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 (2 $S$ ,4 $S$ )-5-fluoroleucine (FLeu1), (2 $S$ ,4 $R$ )-5-fluoroleucine (FLeu2), or 5,5^'-difluoroleucine (diFLeu). ${}^{19}F$ nuclear magnetic resonance (NMR) spectra showed chemical shift ranges spanning more than 9 $\mathrm{ppm}$ . Through-space scalar ${}^{19}F{\text{-}}^{19}F$ couplings between ${\mathrm{CH}}_{2}F$ groups arising from transient fluorine-fluorine contacts are readily manifested in [ ${}^{19}F$ , ${}^{19}F$ ]-TOCSY spectra. The ${}^{19}F$ chemical shifts correlate with the three-bond ${}^{1}H$ - ${}^{19}F$ couplings ( ${}^3{J}_{\mathrm{HF}}$ ), confirming the $\gamma$ -gauche effect as the predominant determinant of the ${}^{19}F$ chemical shifts of the ${\mathrm{CH}}_{2}F$ groups. Different ${}^3{J}_{\mathrm{HF}}$ couplings of different ${\mathrm{CH}}_{2}F$ groups indicate that the rotation of the ${\mathrm{CH}}_{2}F$ groups can be sufficiently restricted in different protein environments to result in the preferential population of a single rotamer. The ${}^3{J}_{\mathrm{HF}}$ couplings also show that ${\mathrm{CH}}_{2}F$ groups populate the different rotameric states differently in the 5,5^'-difluoroleucine residues than in the monofluoroleucine analogues, showing that two ${\mathrm{CH}}_{2}F$ groups in close proximity influence each other's conformation. Nonetheless, the ${}^{19}F$ resonances of the $C^{\delta 1}{H}_{2}F$ and $C^{\delta 2}{H}_{2}F$ groups of difluoroleucine residues can be assigned stereospecifically with good confidence by comparison with the ${}^{19}F$ chemical shifts of the enantiomerically pure fluoroleucines. ${}^{1}H{\text{-}}^{19}F$ nuclear Overhauser effects (NOEs) observed with water indicate hydration with sub-nanosecond residence times.