Leveraging relaxation-optimized ¹H-¹³C_F correlations in 4-¹⁹F-phenylalanine as atomic beacons for probing structure...

NMR spectroscopy of biomolecules provides atomic level information into their structure, dynamics and interactions with their binding partners. However, signal attenuation from line broadening caused by fast relaxation and signal overlap often limits the application of NMR to large macromolecular systems. Here we leverage the slow relaxation properties of ¹³C nuclei attached to ¹⁹F in aromatic ¹⁹F-¹³C spin pairs as well as the spin-spin coupling between the fluorinated ¹³C nucleus and the hydrogen atom at the meta-position to record two-dimensional ¹H-¹³C_F correlation spectra with transverse relaxation-optimized spectroscopy selection on ¹³C_F. To accomplish this, we synthesized [4-¹⁹F¹³C^ζ; 3,5-²H₂^ε] Phe, engineered for optimal relaxation properties, and adapted a residue-specific route to incorporate this residue globally into proteins and a site-specific 4-¹⁹F Phe encoding strategy. This approach resulted in narrow linewidths for proteins ranging from 30 kDa to 180 kDa, enabling interaction studies with small-molecule ligands without requiring specialized ¹⁹F-compatible probes.