Inter-residue through-space scalar ¹⁹F–¹⁹F couplings between CH₂F groups in a protein

Abstract. Using cell-free protein synthesis, the protein G B1-domain (GB1) was prepared with uniform high-level substitution of leucine by (2S,4S)-5-fluoroleucine, (2S,4R)-5-fluoroleucine, or 5,5’-difluoroleucine. ¹⁹F nuclear magnetic resonance (NMR) spectra showed chemical shift ranges spanning more than 9 ppm. Through-space scalar ¹⁹F-¹⁹F couplings between CH₂F groups arising from transient fluorine-fluorine contacts are readily manifested in [¹⁹F,¹⁹F]-TOCSY spectra. The ¹⁹F chemical shifts correlate with the three-bond ¹H–¹⁹F couplings (³J_HF), confirming the γ-gauche effect as the predominant determinant of the ¹⁹F chemical shifts of the CH₂F groups. Different ³J_HF couplings of different CH­₂F groups indicate that the rotation of the CH₂F groups can be sufficiently restricted in different protein environments to result in the preferential population of a single rotamer. The ³J_HF couplings also show that CH₂F groups populate the different rotameric states differently in the 5,5’-difluoroleucine residues than in the monofluoroleucine analogues, showing that two CH₂F groups in close proximity influence each other’s conformation. Nonetheless, the ¹⁹F resonances of the C^δ1H₂F and C^δ2H₂F groups of difluoroleucine residues can be assigned stereospecifically with good confidence by comparison with the ¹⁹F chemical shifts of the enantiomerically pure fluoroleucines. ¹H-¹⁹F NOEs observed with water indicate hydration with subnanosecond residence times.