The major transition state in folding need not involve the immobilization of side chains

During protein folding in which few, if any, definable kinetic intermediate s are observable, the nature of the transition state is central to understa nding the course of the reaction. Current experimental data does not distin guish the relative contributions of side chain immobilization and dehydrati on phenomena to the major rate-limiting transition state whereas this disti nction is central to theoretical models that attempt to simulate the behavi or of proteins during folding. Renaturation of the small proteinase inhibit or cystatin under oxidizing versus reducing conditions is the first experim ental case in which these processes can be studied independently. Using thi s example, we show that sidechain immobilization occurs downstream of the m ajor folding transition state. A consequence of this is the existence of st ates with disordered side chains, which are distinct from kinetic protein f olding intermediates and which lie within the folded state free energy well .