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Abstract Fluorine‐19 NMR spectroscopy has emerged as a powerful tool for studying protein structure, dynamics, and interactions. Of particular interest is the exploitation of trifluoromethyl (tfm) groups, given their high sensitivity and superior transverse relaxation properties, compared to single fluorine atoms. However, biosynthetic incorporation of tfm‐bearing amino acids remains challenging due to cytotoxicity and incompatibility with natural tRNA synthetases. Here, we report on overcoming this challenge using cell‐free synthesis, incorporating trifluoromethyl‐methionine (tfmM) into the protein Cyclophilin A (CypA) with remarkably high efficiency, impossible via biosynthetic means. Importantly, we demonstrate that tfmM CypA binds a native substrate, the N‐terminal domain of HIV‐1 capsid protein (HIV‐1 CA‐NTD), and retains peptidyl prolylcis/transisomerase activity. It also binds the peptide inhibitor Cyclosporine A (CsA) with the same affinity as non‐labeled, wild‐type CypA. Furthermore, we show that¹⁹F isotope shifts and¹⁹F solvent paramagnetic relaxation enhancements (PREs) provide valuable structural information on surface exposure. Taken together, our study illustrates that tfmM can be readily incorporated into proteins at very high levels by cell‐free synthesis without disturbing protein structure and function, significantly expanding the scope of¹⁹F NMR spectroscopy for studying protein structure and dynamics. 

Abstract Studies of protein structure and dynamics are usually carried out in dilute buffer solutions, conditions that differ significantly from the crowded environment in the cell. The double electron‐electron resonance (DEER) technique can track proteins’ conformations in the cell by providing distance distributions between two attached spin labels. This technique, however, cannot access distances below 1.8 nm. Here, we show that Gd^III‐¹⁹F Mims electron‐nuclear double resonance (ENDOR) measurements can cover part of this short range. Low temperature solution and in‐cell ENDOR measurements, complemented with room temperature solution and in‐cell Gd^III‐¹⁹F PRE (paramagnetic relaxation enhancement) NMR measurements, were performed on fluorinated GB1 and ubiquitin (Ub), spin‐labeled with rigid Gd^IIItags. The proteins were delivered into human cells via electroporation. The solution and in‐cell derived Gd^III‐¹⁹F distances were essentially identical and lie in the 1–1.5 nm range revealing that both, GB1 and Ub, retained their overall structure in the Gd^IIIand¹⁹F regions in the cell. 

Comparative study of the performance of different spin labels for¹⁹F electron-nuclear double resonance (ENDOR) for short-range (1.0–1.5 nm) distance measurement in proteins.