Molecular Mechanism of Urea-Induced Protein Denaturation

Abstract

For more than half a century, urea has been used as a strong denaturant in protein folding/unfolding studies. However, the molecular mechanisms of urea-induced protein unfolding still remain unclear. This lack of understanding is to some extent reflects the scarcity of direct thermodynamic information that can be used to characterize interactions of urea with amino acid side chains and the peptide group. We recently demonstrated that volumetric measurements combined with statistical thermodynamic approach may represent a novel and effective way to tackle this problem [Lee, S. & Chalikian, T. V. (2009) J. Phys. Chem. B. 113, 2443-2450]. In this work, we employ high precision acoustic and densimetric techniques to quantify the solvation properties of solutes in the presence of urea. Specifically, we report the partial molar volumes, V°, and adiabatic compressibilities, KS°, of N-acetyl amino acid amides containing all 20 naturally existing amino acid side chains and oligoglycines, (Gly)1-5, at urea concentrations ranging from 0 to 8 M. Using our developed statistical thermodynamic approach, that links volumetric observables of a solute with solute-solvent and solute-cosolvent interactions in binary solvents, we evaluate the binding constants, k, and elementary changes in volume, ΔV, and compressibility, ΔKS, accompanying the replacement of water in the vicinity of the solutes with a urea molecule. While the binding constants are essentially similar for all protein groups, the magnitude and the sign of the determined values of ΔV and ΔKS vary markedly. The latter values reflect the nature of urea interactions with specific functional groups and the concomitant changes in hydration. In general, our results are consistent with a picture in which urea interacts with polar, non-polar and charged groups with comparable affinities, although the underlying forces stabilizing each type of interaction depend on the chemical nature of the interacting group.