Enthalpy Mediated Protein Stabilization by Macromolecular Crowding

Abstract

Understanding the structure and stability of biomolecules in the cellular environment requires knowledge of their interplay with high concentrations of macromolecules, osmolytes, salts and membranes. The macromolecular crowding effect in a cell is usually described by the excluded volume theory based on hardcore repulsions [1]. However, recent studies emphasize the role of further contributions aside from a pure volume effect including enthalpic and solvent effects [2, 3]. We study cosolute effects at high molecular and macromolecular concentrations via a thermodynamic analysis of the thermal unfolding of ubiquitin in the presence of different concentrations of cosolutes (glucose, dextran, poly(ethylene glycol), potassium chloride) [4]. In contrast to the excluded volume theory, we observed enthalpic stabilization and entropic destabilization forces for all tested cosolutes. The enthalpic stabilization mechanism of ubiquitin in macromolecular polysaccharide solutions of dextran was thereby similar to the effects observed in monomeric glucose. The more hydrophobic polymer polyethylene glycol destabilizes native ubiquitin, presumably via unspecific direct interactions with the protein. Thereby, the macromolecular character of PEG and dextran - and hence its excluded volume - is not to the determining factor which influences the folding equilibrium of ubiquitin. Instead, our data suggest a key role for hydration water molecules in the interaction of proteins and cosolutes.