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

Naresh Kumar, Yuichiro Hori, Miyako Nishiura, Kazuya Kikuchi

Research output: Contribution to journalArticlepeer-review

5 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)3694-3701
Number of pages8
JournalChemical Science
Volume11
Issue number14
DOIs
Publication statusPublished - Apr 14 2020
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Chemistry(all)

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