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.