Protein Hydration and Excluded Volume Interactions in Protein Folding and Stability: On the Mechanism of Protein Stabilization by Osmolytes

Abstract

Several osmolytes ranging in size from ∼90-1600 cm3.mol-1 are shown to stabilize the folded states of apo-Mb at pH denaturing conditions. The action of these solutes is consistent with an osmotic stress effect, i.e., with their effect on the chemical potential of water modulating the equilibrium between folded and unfolded states via protein differential hydration. Moreover, the free energy of protein folding depends on solute size. We show that the apparent difference in hydration between molten globule and unfolded states of apo-Mb increases linearly with increasing solute sizes, while the free energy change of protein hydration upon folding decreases with the inverse of molar solute apparent volume. We have analyzed these size effects considering the contribution of excluded volume interactions to protein folding via Monte Carlo simulations of a self-avoiding walk chain in a cubic lattice with hardcore solutes. It is shown that solute-chain self-avoiding interaction decrease the conformational entropy of the chain in proportion to its square radius of gyration, and to the solute volume fraction occupied by the solute. The computational results translate into a difference in the chemical potential between folded and unfolded protein states that remarkably predicts the experimental influence of solute sizes and water chemical potential on the free energy change of apo-Mb refolding induced by osmolytes. Thus, this work may stablish a quantitative link between protein hydration and protein excluded-volume interactions and their effect on the energetic of protein folding.