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

(2,3)-4-Fluorovaline (FVal) is an analogue of valine, where a single CH group is substituted by a CHF group. In the absence of valine, 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 Here, we describe the production and analysis of peptidyl-prolyl isomerase B where all 16 valine residues have been replaced by FVal synthesized with a C-labeled CHF group.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 CHF groups invariably populate staggered rotamers. Most CHF 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. F NMR spectra show a signal dispersion over 25 ppm. The most high-field shifted F resonances correlate with large coupling constants, confirming the impact of the γ- 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 F NMR spectroscopy.