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Free keywords:
CRYSTAL-STRUCTURE; METHYLENETETRAHYDROFOLATE REDUCTASE; STRUCTURAL
FEATURES; NANO-CAGE; COLI; MECHANISM; CONFORMATION; CONFINEMENT;
EXCHANGE; SPECTROSCOPYChaperonins; GroEL; MetF; Single molecule spectroscopy;
Hydrogen/deuterium exchange;
Abstract:
The cylindrical chaperonin GroEL and its cofactor GroES mediate ATP-dependent protein folding in Escherichia colt by transiently encapsulating non-native substrate in a nano-cage formed by the GroEL ring cavity and the lid-shaped GroES. Mechanistic studies of GroEL/ES with heterologous protein substrates suggested that the chaperonin is inefficient, typically requiring multiple ATP-dependent encapsulation cycles with only a few percent of protein folded per cycle. Here we analyzed the spontaneous and chaperoninassisted 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 (beta/alpha)(8) TIM-barrel fold, populates a kinetically trapped folding intermediate(s) (MetF-I) upon dilution from denaturant that fails to convert to the native state, even in the absence of aggregation. GroEUES recognizes MetF-I and catalyzes rapid folding, with 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 its cofactor flavin adenine dinucleotide. Rapid folding required the net negative charge character of the wall of the chaperonin cavity. These findings reveal a remarkable capacity of GroEL/ES to catalyze folding of an endogenous substrate protein that would have coevolved with the chaperonin system. (C) 2020 Elsevier Ltd. All rights reserved.
