(2,4)-5-Fluoroleucine, (2,4)-5-Fluoroleucine, and 5,5'-Difluoroleucine in PpiB: Protein Production, F NMR, and Ligand Sensing Enhanced by the γ-Gauche Effect.

Global substitution of leucine for analogues containing CHF instead of methyl groups delivers proteins with multiple sites for monitoring by F nuclear magnetic resonance (NMR) spectroscopy. The 19 kDa peptidyl-prolyl isomerase B (PpiB) was prepared with uniform high-level substitution of leucine by (2,4)-5-fluoroleucine, (2,4)-5-fluoroleucine, or 5,5'-difluoroleucine. The stability of the samples toward thermal denaturation was little altered compared to the wild-type protein. F nuclear magnetic resonance (NMR) spectra showed large chemical shift dispersions between 6 and 17 ppm. The F chemical shifts correlate with the three-bond H-F couplings (), providing the first experimental verification of the γ-gauche effect predicted by [Feeney, J. 1996, 118, 8700-8706] and establishing the effect as the predominant determinant of the F chemical shifts of CHF groups. Individual CHF groups can be confined to single rotameric states by the protein environment, but most CHF groups exchange between different rotamers at a rate that is fast on the NMR chemical shift scale. Interactions between fluorine atoms in 5,5'-difluoroleucine bias the CHF rotamers in agreement with results obtained previously for 1,3-difluoropropane. The sensitivity of the F chemical shift to the rotameric state of the CHF groups potentially renders them particularly sensitive for detecting allosteric effects.