NIH Research Festival
FARE Award Winner
The chaperonins, a ubiquitous subclass of molecular chaperones, are barrel-shaped, multisubunit assemblies that assist protein folding in an ATP-dependent manner. Although the encapsulation mechanism and accompanying allosteric transitions driven by ATP have been extensively studied, the details of how chaperonins fold proteins remain elusive. Further, encapsulation does not appear to be an absolute requirement for successful re/folding. Here we take advantage of a monomeric, non-aggregating, 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 - to directly demonstrate, using NMR relaxation-based methods, 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, Dark state Exchange Saturation Transfer (DEST) and Carr-Purcell-Meinboom-Gill (CPMG) relaxation dispersion 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 foldase/unfoldase activity is modulated by SH3 deuteration indicating that catalysis of the exchange reaction between folded and intermediate states involved direct interaction of the substrate with the walls of the GroEL chambers. These results provide a basis for how chaperonins, in the absence of co-factors and encapsulation, may be able to passively protect the cell from the deleterious effects of misfolded protein accumulation.
Scientific Focus Area: Biomedical Engineering and Biophysics
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