(2S,4S)-5-Fluoroleucine, (2S,4R)-5-Fluoroleucine, and 5,5′-Difluoroleucine in Escherichia coli PpiB: Protein Production, ¹⁹F NMR, and Ligand Sensing Enhanced by the γ-Gauche Effect

Global substitution of leucine for analogues containing CH2F instead of methyl groups delivers proteins with multiple sites for monitoring by F-19 nuclear magnetic resonance (NMR) spectroscopy. The 19 kDa Escherichia coli peptidyl-prolyl cis-trans isomerase B (PpiB) was prepared with uniform high-level substitution of leucine by (2S,4S)-5-fluoroleucine, (2S,4R)-5-fluoroleucine, or 5,5 '-difluoroleucine. The stability of the samples toward thermal denaturation was little altered compared to the wild-type protein. F-19 nuclear magnetic resonance (NMR) spectra showed large chemical shift dispersions between 6 and 17 ppm. The F-19 chemical shifts correlate with the three-bond H-1-F-19 couplings ((3)J(HF)), providing the first experimental verification of the gamma-gauche effect predicted by [Feeney, J. J. Am. Chem. Soc. 1996, 118, 8700-8706] and establishing the effect as the predominant determinant of the F-19 chemical shifts of CH2F groups. Individual CH2F groups can be confined to single rotameric states by the protein environment, but most CH2F 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 CH2F rotamers in agreement with results obtained previously for 1,3-difluoropropane. The sensitivity of the F-19 chemical shift to the rotameric state of the CH2F groups potentially renders them particularly sensitive for detecting allosteric effects.