 residues) assemble to form an alpha-helical coiled-coil as it is found also in myosin. The last 12 C-terminal residues of one polypeptide chain of LMM-30 and the last 9 C-terminal residues of the other chain are very mobile. The last 8 residues of the two strands are equivalent from the NMR point of view and unfolded; the valine residues in position 255 in the two strands are not equivalent, suggesting an interaction between the two strands, Ser-252, Arg-253, and Asp-254 are completely immobilized in one of the polypeptide strands and partly mobile in the other. Essentially the same pattern is observed in intact myosin. In spite of the large molecular weights of LMM-30 and LMM-30C', it is possible to resolve almost all aromatic residues and to determine the pK values of all the 4 tyrosine and of 9 (out of 10) histidine residues. The tyrosine residues in the two strands are equivalent in the two polypeptide chains and both have a pK of 10.5. The pK values of the histidine residues vary between 5.7 and 7.0.

Amino Acid Sequence↗