Non-additive effects of denaturing and protective cosolvents on protein stability

The conformational stability of the Trpcage protein, in the presence of pure and mixed solutions of two denaturants, urea and guanidinium chloride (GdmCl), and one protective osmolyte, trimethylamine N-oxide (TMAO), are studied using enhanced-sampling all-atomistic molecular dynamics simulations. We find that the pure and the mixed solutions of urea and GdmCl denature Trpcage as a whole, but remarkably, the helical segment ¹NLYIQWL⁷ of Trpcage is stabilized in mixed GdmCl-urea solutions. For this helical segment, we find that urea "over solvates" the peptide backbone by reorganizing water molecules from the peptide side chains to the peptide backbone and GdmCl strongly dehydrates the side chains. The effects of urea and GdmCl on the solvation structure of the peptide are non-additive and urea depletes Gdm⁺ from the surface of the peptide in mixed urea-GdmCl solutions. An intricate thermodynamic balance between these non-additive effects stabilizes the helix in mixed urea-GdmCl solutions. Interestingly, we find that the protective osmolyte TMAO also depletes Gdm⁺ from the peptide-surface in mixed TMAO-GdmCl solutions and the mixture of TMAO and GdmCl is less effective than GdmCl solutions in counteracting the urea-denaturation of the helix.