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.

Multi-stage freezing of HEUR polymer networks with magnetite nanoparticles.

We observe a change in the segmental dynamics of hydrogels based on hydrophobically modified ethoxylated urethanes (HEUR) when hydrophobic magnetite nanoparticles (MNPs) are embedded in the hydrogels. The dynamics of the nanocomposite hydrogels is investigated using dielectric relaxation spectroscopy (DRS) and neutron spin echo (NSE) spectroscopy. The magnetic nanoparticles within the hydrophobic domains of the HEUR polymer network increase the size of these domains and their distance. The size increase leads to a dilution of the polymers close to the hydrophobic domain, allowing higher mobility of the smallest polymer blobs close to the "center". This is reflected in the decrease of the activation energy of the ß-process detected in the DRS data. The increase in distance leads to an increase of the size of the largest hydrophilic polymer blobs. Therefore, the segmental dynamics of the largest blobs is slowed down. At short time scales, i.e. 10(-9) s < τ < 10(-3) s, the suppression of the segmental dynamics is reflected in the α-relaxation processes detected in the DRS data and in the decrease of the relaxation rate Γ of the segmental motion in the NSE data with increasing concentration of magnetic nanoparticles. The stepwise (multi-stage) freezing of the small blobs is only visible for the pure hydrogel at low temperatures. On the other hand, the glass transition temperature (Tg) decreases upon increasing the MNP loading, indicating an acceleration of the segmental dynamics at long time scales (τ∼ 100 s). Therefore, it would be possible to tune the Tg of the hydrogels by varying the MNP concentration. The contribution of the static inhomogeneities to the total scattering function Sst(q) is extracted from the NSE data, revealing a more ordered gel structure than the one giving rise to the total scattering function S(q), with a relaxed correlation length ξNSE = (43 ± 5) Å which is larger than the fluctuating correlation length from a static investigation ξSANS = (17.2 ± 0.3) Å.

Scaling analysis of bio-molecular dynamics derived from elastic incoherent neutron scattering experiments.

Numerous neutron scattering studies of bio-molecular dynamics employ a qualitative analysis of elastic scattering data and atomic mean square displacements. We provide a new quantitative approach showing that the intensity at zero energy exchange can be a rich source of information of bio-structural fluctuations on a pico- to nano-second time scale. Elastic intensity scans performed either as a function of the temperature (back-scattering) and∕or by varying the instrumental resolution (time of flight spectroscopy) yield the activation parameters of molecular motions and the approximate structural correlation function in the time domain. The two methods are unified by a scaling function, which depends on the ratio of correlation time and instrumental resolution time. The elastic scattering concept is illustrated with a dynamic characterization of alanine-dipeptide, protein hydration water, and water-coupled protein motions of lysozyme, per-deuterated c-phycocyanin (CPC) and hydrated myoglobin. The complete elastic scattering function versus temperature, momentum exchange, and instrumental resolution is analyzed instead of focusing on a single cross-over temperature of mean square displacements at the apparent onset temperature of an-harmonic motions. Our method predicts the protein dynamical transition (PDT) at Td from the collective (α) structural relaxation rates of the solvation shell as input. By contrast, the secondary (ß) relaxation enhances the amplitude of fast local motions in the vicinity of the glass temperature Tg. The PDT is specified by step function in the elastic intensity leading from elastic to viscoelastic dynamic behavior at a transition temperature Td.

Interactions of silica nanoparticles with poly(ethylene oxide) and poly(acrylic acid): effect of the polymer molecular weight and of the surface charge.

The properties and the structure of polymer-modified silica nanoparticles were investigated by several characterization methods, with an emphasis on scattering techniques. Both bare and amino functionalized nanoparticles were used. To determine the effect of the charge, the polymer used was either nonionic poly(ethylene oxide) (PEO) or partially deprotonated poly(acrylic acid) (PAA). The particles coated with PEO were investigated by small-angle neutron scattering using the method of external contrast variation to observe the polymer coverage. The quantity adsorbed was found to be increasing with the molecular weight, and the surface type, bare or aminated, did not have a significant influence on the quantity adsorbed. The adsorption of PAA on positively charged aminated particles was investigated by dynamic light scattering and zeta potential measurements. A charge reversal, from positive to negative, was induced by the presence of PAA. Through the derivation of the structure factor, small-angle X-ray scattering provided significant information on the formation of aggregates at low PAA concentrations.

Dynamical transition of protein-hydration water.

