The folding pathway of T4 lysozyme: The high-resolution structure and folding of a hidden intermediate

JOURNAL OF MOLECULAR BIOLOGY

卷:365

Article

Kato, Hidenori ; Feng, Hanqiao ; Bai, Yawen

关键词: intermediate structure;    T4 lysozyme;    hydrogen exchange;    protein folding;    hidden intermediate;   

DOI  :  10.1016/j.jmb.2006.10.047

来源: Elsevier

【 摘 要 】

Folding intermediates have been detected and characterized for many proteins. However, their structures at atomic resolution have only been determined for two small single domain proteins: Rd-apocytochrome b(562) and engrailed homeo domain. T4 lysozyme has two easily distinguishable but energetically coupled domains: the N and C-terminal domains. An early native-state hydrogen exchange experiment identified an intermediate with the C-terminal domain folded and the N-terminal domain unfolded. We have used a native-state hydrogen exchange-directed protein engineering approach to populate this intermediate and demonstrated that it is on the folding pathway and exists after the rate-limiting step. Here, we determined its high-resolution structure and the backbone dynamics by multidimensional NMR methods. We also characterized the folding behavior of the intermediate using stopped-flow fluorescence, protein engineering, and native-state hydrogen exchange. Unlike the folding intermediates of the two single-domain proteins, which have many non-native side-chain interactions, the structure of the hidden folding intermediate of T4 lysozyme is largely native-like. It folds like many small single domain proteins. These results have implications for understanding the folding mechanism and evolution of multi-domain proteins. Published by Elsevier Ltd.

【 授权许可】

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