Hydration changes upon protein unfolding: cosolvent effect analysis

We have characterized the unfolding energetics of ribonuclease a and hen eg g white lysozyme as a function of temperature, pH and concentration of seve ral cosolvents (sucrose, glucose, glycerol, polyethyleneglycol 8000) that a re expected to be preferentially excluded from the surface of native protei ns, and we have calculated the corresponding unfolding changes in preferent ial hydration (Delta Gamma(21)) at 25 degrees C. We find no significant cos olvent concentration effect on the unfolding enthalpy and heat capacity val ues, which suggests that the cosolvents do not interact strongly with the p roteins at the comparatively low cosolvent concentrations employed in this work. In spite of this, the Ar,, values are significantly smaller than theo retical estimates of the unfolding change in the number of water molecules corresponding to first monolayer coverage (Delta N-1), even when, for the p urpose of the Delta N-1 calculation, the solvent accessibility in the unfol ded state is modelled on the basis of compact fragments extracted from fold ed protein structures (T. P. Creamer, R. Srinivasan and G. D. Rose, Biochem istry, 1997, 36, 2832). An analysis in terms of the two-domain (local-bulk) solvent model shows that the low values found for Delta Gamma(21) could be the result of the entrance of rather small amounts of cosolvent in the loc al domain of the native and/or the unfolded protein. In general, the two-do main model suggests that even a weak protein-cosolvent interaction may sign ificantly distort the membrane-free, osmotic stress estimates of the number of water molecules involved in protein conformational changes.