Osmolytes affect hydrophobic collapse and protein folding equilibria. The underlying mechanisms are, however, not well understood. We report large-scale conformational sampling of two hydrophobic polymers with secondary and tertiary amide side chains using extensive molecular dynamics simulations. The calculated free energy of unfolding increases with urea for the secondary amide, yet decreases for the tertiary amide, in agreement with experiment. The underlying mechanism is rooted in opposing entropic driving forces: while urea screens the hydrophobic macromolecular interface and drives unfolding of the tertiary amide, urea's concomitant loss in configurational entropy drives collapse of the secondary amide. Only at sufficiently high urea concentrations bivalent urea hydrogen bonding interactions with the secondary amide lead to further stabilisation of its collapsed state. The observations provide a new angle on the interplay between side chain chemistry, urea hydrogen bonding, and the role of urea in attenuating or strengthening the hydrophobic effect.

The osmolyte trimethylamine-N-oxide (TMAO) is able to increase the thermodynamic stability of folded proteins, counteracting pressure denaturation. Herein, we report experimental solubility data on penta-alanine (pAla) in aqueous TMAO solutions (at pH = 7 and pH = 13) together with molecular simulation data for pAla, penta-serine (pSer), and an elastin-like peptide (ELP) sequence (VPGVG) under varying pH and pressure conditions. The effect of the peptide end groups on TMAO− peptide interactions is investigated by comparing the solvation of zwitterionic and negatively charged pentamers with the solvation of pentamers with charge-neutral C-and N-termini and linear, virtually infinite, peptide chains stretched across the periodic boundaries of the simulation cell. The experiments and simulations consistently show that TMAO is net-depleted from the pAla−water interface, but local accumulation of TMAO is observed just outside the first hydration shell of the peptide. While the same observations are also made in the simulations of the zwitterionic pentamers (Ala, Ser, and ELP) and virtually infinite peptide chains (Ala and ELP), weak preferential binding of TMAO is instead observed for pAla with neutral end groups at a 1 M TMAO concentration and for an ELP pentamer with capped neutral end groups at a 0.55 M TMAO concentration studied in previous work (Y.-T. Liao et al. Proc. Natl. Acad. Sci. USA, 2017USA, , 114, 2479USA, −2484. The above observations made at 1 bar ambient pressure remain qualitatively unchanged at 500 bar and 2 kbar. Local accumulation of TMAO correlates with a reduction in the total number of peptide−solvent hydrogen bonds, independent of the peptide's primary sequence and the applied pressure. By weakening water hydrogen bonds with the protein backbone, TMAO indirectly contributes to stabilizing internal hydrogen bonds in proteins, thus providing a protein stabilization mechanism beyond net depletion.

We report the effect of trimethylamine N-oxide (TMAO) on the solvation of nonpolar solutes in water studied with molecular dynamics (MD) simulations and free-energy calculations. The simulation data indicate the...

The effect of trimethylamine-N-oxide (TMAO) on hydrophobic solvation and hydrophobic interactions of methane have been studied with Molecular Dynamics (MD) simulations in the temperature range between 280 K and 370 K at 1 bar ambient pressure. We observe a temperature transition in the effect of TMAO on the aqueous solubility of methane. At low temperature (280 K), methane is preferentially hydrated, causing TMAO to reduce its solubility in water, while above 320 K, methane preferentially interacts with TMAO, causing TMAO to promote its solubility in water. Based on a statistical-mechanical analysis of the excess chemical potential of methane, we find that the reversible work of creating a repulsive methane cavity opposes the solubility of methane in TMAO/water solution more than in pure water. Below 320 K, this solvent-excluded volume effect overcompensates the contribution of methane-TMAO van der Waals interactions, which promote the solvation of methane and are observed at all temperatures. These van der Waals interactions with the methyl groups of TMAO tip the balance above 320 K where the effect of TMAO on solvent-excluded volume is smaller. We furthermore find that the effective attraction between dissolved methane solutes increases with increasing TMAO concentration. This observation correlates with a reduction in the methane solubility below 320 K, but with an increase in methane solubility at higher temperatures.

Trimethylamine-N-oxide (TMAO) is an osmolyte known for its ability to counteract the pressure denaturation of proteins. Computational studies addressing the molecular mechanisms of TMAO’s osmolyte action have however focused exclusively on its protein-stabilizing properties at ambient pressure, neglecting the changes that may occur under high-pressure conditions where TMAO’s hydration structure changes to that of increased water binding. Here, we present the first study on the combined effect of pressure and TMAO on a mini-protein, TrpCage. The results showed that at high pressures, nonpolar residues packed less tightly and the salt bridge of TrpCage was destabilized. This effect was mitigated by TMAO which was found to be strongly depleted from the protein/water interface at 1 kbar than at 1 bar ambient pressure, thus counterbalancing the thermodynamically unfavorable effect of elevated pressure in the free energy of folding. TMAO was depleted from charged groups, like the salt bridge-forming ones, and accumulated around hydrophobic groups. Still, it stabilized both kinds of interactions. Furthermore, enthalpically favorable TrpCage–water hydrogen bonds were reduced in the presence of TMAO, causing a stronger destabilization of the unfolded state than the folded state. This shifted the protein-folding equilibrium toward the folded state. Therefore, TMAO showed stabilizing effects on different kinds of groups, which were partially enhanced at high pressures.