GroEL traps dimeric and monomeric unfolding intermediates of citrate synthase

Abstract

The prokaryotic molecular chaperone GroE is increasingly expressed under heat shock conditions. GroE protects cells by preventing the irreversible aggregation of thermally unfolding proteins. Here, the interaction of GroE with thermally unfolding titrate synthase (CS) was dissected into several steps that occur before irreversible aggregation, and the conformational states of the unfolding protein recognized by GroEL were determined. The kinetic analysis of CS unfolding revealed the formation of inactive dimeric and monomeric intermediates. GroEL binds both intermediates without affecting the unfolding pathway. Furthermore, the dimeric intermediates are not protected against dissociation in the presence of GroEL. Monomeric CS is stably associated with GroEL, thus preventing further irreversible unfolding steps and subsequent aggregation. During refolding, monomeric CS is encapsulated inside the cavity of GroEL·GroES complexes. Taken together our results suggest that for protection of cells against heat stress both the ability of GroEL to interact with a large variety of nonnative conformations of proteins and the active, GroES-dependent refolding of highly unfolded species are important.