The strategy of applying fluorine NMR to characterize ligand binding to a membrane protein prepared with mixtures of tryptophans substituted with F at different positions on the indole ring was tested. The ¹⁹F NMR behavior of 4‐, 5‐, 6‐, and 7‐fluorotryptophan were directly compared as a function of both micellar environment and fragment size for two overlapping apelin receptor (AR/APJ) segments; one with a single transmembrane (TM) helix and two tryptophan residues, the other with three TM helices and two additional tryptophan residues. Chemical shifts, peak patterns, and nuclear spin relaxation rates were observed to vary as a function of micellar conditions and F substitution position in the indole ring, with the exposure of a given residue to micelle or solvent being the primary differentiating factor. Titration of the 3‐TM AR segment biosynthetically prepared as a mixture of 5‐ and 7‐fluorotryptophan‐containing isoforms by two distinct peptide ligands (apelin‐36 and apela‐32) demonstrated site‐specific ¹⁹F peak intensity changes for one ligand but not the other. In contrast, both ligands perturbed ¹H–¹⁵N HSQC peak patterns to a similar degree. Characterization of multiple fluorotryptophan types for a given set of tryptophan residues, thus, significantly augments the potential to apply ¹⁹F NMR to track otherwise obscure modulation of protein conformation and dynamics without an explicit requirement for mutagenesis or chemical modification.

中文翻译：

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相同残基上的混合氟色氨酸取代基扩展了19F蛋白NMR光谱的多功能性

测试了应用氟NMR表征配体与膜蛋白结合的策略，该膜蛋白是由在吲哚环上不同位置被F取代的色氨酸的混合物制备的。在¹⁹直接比较了4-，5-，6-和7-氟色氨酸的F NMR行为，它是两个重叠的Apelin受体（AR / APJ）片段的胶束环境和片段大小的函数。一个带有一个跨膜（TM）螺旋和两个色氨酸残基，另一个带有三个TM螺旋和两个其他色氨酸残基。观察到化学位移，峰模式和核自旋弛豫率随胶束条件和吲哚环中F取代位置的变化而变化，给定残基暴露于胶束或溶剂是主要的区分因素。通过两种截然不同的肽配体（apelin-36和apela-32）生物合成制备的5-和7-氟色氨酸同工型混合物的3-TM AR节段的滴定显示位点特异性¹⁹一个配体的F峰强度发生变化，而另一种配体没有变化。相反，两个配体对¹ H– ¹⁵ N HSQC峰模式的干扰程度相似。因此，对于给定的一组色氨酸残基，多种氟色氨酸类型的表征大大提高了应用¹⁹ F NMR追踪蛋白质构象和动力学原本晦涩的调节的潜力，而无需明确的诱变或化学修饰。