# 19F-NMR spectroscopy of fluorinated isoleucine analogues in a protein

## TL;DR

This paper uses fluorinated isoleucine analogs with 19F-NMR to study a human protein's structure, finding challenges in stability and spectral heterogeneity.

## Contribution

The study introduces fluorinated isoleucine analogs as NMR probes and compares their effectiveness in protein structure analysis.

## Key findings

- DiFIle was more effective than FIle for fluorinated protein production due to FIle's instability.
- 19F-NMR spectra showed large chemical shifts but were affected by protein conformational heterogeneity and instability.
- Non-uniform cross-peak intensities suggest preferential rotamer populations in fluorinated amino acid side chains.

## Abstract

The R3H domain of the human protein Sµbp-2 was produced with 5-fluoro-L-isoleucine (FIle) and 5,5-difluoro-L-isoleucine (diFIle) as probes for detection by 19F-NMR spectroscopy. The fluorinated protein, produced by cell-free protein synthesis, was obtained more easily with diFIle than FIle as FIle readily hydrolysed at pH 7.5 with the release of fluoride. The 19F-NMR spectra showed large chemical shift ranges but were heterogeneous. The heterogeneities arose from difficulties to fully exclude canonical isoleucine, the presence of multiple conformations and limited stability of the proteins, with the sample made with diFIle being particularly prone to precipitation. 19F resonance assignments were obtained by comparison of the chemical shifts of γ1-protons with those observed in the wild-type protein. Non-uniform cross-peak intensities observed in short-delay 1H,19F correlation experiments suggest incomplete averaging of 3JHF couplings and therefore preferential rotamer populations of the CH2F and CHF2 groups.

The online version contains supplementary material available at 10.1007/s10858-026-00488-z.