How osmolytes influence hydrophobic polymer conformations: A unified view from experiment and theory

Abstract

It is currently the consensus belief that protective osmolytes such as trimethylamine N-oxide (TMAO) favor protein folding by being excluded from the vicinity of a protein, whereas denaturing osmolytes such as urea lead to protein unfolding by strongly binding to the surface. Despite there being consensus on how TMAO and urea affect proteins as a whole, very little is known as to their effects on the individual mechanisms responsible for protein structure formation, especially hydrophobic association. In the present study, we use single-molecule atomic force microscopy and molecular dynamics simulations to investigate the effects of TMAO and urea on the unfolding of the hydrophobic homopolymer polystyrene. Incorporated with interfacial energy measurements, our results show that TMAO and urea act on polystyrene as a protectant and a denaturant, respectively, while complying with Tanford-Wyman preferential binding theory. We provide a molecular explanation suggesting that TMAO molecules have a greater thermodynamic binding affinity with the collapsed conformation of polystyrene than with the extended conformation, while the reverse is true for urea molecules. Results presented here from both experiment and simulation are in line with earlier predictions on a model Lennard-Jones polymer while also demonstrating the distinction in the mechanism of osmolyte action between protein and hydrophobic polymer. This marks, to our knowledge, the first experimental observation of TMAO-induced hydrophobic collapse in a ternary aqueous system. single-molecule force spectroscopy | free energy | preferential binding | TMAO | urea

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

“…Such a result suggests that TMAO is actually enriched rather than depleted at hydrophobic interfaces. This finding is consistent with previous measurements (3,17,31), although it is perhaps puzzling given the fact that TMAO is nearly as effective as glycine at depressing the LCST of the ELP (Fig. 3A).…”

“…Interestingly, Berne and coworkers (17,18) have recently observed similar results for hydrophobic polymers, such as polystyrene and simulated polymers of uncharged Lennard-Jones beads. In particular, they suggested that TMAO accumulates at the surface of extended hydrophobic polymers but still stabilizes collapsed conformations, because TMAO accumulates to an even greater extent at the surface of the collapsed polymer (17,18).…”

“…In particular, they suggested that TMAO accumulates at the surface of extended hydrophobic polymers but still stabilizes collapsed conformations, because TMAO accumulates to an even greater extent at the surface of the collapsed polymer (17,18). However, in contrast to the purely hydrophobic polymers studied by Berne and coworkers (17,18), we have studied ELPs, which are polypeptides with backbone amide groups. In fact, glycine residues, which lack a hydrophobic side chain, account for 40% of the amino acids in the studied ELP systems.…”

“…However, they have proposed a wide array of explanations for this preferential hydration ranging from unfavorable electrostatic interactions (13,14) to "nanocrowder" effects (15, 16). By contrast, other simulations have suggested that TMAO stabilizes folded proteins via direct attractive van der Waals interactions (17,18).Several studies have investigated the effect of TMAO on the solvent. Garde and coworkers (19) concluded that TMAO has minimal impact on hydrophobic interactions, but Paul and Patey (20) suggested that TMAO actually weakens hydrophobic forces.…”

“…However, they have proposed a wide array of explanations for this preferential hydration ranging from unfavorable electrostatic interactions (13,14) to "nanocrowder" effects (15,16). By contrast, other simulations have suggested that TMAO stabilizes folded proteins via direct attractive van der Waals interactions (17,18).…”