Neutron Scattering Contrast Variation Research Articles

Exploiting neutron scattering contrast variation in biological membrane studies.

Biological membranes composed of lipids and proteins are central for the function of all cells and individual components, such as proteins, that are readily studied by a range of structural approaches, including x-ray crystallography and cryo-electron microscopy. However, the study of complex molecular mixtures within the biological membrane structure and dynamics requires techniques that can study nanometer thick molecular bilayers in an aqueous environment at ambient temperature and pressure. Neutron methods, including scattering and spectroscopic approaches, are useful since they can measure structure and dynamics while also being able to penetrate sample holders and cuvettes. The structural approaches, such as small angle neutron scattering and neutron reflectometry, detect scattering caused by the difference in neutron contrast (scattering length) between different molecular components such as lipids or proteins. Usually, the bigger the contrast, the clearer the structural data, and this review uses examples from our research to illustrate how contrast can be increased to allow the structures of individual membrane components to be resolved. Most often this relies upon the use of deuterium in place of hydrogen, but we also discuss the use of magnetic contrast and other elements with useful scattering length values.

Structural Study for Membrane Protein using Solution X-Ray Scattering Contrast Variation

Membrane protein often needs lipid bilayer membrane to stabilize its structure and to fulfill its functions. However, the presence of lipid bilayer membrane makes the structural study on the protein very difficult. Here we proposed to use X-ray scattering contrast variation method in which membrane protein (with lipid membrane) samples will be measured in a series of solutions with various sucrose concentration. X-ray scattering intensity measures the scattering length/capability difference between sample/solute and buffer solution/solvent. Since X-ray scattering lengths of protein and lipid are different and scattering length of sucrose buffer changes along with its concentration, the ratio of X-ray scattering contribution from protein part and lipid part varies along with the sucrose concentration. In such way, we can separate the scattering contribution of protein part from that of lipid. We also simultaneously fit the data sets collected from the sucrose concentration series measurements and obtain the 3-D low resolution structure for the protein and the lipid at the same time. Comparing to neutron scattering contrast variation method, this X-ray contrast method is less expensive and can provide wider q range due to the availability of high flux X-ray sources. A few membrane protein examples will be presented. Figure. SAXS data for membrane protein in various sucrose solution. In this presentation, I will also discuss the capabilities of beamline 12-ID-B of the Advanced Photon Source at Argonne National Laboratory. Acknowledgement: Use of the Advanced Photon Source, an Office of Science User Facility operated for the U.S. Department of Energy (DOE) Office of Science by Argonne National Laboratory, was supportedby the U.S. DOE under Contract No. DE-AC02-06CH11357.

Evaluation of molecular volume change of block copolymer depending on temperature: A SANS study

Amphiphilic Pluronic triblock copolymers form various self-assembled structures such as sphere, cylinder, lamellae and so on, depending on temperature, leading to the increase of hydrophobicity of block copolymers. However, the effective molecular volume change of the block copolymer has not been fully exploited yet, when temperature increases. Here, we have investigated the effective molecular volume change of the block copolymer upon heating by using the contrast variation small angle neutron scattering. The scattering length densities (SLDs) of the block copolymer were experimentally obtained from the neutron scattering contrast variation method between the solvent and the block copolymer at varying temperature. Even though the SLD, which is the intrinsic property of the material, should not be changed by temperature elevation, it was dependent on temperature, indicating that the molecular volume is changed. Therefore, we obtained the increase rate of the molecular volume change of the block copolymer (the effective molecular volume change) from the comparison of the calculated SLD and the standard SLD, which is evaluated by plotting the SANS intensity at the first order Bragg peak as the function of temperature at each volume fraction of D2O and H2O that is about 25.5%–51.3% depending on temperature.

