Endogenous Site-Specific Encoding of Trifluoromethyl-Bearing Phenylalanine and Tryptophan for in-Cell ¹⁹ F NMR

High Resolution Image Download MS PowerPoint Slide Understanding protein structure, dynamics, and interactions in live mammalian cells is essential for elucidating cellular mechanisms in health and disease. Here, we report genetic code expansion (GCE) systems that enable efficient site-specific incorporation of trifluoromethylphenylalanine (tfmF) and trifluoromethyltryptophan (tfmW) into mammalian proteins. While tfmF has previously been encoded in E. coli for electroporation-based in-cell 19 F NMR, we establish the first system for direct tfmF encoding in mammalian cells. Moreover, we developed entirely new GCE tools for tfmW, enabling its incorporation in both E. coli and mammalian cells, the first report of tfmW encoding for 19 F NMR. Using these systems, we expressed fluorinated cyclophilin A in HEK293T cells, compared the sensitivity of the in-cell NMR spectra with those obtained by electroporation, and assessed cyclosporin A binding. This work establishes the first mammalian-cell expression of tfm-labeled proteins, expanding the toolkit of fluorine probes for in-cell NMR.