Structure and kinetics of chemically cross-linked protein gels from small-angle X-ray scattering

Glutaraldehyde (GA) reacts with amino groups in proteins, forming intermolecular cross-links that, at sufficiently high protein concentration, can transform a protein solution into a gel. Although GA has been used as a cross-linking reagent for decades, neither the cross-linking chemistry nor the microstructure of the resulting protein gel have been clearly established. Here we use small-angle X-ray scattering (SAXS) to characterise the microstructure and structural kinetics of gels formed by cross-linking of pancreatic trypsin inhibitor, myoglobin or intestinal fatty acid-binding protein. By comparing the scattering from gels and dilute solutions, we extract the structure factor and the pair correlation function of the gels. The protein gels are spatially heterogeneous, with dense clusters linked by sparse networks. Within the clusters, adjacent protein molecules are almost in contact, but the protein concentration in the cluster is much lower than in a crystal. At the $\sim$1 nm SAXS resolution, the native protein structure is unaffected by cross-linking. The cluster radius is in the range 10 - 50 nm, with the cluster size determined mainly by the availability of lysine amino groups on the protein surface. The development of structure in the gel, on time scales from minutes to hours, appears to obey first-order kinetics. Cross-linking is slower at acidic pH, where the population of amino groups in the reactive deprotonated form is low. These results support the use of cross-linked protein gels in NMR studies of protein dynamics and for modeling NMR relaxation in biological tissue.Comment: 16 pages, 11 figure

Weak self-interactions of globular proteins studied by small-angle X-ray scattering and structure-based modeling

We investigate protein-protein interactions in solution by small-angle X-ray scattering (SAXS) and theoretical modeling. The structure factor for solutions of bovine pancreatic trypsin inhibitor (BPTI), myoglobin (Mb), and intestinal fatty acid-binding protein (IFABP) is determined from SAXS measurements at multiple concentrations, from Monte Carlo simulations with a coarse-grained structure-based interaction model, and from analytic approximate solutions of two idealized colloidal interaction models without adjustable parameters. By combining these approaches, we find that the structure factor is essentially determined by hard-core and screened electrostatic interactions. Other soft short-ranged interactions (van der Waals and solvation-related) are either individually insignificant or tend to cancel out. The structure factor is also not significantly affected by charge fluctuations. For Mb and IFABP, with small net charge and relatively symmetric charge distribution, the structure factor is well described by a hard-sphere model. For BPTI, with larger net charge, screened electrostatic repulsion is also important, but the asymmetry of the charge distribution reduces the repulsion from that predicted by a charged hard-sphere model with the same net charge. Such charge asymmetry may also amplify the effect of shape asymmetry on the protein-protein potential of mean force.Comment: 15 pages, 8 figure

A highly oriented cubic phase formed by lipids under shear

We demonstrate the formation of a macroscopically oriented inverse bicontinuous cubic (QII) lipid phase from a sponge (L3) phase by controlled hydration during shear flow. The L3 phase was the monoolein/ butanediol/water system; the addition of water reduces the butanediol concentration, inducing the formation of a diamond (QIID) cubic phase, which is oriented by the shear flow. The phenomenon was reproduced in both capillary and Couette geometries, indicating that this represents a robust general route for the production of highly aligned bulkQII samples, with applications in nanomaterial templating and protein research

Revealing the mechanisms of hydrogel formation by laccase crosslinking and regeneration of feruloylated arabinoxylan from wheat bran

Feruloylated arabinoxylan (FAX) from cereal brans has large potential to generate multifunctional materials with customized macromolecular and nanostructural architectures and techno-functional properties. Here we investigate the chemical and structural mechanisms of hydrogel formation of wheat bran FAX following enzymatic crosslinking by laccase and a subsequent regeneration procedure involving freeze-drying and resuspension of the crosslinked FAX in different pH buffers, using a battery of biochemical, rheological and physical techniques. The laccase crosslinking induced the conversion of ferulic acid units into a wide diversity of dimeric forms, leading to an increased molecular weight and a closer-packing of the FAX chains. The regeneration step resulted in a remarkable increase in the viscosity and viscoelasticity for all tested pH values. The amount of crystallinity of FAX increased by enzymatic crosslinking, it was however decreased by the regeneration step. The structural characterization revealed that enzymatic crosslinking, in addition to the formation of covalent crosslinks, influences the physical intermolecular interactions between adjacent FAX domains, and the regeneration forms larger clusters with higher dynamic moduli. Our results reveal that both chemical and physical mechanisms influence the network formation and multiscale assembly of wheat bran FAX hydrogels, thus modulating their rheological properties fundamental for their use in food and biomedical applications

Simultaneous and time resolved X-ray scattering and differential Scanning calorimetry experiments (SAXS/WAXD/DSC) using synchrotron radiation

