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

Backgound: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 β-strand and α-helix residues, both near and distant in the sequence.ResultsMutations which break sequence-local α–β 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 α–β 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.ConclusionIn 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

Differential effects of co-chaperonin homologs on cpn60 oligomers

In this study, we have investigated the relationship between chaperonin/co-chaperonin binding, ATP hydrolysis, and protein refolding in heterologous chaperonin systems from bacteria, chloroplast, and mitochondria. We characterized two types of chloroplast cpn60 oligomers, ch-cpn60 composed of α and β subunits (α7β7 ch-cpn60) and one composed of all β subunits (β14 ch-cpn60). In terms of ATPase activity, the rate of ATP hydrolysis increased with protein concentration up to 60 μM, reflecting a concentration at which the oligomers are stable. At high concentrations of cpn60, all cpn10 homologs inhibited ATPase activity of α7β7 ch-cpn60. In contrast, ATPase of β14 ch-cpn60 was inhibited only by mitochondrial cpn10, supporting previous reports showing that β14 is functional only with mitochondrial cpn10 and not with other cpn10 homologs. Surprisingly, direct binding assays showed that both ch-cpn60 oligomer types bind to bacterial, mitochondrial, and chloroplast cpn10 homologs with an equal apparent affinity. Moreover, mitochondrial cpn60 binds chloroplast cpn20 with which it is not able to refold denatured proteins. Protein refolding experiments showed that in such instances, the bound protein is released in a conformation that is not able to refold. The presence of glycerol, or subsequent addition of mitochondrial cpn10, allows us to recover enzymatic activity of the substrate protein. Thus, in our systems, the formation of co-chaperonin/chaperonin complexes does not necessarily lead to protein folding. By using heterologous oligomer systems, we are able to separate the functions of binding and refolding in order to better understand the chaperonin mechanism