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