New instrumentation designed to perform simultaneous time-resolved X-ray scattering experiments at small and wide angles (SAXS/WAXD) as well as differential scanning calorimetry (DSC) has recently been installed at the SAXS beamline of the Laboratório Nacional de Luz Síncrotron. The DSC device proved to be comparable with conventional equipment, allowing temperature variation with rates of up to 60 °C/min with precision of 0.1 °C. The use of a synchrotron radiation source and position sensitive X-ray detectors allows data collection in real time with 30 s resolution. The application of this experimental set-up in the isothermal crystallization and fusion of polymeric materials is given as an example. We present results of experiments with polycaprolactone (PCL) and its blends with chlorinated polyethylene (PCL/PECl), in which the simultaneous appearance of a crystalline structure and lamellar formation can be observed and the rate of process change for different compositions and thermal treatments can be determined. As a concluding remark, we mention that the SAXS/WAXD/DSC simultaneous experiments can also be performed with great advantage in the study of colloids and gel formation, as well as phase transitions in a variety of samples.Neste trabalho apresentamos uma nova instrumentação instalada na linha de SAXS do LNLS. Este equipamento permite a realização de experimentos simultâneos e resolvidos no tempo de espalhamento de raios X a baixos e altos ângulos (SAXS/WAXD) e calorimetria diferencial de varredura (DSC). O dispositivo de DSC mostrou-se comparável a equipamentos convencionais, com taxas de variação de temperatura de até 60 °C/min e uma precisão de 0.1 °C. O uso de uma fonte de radiação síncrotron e de detetores de raios X sensíveis à posição permitiu a obtenção de dados com uma resolução temporal de 30 s. A aplicação deste arranjo experimental no estudo da cristalização isotérmica e da fusão em materiais poliméricos é mostrada para o caso da policaprolactona (PCL) e suas blendas com polietileno clorado (PCL/PECl). As experiências mostraram a formação simultânea da estrutura cristalina e da morfologia lamelar nos diferentes estágios da cristalização assim como mudanças na cinética do processo com o tratamento isotérmico e a composição da blenda. Finalmente cabe destacar que experimentos simultâneos de SAXS/WAXD/DSC permitem o estudo de distintos processos abrangendo não apenas os de cristalização, mas também a formação de colóides e géis ou as transições de fase estruturais em diversos materiais.199206Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Investigation by combined solid-state NMR and SAXS methods of the morphology and domain size in polystyrene-b-polyethylene oxide-b-polystyrene triblock copolymers

The microphase structure of a series of polystyrene-b-polyethylene oxide-b-polystyrene (SEOS) triblock copolymers with different compositions and molecular weights has been studied by solid-state NMR, DSC, wide and small angle X-ray scattering (WAXS and SAXS). WAXS and DSC measurements were used to detect the presence of crystalline domains of polyethylene-oxide (PEO) blocks at room temperature as a function of the copolymer chemical composition. Furthermore, DSC experiments allowed the determination of the melting temperatures of the crystalline part of the PEO blocks. SAXS measurements, performed above and below the melting temperature of the PEO blocks, revealed the formation of periodic structures, but the absence or the weakness of high order reflections peaks did not allow a clear assessment of the morphological structure of the copolymers. This information was inferred by combining the results obtained by SAXS and 1H NMR spin diffusion experiments, which also provided an estimation of the size of the dispersed phases of the nanostructured copolymers. © 2009 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 48: 55–64, 2010FAPES

Influence of Clay Content and Amount of Organic Modifiers on Morphology and Pervaporation Performance of EVA/Clay Nanocomposites

Poly(ethylene-co-vinyl acetate)/organically modified clay nanocomposites were prepared using different clay loadings and by varying the amount of organic modifier. The morphology of the nanocomposites was investigated using small-angle X-ray scattering (SAXS), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). SAXS results displayed intercalation of polymeric chains between the silicate layers in all the cases. The interlayer distance varies slightly between the series. TEM images showed a better dispersion of the clay platelets at lower loading for both series of samples. The pervaporation performances of membranes were analyzed using a chloroform/acetone mixture. Membranes displayed high selectivity. The influence of feed composition on pervaporation was analyzed. The nanoclay content and the influence of free volume on pervaporation performance were also investigated in detail. A drop in selectivity and an increase in permeation rate were observed at higher clay loadings

Intercalation of cationic peptides within Laponite layered clay minerals in aqueous suspensions : The effect of stoichiometry and charge distance matching

Clays can be synthesised to have specific functional properties, which have been exploited in a range of industrial processes. A key characteristic of clay is the presence of a negatively charged surface, surrounded by an oppositely charged rim. Because of that, clays are able to sequester cationic compounds resulting in the formation of ordered layered structures, known as tactoids. Recent research has highlighted the possibility of utilising clay as a drug delivery compound for cationic peptides. Here, we investigate the process of intercalation by using the highly cationic peptide deca-arginine, and the synthetic clay Laponite, in aqueous suspensions with 2.5 wt% Laponite, and varying peptide concentrations. Small-angle X-ray scattering experiments show that tactoids are formed as a function of deca-arginine concentration in the dispersion, and for an excess of peptide, i.e. above a matched charge-ratio between the peptide and clay, the growth of the tactoids is limited, resulting in tactoidal dissolution. Zeta-potential measurements confirm that the observed dissolution is caused by overcharging of the platelets. By employing coarse-grained molecular dynamics simulations based on the continuum model, we are able to predict the tactoid formation, the growth, and the dissolution, in agreement with experimental results. We propose that the present simulation method can be a useful tool to tune peptide and clay characteristics to optimise and determine the extent of intercalation by cationic peptides of therapeutic interest

Novel freeze-dried foams from glutenin- and gliadin-rich fractions

This is the first study on freeze-dried foams prepared from glutenin- and gliadin-rich fractions of wheat gluten and blends thereof. It was found that the foam density and stiffness could be controlled by a suitable choice of the glutenin/gliadin ratio. The glutenin-rich samples had the highest foam densities and the density decreased with increasing gliadin content. The compression modulus also decreased with increasing gliadin content, which was explained by the decrease in foam density, a more open porosity and the more aggregated/polymerized structure in the presence of glutenin. IR and SE-HPLC revealed that the least aggregated foams were those consisting only of the gliadin-rich fraction. Confocal laser scanning microscopy revealed the presence of both HMW-glutenin and gliadin (to a certain extent probably resisting the ethanol extraction process) in the glutenin-rich foams. SAXS indicated that the gliadin-rich fraction contributed with weakly correlated protein aggregates with a characteristic distance of 40–43 Å