MOVEMENT OF THE POSITION OF THE TRANSITION-STATE IN PROTEIN-FOLDING

Hammond behavior, in which two neighboring states move closer to each other along the reaction coordinate as the energy difference between t hem becomes smaller, has previously been observed for the transition s tate of unfolding of barnase. Here, we report Hammond behavior for the small protein chymotrypsin inhibitor 2 (CI2), which folds and unfolds via a single rate-determining transition state and simple two-state k inetics. Mutants have been generated along the entire sequence of the protein and the kinetics of folding and unfolding measured as a functi on of concentration of denaturant. The transition state was found to m ove progressively closer to the folded state on destabilization of the protein by mutation. Different regions of CI2 all show a similar sens itivity to changes in the energy of the transition state. This is in c ontrast to the behavior of barnase on mutation for which the position of the transition state for its unfolding is sensitive to mutation in some regions, especially in its major alpha-helix, but not in others. The transition state for the folding and unfolding of CI2 resembles an expanded version of the folded state and is formed in a concerted man ner, in contrast to that for barnase, in which some regions of structu re are fully formed and others fully unfolded. The reason for the gene ral sensitivity of the position of the transition state of CI2 to muta tion is presumably the relatively uniform degree of structure formatio n in the transition state and the concerted nature of its formation. H ammond behavior was also observed for both CI2 and barnase when the te mperature and denaturation conditions of the unfolding reaction are al tered. For both proteins, the position of the transition state of prot ein folding is more sensitive to changes in energy than is observed fo r the changes of covalent bonds in organic chemistry and enzyme cataly sis. The finding of Hammond behavior for two proteins suggests that it is a general phenomenon in protein folding. The movement of the trans ition state on changing the conditions does have implications in the i nterpretation of computer simulations of protein folding done under ex treme conditions.