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Abstract:

<p>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 C nuclei attached to F in aromatic F-C spin pairs as well as the spin-spin coupling between the fluorinated C nucleus and the hydrogen atom at the meta-position to record two-dimensional H-C correlation spectra with transverse relaxation-optimized spectroscopy selection on C. To accomplish this, we synthesized [4-FC; 3,5-H] Phe, engineered for optimal relaxation properties, and adapted a residue-specific route to incorporate this residue globally into proteins and a site-specific 4-F Phe encoding strategy. This approach resulted in narrow linewidths for proteins ranging from 30&thinsp;kDa to 180&thinsp;kDa, enabling interaction studies with small-molecule ligands without requiring specialized F-compatible probes.</p>