Does trifluoroethanol affect folding pathways and can it be used as a probe of structure in transition states?

Nonaqueous co-solvents, particularly 2,2,2-trifluoroethanol (TFE), have bee n used as tools to study protein folding. By analyzing FKBP12, an alpha/bet a-protein that folds with two-state kinetics, we have been able to address three key questions concerning the use of TFE. First, does TFE perturb the folding pathway? Second, can the observed changes in the rate of folding an d unfolding in TFE be attributed to a change in free energy of a single sta te? Finally, can TFE be used to infer information on secondary structure fo rmation in the transition state? Protein engineering experiments on FKBP12, coupled with folding and unfolding experiments in 0% and 9.6% TFE, conclus ively show that TFE does not perturb the folding pathway of this protein. O ur results also suggest that the changes in folding and unfolding rates obs erved in 9.6% TFE, are due to a global effect of TFE, on the protein, rathe r than the stabilization of any elements of secondary structure in the tran sition state. Thus, studies with TFE and other co-solvents can be accuratel y interpreted only when combined with other techniques.