Urea Interactions with Native and Unfolded Proteins: A Volumetric Study

Abstract

We describe a statistical thermodynamics-based approach to analyzing urea-dependent volumetric properties of proteins. The analysis produces the thermodynamic properties of elementary reactions in which a urea molecule interacts with a protein while also yielding an estimate of the effective solvent-accessible surface areas of the native and unfolded protein states. We carried out high precision measurements of the partial molar volume and adiabatic compressibility of lysozyme, apocytochrome c, ribonuclease A, and α-chymotrypsinogen A at 25°C as a function of urea. The resulting volumetric data were analyzed within the framework of a statistical thermodynamic formalism. Lysozyme remains folded, while apocytochrome c is unfolded between 0 and 8 M urea. In contrast, ribonuclease A and α-chymotrypsinogen A exhibit urea-induced unfolding transitions. Thus, our data permit us to characterize urea-protein interactions in both the native and unfolded states. We interpreted the urea-dependent volumetric properties of urea in terms of equilibrium constant, k, and changes in volume, ΔV0, and compressibility, ΔKT0, for an elementary reaction of urea binding to protein with a concomitant release of two water molecules from its hydration shell. Comparison of the values of k, ΔV0, and ΔKT0 with the similar data obtained for small molecules mimicking protein groups reveals the lack of cooperative effects involved in urea-protein interactions. Urea-dependent volumetric data enable one to evaluate the extent of solvent exposure of protein groups in both the native and unfolded states. We emphasize that the volumetric approach offers a practical way for evaluating the effective solvent accessible surface area of biologically significant fully or partially unfolded polypeptides.