Evidence concerning rate-limiting steps in protein folding from the effects of trifluoroethanol

The refolding kinetics of 13 proteins have been studied in the presence of 2,2,2-trifluoroethanol (TFE), Low concentrations of TFE increased the foldi ng rates of all the proteins, whereas higher concentrations have the opposi te effect. The extent of deceleration of folding correlates closely with si milar effects of guanidine hydrochloride and can be related to the burial o f accessible surface area during folding. For those proteins folding in a t wo-state manner, the extent of acceleration of folding correlates closely w ith the number of local backbone hydrogen bonds in the native structure. Fo r those proteins that fold in a multistate manner, however, the extent of a cceleration is much smaller than that predicted from the data for two-state proteins. These results support the concept that for two-state proteins th e search for native-like contacts is a key aspect of the folding reaction, whereas the rate-determining steps for folding of multistate proteins are a ssociated with the reorganization of stable structure within a collapsed st ate or with the search for native-like interactions within less structured regions.