Intrinsic unfoldase/foldase activity of the chaperonin GroEL directly demonstrated using multinuclear relaxation-based NMR

Abstract

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" GroELbound 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 15 N, 1 H N , and 13 C methyl relaxation dispersion experiments, is composed of a prominent, surface-exposed hydrophobic patch.chaperonins | invisible states | dark state exchange saturation transfer | lifetime line broadening | relaxation dispersion C haperone 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 cat...

General expressions for R1 relaxation for N-site chemical exchange and the special case of linear chains

“…In particular, spontaneous inter-conversion between different protein structures, ligand-binding events, chemical reaction kinetics, and protein folding processes that occur with rate constants between 10 and 10 5 s −1 can be probed by R 1 ρ experiments with rf field strengths ranging from Hz (CEST-limit) to kHz ( R 1 ρ -limit) [2, 3]. Most analyses of experimental data have assumed two-state exchange kinetics, but examples featuring three-site [4, 5], four-site [6, 7] and other [8] topologies illustrate the need to provide theoretical insight into more complex kinetic schemes.…”

Differential conformational modulations of MreB folding upon interactions with GroEL/ES and TRiC chaperonin components

“…The mere binding of the target protein to GroEL has been shown to induce modulation in the chaperonin, and lead to the conformational rearrangement of the target protein213141516. To complete this GroEL binding-induced expansion, the addition of GroES and ATP induces a further compaction of the bound target protein, and improves its refolding into the active conformation1417181920.…”

Chaperone-client interactions: Non-specificity engenders multifunctionality

“…In particular, this unfolding could pull client proteins out of enthalpically stable yet misfolded states. This mechanism has the advantage that it can also apply to proteins that are too large to fit entirely within the folding chamber (43,58,76).…”