Pushing Sensitivity and Specificity Limits in Native Structural Biology: F 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 F-based, 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 H-F cross-polarization (CP), with subsequent F-C double CP providing unique structural information. Remarkably, using H-F-C magnetization transfer allowed the selective detection of 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.