Visualizing Proteins in Mammalian Cells by 19F NMR Spectroscopy

Abstract In‐cell NMR spectroscopy is a powerful tool to investigate protein behavior in physiologically relevant environments. Although proven valuable for disordered proteins, we show that in commonly used 1 H‐ 15 N HSQC spectra of globular proteins, interactions with cellular components often broaden resonances beyond detection. This contrasts 19 F spectra in mammalian cells, in which signals are readily observed. Using several proteins, we demonstrate that surface charges and interaction with cellular binding partners modulate linewidths and resonance frequencies. Importantly, we establish that 19 F paramagnetic relaxation enhancements using stable, rigid Ln(III) chelate pendants, attached via non‐reducible thioether bonds, provide an effective means to obtain accurate distances for assessing protein conformations in the cellular milieu.