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 1H‐15N HSQC spectra of globular proteins, interactions with cellular components often broaden resonances beyond detection. This contrasts 19F 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 19F 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.
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