X-Ray Small Angle Scattering: A New Deconvolution Method for Evaluating Electron Density Distributions from Small Angle Scattering Diagrams

The direct determination of the electron density distributions of multilayered specimens with a small number of unit cells from X-ray small angle scattering experiments via the Q-function method of Hosemann and Bagchi includes the deconvolution of the so-called Q_o-function, the generalized Patterson function of one unit cell. In this paper a new and direct deconvolution method on the basis of Fourier series is presented which is suitable for one-dimensional centrosymmetrical (or antisymmetrical) density distributions. A FORTRAN-program has been written which has an execution time of ca. 20 s on an UNIVAC 1106-computer. The procedure has been successfully tested on some convolution functions generated by membrane-type electron density distributions.     

Small Angle X-Ray Scattering from Lipid-Bound Myelin Basic Protein in Solution

The structure of myelin basic protein (MBP), purified from the myelin sheath in both lipid-free (LF-MBP) and lipid-bound (LB-MBP) forms, was investigated in solution by small angle x-ray scattering. The water-soluble LF-MBP, extracted at pH < 3.0 from defatted brain, is the classical preparation of MBP, commonly regarded as an intrinsically unfolded protein. LB-MBP is a lipoprotein-detergent complex extracted from myelin with its native lipidic environment at pH > 7.0. Under all conditions, the scattering from the two protein forms was different, indicating different molecular shapes. For the LB-MBP, well-defined scattering curves were obtained, suggesting that the protein had a unique, compact (but not globular) structure. Furthermore, these data were compatible with earlier results from molecular modeling calculations on the MBP structure which have been refined by us. In contrast, the LF-MBP data were in accordance with the expected open-coil conformation. The results represent the first direct structural information from x-ray scattering measurements on MBP in its native lipidic environment in solution.     

Small and Wide Angle X-Ray Scattering Studies of Biological Macromolecules in Solution

In this paper, Small and Wide Angle X-ray Scattering (SWAXS) analysis of macromolecules is demonstrated through experimentation. SWAXS is a technique where X-rays are elastically scattered by an inhomogeneous sample in the nm-range at small angles (typically 0.1 - 5°) and wide angles (typically > 5°). This technique provides information about the shape, size, and distribution of macromolecules, characteristic distances of partially ordered materials, pore sizes, and surface-to-volume ratio. Small Angle X-ray Scattering (SAXS) is capable of delivering structural information of macromolecules between 1 and 200 nm, whereas Wide Angle X-ray Scattering (WAXS) can resolve even smaller Bragg spacing of samples between 0.33 nm and 0.49 nm based on the specific system setup and detector. The spacing is determined from Bragg''s law and is dependent on the wavelength and incident angle.In a SWAXS experiment, the materials can be solid or liquid and may contain solid, liquid or gaseous domains (so-called particles) of the same or another material in any combination. SWAXS applications are very broad and include colloids of all types: metals, composites, cement, oil, polymers, plastics, proteins, foods, and pharmaceuticals. For solid samples, the thickness is limited to approximately 5 mm.Usage of a lab-based SWAXS instrument is detailed in this paper. With the available software (e.g., GNOM-ATSAS 2.3 package by D. Svergun EMBL-Hamburg and EasySWAXS software) for the SWAXS system, an experiment can be conducted to determine certain parameters of interest for the given sample. One example of a biological macromolecule experiment is the analysis of 2 wt% lysozyme in a water-based aqueous buffer which can be chosen and prepared through numerous methods. The preparation of the sample follows the guidelines below in the Preparation of the Sample section. Through SWAXS experimentation, important structural parameters of lysozyme, e.g. the radius of gyration, can be analyzed.     

