Modulating Protein Stability and Dynamics by Osmolytes and Electrolytes

Abstract

In chapter 3, the effects of urea and alkylureas were investigated on thermodynamic stability and internal dynamics of heme proteins (Cyt c and Mb). To determine the effects of urea and alkylureas on the internal dynamics of heme proteins, the kinetic and thermodynamic parameters for CO-association reaction of Ferrocyt c and CO-replacement reaction of MbCO by hexacyanoferrate ion were measured under varying concentrations of urea and alkylureas (MU, DMU, EU, TMU) at pH 7.0. As [denaturant] is increased, the rate coefficient of CO-association for Ferrocyt c ( k ass ) first decrease in subdenaturing region and then increase on going from subdenaturing to denaturing milieu, which indicates that the low concentrations of denaturants constrain the internal dynamics of Ferrocyt c . Within the subdenaturing limit, the denaturant-mediated constrained dynamics of Ferrocyt c is found to be more for urea and least for TMU. However, within the subdenaturing limit, such denaturant-mediated constrained dynamics is not observed for Mb. Intermolecular docking between horse Cyt c and denaturant molecule (urea, MU, DMU, EU and TMU) reveals that polyfunctional interactions between the denaturant and different groups of and#937;-loop of Cyt c and other part of protein decrease with an increase of alkyl group on urea molecule, which suggests that the decrease in the extent of restricted dynamics of and#937;-loop with a corresponding increase of alkyl groups on urea molecule is due to the decrease of denaturant-mediated cross-linking interactions. These denaturant mediated interactions are expected to reduce the entropy of Ferrocyt c . Analysis of rate temperature data shows a progressive decrease in entropy of Ferrocyt c in the native to subdenaturing region.