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

ABSTRACT 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 19 F-based, 1 H-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 1 H- 19 F cross-polarization (CP), with subsequent 19 F- 13 C double CP providing unique structural information. Remarkably, using 1 H- 19 F 13 C magnetization transfer allowed selective detection of 13 C 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.