Mixed Fluorotryptophan Substitutions at the Same Residue Expand the Versatility of ¹⁹F Protein NMR Spectroscopy
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 with mixtures of 5‐ and 7‐fluorotryptophan 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 heteronuclear single quantum coherence experiment peak patterns to a similar degree. Characterization of fluorotryptophan mixtures 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.
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