Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 109, Issue 44, Pages 17777-17782Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1201799109
Keywords
excited protein states; protein folding intermediate
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Funding
- National Institutes of Health [P30GM092369]
- University of Connecticut Health Center
- Canadian Institutes of Health Research (CIHR)
- CIHR
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Studies of protein folding and the intermediates that are formed along the folding pathway provide valuable insights into the process by which an unfolded ensemble forms a functional native conformation. However, because intermediates on folding pathways can serve as initiation points of aggregation (implicated in a number of diseases), their characterization assumes an even greater importance. Establishing the role of such intermediates in folding, misfolding, and aggregation remains a major challenge due to their often low populations and short lifetimes. We recently used NMR relaxation dispersion methods and computational techniques to determine an atomic resolution structure of the folding intermediate of a small protein module-the FF domain-with an equilibrium population of 2-3% and a millisecond lifetime, 25 degrees C. Based on this structure a variant FF domain has been designed in which the native state is selectively destabilized by removing the carboxyl-terminal helix in the native structure to produce a highly populated structural mimic of the intermediate state. Here, we show via solution NMR studies of the designed mimic that the mimic forms distinct conformers corresponding to monomeric and dimeric (K-d = 0.2 mM) forms of the protein. The conformers exchange on the seconds timescale with a monomer association rate of 1.1.10(4) M-1 s(-1) and with a region responsible for dimerization localized to the amino-terminal residues of the FF domain. This study establishes the FF domain intermediate as a central player in both folding and misfolding pathways and illustrates how incomplete folding can lead to the formation of higher-order structures.
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