Mixed Fluorotryptophan Substitutions at the Same Residue Expand the Versatility of ¹⁹F Protein NMR Spectroscopy

Abstract

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 F 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 F peak intensity changes for one ligand but not the other. In contrast, both ligands perturbed H- N 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 F NMR to track otherwise obscure modulation of protein conformation and dynamics without an explicit requirement for mutagenesis or chemical modification.

Simultaneous Ligand and Receptor Tracking through NMR Spectroscopy Enabled by Distinct 19F Labels

“…Comparing the TM1-3:F*-ap-13 interaction to our prior studies, titration of AR TM1-3 with apelin-36 led to 19 F signal attenuation most prevalently at the W95 site without observable chemical shift perturbation at any of the four Trp residues in TM1-3 [33]. This is in distinct contrast to the present situation with F*-ap-13, where chemical shift perturbation was clear for both W24 and W85.…”

“…It should be noted that F-Trp chemical shifts may vary further than what has been observed in AR fragments [39], thus some optimization may be required to ensure that the class of F-Trp labeling used does not overlap in 19 F chemical shift with 2,4,5F-Phe. This must also be balanced with the fact that, as we have noted previously [33], different F-Trp types give rise to varying degrees of chemical shift overlap or dispersion at a given Trp site in the protein. Here, no overlap was encountered between ligand and receptor in any case, providing clear and unambiguous tracking of the effects of titration upon both species.…”

“…The 19 F chemical shift ranges observed for the 2,4,5F-Phe in buffer, micelles, and in titration with AR fragments (Figure 8), appear generally distinct from those of any of the F-Trp species routinely biosynthetically incorporated [33,34,38]. It should be noted that F-Trp chemical shifts may vary further than what has been observed in AR fragments [39], thus some optimization may be required to ensure that the class of F-Trp labeling used does not overlap in 19 F chemical shift with 2,4,5F-Phe.…”

“…We have previously directly applied 19 F NMR spectroscopy to study ligand binding to apelin receptor fragments [33]. In these prior studies, we took advantage of the ability to biosynthetically incorporate fluorine-labeled Trp (F-Trp) residues [34].…”

“…The low prevalence of Trp means that 19 F labels are inherently relatively sparse, allowing for straightforward monitoring of well-defined sites. Through site-directed mutagenesis, signal assignment of four Trp residues covering the first three transmembrane (TM) segments of the AR was readily possible [33]. Specifically, two constructs were examined (Figure 2): (1) AR55, comprising residues 1-55 of the AR including the N-terminal tail and first TM segment [21]; and, (2) the TM1-3 fragment, comprising residues 1-138 covering the first 3 TM segments and N-terminal tail [35].…”