High-energy channeling in protein folding.

Recent controversy about the role of populated intermediates in protein folding emphasizes the need to better characterize other events on the folding pathway. A complication is that these involve high-energy states which are difficult to target experimentally since they do not accumulate kinetically. Here, we explore the energetics of high-energy states and map out the shape of the free-energy profile for folding of the two-state protein U1A. The analysis is based on nonlinearities in the GdnHCl dependence of the activation energy for unfolding, which we interpret in terms of structural changes of the protein-folding transition state. The result suggests that U1A folds by high-energy channeling where most of the conformational search takes place isoenergetically at transition-state level. This is manifested in a very broad and flat activation barrier, the top of which covers more than 60% of the reaction coordinate. The interpretation favors a folding mechanism where the pathway leading to the native protein is determined by the sequence's ability to stabilize productive transition states.