The folding pathway of T4 lysozyme: An on-pathway hidden folding intermediate

JOURNAL OF MOLECULAR BIOLOGY

卷:365

Article

Kato, Hidenori ; Vu, Ngoc-Diep ; Feng, Hanqiao ; Zhou, Zheng ; Bai, Yawen

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

DOI  :  10.1016/j.jmb.2006.10.048

来源: Elsevier

【 摘 要 】

T4 lysozyme has two easily distinguishable but energetically coupled domains: the N and C-terminal domains. In earlier studies, an amide hydrogen/deuterium exchange pulse-labeling experiment detected a stable submillisecond intermediate that accumulates before the rate-limiting transition state. It involves the formation of structures in both the N and C-terminal regions. However, a native-state hydrogen exchange experiment.Y subsequently detected an equilibrium intermediate that only involves the formation of the C-terminal domain. Here, using stopped-flow circular dichroism and fluorescence, amide hydrogen exchange-folding competition, and protein engineering methods, we re-examined the folding pathway of T4-lysozyme. We found no evidence for the existence of a stable folding intermediate before the rate-limiting transition state at neutral pH. In addition, using native-state hydrogen exchange-directed protein engineering, we created a mimic of the equilibrium intermediate. We found that the intermediate mimic folds with the same rate as the wild-type protein, suggesting that the equilibrium intermediate is an on-pathway intermediate that exists after the rate-limiting transition state. Published by Elsevier Ltd.

【 授权许可】

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