| Abstrakt: |
We have characterized the unfolding energetics of ribonuclease a and hen egg white lysozyme as a function of temperature, pH and concentration of several cosolvents (sucrose, glucose, glycerol, polyethyleneglycol 8000) that are expected to be preferentially excluded from the surface of native proteins, and we have calculated the corresponding unfolding changes in preferential hydration (ΔΓ21) at 25°C. We find no significant cosolvent concentration effect on the unfolding enthalpy and heat capacity values, which suggests that the cosolvents do not interact strongly with the proteins at the comparatively low cosolvent concentrations employed in this work. In spite of this, the ΔΓ21 values are significantly smaller than theoretical estimates of the unfolding change in the number of water molecules corresponding to first monolayer coverage (ΔN1), even when, for the purpose of the ΔN1 calculation, the solvent accessibility in the unfolded state is modelled on the basis of compact fragments extracted from folded protein structures (T. P. Creamer, R. Srinivasan and G. D. Rose, Biochemistry, 1997, 36, 2832). An analysis in terms of the two-domain (local-bulk) solvent model shows that the low values found for ΔΓ21 could be the result of the entrance of rather small amounts of cosolvent in the local domain of the native and/or the unfolded protein. In general, the two-domain model suggests that even a weak protein–cosolvent interaction may significantly distort the membrane-free, osmotic stress estimates of the number of water molecules involved in protein conformational changes. |