Scanning Microfocus Small Angle X-ray Scattering Study of the Avian Eggshell

Synchrotron microfocus small angle X-ray scattering was used to investigate the nanostructure and microscopic variation of eggshells. It uses a microbeam allowing the ability to probe interactions between the organic and inorganic components at nanometer level and is ideal for mapping over small areas to obtain a detailed analysis of structural variations. Thin sections of eggshells were scanned from the shell membrane （inner） to the cuticle （outer） surface. The data collected was used to produce two-dimensional maps showing microscopic changes within the different layers of the eggshell. The structural alterations ap- parently could have implications at the macroscopic level of the resulting eggshell. As the organic matrix is embedded within the eggshell this may contribute to the variations observed in calcite crystal form and texture, Structural information obtained about a biomaterial at different length scales is important in relating the structure to its functional properties. This knowledge and the principles behind the formation of biomaterials could be used in the attempt of bioengineering new systems.     

Anomalous Small Angle X-Ray Scattering Simulations: Proof of Concept for Distance Measurements for Nanoparticle-Labelled Biomacromolecules in Solution

Anomalous small angle X-ray scattering can in principle be used to determine distances between metal label species on biological molecules. Previous experimental studies in the past were unable to distinguish the label-label scattering contribution from that of the molecule, because of the use of atomic labels; these labels contribute only a small proportion of the total scattering signal. However, with the development of nanocrystal labels (of 50–100 atoms) there is the possibility for a renewed attempt at applying anomalous small angle X-ray scattering for distance measurement. This is because the contribution to the scattered signal is necessarily considerably stronger than for atomic labels. Here we demonstrate through simulations, the feasibility of the technique to determine the end-to-end distances of labelled nucleic acid molecules as well as other internal distances mimicking a labelled DNA binding protein if the labels are dissimilar metal nanocrystals. Of crucial importance is the ratio of mass of the nanocrystals to that of the labelled macromolecule, as well as the level of statistical errors in the scattering intensity measurements. The mathematics behind the distance determination process is presented, along with a fitting routine than incorporates maximum entropy regularisation.     

Characterization of the Decaheme c-Type Cytochrome OmcA in Solution and on Hematite Surfaces by Small Angle X-Ray Scattering and Neutron Reflectometry

The outer membrane protein OmcA is an 85 kDa decaheme c-type cytochrome located on the surface of the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1. It is assumed to mediate shuttling of electrons to extracellular acceptors that include solid metal oxides such as hematite (α-Fe₂O₃). No information is yet available concerning OmcA structure in physiologically relevant conditions such as aqueous environments. We purified OmcA and characterized its solution structure by small angle x-ray scattering (SAXS), and its interaction at the hematite-water interface by neutron reflectometry. SAXS showed that OmcA is a monomer that adopts a flat ellipsoidal shape with an overall dimension of 34 × 90 × 65 ų. To our knowledge, we obtained the first direct evidence that OmcA undergoes a redox state-dependent conformational change in solution whereby reduction decreases the overall length of OmcA by ∼7 Å (the maximum dimension was 96 Å for oxidized OmcA, and 89 Å for NADH and dithionite-reduced OmcA). OmcA was also found to physically interact with electron shuttle molecules such as flavin mononucleotide, resulting in the formation of high-molecular-weight assemblies. Neutron reflectometry showed that OmcA forms a well-defined monomolecular layer on hematite surfaces, where it assumes an orientation that maximizes its contact area with the mineral surface. These novel insights into the molecular structure of OmcA in solution, and its interaction with insoluble hematite and small organic ligands, demonstrate the fundamental structural bases underlying OmcA's role in mediating redox processes.     

