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Talanta

Talanta

IF: 5.6
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Enhanced imaging of protein-specific palmitoylation with HCR-based cis-membrane multi-FRET.

Published:19 July 2023 DOI: 10.1016/j.talanta.2023.124972 PMID: 37487269
Yixin Fu , Husun Qian , Yujun Yang , Junjie Li , Guoming Xie

Abstract

Palmitoylation plays an important role in modulating protein trafficking, stability, and activity. The major predicament in protein palmitoylation study is the lack of specific and sensitive tools to visualize protein-specific palmitoylation. Although FRET approach was explored by metabolically labeled palmitic acid and antibody recognized target protein. The trans-membrane strategy suffers from low FRET efficiency due to the donor and acceptor located at different sides of membrane. Herein, we proposed a cis-membrane multi–fluorescence resonance energy transfer (multi–FRET) for amplified visualization of specific palmitoylated proteins through metabolic labeling and targeted recognition. The azido-palmitic acid (azido-PA) was metabolically incorporated into cellular palmitoylated proteins, followed by reacting with dibenzylcylooctyne-modified Cy5 (DBCO-Cy5) through copper-free click chemistry. The protein probe was attached to targeted protein by specific peptide recognition, which initiates a hybridization chain reaction (HCR) amplification process. The cis-membrane labeling method enables effective intramolecular donor-acceptor distance and allow to increase FRET efficiency. Simultaneously, HCR amplification triggered multi–FRET phenomenon with significantly improved FRET efficiency. With the superiority, this strategy has achieved the enhanced FRET imaging of palmitoylated PD-L1 and visualizing the palmitoylation changes of on PD-L1 under drug treatment. Furthermore, the established method successfully amplified visualization of PD-L1 palmitoylation in vivo and mice tumor slice. We envision the approach would provide a useful platform to investigate the effects of palmitoylation on the protein structure and function.

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