Solution thermodynamic approach to analyze protein stability in aqueous solutions.

Protein thermal stability was analyzed by a solution thermodynamic approach. The small energetic differences in hydrogen-bonds (HB) among amino acid resdues and water molecules were proved to be amplified by the large number of HB involved to bring about the equilibrium shift from folding to unfolding of proteins. In aqueous solutions, water activity (A) plays a key role in protein stability. Therefore, A was precisely determined for various solutions and its relationship with solution structure was discussed. Wyman-Tanford analysis based on A showed linear regressions, without exception, between protein unfolding-ratio and A for lysozyme, ribonuclease A, and α-chymotrypsinogen A in various solutions with sugars, osmolytes, alcohols, and protein denaturant. From this linear regression, the free energy difference, ΔΔG, for a protein in a solution and in pure water, was easily obtained. Protein stability in a solution was proved to be determined by a balance between hydration and solute-binding effects to the protein and also by solution structure, which indirectly affects the hydrophobic interaction in a protein molecule. Temperature dependence of HB on protein stability suggested its interrelationship with hydrophobic interaction.