RESUMO
Alcohols and urea are widely used as effective protein denaturants. Among monohydric alcohols, 2,2,2-trifluoroethanol (TFE) has large cosolvent effects as a helix stabilizer in proteins. In contrast, urea efficiently denatures ordered native structures, including helices, into coils. These opposing cosolvent effects of TFE and urea are well known, even though both preferentially bind to proteins; however, the underlying molecular mechanism remains controversial. Cosolvent-dependent relative stability between native and denatured states is rigorously related to the difference in preferential binding parameters (PBPs) between these states. In this study, GCN4-p1 with two-stranded coiled coil helices was employed as a model protein, and molecular dynamics simulations for the helix dimer and isolated coil were conducted in aqueous solutions with 2 M TFE and urea. As 2 M cosolvent aqueous solutions did not exhibit clustering of cosolvent molecules, we were able to directly investigate the molecular origin of the excess PBP without considering the enhancement effect of PBPs arising from the concentration fluctuations. The calculated excess PBPs of TFE for the helices and those of urea for the coils were consistent with experimentally observed stabilization of helix by TFE and that of coil by urea. The former was caused by electrostatic interactions between TFE and side chains of the helices, while the latter was attributed to both electrostatic and dispersion interactions between urea and the main chains. Unexpectedly, reverse-micelle-like orientations of TFE molecules strengthened the electrostatic interactions between TFE and the side chains, resulting in strengthening of TFE solvation.