Molecular basis of the osmolyte effect on protein stability: a lesson from the mechanical unfolding of lysozyme.

ABSTRACT

Osmolytes are a class of small organic molecules that shift the protein folding equilibrium. For this reason, they are accumulated by organisms under environmental stress and find applications in biotechnology where proteins need to be stabilized or dissolved. However, despite years of research, debate continues over the exact mechanisms underpinning the stabilizing and denaturing effect of osmolytes. Here, we simulated the mechanical denaturation of lysozyme in different solvent conditions to study the molecular mechanism by which two biologically relevant osmolytes, denaturing (urea) and stabilizing (betaine), affect the folding equilibrium. We found that urea interacts favorably with all types of residues via both hydrogen bonds and dispersion forces, and therefore accumulates in a diffuse solvation shell around the protein. This not only provides an enthalpic stabilization of the unfolded state, but also weakens the hydrophobic effect, as hydrophobic forces promote the association of urea with nonpolar residues, facilitating the unfolding. In contrast, we observed that betaine is excluded from the protein backbone and nonpolar side chains, but is accumulated near the basic residues, yielding a nonuniform distribution of betaine molecules at the protein surface. Spatially resolved solvent-protein interaction energies further suggested that betaine behaves in a ligand- rather than solvent-like manner and its exclusion from the protein surface arises mostly from the scarcity of favorable binding sites. Finally, we found that, in the presence of betaine, the reduced ability of water molecules to solvate the protein results in an additional enthalpic contribution to the betaine-induced stabilization.