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

F 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 F chemical shifts has been traditionally carried out using mutagenesis. Here we show 2D F-C correlation experiments that allow efficient assignment of the F chemical shifts. We have compared several rotational-echo double-resonance-based pulse sequences and F-C cross polarization (CP) for 2D F-C correlation. We found that direct transferred-echo double-resonance (TEDOR) transfer from F to C and vice versa outperforms out-and-back coherence transfer schemes. F detection gives twofold higher sensitivity over C detection for the 2D correlation experiment. At MAS frequencies of 25-35 kHz, double-quantum F-C 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 F-C correlation experiments on the model compounds t-Boc-4F-phenylalanine and GB1. Application of the 2D F-C TEDOR correlation experiment to the tetrameric influenza BM2 transmembrane peptide shows intermolecular C-F 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.