期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 112, 期 29, 页码 8817-8823出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1510083112
关键词
chaperonins; invisible states; dark state exchange saturation transfer; lifetime line broadening; relaxation dispersion
资金
- National Institute of Diabetes and Digestive and Kidney Diseases/National Institutes of Health (NIDDK/NIH)
- AIDS Targeted Antiviral Program of the NIH
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 Carr-Purcell-Meinboom-Gill 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 invisible 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 N-15, H-1(N), and C-13(methyl) relaxation dispersion experiments, is composed of a prominent, surface-exposed hydrophobic patch.
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