Assessment of Attainable Distances in Proteins by Through‐Space ¹⁹ F– ¹⁹ F Couplings

ABSTRACT 19 F NMR is becoming an increasingly powerful method for studying the conformation and ligand binding of proteins in the solid state and in solution. In liquids NMR, surprisingly, reports on through‐space 19 F– 19 F coupling remain scarce. Here, we present the first 19 F– 19 F NOESY study designed to calibrate inter‐fluorine distances in proteins. Three difluorinated tryptophan analogues (4,5‐F 2 ‐Trp, 4,6‐F 2 ‐Trp, 5,6‐F 2 ‐Trp) were synthesized and site‐specifically incorporated into human cyclophilin A (CypA). Series of 19 F– 19 F NOESY build‐up experiments were recorded for each labeled protein, and the extracted cross‐relaxation rates were compared to fixed reference distances. While small distances in the 2.5–3.0 Å range were easily accessible, the upper limit at 14.1 T is ∼5 Å. These results provide evidence that short distances can be extracted from 19 F– 19 F NOESY spectra, expanding the toolbox for studying protein‐protein or protein‐ligand interactions by NMR.