2,2,2-Trifluoroethanol (TFE) is a well-known protein α-helix stabilizer; nevertheless, after much investigation, no consensus has been reached on TFE’s stabilizing mechanism. TFE alters the structure of water and affects its dielectric properties, but also competes with it for hydrogen bonds with the backbone and side chains of proteins. Thus, indirect and direct mechanisms of TFE activity have been proposed. The direct mode is especially appealing: TFE establishes hydrogen bonds with the carbonyls of the peptides’ backbones, eliminating water, apparently protecting the intra-helix hydrogen bond. Because these interactions occur simultaneously with other changes in the solution structure, it is difficult to disentangle the contribution of direct vs. indirect processes to the TFE stabilizing effect. Here, we perform extensive enhanced sampling simulations of the (AAQAA)3 peptide in mixtures of water and TFE at various concentrations. Minimum-distance distribution functions (MDDFs) and the Kirkwood-Buff (KB) theory of solutions are used to understand the molecular and thermodynamic basis of the TFE mechanism of α-helix stabilization. The simulations confirm the stabilizing role of TFE on the helical content of the peptide and that the helical structures are preferentially solvated by TFE. TFE effectively interacts with the protein backbone, excluding water, in agreement with the direct-interaction model. Yet, simulations allow alchemical experiments to be performed, and thus we modified the intermolecular backbone-TFE interactions to prevent the putatively stabilizing hydrogen-bond. Surprisingly, the peptide’s helical content increased, showing that these direct contacts have a denaturing effective contribution. At the same time, the preferential interaction parameters remain basically constant in the absence of the TFE-backbone hydrogen bonds. Therefore, the model of TFE helix stabilization based on the protection of backbone hydrogen bonds by hydrogen-bonding the backbone’s carbonyl is not supported by evidence. We show that TFE non-specific interactions with the helical conformations are stronger than with the coil states, excluding water.
A. F. Pereira, V. Piccoli, L. Martínez, Trifluoroethanol direct interactions with protein backbones destabilize alpha-helices J. Mol. Liq. 365 (2022) 120209.