Osmolyte effects on water diffusion: Urea induces changes in the entropic barrier, TMAO in the energetic barrier

Osmolytes have attracted significant interest for their effects on protein structures. Prototypical examples are urea and trimethylamine N-oxide (TMAO), which destabilize and stabilize folded proteins, respectively. Whether the origin of these effects lies in direct osmolyte-protein interactions or indirect perturbations of the water solvent is still not fully clear. In this Communication, the details of the latter mechanism are investigated both theoretically and experimentally by examining how the two osmolytes influence water diffusion as a function of their concentration up to 8M. Addition of both urea and TMAO slow water diffusion, the latter more strongly. Importantly, the associated activation energies, characterized in detail here for the first time, show dramatically different driving forces underlying these behaviors: urea modestly lowers the water diffusion activation energy while TMAO strongly increases it.