Simultaneous Site- and Residue-Specific Incorporation of Fluorinated Amino Acids for In-Cell Structural Biology by 19F Magic Angle Spinning NMR Spectroscopy.

Magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy is uniquely positioned to yield atomic-level structural and dynamic information on large proteins and protein assemblies in cellular environments. Yet, in-cell MAS NMR applications using the most common biomolecular reporter nuclei-1H, 13C, and 15N-remain hampered by a large cellular background, inherently low sensitivity, and limited resolution. In contrast, 19F is a high-receptivity nucleus that is naturally absent in biological molecules and is an excellent probe for in-cell MAS NMR. Here, we report a 19F-labeling strategy that combines site-specific incorporation of 4-(trifluoromethyl)-l-phenylalanine (tfmF) via genetic code expansion with fluorination of all tryptophan residues at the 7 position of the indole ring. We apply this approach to the N-terminal domain of the nucleocapsid protein from SARS-CoV-2, NNTD. For a microcrystalline 7F-Trp,tfmF28,U-15N-NNTD sample, 19F-19F correlations between the CF3 group of the tfmF and the F atoms of the three 7F-Trp residues are detected in 2D dipolar-based experiments, spanning a distance range from 10 to 21 Å. Additionally, for 7F-Trp,tfmF28,U-15N-NNTD electroporated into A2780 cells, the increased spectral resolution and sensitivity afforded by the incorporation of the CF3 group enable the acquisition of high-quality 19F in-cell MAS NMR spectra within a few hours. The simultaneous labeling with an aromatic fluorine atom and a CF3 group expands the toolbox of 19F MAS NMR for cellular structural biology.