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Abstract

The chaperonin GroEL, a cylindrical complex consisting of two stacked heptameric rings, and its lid-like cofactor GroES form a nano-cage in which a single polypeptide chain is transiently enclosed and allowed to fold unimpaired by aggregation. GroEL and GroES undergo an ATP-regulated interaction cycle that serves to close and open the folding cage. Recent reports suggest that the presence of non-native substrate protein alters the GroEL/ES reaction by shifting it from asymmetric to symmetric complexes. In the asymmetric reaction mode, only one ring of GroEL is GroES bound and the two rings function sequentially, coupled by negative allostery. In the symmetric mode, both GroEL rings are GroES bound and are folding active simultaneously. Here, we find that the results of assays based on fluorescence resonance energy transfer recently used to quantify symmetric complexes depend strongly on the fluorophore pair used. We therefore developed a novel assay based on fluorescence cross-correlation spectroscopy to accurately measure GroEL:GroES stoichiometry. This assay avoids fluorophore labeling of GroEL and the use of GroEL cysteine mutants. Our results show that symmetric GroEL:GroES(2) complexes are substantially populated only in the presence of non-foldable model proteins, such as alpha-lactalbumin and a-casein, which "over-stimulate" the GroEL ATPase and uncouple the negative GroEL inter-ring allostery. In contrast, asymmetric complexes are dominant both in the absence of substrate and in the presence of foldable substrate proteins. Moreover, uncoupling of the GroEL rings and formation of symmetric GroEL:GroES2 complexes is suppressed at physiological ATP:ADP concentration. We conclude that the asymmetric GroEL:GroES complex represents the main folding active form of the chaperonin. (C) 2015 Elsevier Ltd. All rights reserved.