Pushing Sensitivity and Specificity Limits in Native Structural Biology: 19F Multinuclear Dynamic Nuclear Polarization with Magic Angle Spinning

Understanding protein structures and their interactions within natural cellular environments is essential for deciphering cellular processes and advancing therapeutic development. Obtaining atomic-level information about protein structural changes in cellular contexts poses a significant challenge. Here, we introduce a ¹⁹F-based, ¹H-assisted dynamic nuclear polarization (DNP) magic angle spinning (MAS) NMR approach that offers exceptionally high sensitivity and specificity, enabling background-free detection of target proteins in mammalian cells for atomic-level structural analysis. We demonstrate this methodology in A2780 cells for the human Cyclophilin A (CypA) protein, with a single fluorine atom incorporated in the sole tryptophan residue. We achieved significant sensitivity gains through ¹H–¹⁹F cross-polarization (CP), with subsequent ¹⁹F–¹³C double CP providing unique structural information. Remarkably, using ¹H–¹⁹F–¹³C magnetization transfer allowed the selective detection of ¹³C signals from CypA residues up to 6 Å away from the fluorine label. Taken together, our study establishes a framework for investigating protein structure, dynamics, and interactions in mammalian cells by DNP MAS NMR.