A surprisingly simple three-state generic process for reversible protein denaturation by trifluoroethanol.

Despite the rich knowledge of the influence of 2,2,2-trifluoroethanol (TFE) on the structure and conformation of peptides and proteins, the mode(s) of TFE-protein interactions and the mechanism by which TFE reversibly denatures a globular protein remain elusive. This study systematically examines TFE-induced equilibrium transition curves for six paradigmatic globular proteins by using basic fluorescence and circular dichroism measurements under neutral pH conditions. The results are remarkably simple. Low TFE invariably unfolds the tertiary structure of all proteins to produce the obligate intermediate (I) which retains nearly all of native-state secondary structure, but enables the formation of extra α-helices as the level of TFE is raised higher. Inspection of the transitions at once reveals that the tertiary structure unfolding is always a distinct process, necessitating the inclusion of at least one obligate intermediate in the TFE-induced protein denaturation. It appears that the intermediate in the minimal unfolding mechanism N⇌I⇌D somehow acquires higher α-helical propensity to generate α-helices in excess of that in the native state to produce the denatured state (D), also called the TFE state. The low TFE-populated intermediate I may be called a universal intermediate by virtue of its α-helical propensity. Contrary to many earlier suggestions, this study dismisses molten globule (MG)-like attribute of I or D.