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GroEL-assisted protein folding studied by hydrogen exchange and mass spectrometry
The chaperonin GroEL is a double-ring structure with a central cavity that provides an environment for efficient folding of proteins in vivo. A combination of amide hydrogen exchange and mass spectrometry has been used to probe the conformational changes of GroEL and mitochondrial malate dehydrogenase (MDH), a GroEL substrate, in an assisted folding reaction. ^ The structure of MDH during various stages of assisted folding was probed by hydrogen exchange. Substantial residue structure was observed in the NAD + binding motif of non-native MDH bound to SR1, a single ring mutant of GroEL, indicating that MDH was not completely unfolded when bound to GroEL. Release of this form of MDH into the central cavity for initiation of folding resulted in a decrease of hydrogen exchange protection. This was attributed to loss of shielding by SR1. Subsequent folding of MDH inside the SR1 cavity occurred via two sequential, highly cooperative steps. ^ Structural changes of SR1 induced by MDH binding were investigated by comparing the hydrogen exchange kinetics of SR1 in the presence or absence of MDH. Results suggested that the majority of the SR1 subunits were in contact with MDH. This finding provides the first experimental evidence for multivalent binding of non-native substrate proteins by GroEL, suggesting a structural basis for unfolding of the substrate protein upon its release into the GroEL cavity. ^ Unfolding and disassembly of GroEL in guanidine hydrochloride was studied by hydrogen exchange. It was found that the GroEL apical and intermediate domains unfolded substantially faster than the equatorial domain, while unfolding of the equatorial domain was coupled to disassembly of the GroEL ring structure. This study points to features that may be important in the folding and assembly of the GroEL macroassembly. ^
Chen, Jiwen, "GroEL-assisted protein folding studied by hydrogen exchange and mass spectrometry" (2000). ETD collection for University of Nebraska - Lincoln. AAI9976979.