Two-dimensional ¹⁹F-¹³C correlation NMR for ¹⁹F resonance assignment of fluorinated proteins

¹⁹F solid-state NMR is an excellent approach for measuring long-range distances for structure determination and for studying molecular motion. For multi-fluorinated proteins, assignment of ¹⁹F chemical shifts has been traditionally carried out using mutagenesis. Here we show 2D ¹⁹F-¹³C correlation experiments that allow efficient assignment of the ¹⁹F chemical shifts. We have compared several rotational-echo double-resonance-based pulse sequences and ¹⁹F-¹³C cross polarization (CP) for 2D ¹⁹F-¹³C correlation. We found that direct transferred-echo double-resonance (TEDOR) transfer from ¹⁹F to ¹³C and vice versa outperforms out-and-back coherence transfer schemes. ¹⁹F detection gives twofold higher sensitivity over ¹³C detection for the 2D correlation experiment. At MAS frequencies of 25-35 kHz, double-quantum ¹⁹F-¹³C CP has higher coherence transfer efficiencies than zero-quantum CP. The most efficient TEDOR transfer experiment has higher sensitivity than the most efficient double-quantum CP experiment. We demonstrate these 2D ¹⁹F-¹³C correlation experiments on the model compounds t-Boc-4F-phenylalanine and GB1. Application of the 2D ¹⁹F-¹³C TEDOR correlation experiment to the tetrameric influenza BM2 transmembrane peptide shows intermolecular ¹³C-¹⁹F cross peaks that indicate that the BM2 tetramers cluster in the lipid bilayer in an antiparallel fashion. This clustering may be relevant for the virus budding function of this protein.