Insights into the stability of native and partially folded states of ubiquitin: Effects of cosolvents and denaturants on the thermodynamics of protein folding

The thermodynamics of the native <---->A state and native-unfolded transitions for ubiquitin have been characterized in detail using the denaturants methanol and guanidinium chloride (Gdn.HCl) both separately and in combination. Gdn.HCl destabilizes the partially folded alcohol-induced A state such that the effects of alcoholic solvents on the native-unfolded transition can be investigated directly via a two-state model. The combined denaturing effects of methanol and Gdn.HCl appear to conform to a simple additive model. We show that ubiquitin folds and unfolds cooperatively in all cases, forming the same "native" state; however, the thermodynamics of the N <---->U transition change dramatically between alcoholic and Gdn.HCl solutions, with folding in aqueous methanol associated with large negative enthalpy and entropy terms at 298 K with a gradual falloff in Delta Cp at higher methanol concentrations, as previously reported for the N <---->A transition (Woolfson, D. N., Cooper, A., Harding, M. M., Williams, D. H., and Evans, P. A. (1993) J. Mol. Biol. 229, 502-511.). Both the N <---->U and the N <---->A transitions are enthalpy driven to a similar extent. We conclude that under these conditions van der Waals interactions in the packing of the nonpolar protein core, which is common to both the N <---->U and the N <---->A transitions, appear to drive folding in the absence of entropic effects associated with release of ordered solvent (hydrophobic effect). Solvent transfer studies of hydrocarbons into alcoholic solvents, with and without Gdn.HCl, are consistent with a large enthalpic driving force for burial of a nonpolar surface, with a linear dependence of protein stability (DeltaG(N <---->U)) on cosolvent concentration reflected in a similar linear dependence of hydrocarbon solubility. The data demonstrate that the hydrophobic effect is not a prerequisite for specific stabilization of the native state or the A state and that van der Waals packing of the nonpolar core appears to be the dominant factor in stabilization of the native state.