Kinetics of unfolding and folding from amide hydrogen exchange in native ubiquitin

Amide hydrogen (NH) exchange is one of the few experimental techniques with the potential for determining the thermodynamics and kinetics of conformat ional motions at nearly every residue in native proteins. Quantitative inte rpretation of NH exchange in terms of molecular motions relies on a simple two-state kinetic model: at any given slowly exchanging NH, a closed or exc hange-incompetent conformation is in equilibrium with an open or exchange-c ompetent conformation. Previous studies have demonstrated the accuracy of t his model in measuring conformational equilibria by comparing exchange data with the thermodynamics of protein unfolding. We report here a test of the accuracy of the model in determining the kinetics of conformational change s in native proteins. The kinetics of folding and unfolding for ubiquitin h ave been measured by conventional methods and compared with those derived f rom a comprehensive analysis of the pH dependence of exchange in native ubi quitin. Rate constants for folding and unfolding from these two very differ ent types of experiments show good agreement. The simple model for NH excha nge thus appears to be a robust framework for obtaining quantitative inform ation about molecular motions in native proteins.