Solution Structure of an Intramembrane Aspartyl Protease via Small Angle Neutron Scattering

Intramembrane aspartyl proteases (IAPs) comprise one of four families of integral membrane proteases that hydrolyze substrates within the hydrophobic lipid bilayer. IAPs include signal peptide peptidase, which processes remnant signal peptides from nascent polypeptides in the endoplasmic reticulum, and presenilin, the catalytic component of the γ-secretase complex that processes Notch and amyloid precursor protein. Despite their broad biomedical reach, basic structure-function relationships of IAPs remain active areas of research. Characterization of membrane-bound proteins is notoriously challenging due to their inherently hydrophobic character. For IAPs, oligomerization state in solution is one outstanding question, with previous proposals for monomer, dimer, tetramer, and octamer. Here we used small angle neutron scattering (SANS) to characterize n-dodecyl-β-D-maltopyranoside (DDM) detergent solutions containing and absent a microbial IAP ortholog. A unique feature of SANS is the ability to modulate the solvent composition to mask all but the enzyme of interest. The signal from the IAP was enhanced by deuteration and, uniquely, scattering from DDM and buffers were matched by the use of both tail-deuterated DDM and D₂O. The radius of gyration calculated for IAP and the corresponding ab initio consensus model are consistent with a monomer. The model is slightly smaller than the crystallographic IAP monomer, suggesting a more compact protein in solution compared with the crystal lattice. Our study provides direct insight into the oligomeric state of purified IAP in surfactant solution, and demonstrates the utility of fully contrast-matching the detergent in SANS to characterize other intramembrane proteases and their membrane-bound substrates.     

Monomeric Amyloid Beta Peptide in Hexafluoroisopropanol Detected by Small Angle Neutron Scattering

Small proteins like amyloid beta (Aβ) monomers are related to neurodegenerative disorders by aggregation to insoluble fibrils. Small angle neutron scattering (SANS) is a nondestructive method to observe the aggregation process in solution. We show that SANS is able to resolve monomers of small molecular weight like Aβ for aggregation studies. We examine Aβ monomers after prolonged storing in d-hexafluoroisopropanol (dHFIP) by using SANS and dynamic light scattering (DLS). We determined the radius of gyration from SANS as 1.0±0.1 nm for Aβ_1–40 and 1.6±0.1 nm for Aβ_1–42 in agreement with 3D NMR structures in similar solvents suggesting a solvent surface layer with 5% increased density. After initial dissolution in dHFIP Aβ aggregates sediment with a major component of pure monomers showing a hydrodynamic radius of 1.8±0.3 nm for Aβ_1–40 and 3.2±0.4 nm for Aβ_1–42 including a surface layer of dHFIP solvent molecules.     

Wide-Angle X-Ray Solution Scattering for Protein-Ligand Binding: Multivariate Curve Resolution with Bayesian Confidence Intervals

A new way to use wide-angle x-ray solution scattering to study protein-ligand binding is presented. First, scattering patterns are measured at different protein and ligand concentrations. Multivariate curve resolution based on singular value decomposition and global analysis is applied to estimate the binding affinities and reference patterns (i.e., the scattering patterns of individual components). As validated by simulation, Bayesian confidence intervals provide accurate uncertainty estimates for the binding free energies and reference patterns. Experimental results from several protein-ligand systems demonstrate the feasibility of the approach, which promises to expand the role of wide-angle x-ray scattering as a quantitative biophysical tool.     

Human Microtubule-Associated-Protein Tau Regulates the Number of Protofilaments in Microtubules: A Synchrotron X-Ray Scattering Study

Microtubules (MTs), a major component of the eukaryotic cytoskeleton, are 25 nm protein nanotubes with walls comprised of assembled protofilaments built from αβ heterodimeric tubulin. In neural cells, different isoforms of the microtubule-associated-protein (MAP) tau regulate tubulin assembly and MT stability. Using synchrotron small angle x-ray scattering (SAXS), we have examined the effects of all six naturally occurring central nervous system tau isoforms on the assembly structure of taxol-stabilized MTs. Most notably, we found that tau regulates the distribution of protofilament numbers in MTs as reflected in the observed increase in the average radius 〈R^MT〉 of MTs with increasing Φ, the tau/tubulin-dimer molar ratio. Within experimental scatter, the change in 〈R^MT〉 seems to be isoform independent. Significantly, 〈R^MT〉 was observed to rapidly increase for 0 < Φ < 0.2 and saturate for Φ between 0.2-0.5. Thus, a local shape distortion of the tubulin dimer on tau binding, at coverages much less than a monolayer, is spread collectively over many dimers on the scale of protofilaments. This implies that tau regulates the shape of protofilaments and thus the spontaneous curvature C_o^MT of MTs leading to changes in the curvature C^MT (=1/R^MT). An important biological implication of these findings is a possible allosteric role for tau where the tau-induced shape changes of the MT surface may effect the MT binding activity of other MAPs present in neurons. Furthermore, the results, which provide insight into the regulation of the elastic properties of MTs by tau, may also impact biomaterials applications requiring radial size-controlled nanotubes.     

