The development of tertiary interactions during the folding of a large protein.

We have used protein engineering and relaxation kinetics to examine the order in which secondary structure elements assemble during folding. Aliphatic contacts in the core of a large domain within the monomeric protein phosphoglycerate kinase (PGK) were disrupted in order to map the development of interactions between beta-strand and alpha-helix residues, both near and distant in the sequence. Mutations which break sequence-local alpha-beta contacts destabilize the first identifiable intermediate in folding, showing that these contacts develop early in the folding pathway. In contrast, the removal of sequence-distant alpha-beta interactions has little effect at this stage, but reduces the rate at which the intermediate converts to the native state. Thus, contacts between these remote segments of secondary structure start to form later on in the process, during the rate-limiting transition. In the case of this large protein domain, our results support the hypothesis that folding proceeds by a hierarchic pathway. Interactions form rapidly between sequence-local groups to produce microdomains before the establishment of the long-range contacts necessary to define the global fold, which proceeds through a highly hydrated transition state.