High‐Efficiency Trifluoromethyl‐Methionine Incorporation into Cyclophilin A by Cell‐Free Synthesis for ¹⁹F NMR Studies

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 prolyl cis / trans isomerase activity. It also binds the peptide inhibitor Cyclosporine A (CsA) with the same affinity as non‐labeled, wild‐type CypA. Furthermore, we show that 19 F isotope shifts and 19 F 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 of 19 F NMR spectroscopy for studying protein structure and dynamics.