Multiple structural transitions of the GroEL subunit are sensitive to intermolecular interactions with cochaperonin and refolding polypeptide.

In this study we attempted to determine the specific roles of the numerous conformational changes that are observed in the bacterial chaperonin GroEL, by performing stopped-flow experiments on GroEL R231W in the presence of a refolding substrate protein. The apparent rate of one kinetic phase was decreased by approximately 25% in the presence of prebound unfolded malate dehydrogenase while another phase was suppressed completely under the same conditions, reflecting different effects of the unfolded protein on multiple structural transitions within GroEL. The addition of cochaperonin GroES counteracts the effect of the bound substrate protein in the former case, but had no effect on the latter, more extensive suppression. Using a chemically modified form of GroEL R231W which is incapable of releasing substrate proteins at low temperatures, we identified a conformational transition that is implicated in the release of substrate proteins. Parts of the actual process of substrate protein release were also observed through fluorescence resonance energy transfer experiments involving GroEL and labeled substrate protein. Analysis of the energy transfer data revealed an interesting relationship between substrate protein displacement and a specific structural transition in the GroEL apical domain.