GroEL interaction and folding of MetF studied by hydrogen/deuterium exchange – mass spectrometry

The cylindrical chaperonin GroEL and its cofactor GroES mediate ATP-dependent protein folding in E. coli by transiently encapsulating non-native substrate in a nano-cage formed by the GroEL ring cavity and the lid-shaped GroES. We analyzed the spontaneous and chaperonin-assisted folding of the essential enzyme 5,10-methylenetetrahydrofolate reductase (MetF) of E. coli, an obligate GroEL/ES substrate. We found that MetF, a homotetramer of 33 kDa subunits with (8) TIM-barrel fold, is unable to fold spontaneously, even in the complete absence of aggregation, and populates a kinetically trapped folding intermediate(s) (MetF-I). GroEL/ES recognized MetF-I and catalyzed its folding with high efficiency, at a half-time of ~4 s and with more than 50% of protein folded in a single round of encapsulation. Analysis by hydrogen/deuterium exchange at peptide resolution showed that the MetF subunit folds to completion in the GroEL/ES nano-cage and binds the co-factor FAD.