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Ultra-Photosensitization of Fluorescent Protein Chromophores towards Photocatalytic Proximity Labeling and Proteomic Profiling

ABSTRACT: Due to relatively lower reactive oxygen species (ROS) quantum yield, photosensitizing fluorescent proteins (FP) (e.g. KillerRed) have been shown not suitable for photo-catalytic protein proximity labeling and downstream proteomic profiling applications. To acquire such functions, we engineer FP chromophores in a 12×12 combinatorial matrix of synthetic analogues, yielding maximally over 1000-fold enhancement of reactive oxygen species (ROS) production. We show the ROS yield is correlated to the directionality of “push-pull” effect of FP chromophores. After conjugation to HaloTag, the ultra-photosensitized FP mimics (namely upsFP) herein realizes photo-catalytic protein proximity labeling using nucleophilic amino substrates. A set of photochemical characterizations, theoretical calculation, and tandem mass spectrometry collectively reveals radical mediated labeling with expanded reactivity towards protein residues via type I photosensitization pathway. Finally, we showcase a proteomic profiling application by 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 3 orders of magnitude ultra- photosensitization enables photocatalytic proximity labeling and profiling functions for biomimetic fluorescent proteins.

ORGANISM(S): Homo Sapiens

SUBMITTER: Lihua Zhang

PROVIDER: PXD055096 | iProX | Fri Aug 23 00:00:00 BST 2024

REPOSITORIES: iProX

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