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 N， HN， and Cmethyl relaxation dispersion experiments， is composed of a prominent， surface-exposed hydrophobic patch.