The effect of denaturants on protein thermal stability analyzed through a theoretical model considering multiple binding sites.

A novel mathematical development applied to protein ligand binding thermodynamics is proposed, which allows the simulation, and therefore the analysis of the effects of multiple and independent binding sites to the Native and/or Unfolded protein conformations, with different binding constant values. Protein stability is affected when it binds to a small number of high affinity ligands or to a high number of low affinity ligands. Differential scanning calorimetry (DSC) measures released or absorbed energy of thermally induced structural transitions of biomolecules. This paper presents the general theoretical development for the analysis of thermograms of proteins obtained for n-ligands bound to the native protein and m-ligands bound to their unfolded form. In particular, the effect of ligands with low affinity and with a high number of binding sites (n and/or m > 50) is analyzed. If the interaction with the native form of the protein is the one that predominates, they are considered stabilizers and if the binding with the unfolded species predominates, it is expected a destabilizing effect. The formalism presented here can be adapted to fitting routines in order to simultaneously obtain the unfolding energy and ligand binding energy of the protein. The effect of guanidinium chloride on bovine serum albumin thermal stability, was successfully analyzed with the model considering low number of middle affinity binding sites to the native state and a high number of weak binding sites to the unfolded state.

Dual substrate/solvent- roles of water and mixed reaction-diffusion control of ß-Galactosidase catalyzed reactions in PEG-induced macromolecular crowding conditions.

Here we studied the effect of molecular crowding on the hydrolysis of ortho- and para-nitrophenyl-ß-D-galactopyranosides (ONPG, PNPG) catalysed by Escherichia coli ß-Galactosidase in the presence of 0-35%w/v 6kD polyethyleneglycol (PEG6000). The Eadie-Hofstee data analysis exhibited single straight lines for PNPG at all [PEG6000] as well as for ONPG in the absence of PEG6000 so a Michaelian model was applied to calculate the kinetic parameters KM and kcat (catalytic rate constant) values. However, for ONPG hydrolysis in the presence of PEG6000, the two slopes visualized in Eadie-Hofstee plots leaded to apply a biphasic kinetic model to fit initial rate vs. [ONPG] plots hence calculating two apparent KM and two kcat values. Since the rate limiting-step of the enzymatic hydrolysis mechanism of ONPG, but not of PNPG, is the water-dependent one, the existence of several molecular water populations differing in their energy and/or their availability as reactants may explain the biphasic kinetics in the presence of PEG6000. With PNPG, KM as well as kcat varied with [PEG6000] like a parabola opening upward with a minimum at 15 %w/v [PEG6000]. In the case of ONPG, one of the components became constant while the other component exhibited a slight increasing tendency in kcat plus high and [PEG6000]-dependent increasing KM values. Sedimentation velocity analysis demonstrated that PEG6000 impaired the diffusion of ß-Gal but not that of substrates. In conjunction, kinetic data reflected complex combinations of PEG6000-induced effects on enzyme structure, water structure, thermodynamic activities of all the chemical species participating in the reaction and protein diffusion.

ß-sheet to α-helix conversion and thermal stability of ß-Galactosidase encapsulated in a nanoporous silica gel.

The effect on protein conformation and thermal stability was studied for ß-Galactosidase (ß-Gal) encapsulated in the nanopores of a silicate matrix (Eß-Gal). Circular dichroism spectra showed that, compared with the enzyme in buffer (Sß-Gal), Eß-Gal exhibited a higher content of α-helix structure. Heating Eß-Gal up to 75 °C caused a decrease in the content of ß-sheet structure and additional augments on Eß-Gal components attributed to helical content, instead of the generalized loss of the ellipticity signal observed with Sß-Gal. Steady state fluorescence spectroscopy analysis evidenced an Eß-Gal structure less compact and more accessible to solvent and also less stable against temperature increase. While for Sß-Gal the denaturation midpoint (Tm) was 59 °C, for Eß-Galit was 48 °C. The enzymatic activity assays at increasing temperatures showed that in both conditions, the enzyme lost most of its hydrolytic activity against ONPG at temperatures above 65 °C and Eß-Gal did it even at lower T values. Concluding, confinement in silica nanopores induced conformational changes on the tertiary/cuaternary structure of Eß-Gal leading to the loss of thermal stability and enzymatic activity.

Environmental Topology and Water Availability Modulates the Catalytic Activity of ß-Galactosidase Entrapped in a Nanosporous Silicate Matrix.

In the present work we studied the catalytic activity of E. coli ß-Gal confined in a nanoporous silicate matrix (Eß-Gal) at different times after the beginning of the sol-gel polymerization process. Enzyme kinetic experiments with two substrates (ONPG and PNPG) that differed in the rate-limiting steps of the reaction mechanism for their ß-Gal-catalyzed hydrolysis, measurements of transverse relaxation times (T2) of water protons through 1H-NMR, and scanning electron microscopy analysis of the gel nanostructure, were performed. In conjunction, results provided evidence that water availability is crucial for the modulation observed in the catalytic activity of ß-Gal as long as water participate in the rate limiting step of the reaction (only with ONPG). In this case, a biphasic rate vs. substrate concentration was obtained exhibiting one phase with catalytic rate constant (kcA), similar to that observed in solution, and another phase with a higher and aging-dependent catalytic rate constant (kcB). More structured water populations (lower T2) correlates with higher catalytic rate constants (kcB). The T2-kcB negative correlation observed along the aging of gels within the 15-days period assayed reinforces the coupling between water structure and the hydrolysis catalysis inside gels.