Protein Vivisection Reveals Elusive Intermediates in Folding

JOURNAL OF MOLECULAR BIOLOGY

卷:397

Article

Zheng, Zhongzhou¹  Sosnick, Tobin R.^1,2 

[1] Univ Chicago, Dept Biochem & Mol Biol, Chicago, IL 60637 USA

[2] Univ Chicago, Inst Biophys Dynam, Computat Inst, Chicago, IL 60637 USA

关键词: Psi-analysis;    ubiquitin;    protein engineering;    native-state hydrogen exchange;    NMR;   

DOI  :  10.1016/j.jmb.2010.01.056

来源: Elsevier

【 摘 要 】

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.

【 授权许可】

Free