Thin layers of water on biomolecular and other nanostructured surfaces can be supercooled to temperatures not accessible with bulk water. Chen et al. [Proc. Natl. Acad. Sci. U.S.A. 103, 9012 (2006)]10.1073/pnas.0602474103 suggested that anomalies near 220 K observed by quasielastic neutron scattering can be explained by a hidden critical point of bulk water. Based on more sensitive measurements of water on perdeuterated phycocyanin, using the new neutron backscattering spectrometer SPHERES, and an improved data analysis, we present results that show no sign of such a fragile-to-strong transition. The inflection of the elastic intensity at 220 K has a dynamic origin that is compatible with a calorimetric glass transition at 170 K. The temperature dependence of the relaxation times is highly sensitive to data evaluation; it can be brought into perfect agreement with the results of other techniques, without any anomaly.

Temperature dependence on structure and dynamics of Bovine Pancreatic Trypsin Inhibitor (BPTI): a neutron scattering study.

We have studied the influence of temperature on the structure of BPTI in solution by small angle neutron scattering. We have investigated the variation of the radius of gyration and the modification of the shape of BPTI between ambient temperature and 368 K. Results have shown an increase of the radius of gyration from 10.9 A at ambient temperature up to 13.3 A at 368 K. Global and internal dynamics of BPTI in solution were studied by quasielastic neutron scattering. The analysis of neutron data in terms of intermediate scattering function reveals two relaxation times tau(1) and tau(2) related respectively to global translational diffusive motions and to internal motions of protein. Motions of protons belonging to lateral chains of residues located at the surface of the protein have been detected. The results are compared to the recently published results concerning the influence of pressure on structure and dynamics of BPTI in solution [Appavou MS et al. Biochimica et Biophysica Acta, 1764, 2006, pp 414-423].

Dynamics of well-folded and natively disordered proteins in solution: a time-of-flight neutron scattering study.

Casein proteins belong to the class of natively disordered proteins. The existence of disordered biologically active proteins questions the assumption that a well-folded structure is required for function. A hypothesis generally put forward is that the unstructured nature of these proteins results from the functional need of a higher flexibility. This interplay between structure and dynamics was investigated in a series of time-of-flight neutron scattering experiments, performed on casein proteins, as well as on three well-folded proteins with distinct secondary structures, namely, myoglobin (alpha), lysozyme (alpha/beta) and concanavalin A (beta). To illustrate the subtraction of the solvent contribution from the scattering spectra, we used the dynamic susceptibility spectra emphasizing the high frequency part of the spectrum, where the solvent dominates. The quality of the procedure is checked by comparing the corrected spectra to those of the dry and hydrated protein with negligible solvent contamination. Results of spectra analysis reveal differences in motional amplitudes of well-folded proteins, where beta-sheet structures appear to be more rigid than a cluster of alpha-helices. The disordered caseins display the largest conformational displacements. Moreover their global diffusion rates deviate from the expected dependence, suggesting further large-scale conformational motions.

Influence of pressure on structure and dynamics of bovine pancreatic trypsin inhibitor (BPTI): small angle and quasi-elastic neutron scattering studies.

We have studied the influence of pressure on structure and dynamics of a small protein belonging to the enzymatic catalysis: the bovine pancreatic trypsin inhibitor (BPTI). Using a copper-beryllium high-pressure cell, we have performed small angle neutron scattering (SANS) experiment on NEAT spectrometer at HMI (Berlin, Germany). In the SANS configuration, the evolution of the radius of gyration and of the shape of the protein under pressures up to 6,000 bar has been studied. When increasing pressure from atmospheric pressure up to 6,000 bar, the pressure effects on the global structure of BPTI result on a reduction of the radius of gyration from 13.4 A down to 12.0 A. Between 5,000 and 6,000 bar, some transition already detected by FTIR [N. Takeda, K. Nakano, M. Kato, Y. Taniguchi, Biospectroscopy, 4, 1998, pp. 209-216] is observed. The pressure effect is not reversible because the initial value of the radius of gyration is not recovered after pressure release. By extending the range of wave-vectors to high q, we have observed a change of the form factor (shape) of the BPTI under pressure. At atmospheric pressure BPTI exhibits an ellipsoidal form factor that is characteristic of the native state. When the pressure is increased from atmospheric pressure up to 6,000 bar, the protein keeps its ellipsoidal shape. The parameters of the ellipsoid vary and the transition detected between 5,000 and 6,000 bar in the form factor of BPTI is in agreement with the FTIR results. After pressure release, the form factor of BPTI is characteristic of an ellipsoid of revolution with a semi-axis a, slightly elongated with respect to that of the native one, indicating that the pressure-induced structural changes on the protein are not reversible. The global motions and the internal dynamics of BPTI protein have been investigated in the same pressure range by quasi-elastic neutron scattering experiments on IN5 time-of-flight spectrometer at ILL (Grenoble, France). The diffusion coefficients D and the internal relaxation times of BPTI deduced from the analysis of the intermediate scattering functions show a slowing down of protein dynamics when increasing pressure.