Interactions of Urea with the Folded and Unfolded States of Proteins

Abstract

Urea is a strong protein denaturant, yet, the mechanism of action of urea on the protein unfolding process is still largely unknown. To tackle this problem, we determined the partial molar volumes, Vo, and adiabatic compressibilities, KoS, of a set of model proteins to examine their interactions with urea. Specifically, we measured the partial molar volume and adiabatic compressibility of ribonuclease A, α-chymotrypsinogen A, lysozyme, and apocytochrome c in aqueous solutions of urea at concentrations between 0 and 8M. At pH 2 and pH3, ribonuclease A and α-chymotrypsinogen A, respectively, exhibit a two-state transition of unfolding over the range of urea concentrations studied. Even in 8M urea, lysozyme retains its native conformation and apocytochrome c remains unfolded at pH 7. The fact that lysozyme and apocytochrome c do not undergo any conformational transitions in the presence of urea provides an opportunity to study the interactions of urea with proteins in the native and unfolded states within the entire range of experimentally accessible urea concentrations. We analyze our resulting volumetric data within the framework of a statistical thermodynamic model in which each instance of urea interaction with a protein is viewed as a binding reaction that is accompanied by release of two water molecules. From this analysis, we calculate the association constants, k, as well as changes in volume, ΔVO, and adiabatic compressibility, ΔKSO, accompanying each urea-protein association event in an ideal solution. By comparing these parameters with similar characteristics determined for low-molecular weight analogues of proteins, we quantify the extent of cooperative effects involved in interactions of urea with any of the proteins studied in this work.