利用 19F-13C 二维核磁共振分析同源二聚体氟乙酸脱卤酶的构象状态。

IF 4.2 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY RSC Chemical Biology Pub Date : 2024-10-10 DOI:10.1039/d4cb00176a
Motasem Suleiman, Geordon A Frere, Ricarda Törner, Lauren Tabunar, Gaurav Vijay Bhole, Keith Taverner, Nobuyuki Tsuchimura, Dmitry Pichugin, Roman J Lichtenecker, Oleksandr Vozny, Patrick Gunning, Haribabu Arthanari, Adnan Sljoka, Robert S Prosser
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引用次数: 0

摘要

色氨酸对蛋白质的稳定性、异构性和催化作用起着至关重要的作用。利用氟(19F)核磁共振(NMR),可以通过氟色氨酸报告来研究蛋白质构象动力学和结构-活性关系(SARs)。色氨酸类似物(如 4-、5-、6-或 7-氟色氨酸)可通过抑制内源性色氨酸的生物合成,在异源表达过程中被常规加入蛋白质中。基于与 5-氟色氨酸相关的巨大 19F 化学位移分散,我们介绍了一种使用直接生物合成前体 5-氟蒽酸-(苯基-13C6)掺入 13C 富集 5-氟色氨酸的方法。同源二聚体酶氟乙酸脱卤酶(FAcD)是一种嗜热的α/β水解酶,负责水解氟乙酸中的C-F键。由此产生的二维 19F-13C(横向弛豫优化光谱)TROSY 异核相关光谱可完全解析 apo 酶和饱和底物类似物溴乙酸酯的 FAcD 中的全部 9 个色氨酸残基。(19F,13C)相关光谱还揭示了 apo 样品中的许多次要共振。通过计算刚度传递异构分析验证了每个色氨酸残基在异构通讯中的作用,在这种情况下,该分析探讨了所有可能的色氨酸对之间的相对原体间通讯。
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Characterization of conformational states of the homodimeric enzyme fluoroacetate dehalogenase by 19F-13C two-dimensional NMR.

Tryptophan plays a critical role in proteins by contributing to stability, allostery, and catalysis. Using fluorine (19F) nuclear magnetic resonance (NMR), protein conformational dynamics and structure-activity relationships (SARs) can be studied via fluorotryptophan reporters. Tryptophan analogs such as 4-, 5-, 6-, or 7-fluorotryptophan can be routinely incorporated into proteins during heterologous expression by arresting endogenous tryptophan biosynthesis. Building upon the large 19F chemical shift dispersion associated with 5-fluorotryptophan, we introduce an approach to the incorporation of 13C-enriched 5-fluorotryptophan using a direct biosynthetic precursor, 5-fluoroanthranilic acid-(phenyl-13C6). The homodimeric enzyme fluoroacetate dehalogenase (FAcD), a thermophilic alpha/beta hydrolase responsible for the hydrolysis of a C-F bond in fluoroacetate, was expressed and biosynthetically labeled with (phenyl-13C6) 5-fluorotryptophan. The resulting two-dimensional 19F-13C (transverse relaxation optimized spectroscopy) TROSY heteronuclear correlation spectra provide complete resolution of all 9 tryptophan residues in the apo enzyme and FAcD saturated with the substrate analog bromoacetate. The (19F,13C) correlation spectra also reveal a multitude of minor resonances in the apo sample. The role of each tryptophan residue in allosteric communication was validated with computational rigidity transmission allostery analysis, which in this case explores the relative interprotomer communication between all possible tryptophan pairs.

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来源期刊
CiteScore
6.10
自引率
0.00%
发文量
128
审稿时长
10 weeks
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