Folding of malate dehydrogenase inside the GroEL–GroES cavity

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The chaperonin GroEL binds nonnative substrate protein in the hydrophobic central cavity of an open ring. ATP and GroES binding to the same ring converts this cavity into an encapsulated, hydrophilic chamber that mediates productive folding. A 'rack' mechanism of initial protein unfolding proposes that, upon GroES and ATP binding, the polypeptide is stretched between the binding sites on the twisting apical domains of GroEL before complete release into the chamber. Here, the structure of malate dehydrogenase (MDH) subunit during folding is monitored by deuterium exchange, peptic fragment production and mass spectrometry. When bound to GroEL, MDH exhibits a core of partially protected secondary structure that is only modestly deprotected upon ATP and GroES binding. Moreover, deprotection is broadly distributed throughout MDH, suggesting that it results from breaking hydrogen bonds between MDH and the cavity wall or global destabilization, as opposed to forced mechanical unfolding.

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Figure 1: Experimental scheme for examining secondary structure of MDH subunit during its chaperonin-mediated folding.
Figure 2: Mass spectra showing extent of deuterium incorporation into MDH after various times of SR1-assisted folding, measured after pulse-labeling in D2O for 1 s.
Figure 3: Four regions of MDH in the MDH–SR1 binary complex that exhibit the greatest degree of protection from deuterium exchange.
Figure 4: Retention of exchange protection in the four regions of MDH exhibiting greatest protection in the MDH–SR1 binary complex upon addition of ATP/GroES.
Figure 5: MDH refolded inside SR1–GroES lacks native protection from exchange only at the dimer interface.


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This work was supported by grants from the NIH, the Nebraska Center for Mass Spectrometry and the Howard Hughes Medical Institute. We thank members of the Horwich lab for helpful discussion and W. Fenton for critical reading of the manuscript.

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Correspondence to Arthur L. Horwich.

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Chen, J., Walter, S., Horwich, A. et al. Folding of malate dehydrogenase inside the GroEL–GroES cavity. Nat Struct Mol Biol 8, 721–728 (2001) doi:10.1038/90443

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