Translocase Activity and Asymmetric Model Membranes Probed by Neutron Scattering

Biological membranes are almost universally asymmetric with individual leaflets containing different lipid species at varying concentrations. This asymmetry may play a key role in biological functions such as protein-membrane interactions, membrane trafficking, and cellular signaling. Traditional model membrane systems are comprised of symmetric membrane leaflets and though progress has been made in developing asymmetric model membranes, these systems are unstable and easily contaminated. We have developed an actively asymmetric biomembrane model system by purifying and reconstituting the well-characterized E. coli phospholipid ABC transporter MsbA into proteoliposomes that mimic the phospholipid composition of a physiological bacterial membrane. Subsequent MsbA-mediated phospholipid translocation and resultant membrane asymmetry was studied using small-angle neutron scattering (SANS). SANS is exceptionally well suited for studying the phospholipid distribution perpendicular to the bilayer plane due to its nondestructive nature and sub-nanometer resolution. Additionally, the hydrogen/deuterium (H/D) neutron scattering contrast variation from isotopic phospholipid labeling enables us to track the translocation of lipid membrane components without introducing bulky fluorescence or spin labels. In this study, the MsbA-mediated translocation of the phospholipid phosphatidylethamolamine (PE) between proteoliposome leaflets and the formation of asymmetric bilayers was studied using SANS for the first time. The sustained asymmetry in these model protocells will be central for future studies on biomimetic membrane systems and their role in diverse membrane-directed biological processes.

Small angle neutron scattering contrast variation reveals heterogeneities of interactions in protein gels.

We propose a quantitative approach to probe the spatial heterogeneities of interactions in macromolecular gels, based on a combination of small angle X-ray (SAXS) and neutrons (SANS) scattering. We investigate the structure of model gluten protein gels and show that the gels display radically different SAXS and SANS profiles when the solvent is (at least partially) deuterated. The detailed analysis of the SANS signal as a function of the solvent deuteration demonstrates heterogeneities of sample deuteration at different length scales. The progressive exchange between the protons (H) of the proteins and the deuteriums (D) of the solvent is inhomogeneous and 60 nm large zones that are enriched in H are evidenced. In addition, at low protein concentration, in the sol state, solvent deuteration induces a liquid/liquid phase separation. Complementary biochemical and structure analyses show that the denser protein phase is more protonated and specifically enriched in glutenin, the polymeric fraction of gluten proteins. These findings suggest that the presence of H-rich zones in gluten gels would arise from the preferential interaction of glutenin polymers through a tight network of non-exchangeable intermolecular hydrogen bonds.

Colloidal dispersions of octadecyl grafted silica spheres in toluene: A global analysis of small angle neutron scattering contrast variation and concentration dependence measurements

In this paper we report measurements of the form factor and the structure factor of a sterically stabilized colloidal dispersion consisting of silica spheres coated with octadecane in toluene by small angle neutron scattering (SANS). The phase diagram of this system shows the liquid-liquid coexistence line and also a jamming transition at higher concentrations, where the jamming line intersects the coexistence line roughly at the critical point. We have performed SANS experiments at a temperature well above the transition temperature and at various volume fractions phi, spanning from the very dilute regime (phi=0.2%) to the critical concentration (phi=16%) and the highly viscous regime (phi=39.2%). Except for the very dilute regime, we observe a structure factor S(q) in all other cases. We fitted our data over the whole concentration regime using a global fitting routine with a core-shell model for the form factor P(q), taking into account the structure factor, which we describe with the Robertus model for an adhesive polydisperse core-shell particle. At a volume fraction of phi=5% a SANS contrast variation experiment has been performed. From that the product of the volume of the shell and the amount of solvent within the corona of our core-shell particle could be determined. At the most probable shell thickness of 2.3 nm a solvent content of about 50% within the corona was found. Moreover we could conclude that the core is not interpenetrated by solvent molecules. From the contrast variation experiment followed that the structure factor at zero average contrast exhibits a strong q dependence, which is an effect of an inhomogeneous particle in combination with a size distribution.

Characterization of Nanoporous Low-k Thin Films by Small-Angle Neutron Scattering Contrast Variation

Small-angle neutron scattering contrast variation is applied to characterization of nanoporous low-dielectric constant (low-k) thin films. Films are exposed to saturated solvent vapor in air, whereby the pores fill with liquid by capillary condensation. The pores are filled with mixtures of hydrogen- and deuterium-containing solvents to vary the neutron contrast with the matrix (wall). The composition of the solvent mixture is systematically varied to identify a composition that minimizes the scattered intensity (contrast match point). From the contrast match point composition, film characteristics including matrix density and homogeneity are assessed. Four spin-on low-k materials including a methylsilsesquioxane, an organic polymer, a xerogel, and a hydrogensilsesquioxane are characterized by the new technique. Calculated matrix mass densities are compared to independent density measurements obtained by an established specular X-ray reflectivity technique. We find no evidence of "closed pores", defined here as pores inaccessible to the probe solvent, in any of the materials studied.

