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

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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
Chemical Science ( IF 7.6 ) Pub Date : 2020/03/09 , DOI: 10.1039/d0sc00499e
Naresh Kumar ₁ , Yuichiro Hori _{1,

2} , Miyako Nishiura ₁ , Kazuya Kikuchi _{1,

2,

3}

Affiliation

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 (S_N2 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.

中文翻译：

使用反应性荧光配体对 PYP 标签蛋白进行快速免洗标记，为长期细胞成像研究提供稳定的荧光蛋白缀合物

使用蛋白质标签或化学探针将蛋白质转化为荧光缀合物的共价标记系统是进行实时成像和脉冲追踪研究的强大工具，可以研究蛋白质在复杂生物系统中的时空作用。_{在本研究中，我们用弱荧光 α,β-不饱和酮（共轭加成）和 α-卤甲基酮（S N} 2反应）受体共价修饰 PYP-标签蛋白的特定亲核半胱氨酸残基，以提供高荧光 PYP-标签-二甲氨基香豆素 (DMAC) 缀合物，其配体通过稳定的硫醚接头与 PYP 蛋白共价结合。研究发现，氯甲基酮衍生的 DMAC-CMK 试剂可为细胞系统中标记 PYP 标签提供最佳的动力学和稳定性特征，体外研究表明PYP-DMAC-CMK 缀合物表现出优异的光稳定性和细胞稳定性特征，使它们能够可用于细胞系统中的长期蛋白质成像研究。使用这种免洗荧光标记 PYP-tag-DMAC 系统来可视化正在进行有丝分裂的分裂细胞以及对与细胞核中染色质结合的 PYP-tag 融合端粒结合蛋白进行成像的潜力已得到证明。

更新日期：2020-04-08

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