Effect of solute-solvent interactions on protein stability and ligand binding

My doctoral thesis is aimed at characterizing the effect of solute–solvent interactions on protein stability and ligand binding events. To quantify non-specific protein–solvent interactions, we measured and analyzed partial molar volume and compressibility of proteins within a framework of statistical thermodynamic formalisms. We characterized the binding of tri-N-acetylglucosamine [(GlcNAc)3] and cAMP to lysozyme and cAMP-binding domain of EPAC1, respectively. Our volumetric measurements reveal that the protein–ligand complexes are less dynamic compared to their unbound states while complex formation is accompanied by the release of water of hydration to the bulk. To characterize protein–urea interactions, we determined and analyzed volumetric properties of four globular proteins at urea concentrations ranging from 0 to 8 M. We interpreted urea-dependent volumetric measurements in terms of the solvent exchange model in which the binding of urea to a protein proceeds with a release of two waters of hydration. Comparison of urea-dependent volumetric data for a folded protein with the similar data obtained on small molecules mimicking protein groups suggests the lack of cooperative effects involved in protein–urea interactions. Changes in volumetric properties associated with urea-induced unfolding transitions are consistent with solvent-accessible surface areas of unfolded proteins being roughly twice as large as those of their respective native states. Specifically, we reported the first experimental characterization of the thermodynamic profile of urea binding to a native protein. We measured the volumetric parameters of lysozyme as a function of urea within a temperature range of 18 to 45 ◦C. Based on the van’t Hoff analysis of the temperature dependence of the equilibrium constants for protein–urea binding events we evaluated the full thermodynamic profile of protein–urea interactions. Finally, we combined the experimental and theoretical approaches to investigate the influence of the urea on the binding of (GlcNAc)3 to lysozyme. Analysis of urea dependence of the binding free energy reveals that the protein–ligand binding reaction is governed by a close interplay between the free energy contributions of the excluded volume effect and direct solute–solvent interactions.Ph.D