γ effects identify preferentially populated rotamers of CH 2 F groups: side-chain conformations of fluorinated valine analogues in a protein

Using cell-free protein synthesis, the protein G B1 domain (GB1) was prepared with uniform high-level substitution of valine by (2 S ,3 S )-4-fluorovaline, (2 S ,3 R )-4-fluorovaline or 4,4'-difluorovaline. The 19 F nuclear magnetic resonance (NMR) signals are distributed over a wide spectral range. The fluorinated samples maintain the relative 1 H chemical shifts of the wild-type protein, opening a convenient route to assign the 19 F-NMR signals. For the singly fluorinated residues, the 13 C chemical shifts of the remaining CH 3 group are subject to a γ effect that depends on the population of different rotameric states of the CH 2 F group and correlates with 3 J FC coupling constants. In addition, the preferentially populated rotamers are reflected by the γ -gauche effect on 19 F chemical shifts, which correlates with 3 J HF couplings. Some of the side-chain conformations determined by these restraints position the fluorine atom near a backbone carbonyl group, a non-intuitive finding that has previously been observed in the high-resolution crystal structure of a different protein. Through-space scalar 19 F– 19 F couplings due to transient fluorine–fluorine contacts are observed between residues 39 and 54.