Kinetic and Thermodynamic Studies On the Effect of Chaotropic and Kosmotropic Cosolvents on Proteins

Abstract

To find out the effects of alcohols on the low-frequency local motions that control slow changes in structural dynamics of native-like compact-states of proteins, the effects of alcohols on structural fluctuation of M80-containing and#937;-loop have been evaluated by measuring the rate coefficients for slow thermally-driven CO-dissociation reaction of a natively-folded carbonmonoxycytochrome c (NCO) under varying concentrations of alcohols (methanol, ethanol, 1-propanol, 2-propanol, 3and#61616;-butanol, and 2,2,2-trifluoroethanol (TFE)) at pH 7.0. As alcohols concentration is increased within the subdenaturing limit of denaturant, the rate coefficients for CO-dissociation reaction decrease, indicating that subdenaturing concentrations of alcohols decrease the spatial displacement of thermal motion of and#937;-loop. The spatial displacement of thermal motion of the and#937;-loop is decreased most for TFE and 1-propanol and least for methanol. This finding indicates that the thermal motion of the protein in the subdenaturing limit of alcohols is controlled by the hydrophobicity of alcohol as well as by some specific effects of alcohols. Thermal denaturation studies of ferrocytochrome c (Ferrocyt c) and myoglobin (Mb) at pH 7.0 and lysozyme (Lyz) at pH 2.3 in the presence of various concentrations of these alcohols suggest that alcohols decrease the thermal stabilities of native and partially denatured proteins. The stabilization free energy (and#916;and#916;G) of Ferrocyt c and Lyz in alcohols solution was calculated from the slope of the Wyman-Tanford (WT) plot and water activity. The m-values obtained from the slope of and#916;and#916;G vs [alcohols] plots were found to be more negative for longer and linear chain alcohols, consistent with destabilization of proteins by alcohols through the disturbance of hydrophobic interactions and hydrogen-bonding. Compatible osmolyte such as glycine betaine (GB) and low concentrations of chaotropic denaturants such as guanidine hydrochloride (GdnHCl) and urea decrease the motional freedom of native Ferrocyt c at pH 7.0.