INAUGURAL ARTICLE by a Recently Elected Academy Member:Intrinsic unfoldase/foldase activity of the chaperonin GroEL directly demonstrated using multinuclear relaxation-based NMR

The prototypical chaperonin GroEL assists protein folding through an ATP-dependent encapsulation mechanism. The details of how GroEL folds proteins remain elusive, particularly because encapsulation is not an absolute requirement for successful re/folding. Here we make use of a metastable model protein substrate, comprising a triple mutant of Fyn SH3, to directly demonstrate, by simultaneous analysis of three complementary NMR-based relaxation experiments (lifetime line broadening, dark state exchange saturation transfer, and Carr–Purcell–Meinboom–Gill relaxation dispersion), that apo GroEL accelerates the overall interconversion rate between the native state and a well-defined folding intermediate by about 20-fold, under conditions where the “invisible” GroEL-bound states have occupancies below 1%. This is largely achieved through a 500-fold acceleration in the folded-to-intermediate transition of the protein substrate. Catalysis is modulated by a kinetic deuterium isotope effect that reduces the overall interconversion rate between the GroEL-bound species by about 3-fold, indicative of a significant hydrophobic contribution. The location of the GroEL binding site on the folding intermediate, mapped from ¹⁵N, ¹H_N, and ¹³C_methyl relaxation dispersion experiments, is composed of a prominent, surface-exposed hydrophobic patch.Chaperone networks have evolved to correctly fold native proteins and protect against the damaging effects of misfolding and aggregation on protein homeostasis (1, 2). The chaperonins, a ubiquitous subclass of chaperones, are barrel-shaped, multisubunit assemblies composed of two ring cavities, transiently capped by either an extrinsic cochaperone or a built-in lid domain, which assist protein folding in an ATP-dependent manner (3–6). Although the encapsulation mechanism and accompanying allosteric transitions driven by ATP have been extensively studied, the details of how chaperonins fold proteins remain elusive (3, 6, 7). Further, encapsulation does not appear to be an absolute requirement for successful re/folding (8). Moreover, hydrogen/deuterium exchange experiments on several protein substrates (9–12) and fluorescence-based refolding experiments (13) suggest that the prototypical chaperonin GroEL may possess intrinsic unfoldase activity. Here we take advantage of a monomeric, nonaggregating, well-defined system—a triple mutant of the Fyn SH3 domain that exists in dynamic equilibrium between the major native state and a sparsely populated folding intermediate (14, 15)—to directly demonstrate, using NMR relaxation-based methods (16), the ability of apo GroEL to accelerate the interconversion between these two states by almost three orders of magnitude. Simultaneous analysis of lifetime line-broadening (17), dark state exchange saturation transfer (DEST) (18), and Carr–Purcell–Meinboom–Gill (CPMG) relaxation dispersion (19) data permitted us to determine the catalytic rate constants and ascertain the location of the GroEL binding site on the folding intermediate under conditions where the population of GroEL-bound native and intermediate states is less than 1%. Further, GroEL unfoldase/foldase activity is modulated by SH3 deuteration, indicating that catalysis of the exchange reaction between folded and intermediate states involves direct interaction of the substrate with the walls of the GroEL chambers. These results provide a basis for how chaperonins, in the absence of cofactors and encapsulation, may be able to passively protect the cell from the deleterious effects of misfolded protein accumulation.