Phase Separation in Binary Mixtures of Bipolar and Monopolar Lipid Dispersions Revealed by H NMR Spectroscopy, Small Angle X-Ray Scattering, and Molecular Theory

Binary mixtures of C₂₀BAS and POPC membranes were studied by solid-state ²H NMR spectroscopy and small angle x-ray scattering (SAXS) over a wide range of concentrations and at different temperatures. Three specifically deuterated C₂₀BAS derivatives—[1′,1′,20′,20′-²H₄]C₂₀BAS, [2′,2′,19′,19′-²H₄]C₂₀BAS, and [10′,11′-²H₂]C₂₀BAS—combined with protiated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), as well as membranes containing POPC-d₃₁ and fully protiated bolalipid, were used in NMR experiments to obtain structural information for the mixtures. The ²H NMR spectra of [10′,11′-²H₂]C₂₀BAS/POPC membrane dispersions reveal that the bolalipid is predominantly in the transmembrane conformation at high bolalipid concentrations (100, 90, and 70 mol %). At ≤50 mol % C₂₀BAS, smaller quadrupolar couplings appear in the spectra, indicating the presence of U-shaped conformers. The proportion of U-shaped bolalipids increases as the amount of POPC in the membrane increases; however, the transmembrane component remains the dominant bolalipid conformation in the membrane even at 45°C and 10 mol % C₂₀BAS, where it accounts for ∼50% of the bolalipid population. The large fraction of C₂₀BAS transmembrane conformers, regardless of the C₂₀BAS/POPC ratio, together with the findings from molecular mean-field theory calculations, suggests the coexistence of phase-separated bolalipid-rich domains and POPC-rich domains. A single lamellar repeat distance was observed in SAXS experiments corresponding to the average repeat spacing expected for C₂₀BAS- and POPC-rich domains. These observations are consistent with the presence of microphase-separated domains in the mixed membrane samples that arise from POPC-C₂₀BAS hydrophobic mismatch.     

Characterization of the Binding of Gallium, Platinum, and Uranium to Pseudomonas fluorescens by Small-Angle X-Ray Scattering and Transmission Electron Microscopy

Small-angle X-ray scattering (SAXS) was used to determine the binding of Ga, U, and Pt to Pseudomonas fluorescens in aqueous buffer. Atomic absorption spectrophotometry was used to quantify the heavy metals during bulk analysis, whereas transmission electron microscopy of whole mounts and thin sections was used to determine the locations of the cell-bound metal precipitates, as well as their sizes and physical structures. Energy-dispersive X-ray spectroscopy confirmed the compositions and identities of the precipitates and helped show that they were associated primarily with the envelope layers of the bacteria. Unlike Ga and Pt, which were located only at the cell surface, U was also found intracellularly in ~ 10% of the cells. This cytoplasmic location ultimately killed and lysed the cells. Surface-bound Ga and U were spread over the entire cell envelope (outer membrane-peptidoglycan-plasma membrane complex), whereas Pt was associated only with the lipopolysaccharide-rich, external face of the outer membrane. SAXS confirmed these data and showed that the bacteria were metal-enshrouded particles that were 1.0 to 1.5 μm in diameter. SAXS also provided a statistically significant representation of the bound metal precipitates, which ranged in size from 10 nm to 1 μm. The correlation between the microscopic data and the scattering data was extremely good. Since SAXS is performed in an aqueous milieu, it yields a more representative picture of the physical state of the metal bound to cell surfaces.     

