HaloTag-Based Hybrid Targetable and Ratiometric Sensors for Intracellular Zinc.,ACS Chemical Biology

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HaloTag-Based Hybrid Targetable and Ratiometric Sensors for Intracellular Zinc.
ACS Chemical Biology ( IF 3.5 ) Pub Date : 2020-01-24 , DOI: 10.1021/acschembio.9b00872
Melissa L Zastrow ₁ , Zhen Huang ₁ , Stephen J Lippard ₁

Affiliation

We report a new series of small molecule-protein hybrid zinc sensors that combine genetic targetability with the spectroscopic profile of synthetic fluorophores. We functionalized the zinc sensor ZinPyr-1 (ZP1) with a chloroalkane linker (ZP1-12Cl) that reacts specifically with the engineered protein HaloTag. The resulting construct, ZP1-HaloTag, binds zinc ions with a threefold fluorescence enhancement. Through exploitation of the protein synthesis machinery of live cells, the HaloTag protein component was expressed, and the ZP1-HaloTag hybrid was assembled upon bath application of ZP1-12Cl. After fusion of HaloTag with targeting peptides or proteins, the resulting hybrid sensor could be directed to specific subcellular locales, including the nucleus, mitochondrial outer membrane, and endoplasmic reticulum. Furthermore, HaloTag was linked with the red fluorescent protein mCherry, permitting formation of a two-fluorophore system that provides not only targetable but also ratiometric sensing of cellular zinc. This system reversibly detects both exogenous and endogenous mobile Zn2+ in response to reactive nitrogen species in live HeLa cells. HaloTag-based hybrid zinc sensors offer new opportunities for visualizing and quantifying biological mobile zinc at discrete subcellular compartments.

中文翻译：

基于 HaloTag 的混合靶向和比例传感器，用于细胞内锌。

我们报告了一系列新的小分子-蛋白质混合锌传感器，它将遗传靶向性与合成荧光团的光谱特征结合起来。我们使用氯烷连接基 (ZP1-12Cl) 对锌传感器 ZinPyr-1 (ZP1) 进行功能化，该连接基与工程蛋白 HaloTag 发生特异性反应。由此产生的构建体 ZP1-HaloTag 与锌离子结合，荧光增强三倍。通过利用活细胞的蛋白质合成机制，表达 HaloTag 蛋白质成分，并在 ZP1-12Cl 浴中组装 ZP1-HaloTag 杂交体。 HaloTag 与靶向肽或蛋白质融合后，所得混合传感器可以定向到特定的亚细胞区域，包括细胞核、线粒体外膜和内质网。此外，HaloTag 与红色荧光蛋白 mCherry 连接，从而形成双荧光团系统，该系统不仅提供细胞锌的靶向性而且还提供比例传感。该系统可逆地检测外源性和内源性移动 Zn2+，以响应活 HeLa 细胞中的活性氮。基于 HaloTag 的混合锌传感器为在离散的亚细胞区室中可视化和量化生物移动锌提供了新的机会。

更新日期：2020-01-26

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