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Intrinsic unfoldase/foldase activity of the chaperonin GroEL directly demonstrated using multinuclear relaxation-based NMR
The prototypical chaperonin GroEL assists protein folding through an ATP-dependent encapsulation mechanism. The details of how GroEL folds proteins remain elusive, particularly because encapsulation is not an absolute requirement for successful re/folding. Here we make use of a metastable model prot...
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Published in: | Proceedings of the National Academy of Sciences - PNAS 2015-07, Vol.112 (29), p.8817-8823 |
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Main Authors: | , , |
Format: | Article |
Language: | English |
Subjects: | |
Citations: | Items that this one cites Items that cite this one |
Online Access: | Get full text |
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Summary: | The prototypical chaperonin GroEL assists protein folding through an ATP-dependent encapsulation mechanism. The details of how GroEL folds proteins remain elusive, particularly because encapsulation is not an absolute requirement for successful re/folding. Here we make use of a metastable model protein substrate, comprising a triple mutant of Fyn SH3, to directly demonstrate, by simultaneous analysis of three complementary NMR-based relaxation experiments (lifetime line broadening, dark state exchange saturation transfer, and CarrâPurcellâMeinboomâGill relaxation dispersion), that apo GroEL accelerates the overall interconversion rate between the native state and a well-defined folding intermediate by about 20-fold, under conditions where the âinvisibleâ GroEL-bound states have occupancies below 1%. This is largely achieved through a 500-fold acceleration in the folded-to-intermediate transition of the protein substrate. Catalysis is modulated by a kinetic deuterium isotope effect that reduces the overall interconversion rate between the GroEL-bound species by about 3-fold, indicative of a significant hydrophobic contribution. The location of the GroEL binding site on the folding intermediate, mapped from ¹âµN, ¹H N, and ¹³C ââââyâ relaxation dispersion experiments, is composed of a prominent, surface-exposed hydrophobic patch.
Chaperones are integral components of the cellular machinery that assist protein folding and protect against misfolding and aggregation. A bottleneck in understanding how chaperones work is that the relevant functional states are too sparsely populated and dynamic to be observed using conventional biophysical methods. NMR is uniquely suited to detect and provide atomic resolution functional information on such âinvisibleâ states. Here we quantitate the kinetics of the chaperone GroEL binding to a protein substrate that exists in a metastable equilibrium between the native state and a sparsely populated folding intermediate, under conditions where the GroEL-bound states are not directly observable. We show that in the absence of cofactors, GroEL possesses substantial intrinsic un/foldase activity that is mediated by hydrophobic interactions. |
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ISSN: | 0027-8424 1091-6490 |
DOI: | 10.1073/pnas.1510083112 |