Equilibrium and kinetic hydrogen exchange experiments show that cytochrome c is composed of five foldon units that continually unfold and refold even under native conditions. Folding proceeds by the stepwise assembly of the foldon units rather than one amino acid at a time. The folding pathway is determined by a sequential stabilization process; previously formed foldons guide and stabilize subsequent foldons to progressively build the native protein. Four other proteins have been found to show similar behavior. These results support stepwise protein folding pathways through discrete intermediates.cytochrome c ͉ hydrogen exchange ͉ stability labeling T o understand how proteins fold to their native state, it will be necessary to define the structure of the intermediate forms that they pass through, as for any other biochemical mechanism. This effort has proven to be exceptionally difficult. Partially folded kinetic intermediates live for Ͻ1 s, and they cannot be isolated and studied by the usual structural methods. Spectroscopic observations widely used to detect fast kinetic folding events provide very little structural detail and can be misleading in respect to structure formation. Theoretical methods are not yet able to simulate the folding of sizeable proteins.These problems can be avoided by studying proteins under native conditions. Under these conditions proteins predominantly occupy their lowest free energy state (1) but they must repeatedly unfold and refold, thermodynamically cycling through all possible higherenergy forms. The high-energy forms generally exist only at minuscule levels and therefore are invisible to most methods, which perceive only the overwhelmingly populated native state. These high-energy forms can be detected by hydrogen exchange (HX) methods because the predominant native structure makes no contribution to the HX rates that one measures. Hydrogens that are protected in the native protein can exchange with solvent only when their protecting H bonds are transiently broken as the protein searches through its higher-energy forms.HX measurements provide amino acid-resolved information that can in favorable cases define the structure of partially folded intermediate forms, their equilibrium and kinetic parameters, and their interconversions. Results obtained for cytochrome c (Cyt c) show that it is composed of five subglobally cooperative unfolding͞ refolding units, called foldons, color-coded in Fig. 1. This article integrates our research and prior information for Cyt c and other proteins. All of these results consistently show that proteins at equilibrium are composed of foldon building blocks and that they fold kinetically by the stepwise assembly of their foldon units, guided by the same factors that determine the native state. Materials and MethodsWT equine Cyt c (type VI from Sigma) was further purified as required. The pseudo-WT (pWT) protein and its mutants were expressed in Escherichia coli and purified as described (2). The pWT protein has no N-terminal acetylation; also, tw...

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