期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 110, 期 28, 页码 11361-11366出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1305715110
关键词
w supramolecular machine; protein-protein interactions; conformational sampling
资金
- National Institute of Diabetes and Digestive and Kidney Diseases
- NIH
- AIDS Targeted Antiviral Program
- NIH Director
The mechanism whereby the prototypical chaperonin GroEL performs work on substrate proteins has not yet been fully elucidated, hindered by lack of detailed structural and dynamic information on the bound substrate. Previous investigations have produced conflicting reports on the state of GroEL-bound polypeptides, largely due to the transient and dynamic nature of these complexes. Here, we present a unique approach, based on combined analysis of four complementary relaxation-based NMR experiments, to probe directly the dark NMR-invisible state of the model, intrinsically disordered, polypeptide amyloid beta (A beta 40) bound to GroEL. The four NMR experiments, lifetime line-broadening, dark-state exchange saturation transfer, relaxation dispersion, and small exchange-induced chemical shifts, are dependent in different ways on the overall exchange rates and populations of the free and bound states of the substrate, as well as on residue-specific dynamics and structure within the bound state as reported by transverse magnetization relaxation rates and backbone chemical shifts, respectively. Global fitting of all the NMR data shows that the complex is transient with a lifetime of <1 ms, that binding involves two predominantly hydrophobic segments corresponding to predicted GroEL consensus binding sequences, and that the structure of the bound polypeptide remains intrinsically and dynamically disordered with minimal changes in secondary structure propensity relative to the free state. Our results establish a unique method to observe NMR-invisible dynamic states of GroEL-bound substrates and to describe at atomic resolution the events between substrate binding and encapsulation that are crucial for understanding the normal and stress-related metabolic function of chaperonins.
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