Mechanism of GroEL/GroES assisted protein folding

Chaperonins, such as E. coli GroEL, are large oligomeric ring structures that assist protein folding. Both in vitro and in vivo, GroEL and its cochaperonin GroES are responsible for the folding of a broad spectrum of unrelated proteins. We investigated the mechanism by which GroEL assists the folding of these substrates.; Biochemical analyses indicate that GroEL undergoes cycles of substrate binding to its apical domains, encapsulation within its central cavity, and release into the solution. Studies of GroEL “minichaperones” containing only the apical substrate binding subdomain have questioned the functional importance of substrate encapsulation within GroEL-GroES complexes. Minichaperones were reported to assist folding despite the fact that they are monomeric and therefore cannot form a central cavity. We compared directly the folding activity of minichaperones with that of the full GroEL-GroES system. Our result argue that the full GroEL folding activity requires the intact GroEL-GroES complex, and in light of previous studies, underscore the importance of substrate encapsulation for providing a folding environment distinct from the bulk solution.; To understand the basis and limits of GroEL's substrate specificity, we have established an in vivo screen that allows us to select for GroEL/GroES mutants with enhanced abilities to promote folding of a specific substrate, green fluorescence protein (GFP). We identified mutant chaperonins that strongly improves the folding efficiency of GFP. This enhanced ability to chaperone GFP folding comes at the expense of the ability of the mutants to assist folding of other substrates, implying that GroEL is evolutionarily tailored to be a general chaperone for many substrates, instead of being optimized for specific proteins. The substrate-optimized chaperonins provide useful reagent for dissecting the mechanism of chaperonin-mediated protein folding. Finally, these substrate-optimized mutants may also have practical applications for the improved production of active proteins.