Adel Aschi

Concentration regimes and denaturation effects on the conformational changes of α-chymotrypsin by viscosity and dynamic light scattering measurements

This work focused on the conformational changes of α-chymotrypsin from bovine pancreas for various concentrations as well as the effects of chemical denaturation and organic solvents using both of viscosity and dynamic light scattering techniques. A wide range of concentrations varying between 0.1 and 30 mg/ml is tested to determine the critical concentration c** that separates the extremely dilute regime to the dilute regime and the overlapping concentration c* that separates the dilute regime to the semi-dilute regime. The knowledge of α-chymotrypsin folding process is followed for a range of chemical denaturant concentrations varying from 1 to 6 M. We showed that the α-chymotrypsin folds through a cooperative two-state transition without detectable kinetic intermediates and the formation of 50% unfolded happens for 5.5 M of urea or guanidinium chloride (GdmCl) concentrations. The action modes of the acetonitrile on the conformational changes of α-chymotrypsin are also achieved for concentrations varying between 10 and 50%.

Polyacrylic acids–bovine serum albumin complexation: Structure and dynamics

The study of the mixture of BSA with polyacrylic acids at different masses versus pH allowed highlighting the existence of two regimes of weak and strong complexation. These complexes were studied in diluted regime concentration, by turbidimetry, dynamic light scattering (DLS), zeta-potential measurements and nuclear magnetic resonance (NMR). We have followed the pH effect on the structure and properties of the complex. This allowed refining the interpretation of the phase diagram and understanding the observed phenomena. The NMR measurements allowed probing the dynamics of the constituents versus the pH. The computational method was used to precisely determine the electrostatic potential of BSA and how the polyelectrolyte binds to it at different pH.

Complex formation between ovalbumin and strong polyanion PSSNa: Study of structure and properties

The mixture system of long-chain polyelectrolyte complexed with a globular protein was investigated based on dynamic light scattering and turbidimetric measurements. We have discussed at different pH values the influence of high salt concentration and mass ratio (protein:PSSNa) on the behavior of the mixture. In dilute concentration regime, the PSSNa chain contracts at pHc by patch binding. We found two critical values of mass ratio: The first corresponds to the maximum shrinking of PSSNa. The second indicates the system that became more stable where the number of proteins attached to the PSSNa chain was constant. The screen of electrostatic interaction shows a high contribution of hydrophobic interaction at large salt concentration to form the coacervates. By building phase diagram, the continuity of pHφ1 in over whole range of salt concentrations and the widening of pH window (pHφ1-pHφ2) were observed. At certain salt concentrations, we can obtain the coexistence of two types of complex particles formed by electrostatic and hydrophobic interactions.

Impact of macromolecular crowding on structure and properties of pepsin and trypsin

The change of conformation of pepsin and trypsin in absence and presence of a high molecular crowding agent has been characterized using dynamic light scattering (DLS). Structural properties were investigated as a function of chemical denaturant concentrations, the guanidine hydrochloride (GdmCl). The results showed that Ficoll 400, macromolecular crowding, has a strong effect on the chemical denaturation process of these two proteins. The changes of measured hydrodynamic radius are attributed to the enhancement effect of the crowder agent due to the excluded volume effects. The data proved that the large size of a macromolecular crowder plays a crucial role on the conformation of a protein in its unfolded states. The values of interactions Parameter kd of complex particles and a number of proteins npr attached on the Ficoll 400 measured in different GdmCl concentrations. The effect of aging on the structure of complex are studied by small angle light scattering (SALS).

Investigation of Poly(vinyl pyrrolidone) in methanol by dynamic light scattering and viscosity techniques

Abstract The behavior of poly(vinyl pyrrolidone) (PVP) in methanol was examined using several independent methods. The hydrodynamic radius (Rh) of individual samples, over a range of molecular weights (10,000-360,000), was determined using dynamic light scattering (DLS) measurements. Dynamic Light Scattering (DLS) techniques directly probe such dynamics by monitoring and analyzing the pattern of fluctuations of the light scattered from polymer molecules. Some viscosity measurements were also performed to complete the DLS measurements and to provide more information on the particle structure. The results obtained with PVP-methanol system showed that plotting the variation of intrinsic viscosity versus the logarithm of the molecular mass of this polymer, we observe one crossover point. This crossover point appears when we reach the Θ-solvent behavior and delimit two molecular mass regions. The second order least-squares regression was used as an approach and was in excellent agreement with viscometric experimental results.

Structure and properties of native and unfolded lysing enzyme from T. harzianum: Chemical and pH denaturation.

The effect of chemical denaturants and pH on the change of the conformation of the protein Lysing Enzyme from Trichoderma Harzianum has been investigated by dynamic light scattering (DLS) and turbidimetry. Chemical denaturants are frequently used to describe the mechanisms of folding and transition states. We have analyzed the pH effect on the properties and particle size of the protein. The compaction factor CI has shown that the protein is weakly disordered. The molecular dynamics simulations confirm, at neutral pH, that the protein has a low net charge and high hydrophobicity.

Exploiting Complex Formation between Polysaccharides and Protein Microgels To Influence Particle Stabilization of W/W Emulsions

Protein particles were complexed with polysaccharides, and the effect on their capacity to stabilize water-in-water (W/W) emulsions was investigated. Protein microgels were formed by heating aqueous solutions of whey protein isolate. The microgels were subsequently mixed with anionic or cationic polysaccharides: κ-carrageenan (κ-car) or chitosan, respectively. The molar mass and radius of the complexes formed in dilute microgel suspensions (40 mg/L) were characterized by light scattering techniques as a function of the pH and the composition. The structure and stability of complexes formed at a higher microgel concentration (3 g/L) were studied by confocal laser scanning microscopy. It was found that small stable complexes can be formed with κ-car between pH 4.3 and pH 5.5 and with chitosan between pH 4.1 and pH 6.5, that is, both below and above the isoionic point of the microgels (pI = 5.0). Complexation with polysaccharides stabilized aqueous suspensions of microgels in the pH range where they flocculated in the absence of polysaccharides (4.3-5.5). W/W emulsions were produced by mixing dextran and poly(ethylene oxide) solutions. Microgels added to these emulsions spontaneously form a layer around the dispersed droplets, which inhibits coalescence to different extents depending on the conditions. The effect of complexation on the structure of the emulsions was investigated as a function of the pH. It is shown that stable liquid-like emulsions can be obtained in the pH range where emulsions containing only microgels flocculate.

Core-shell particles formed by β-lactoglobulin microgel coated with xyloglucan.

Core-shell particles were formed by mixing in aqueous solution the neutral polysaccharide xyloglucan (XG) with microgels. The last one was obtained by heating the whey protein β-lactoglobulin (β-LG) in the presence of CaCl2. XG adsorbed spontaneously unto the microgels at pH<5.6. The amount of bound XG per protein was determined using a combination of centrifugation and size exclusion chromatography. It increased linearly with increasing XG concentration. The fraction of XG that adsorbed increased with decreasing pH. The formation of the XG shell inhibited large scale flocculation of the particles, that causes precipitation for naked microgels, close to their isoionic point. The thickness of the XG shell was estimated by measurement of the hydrodynamic radius using dynamic light scattering. The extent of binding depended on the pH history during mixing showing that the protein/XG complex was not in thermodynamic equilibrium.