Thermodynamic stabilization of substrate protein within the GroEL:ES chaperonin cage

The adenosine triphosphate (ATP)-regulated GroEL/ES chaperonin of Escherichia coli promotes protein folding by transiently encapsulating unfolded substrate protein in a nano-cage formed by the cylindrical GroEL and the lid-shaped GroES. How this confinement modulates the folding energy landscape, allowing accelerated protein folding unimpaired by aggregation, remains incompletely understood. Prompted by recent observations suggesting that the chaperonin cavity may favor unfolding [I. Korobko et al., Proc. Natl. Acad. Sci. U.S.A. 119, e2213170119 (2022)], here we reinvestigated the effect of encapsulation on protein thermodynamic stability. To allow thermal unfolding of an encapsulated substrate protein, we generated a destabilized variant of maltose-binding protein (MBP) by introducing four mutations, quadruple mutant-MBP (QM-MBP), lowering the melting temperature (T_m) from 63.6 °C to 44.3 °C. GroEL/ES accelerated the slow spontaneous folding of QM-MBP ~20-fold. The protein reached a native-like structure while encapsulated inside GroEL:ES, as demonstrated using hydrogen/deuterium exchange and single molecule fluorescence resonance energy transfer analyses in presence and absence of the ligand maltose. Importantly, confinement in the thermally stable chaperonin shifted the cooperative unfolding transition of QM-MBP to a T_m of 52.4 °C, corresponding to a stabilization by 4.6 kcal mol^-1. In-cage thermal unfolding was reversible, indicating that the environment of the chaperonin cavity is conducive to folding. These findings suggest that a thermodynamically stabilizing effect of the GroEL:ES cage contributes to promoting protein folding.