1000 fold Ultra‐Photosensitized Fluorescent Protein Mimics Toward Photocatalytic Proximity Labeling and Proteomic Profiling Functions

Abstract Photosensitizing fluorescent proteins (FP) (e.g. KillerRed) have been shown not capable of photo‐catalytic protein proximity labeling for downstream proteomic profiling applications. To acquire such a function, FP chromophores are engineered in a 12 × 12 combinatorial matrix of synthetic analoges, achieving up to 1000 fold enhancement of reactive oxygen species (ROS) production compared to the natural FPs. FP chromophores are shown with larger dipole moments exhibit higher ROS yield toward protein labeling. By conjugating the ultra‐photosensitized FP chromophore to HaloTag (namely upsFP tag), its photo‐catalytic protein proximity labeling function is demonstrated using nucleophilic amino substrates. Through photochemical characterizations, theoretical calculation, and tandem mass spectrometry, a radical‐mediated labeling mechanism is revealed with expanded reactivity toward diverse protein residues via a type I photosensitization pathway. Finally, a proteomic profiling application is showcased using the upsFP tag to resolve the dynamic interactome variations upon TAR DNA‐binding protein 43 (TDP43) phase separation and suborganellar translocation. Together, this work demonstrates three orders of magnitude ultra‐photosensitization of fluorescent protein chromophore enables photocatalytic protein proximity labeling and profiling functions that are impractical for natural fluorescent proteins.