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

## TL;DR

This paper introduces new methods to incorporate fluorinated amino acids into proteins in mammalian cells for NMR studies.

## Contribution

The first system for encoding trifluoromethylphenylalanine and trifluoromethyltryptophan in mammalian cells for in-cell 19F NMR.

## Key findings

- Fluorinated cyclophilin A was successfully expressed in HEK293T cells.
- In-cell NMR spectra sensitivity was compared with electroporation-based methods.
- Cyclosporin A binding was assessed using the fluorinated proteins.

## Abstract

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 19F 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 19F 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.