Interactions of Urea with Native and Unfolded Proteins: A Volumetric Study

We describe a statistical thermodynamic approach to analyzing urea-dependent volumetric properties of proteins. We use this approach to analyze our urea-dependent data on the partial molar volume and adiabatic compressibility of lysozyme, apocytochrome <i>c</i>, ribonuclease A, and α-chymotrypsinogen A. The analysis produces the thermodynamic properties of elementary urea–protein association reactions while also yielding estimates of the effective solvent-accessible surface areas of the native and unfolded protein states. Lysozyme and apocytochrome <i>c</i> do not undergo urea-induced transitions. The former remains folded, while the latter 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 the proteins in terms of the equilibrium constant, <i>k</i>, and changes in volume, Δ<i>V</i>₀, and compressibility, Δ<i>K</i>_T0, for a reaction in which urea binds to a protein with a concomitant release of two waters of hydration to the bulk. Comparison of the values of <i>k</i>, Δ<i>V</i>₀, and Δ<i>K</i>_T0 with the similar data obtained on small molecules mimicking protein groups reveals lack of cooperative effects involved in urea–protein interactions. In general, the volumetric approach, while providing a unique characterization of cosolvent–protein interactions, offers a practical way for evaluating the effective solvent accessible surface area of biologically significant fully or partially unfolded polypeptides