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Abstract ¹⁹F NMR spectroscopy is an attractive and growing area of research with broad applications in biochemistry, chemical biology, medicinal chemistry, and materials science. We have explored fast magic angle spinning (MAS)¹⁹F solid‐state NMR spectroscopy in assemblies of HIV‐1 capsid protein. Tryptophan residues with fluorine substitution at the 5‐position of the indole ring were used as the reporters. The¹⁹F chemical shifts for the five tryptophan residues are distinct, reflecting differences in their local environment. Spin‐diffusion and radio‐frequency‐driven‐recoupling experiments were performed at MAS frequencies of 35 kHz and 40–60 kHz, respectively. Fast MAS frequencies of 40–60 kHz are essential for consistently establishing¹⁹F–¹⁹F correlations, yielding interatomic distances of the order of 20 Å. Our results demonstrate the potential of fast MAS¹⁹F NMR spectroscopy for structural analysis in large biological assemblies. 

Obtaining atomic-level information on components in the cell is a major focus in structural biology. Elucidating specific structural and dynamic features of proteins and their interactions in the cellular context is crucial for understanding cellular processes. We introduce¹⁹F dynamic nuclear polarization (DNP) combined with fast magic-angle-spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy as a powerful technique to study proteins in mammalian cells. We demonstrate our approach on the severe acute respiratory syndrome coronavirus 2 5F-Trp-N^NTDprotein, electroporated into human cells. DNP signal enhancements of 30- to 40-fold were observed, translating into over 1000-fold experimental time savings. High signal-to-noise ratio spectra were acquired on nanomole quantities of a protein in cells in minutes. 2D¹⁹F-¹⁹F dipolar correlation spectra with remarkable sensitivity and resolution were obtained, exhibiting¹⁹F-¹⁹F cross peaks associated with fluorine atoms as far as ~10 angstroms apart. This work paves the way for¹⁹F DNP-enhanced MAS NMR applications in cells for probing protein structure, dynamics, and ligand interactions.