γ effects identify preferentially populated rotamers of CH$_2$ F groups: side-chain conformations of fluorinated valine analogues in a protein

Abstract

Using cell-free protein synthesis, the protein G B1 domain (GB1) was prepared with uniform highlevel substitution of valine by (2S,3S)-4-fluorovaline, (2S,3R)-4-fluorovaline or 4,4’-difluorovaline. The 19F nuclear magnetic resonance (NMR) signals are distributed over a wide spectral range. The fluorinated samples maintain the relative 1H chemical shifts of the wild-type protein, opening a convenient route to assign the 19FNMR signals. For the singly fluorinated residues, the 13C chemical shifts of the remaining CH3 group are subject to a γ effect that depends on the population of different rotameric states of the CH2F group and correlates with 3JFC coupling constants. In addition, the preferentially populated rotamers are reflected by the γ -gauche effect on 19F chemical shifts, which correlates with 3JHF 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 19F–19F couplings due to transient fluorine–fluorine contacts are observed between residues 39 and 54.