Abstract
Many essential proteins cannot fold without help from chaperonins, like the GroELS system of Escherichia coli. How chaperonins accelerate protein folding remains controversial. Here we test key predictions of both passive and active models of GroELS-stimulated folding, using the endogenous E. coli metalloprotease PepQ. While GroELS increases the folding rate of PepQ by over 15-fold, we demonstrate that slow spontaneous folding of PepQ is not caused by aggregation. Fluorescence measurements suggest that, when folding inside the GroEL-GroES cavity, PepQ populates conformations not observed during spontaneous folding in free solution. Using cryo-electron microscopy, we show that the GroEL C-termini make physical contact with the PepQ folding intermediate and help retain it deep within the GroEL cavity, resulting in reduced compactness of the PepQ monomer. Our findings strongly support an active model of chaperonin-mediated protein folding, where partial unfolding of misfolded intermediates plays a key role.
Highlights
Many essential proteins cannot fold without help from chaperonins, like the GroELS system of Escherichia coli
Formation of the GroELGroES complex first requires that a GroEL ring bind ATP, which triggers a series of conformational rearrangements of the GroEL ring, permitting GroES to bind and resulting in the encapsulation of the substrate protein
In the presence of the cycling GroEL-GroES system, PepQ folds with an observed half-time of B1 min to a final yield of 80–90% (Fig. 1b)
Summary
Many essential proteins cannot fold without help from chaperonins, like the GroELS system of Escherichia coli. GroELS stimulates protein folding because these kinetically trapped intermediates benefit from protection against aggregation and from additional, and essential, corrective actions provided by the chaperonin[3,16] The mechanism of this corrective action remains controversial, but has been suggested to come from either (1) repetitive unfolding and iterative annealing[17,18] or (2) smoothing of a substrate protein’s free energy landscape as a result of confinement inside the GroEL-GroES cavity, where either steric constraints and/or interactions within the chamber prevent unproductive folding pathways in favour of productive ones[3,15,16]. Subsequent encapsulation of the partially unfolded folding PepQ monomer within the GroEL/ES chamber fundamentally alters the folding trajectory of the protein, resulting in a faster and more efficient search for the native state
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