A synthetic holliday junction is sandwiched between two tetrameric Mycobacterium leprae RuvA structures in solution: new insights from neutron scattering contrast variation and modelling

The interaction between homologous DNA molecules in recombination and DNA repair leads to the formation of crossover intermediates known as Holliday junctions. Their enzymatic processing by the RuvABC system in bacteria involves the formation of a complex between RuvA and the Holliday junction. To study the solution structure of this complex, contrast variation by neutron scattering was applied to Mycobacterium leprae RuvA (MleRuvA), a synthetic analogue of a Holliday junction with 16 base-pairs in each arm, and their stable complex. Unbound MleRuvA was octameric in solution, and formed an octameric complex with the DNA junction. The radii of gyration at infinite contrast were determined to be 3.65 nm, 2.74 nm and 4.15 nm for MleRuvA, DNA junction and their complex, respectively, showing that the complex was structurally more extended than MleRuvA. No difference was observed in the presence or absence of Mg2+. The large difference in RG values for the free and complexed protein in 65% 2H2O, where the DNA component is “invisible”, showed that a substantial structural change had occurred in complexed MleRuvA. The slopes of the Stuhrmann plots for MleRuvA and the complex were 19 and 15 or less (× 10−5), respectively, indicating that DNA passed through the centre of the complex. Automated constrained molecular modelling based on the Escherichia coli RuvA crystal structure demonstrated that the scattering curve of octameric MleRuvA in 65% and 100% 2H2O is explained by a face-to-face association of two MleRuvA tetramers stabilised by salt-bridges. The corresponding modelling of the complex in 65% 2H2O showed that the two tetramers are separated by a void space of about 1–2 nm, which can accommodate the width of B-form DNA. Minor conformational changes between unbound and complexed MleRuvA may occur. These observations show that RuvA plays a more complex role in homologous recombination than previously thought.

A core-shell model of calcium phosphate nanoclusters stabilized by beta-casein phosphopeptides, derived from sedimentation equilibrium and small-angle X-ray and neutron-scattering measurements.

Calcium phosphate nanoclusters were prepared under standardised conditions using 10 mg ml(-1) of the 25-amino-acid N-terminal tryptic phosphopeptide of bovine beta-casein as a stabilising agent. The Mr determined by sedimentation equilibrium was 197,600+/-13,700 and the apparent radius of gyration determined by X-ray scattering was 2.80+/-0.05 nm. A small-angle neutron scattering contrast variation study in 1H2O/2H2O mixtures was performed and gave radii of gyration at the calculated match points for the calcium phosphate (88.2% 2H2O) and phosphopeptide (41.3% 2H2O) of 3.39+/-0.08 nm and 1.85+/-0.05 nm, respectively. Measurements at larger scattering wave vector showed a subsidiary maximum at about Q = 1.6 nm(-1). The results are consistent with a model of the nanoclusters comprising a spherical core of 355+/-20 CaHPO4 x 2 H2O units, density 2.31 g ml(-1) and radius 2.30+/-0.05 nm surrounded by 49+/-4 peptide chains with a partial specific volume of 0.7 cm3 g(-1), forming a tightly packed shell with an outer radius of 4.04+/-0.15 nm. This model suggests that the phosphopeptide is able to arrest the process of growth of the precipitating phase of calcium phosphate at its earliest stages. A similar role for whole casein could be vital to the normal functioning of the mammary gland during milk secretion.

Partial structure factors in colloidal silica mixtures determined with small-angle neutron scattering contrast variation

Small-angle neutron scattering experiments at various contrasts were performed on concentrated mixtures of colloidal spheres differing in size. Both colloidal components consisted of fairly monodisperse silica cores coated with a layer of octadecyl chains. Cyclohexane was used as dispersing medium; variation of the contrast was achieved by using mixtures of 1H -cyclohexane and 2H -cyclohexane. Scattered intensities were measured at three volume fractions up to 0.4, at equal partial volume fractions. The different contrast dependence of the scattering amplitudes of both colloids allowed us to calculate partial structure factors. This was done using a method which has not been reported previously. Describing the intraparticle structures with layered-sphere models, and using a decoupling approximation, three partials were obtained from a system of linear equations. The scattering curves at the various contrasts constitute a consistent data set, at all volume fractions. Although the separate components interact like hard spheres, the partial structure factors in the mixture reflect marked deviations from hard-sphere behavior. Their nature can be qualitatively explained with an attractive interaction between unequal particles. This is confirmed with simple model calculations.