Journal
JOURNAL OF MOLECULAR BIOLOGY
Volume 397, Issue 3, Pages 777-788Publisher
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2010.01.056
Keywords
Psi-analysis; ubiquitin; protein engineering; native-state hydrogen exchange; NMR
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Funding
- U.S. Department of Energy, Basic Energy Sciences, Office of Science [W-31-109-ENG-38]
- National Institutes of Health [RR-08630]
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Although most folding intermediates escape detection, their characterization is crucial to the elucidation of folding mechanisms. Here, we outline a powerful strategy to populate partially unfolded intermediates: A buried aliphatic residue is substituted with a charged residue (e.g., Leu -> Glu(-)) to destabilize and unfold a specific region of the protein. We applied this strategy to ubiquitin, reversibly trapping a folding intermediate in which the beta 5-strand is unfolded. The intermediate refolds to a native-like structure upon charge neutralization under mildly acidic conditions. Characterization of the trapped intermediate using NMR and hydrogen exchange methods identifies a second folding intermediate and reveals the order and free energies of the two major folding events on the native side of the rate-limiting step. This general strategy may be combined with other methods and have broad applications in the study of protein folding and other reactions that require trapping of high-energy states. (c) 2010 Elsevier Ltd. All rights reserved.
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