High-Performance 19F-Tags in Detecting Protein “Hot Spots” in Live Mammalian Cells

The understanding of weak protein interactions in native intracellular environments remains challenging due to macromolecular crowding, transient associations, and the highly heterogeneous chemical and spatial landscape of live cells. In-cell 19F NMR has been shown to be a promising tool for the characterization of intracellular interactions and dynamics of proteins, but the quality and sensitivity of 19F-reporters in detecting intracellular protein interactions remain largely unexplored. Here, we systematically designed and synthesized ten 19F-tags (T1−T10) with diverse hydrophobicity and rigidity. The 19F-tag was site-specifically installed in ubiquitin via thiol conjugation chemistry. We then examined the protein−19F-tag conjugates across various crowding media including chemical reagents, proteins, and cell lysates. Our findings reveal that the chemical structure of 19F-tags is critical for accurately reflecting the intrinsic behavior of the target protein. Unexpectedly, certain 19F-tags themselves, despite their small sizes, tend to interact transiently with commonly used crowding media (such as BSA), thereby confounding authentic protein interactions. Crucially, the live-cell NMR assay demonstrated that the 19F-tag T8 (3-chloro-2-(phenylsulfonyl)-5-(trifluoromethyl)pyridine) (PSPy-m3F), featuring a shorter linker tether and a balanced amphiphilic profile, serves as an optimal 19F NMR reporter. It effectively resolves ubiquitin “hot spots” that are highly sensitive to weak intracellular interactions, providing a useful high-resolution tool for in-cell structural biology.