Hammond behavior versus ground state effects in protein folding: evidence for narrow free energy barriers and residual structure in unfolded states

Apparent transition state movement upon mutation or changes in solvent conditions is frequently observed in protein folding and is often interpreted in terms of Hammond behavior. This led to the conclusion that barrier regions in protein folding are broad maxima on the free energy landscape. Here, we use the concept of self-interaction and cross-interaction parameters to test experimental data of 21 well-characterized proteins for Hammond behavior. This allows us to characterize the origin of transition state movements along different reaction coordinates. Only one of the 21 proteins shows a small but coherent transition state movement in agreement with the Hammond postulate. In most proteins the structure of the transition state is insensitive to changes in protein stability. The apparent change in the position of the transition state upon mutation, which is frequently observed in phi-value analysis, is in most cases due to ground-state effects caused by structural changes in the unfolded state. This argues for significant residual structure in unfolded polypeptide chains of many proteins. Disruption of these residual interactions by mutation often leads to decreased folding rates, which implies that these interactions are still present in the transition state. The failure to detect Hammond behavior shows that the free energy barriers encountered by a folding polypeptide chain are generally rather narrow and robust maxima for all experimentally explorable reaction coordinates.