THE DEVELOPMENT OF TERTIARY INTERACTIONS DURING THE FOLDING OF A LARGE PROTEIN

Background: We have used protein engineering and relaxation kinetics t o examine the order in which secondary structure elements assemble dur ing folding. Aliphatic contacts in the core of a large domain within t he monomeric protein phosphoglycerate kinase (PGK) were disrupted in o rder to map the development of interactions between beta-strand and al pha-helix residues, both near and distant in the sequence. Results: Mu tations which break sequence-local alpha-beta contacts destabilize the first identifiable intermediate in folding, showing that these contac ts develop early in the folding pathway. In contrast, the removal of s equence-distant alpha-beta interactions has little effect at this stag e, but reduces the rate at which the intermediate converts to the nati ve state. Thus, contacts between these remote segments of secondary st ructure start to form later on in the process, during the rate-limitin g transition, Conclusions: In the case of this large protein domain, o ur results support the hypothesis that folding proceeds by a hierarchi c pathway. interactions form rapidly between sequence-local groups to produce microdomains before the establishment of the long-range contac ts necessary to define the global fold, which proceeds through a highl y hydrated transition state. (C) Current Biology Ltd