Expanding the tool box for native structural biology: ¹⁹F dynamic nuclear polarization with fast magic angle spinning

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 introduce ¹⁹F 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^NTD protein, 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. 2D ¹⁹F-¹⁹F dipolar correlation spectra with remarkable sensitivity and resolution were obtained, exhibiting ¹⁹F-¹⁹F cross peaks associated with fluorine atoms as far as ~10 angstroms apart. This work paves the way for ¹⁹F DNP-enhanced MAS NMR applications in cells for probing protein structure, dynamics, and ligand interactions.