2-Fluorotyrosine is a valuable but understudied amino acid for protein-observed ¹⁹F NMR

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 pK_a 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 pK_a 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.