1.3 Å Crystal Structure of E. coli Peptidyl–Prolyl Isomerase B with Uniform Substitution of Valine by (2S,3S)-4-Fluorovaline Reveals Structure Conservation and Multiple Staggered Rotamers of CH2F Groups

(2S,3S)-4-Fluorovaline (FVal) is an analogue of valine, where a single CH₃ group is substituted by a CH₂F group. In the absence of valine, E. coli valyl-tRNA synthetase uses FVal as a substitute, enabling the production of proteins uniformly labeled with FVal. Here, we describe the production and analysis of E. coli peptidyl–prolyl isomerase B where all 16 valine residues have been replaced by FVal synthesized with a ¹³C-labeled CH₂F group. Although the melting temperature is lower by about 11 °C relative to the wild-type protein, the three-dimensional protein structure is almost completely conserved, as shown by X-ray crystallography. The CH₂F groups invariably populate staggered rotamers. Most CH₂F groups populate two different rotamers. The increased space requirement of fluorine versus hydrogen does not prohibit rotamers that position fluorine next to a backbone carbonyl carbon. ¹⁹F NMR spectra show a signal dispersion over 25 ppm. The most high-field shifted ¹⁹F resonances correlate with large ³J_HF coupling constants, confirming the impact of the γ-gauche effect on the signal dispersion. The present work is the second experimental verification of the effect and extends its validity to fluorovaline. The abundance of valine in proteins and structural conservation with FVal renders this valine analogue attractive for probing proteins by ¹⁹F NMR spectroscopy.