Incorporation of 19F into proteins allows for the study of their molecular interactions via NMR. The study of 19F labeled aromatic amino acids has largely focused on 4-,5-, or 6-fluorotryptophan, 4-fluorophenylalanine, (4,5, or 6FW) or 3-fluorotyrosine (3FY), whereas 2-fluorotyrosine (2FY) has remained largely understudied. Here we report a comparative analysis with different fluorinated amino acids. We first report the NMR chemical shift responsiveness of five aromatic amino acid mimics to changes in solvent polarity and find that the most responsive, a mimic of 3FY, has a 2.9-fold greater change in chemical shift compared to the other amino acid mimics in aprotic solvents including the 2FY mimic. We also probed the utility of 2FY for 19F NMR by measuring its NMR relaxation properties in solution and the chemical shift anisotropy (CSA) of a polycrystalline sample of the amino acid by magic angle spinning. Using protein-observed fluorine NMR (PrOF NMR), we compared the influence of 2FY and 3FY incorporation on stability and pKa perturbation when incorporated into the KIX domain of CBP/p300. Lastly, we investigated the 19F NMR response of both 2FY and 3FY-labeled proteins to a protein-protein interaction partner, MLL, and discovered that 2FY can report on allosteric interactions that are not observed with 3FY-labeling in this protein complex. The reduced perturbation to pKa and similar but reduced CSA of 2FY to 3FY supports 2FY as a suitable alternative amino acid for incorporation into large proteins for 19F NMR analysis.

中文翻译：

---

对于蛋白质观察到的19 F NMR，2-氟酪氨酸是一种有价值的但未被充分研究的氨基酸。

将19F掺入蛋白质可以通过NMR研究其分子相互作用。19F标记的芳香族氨基酸的研究主要集中在4-，5-或6-氟色氨酸，4-氟苯丙氨酸（4,5或6FW）或3-氟酪氨酸（3FY），而2-氟酪氨酸（2FY）仍然在很大程度上未被研究。在这里，我们报告了不同氟化氨基酸的比较分析。我们首先报告了五种芳香族氨基酸模拟物对溶剂极性变化的NMR化学位移响应性，发现与非质子质子中的其他氨基酸模拟物相比，响应性最强的3FY模拟物的化学位移变化高2.9倍溶剂，包括2FY模拟物。我们还通过测量2FY在溶液中的NMR弛豫特性以及通过魔术角旋转法测量氨基酸的多晶样品的化学位移各向异性（CSA），来探索2FY在19F NMR中的实用性。使用蛋白质观察的氟NMR（PrOF NMR），我们比较了2FY和3FY掺入对CBP / p300的KIX域中稳定性和pKa扰动的影响。最后，我们研究了2FY和3FY标记的蛋白质对蛋白质-蛋白质相互作用伴侣MLL的19F NMR反应，并发现2FY可以报告在该蛋白质复合物中未观察到用3FY标记观察到的变构相互作用。对pKa的减少的干扰以及类似的但2FY的CSA减少至3FY的CSA，支持2FY作为适合掺入大蛋白中进行19F NMR分析的合适替代氨基酸。