TMAO: Protecting Proteins from Feeling the Heat

Osmolytes are ubiquitous in the cell and play an important role in controlling protein stability under stress. The natural osmolyte trimethylamine N-oxide (TMAO) is used by marine animals to counteract the effect of pressure denaturation at large depths. The molecular mechanism of TMAO stabilization against pressure and urea denaturation has been extensively studied, but the effect of TMAO against high temperature has not been addressed. To delineate the effect of TMAO on folded and unfolded ensembles at different temperatures, we study a mutant of the well-characterized, fastfolding model protein B (PRB). We have carried out extensive, >190 µs in total, all-atom simulations of thermal folding/unfolding of PRB at multiple temperatures and concentrations of TMAO. The simulations captured folding and unfolding events and show an increased stability of PRB in presence of TMAO. At higher TMAO concentration, intermediate ensembles are gradually more favored over the unfolded state. Quantifying TMAO-water interactions revealed that at a low concentration threshold, TMAO forms a shell near but not at the protein surface, disrupting the water network and increasing hydration of the protein to help stabilize it. Intriguingly, we found that there are intermittent interactions between TMAO and certain protein side chains with preferred TMAO orientations. Although previous studies have proposed such interactions, the long time scales we study here help to highlight the protein’s sensitivity to local environment, particularly hydration, and raise questions about how even transient interactions could couple protein stability to TMAO effects.