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 detec-tion 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 incor-porated 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 in-vestigating protein structure, dynamics, and interactions in mammalian cells by DNP MAS NMR.
Movellan et al. (Sat,) studied this question.
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