Trimethylamine N -oxide stabilizes proteins via a distinct mechanism compared with betaine and glycine

Abstract

We report experimental and computational studies investigating the effects of three osmolytes, trimethylamine N-oxide (TMAO), betaine, and glycine, on the hydrophobic collapse of an elastin-like polypeptide (ELP). All three osmolytes stabilize collapsed conformations of the ELP and reduce the lower critical solution temperature (LSCT) linearly with osmolyte concentration. As expected from conventional preferential solvation arguments, betaine and glycine both increase the surface tension at the air-water interface. TMAO, however, reduces the surface tension. Atomically detailed molecular dynamics (MD) simulations suggest that TMAO also slightly accumulates at the polymer-water interface, whereas glycine and betaine are strongly depleted. To investigate alternative mechanisms for osmolyte effects, we performed FTIR experiments that characterized the impact of each cosolvent on the bulk water structure. These experiments showed that TMAO red-shifts the OH stretch of the IR spectrum via a mechanism that was very sensitive to the protonation state of the NO moiety. Glycine also caused a red shift in the OH stretch region, whereas betaine minimally impacted this region. Thus, the effects of osmolytes on the OH spectrum appear uncorrelated with their effects upon hydrophobic collapse. Similarly, MD simulations suggested that TMAO disrupts the water structure to the least extent, whereas glycine exerts the greatest influence on the water structure. These results suggest that TMAO stabilizes collapsed conformations via a mechanism that is distinct from glycine and betaine. In particular, we propose that TMAO stabilizes proteins by acting as a surfactant for the heterogeneous surfaces of folded proteins.osmolytes | protein folding | mechanism | spectroscopy | MD simulations M any organisms use small organic osmolytes to stabilize proteins in harsh environments, such as when the salinity is highly variable (1). In particular, trimethylamine N-oxide (TMAO) is known to counteract the denaturing effects of urea as well as salts, and it is present at high concentrations in some aquatic organisms (2). Its effects are often compared with the ions on the left side of the Hofmeister series, which help stabilize the native, folded structures of proteins (Fig. 1).Because of their fundamental biophysical importance, many studies have investigated the behavior and effects of osmolytes. In particular, Timasheff and coworkers (3, 4) proposed that osmolyte effects result from the relative partitioning of these molecules between the bulk solution and the protein-water interface. Stabilization should occur when osmolytes are depleted from the protein-water interface, but proteins will unfold when osmolytes accumulate at this interface. Accordingly, osmolyte effects are often interpreted in terms of an effective protein-water "surface tension." In fact, despite the significant differences between protein surfaces and air-water interfaces, osmolyte effects are often, although not always, consistent with their effect on the air-water interfa...

Cosolutes, Crowding, and Protein Folding Kinetics

“…Recent studies indicate stabilization mechanisms may vary between stabilizing osmolytes, emphasizing the need for a kinetic investigation. 93,94 …”

Unifying the Ambivalent Mechanisms of Protein-Stabilising Osmolytes

“…Over the years, this model of preferential exclusion of osmolytes from protein surface has received extensive supports in multiple investigations. [8][9][10][11][12][13][14] However, some recent investigations [15][16][17][18] involving the quintessential osmolyte TMAO are beginning to hint at its more complex and heterogenous molecular picture of the proteinstabilizing role, showing signs of deviation from the tenets of popular osmophobic model.…”

A generalized purification step for viral particles using mannitol flocculation

“…Denaturing osmolytes, like urea, are well‐known for unfolding proteins. It is hypothesized that protecting osmolytes are depleted at the protein surface, with a possible exception of trimethyl amine N ‐oxide (TMAO). Osmolytes in hydrophobic polymer solutions have been shown to stabilize the collapsed state .…”

“…It is hypothesized that protecting osmolytes are depleted at the protein surface, with a possible exception of trimethyl amine N ‐oxide (TMAO). Osmolytes in hydrophobic polymer solutions have been shown to stabilize the collapsed state . Denaturing osmolytes prefer the elongated polymer state and denaturing osmolytes accumulate on the protein surface .…”

“…Osmolytes in hydrophobic polymer solutions have been shown to stabilize the collapsed state. 19,20 Denaturing osmolytes prefer the elongated polymer state 20 and denaturing osmolytes accumulate on the protein surface. 18 Osmolytes also have an interesting effect on surface charge.…”