Mechanisms of protein stabilization by osmolytes: The transfer model, surface tension theory, and excluded volume

This dissertation provides a quantitative test of the mechanisms proposed to be responsible for the stabilization of proteins by naturally occurring osmolytes. The accumulation of small organic molecules, osmolytes, is one strategy of biochemical adaptation that alters the physiological state of the cellular milieu in order to counter specific environmental stresses (temperature extremes, high osmotic pressure, desiccation, the presence of intracellular urea) that challenge the survival of many organisms.; A key observation by Timasheff demonstrated that solutions of osmolytes that confer protection to macromolecular structure and function cause an enrichment of water in the vicinity of the protein surface. This enrichment of water, preferential hydration, could arise from (1) the various chemical affinities of residues on the protein surface that net a thermodynamically unfavorable interaction with the osmolyte solution, (2) an increase in the surface tension of water due to the presence of osmolytes, or (3) steric exclusion of osmolytes due to the difference in size between organic osmolytes and water molecules.; To identify which of these mechanisms are predominantly responsible for preferential hydration of protein surfaces concomitant with the stabilization of proteins by osmolytes, the Gibbs transfer free energy model, surface tension theory, and the excluded volume model are utilized to provide quantitative predictions of the experimental stability of proteins in aqueous osmolyte solution. The Transfer Model provides thermodynamic affinities of the peptide backbone and residue side chains for the osmolyte containing solution. The effect of osmolytes on the solution surface tension and a thermodynamic basis for quantifying excluded volume effects of osmolytes is also provided. The free energy predictions of these models are framed into the context of a thermodynamic cycle so that each model can be quantitatively compared on an equal basis with the experimental free energy measurements of protein denaturation and folding.; It is demonstrated that the transfer model is in remarkable agreement with the experimental protein stabilities in the presence of osmolytes and provides the best description of protein-osmolyte interactions because this model is able to describe the chemical heterogeneity of the protein surface. Surface tension theory and excluded volume view the protein surface as chemically inert and homogeneous and therefore are not able to correctly describe the interactions between proteins and osmolytes.