TMAO: Protecting Proteins from Feeling the Heat

Abstract

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 unlike the case of other osmolytes the ability of TMAO to protect proteins from high temperature has not been quantified. To reveal the effect of TMAO on folded and unfolded protein ensembles and the hydration shell at different temperatures, we study a mutant of the well-characterized, fast-folding model protein B (PRB). We carried out >190 µs in total all-atom simulations of thermal folding/unfolding of PRB at multiple temperatures and concentrations of TMAO. The simulations show increased thermal stability of PRB in presence of TMAO. Partly structured, compact ensembles are favored over the unfolded state. TMAO forms two shells near the protein: an outer shell away from the protein surface alters hydrogen bond lifetimes of water molecules and increases hydration of the protein to help stabilize it; a less-populated inner shell with opposite TMAO orientation closer to the protein surface binds exclusively to basic side chains. The cooperative co-solute effect of the inner and outer shell TMAO has a small number of TMAO molecules ‘herding’ water molecules into two hydration shells at or near the protein surface. The stabilizing effect of TMAO on our protein saturates at 1 M despite higher TMAO solubility, so there may be little evolutionary pressure for extremophiles to produce higher intracellular TMAO concentrations if true in general.