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Rapid no-wash labeling of PYP-tag proteins with reactive fluorogenic ligands affords stable fluorescent protein conjugates for long-term cell imaging studies

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Abstract

Covalent labeling systems that employ protein-tags or chemical probes to convert proteins into fluorescent conjugates are powerful tools for carrying out real time imaging and pulse-chase tracking studies that enable the spatiotemporal role of proteins in complex biological systems to be investigated. In this study, we have covalently modified a specific nucleophilic cysteine residue of the PYP-tag protein with weakly fluorescent α,β-unsaturated ketone (conjugate addition) and α-halomethyl ketone (SN2 reaction) acceptors to afford highly fluorescent PYP-tag-dimethylaminocoumarin (DMAC) conjugates, whose ligands are covalently bound to the PYP-protein through stable thioether linkers. A chloromethylketone derived DMAC-CMK reagent was found to afford the best kinetic and stability profile for labeling the PYP-tag in cellular systems, with in vitro studies demonstrating that PYP-DMAC-CMK conjugates exhibit excellent photostability and cellular stability profiles which enables them to be used for long-term protein imaging studies in cellular systems. The potential of using this no wash fluorescent labeling PYP-tag-DMAC system to visualise dividing cells undergoing mitosis and for imaging a PYP-tag fused telomere binding protein bound to chromatin in cell nuclei has been demonstrated.

Graphical abstract: Rapid no-wash labeling of PYP-tag proteins with reactive fluorogenic ligands affords stable fluorescent protein conjugates for long-term cell imaging studies

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Article information


Submitted
28 Jan 2020
Accepted
07 Mar 2020
First published
09 Mar 2020

This article is Open Access
All publication charges for this article have been paid for by the Royal Society of Chemistry

Chem. Sci., 2020, Advance Article
Article type
Edge Article

Rapid no-wash labeling of PYP-tag proteins with reactive fluorogenic ligands affords stable fluorescent protein conjugates for long-term cell imaging studies

N. Kumar, Y. Hori, M. Nishiura and K. Kikuchi, Chem. Sci., 2020, Advance Article , DOI: 10.1039/D0SC00499E

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