Rare fluctuations of native proteins sampled by equilibrium hydrogen exchange

Equilibrium hydrogen-exchange measurements are a powerful tool for investigating the structures, stabilities, and dynamics of native and nonnative states of proteins.1-4 Previous analyses of hydrogen-exchange data have provided considerable insight into such properties for a variety of proteins, but the results have generally been qualitative in nature,1,3 although more quantitative thermodynamic analyses have been proposed.4-7 Here we present a model for the exchange of amide hydrogens with solvent water and determine the structures of species populated during the rare fluctuations of the native state that are required for the exchange. The method is based on an exploration of conformational space using Monte Carlo sampling biased by experimental data.8 The data involved are the experimental protection (P) factors, which represent the ratios of the exchange rates of amide hydrogens in unstructured polypeptides to those of the same residues in the native protein.