The folding pathway of T4 lysozyme: the high-resolution structure and folding of a hidden intermediate |
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Authors: | Kato Hidenori Feng Hanqiao Bai Yawen |
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Affiliation: | Laboratory of Biochemistry, Center for Cancer Research, National Cancer Institute, Building 37, Room 6114E, Bethesda, MD 20892, USA. |
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Abstract: | 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 multi-dimensional 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. |
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Keywords: | ENHD, engrailed homeodomain NOE, nuclear Overhauser effect HSQC, heteronuclear single quantum coherence |
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