Endogenous Site-Specific Encoding of Trifluoromethyl-Bearing Phenylalanine and Tryptophan for in-Cell 19 F NMR
Year: 2026
Type: article
Abstract: 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.
DOI: 10.1021/jacs.5c18349
Language: English
Subfield: Spectroscopy
Field: Chemistry
Domain: Physical Sciences
Open Access status: hybrid
Estimated APC paid: $4,500
Funders: National Science Foundation, American Heart Association, Oregon State University, Eusko Jaurlaritza, Euskal Herriko Unibertsitatea, National Institutes of Health, European Regional Development Fund, Agencia Estatal de Investigación, National Institute of General Medical Sciences, Division of Chemistry
Awards: G2262748287, Ideal eukaryotic tetrazine ligations for imaging protein dynamics in live cells, G3480869486, G4033406476, G425668148, G451917667, Development of Fluorine Nuclear Magnetic Resonance (NMR) Spectroscopy as a Versatile Probe of Structure and Chemical Environment in Proteins, NSF/MCB-BSF The virtues of lanthanides and fluorine for tracking in-cell protein conformation: a marriage of NMR and EPR, G5943232429, G5967599077, G6024828775, Ion Mobility Time-of-flight Mass Spectrometry System, G7084143925, G7806286829, G8266294959, The GCE4All Center: Unleashing the Potential of Genetic Code Expansion for Biomedical Research, G937381806
OpenAlex ID: W7128821997