Cell

Biomolecule

Viability assay

Nuclear magnetic resonance spectroscopy

Stereochemistry

Biology

Fluorine-19 NMR

Biophysics

Engineering

Heteronuclear single quantum coherence spectroscopy

Endogeny

Chemistry

Sensitivity (control systems)

Electronic engineering

Metabolism

Cellular metabolism

Biochemistry

Journal of the American Chemical Society

NMR has the resolution and specificity to determine atomic-level protein structures of isotopically labeled proteins in complex environments, and with the sensitivity gains conferred by dynamic nuclear polarization (DNP), NMR has the sensitivity to detect proteins at their endogenous concentrations. However, DNP sensitivity enhancements are critically dependent on experimental conditions and sample composition. While some of these conditions are theoretically compatible with cellular viability, the effects of others on cellular sample integrity are unknown. Uncertainty about the integrity of cellular samples limits the utility of experimental outputs of in-cell experiments. Using several measures, we establish conditions that support DNP enhancements that can enable detection of micromolar concentrations of proteins in experimentally tractable times that are compatible with cellular viability. Taken together, we establish DNP-assisted MAS NMR as a technique for structural investigations of biomolecules in intact viable cells that can be phenotyped both before and after NMR experiments.

In-Cell Sensitivity-Enhanced NMR of Intact Viable Mammalian Cells