Excited-State Intramolecular Proton Transfer Enables Self-Reporting Fluorescent Photoaffinity Labeling in Live Cells

Area of Science:

• Biochemistry
• Chemical Biology
• Molecular Biology

Background:

• Photoaffinity labeling (PAL) is crucial for studying transient biomolecular interactions.
• Conventional PAL methods often involve reactive intermediates causing non-specific reactions and varied cross-linked products.

Purpose of the Study:

• To develop a novel photoaffinity labeling (PAL) strategy addressing limitations of existing methods.
• To introduce a self-reporting and spatially precise PAL platform for mapping intracellular interactions.

Main Methods:

• Utilized excited-state intramolecular proton transfer (ESIPT) chemistry based on 2-(2'-hydroxyphenyl)benzimidazole (HBI).
• Employed HBI for picosecond enol-to-keto tautomerization to generate a transient Michael-acceptor electrophile.
• Validated the platform through chemoselective cysteine capture and in situ reporting via keto-band emission.

Main Results:

• Demonstrated intrinsic spatial fidelity and reduced solvent quenching due to reversible ground-state return in the absence of nucleophiles.
• Successfully applied the platform for proximity-directed capture of a formylglycine-generating enzyme (FGE)/substrate pair.
• Performed de novo interactome profiling of tau K18-P301L, identifying eight cellular effectors, including four novel ones.

Conclusions:

• ESIPT photochemistry offers a minimally perturbing, self-reporting PAL platform for intracellular interaction mapping.
• The developed method enhances spatial fidelity and reduces non-specific labeling compared to conventional techniques.
• This platform provides a powerful tool for investigating complex cellular interaction networks in native environments.