Structure of Supercritical Fluid Krypton at Small Scattering Angle Using Parallel Molecular Dynamics Simulation

Abstract

We present a parallel algorithm for molecular dynamics involving short-range two- and three-body potentials and the pair-correlation function, g(r). The method is based on a spatial decomposition of the simulation box that takes advantage of a linked-cell list, and allows a load balanced partition of the computations of both the forces and g(r) over the processors. The tests of the program is conducted by evaluating the efficiency for both the thermalization phase and the production phase of the simulation. This method is successfully applied to the calculation of the direct correlation function of fluid krypton at small scattering angle along the T = 297 K supercritical isotherm.     

Fibre X-Ray and Conformational Study of the Binding of Metal Ions on DNA

Abstract

Results obtained from X-ray diffraction as well as from conformational analysis of Ag-DNA fibres are presented. For small percentages of Ag⁺ bound and high humidity, the B-DNA form is maintained. As the percentage of Ag⁺ is increased, the helical parameters of the B-DNA are modified. These modifications are directly related to the percentage of G—C bases. The periodicity of the DNA fibres are perturbed as Ag⁺ is mainly bound to G—C pairs and, thus, only the equatorial diffracted intensities can be compared to values calculated from molecular models. It is shown, by this way, that the first binding site is located on N7 of G. A second site is situated between N3 and N1 of the G—C pair, at the place of a hydrogen bond. A molecular model of the Ag-DNA complex is proposed and shown to be in agreement with experimental data. Results obtained allow to get some information on the binding of other ions such as Cu²⁺ and Hg²⁺ which give very little modification of the fibre X-ray patterns.     

All-Atom Ensemble Modeling to Analyze Small-Angle X-Ray Scattering of Glycosylated Proteins

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Highlights? MW determined from zero angle scattering is accurate for glycoproteins ? All-atom modeling with SAXS data can identify structural features of glycans ? SAXS ab initio reconstructions provide little structural information for glycans     

The Structure of High Molecular Weight Ribonucleic Acid in Solution: A Small-Angle X-Ray Scattering Study

Small-angle x-ray scattering studies on an absolute scale have been carried out on isotropic solutions of high molecular weight RNA obtained from ascites tumor cells, E. coli, and yeast. It was found that in all three cases the RNA is composed of short rigid rods (50 to 150Å in length) joined by small flexible regions. The rods account for almost the entire structure (at least 90 per cent); their radius of gyration about the axis and their mass per unit length are similar to those of DNA, suggesting a double-stranded helical structure. The rods are joined in an array forming the compact RNA molecule. On thermal degradation, the molecular superstructure disappears while the rods persist.     

Evidence That Calmodulin Binding to the Dopamine D2 Receptor Enhances Receptor Signaling

The Ca²⁺ sensor calmodulin (CaM) regulates numerous proteins involved in G protein-coupled receptor (GPCR) signaling. CaM binds directly to some GPCRs, including the dopamine D₂ receptor. We confirmed that the third intracellular loop of the D₂ receptor is a direct contact point for CaM binding using coimmunoprecipitation and a polyHis pull-down assay, and we determined that the D₂-like receptor agonist 7-OH-DPAT increased the colocalization of the D₂ receptor and endogenous CaM in both 293 cells and in primary neostriatal cultures. The N-terminal three or four residues of D₂-IC3 were required for the binding of CaM; mutation of three of these residues in the full-length receptor (I210C/K211C/I212C) decreased the coprecipitation of the D₂ receptor and CaM and also significantly decreased D₂ receptor signaling, without altering the coupling of the receptor to G proteins. Taken together, these findings suggest that binding of CaM to the dopamine D₂ receptor enhances D₂ receptor signaling.