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

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 (FLeu1), (2S,4R)-5-fluoroleucine (FLeu2), or 5,5′-difluoroleucine (diFLeu). 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 (3JHF), confirming the γ3-gauche effect as the predominant determinant of the 19F chemical shifts of the CH2F groups. Different 3JHF 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 3JHF 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 Cδ1H2F and Cδ2H2F 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 nuclear Overhauser effects (NOEs) observed with water indicate hydration with sub-nanosecond residence times.