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submitted on 17.07.2020 and posted on 17.07.2020by Mayank Boob, Shahar Sukenik, Martin Gruebele, Taras Pogorelov
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.