The substitution of a single hydrogen atom in a protein by fluorine yields a site-specific probe for sensitive detection by ¹⁹F nuclear magnetic resonance (NMR) spectroscopy, where the absence of background signal from the protein facilitates the detection of minor conformational species. We developed genetic encoding systems for the site-selective incorporation of 4-fluorotryptophan, 5-fluorotryptophan, 6-fluorotryptophan, and 7-fluorotryptophan in response to an amber stop codon and used them to investigate conformational heterogeneity in a designed amino acid binding protein and in flaviviral NS2B-NS3 proteases. These proteases have been shown to present variable conformations in X-ray crystal structures, including flips of the indole side chains of tryptophan residues. The ¹⁹F NMR spectra of different fluorotryptophan isomers installed at the conserved site of Trp83 indicate that the indole ring flip is common in flaviviral NS2B-NS3 proteases in the apo state and suppressed by an active-site inhibitor.

中文翻译：

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氟-L-色氨酸的基因编码，用于通过核磁共振波谱对蛋白质构象异质性进行位点特异性检测

用氟取代蛋白质中的单个氢原子可产生位点特异性探针，可通过¹⁹ F 核磁共振 (NMR) 光谱进行灵敏检测，其中蛋白质背景信号的缺失有助于检测次要构象物种。我们开发了基因编码系统，用于响应琥珀终止密码子而选择性地掺入 4-氟色氨酸、5-氟色氨酸、6-氟色氨酸和 7-氟色氨酸，并用它们来研究设计的氨基酸结合蛋白中的构象异质性和存在于黄病毒 NS2B-NS3 蛋白酶中。这些蛋白酶已被证明在 X 射线晶体结构中呈现可变构象，包括色氨酸残基吲哚侧链的翻转。 Trp83 保守位点上不同氟色氨酸异构体的¹⁹ F NMR谱表明，吲哚环翻转在 apo 状态的黄病毒 NS2B-NS3 蛋白酶中很常见，并被活性位点抑制剂抑制。