The HaloTag as a general scaffold for far-red tunable chemigenetic indicators
The HaloTag as a general scaffold for far-red tunable chemigenetic indicators
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DOI：
10.1038/s41589-021-00775-w
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发表时间：
2021-04-01
影响因子：
14.8
通讯作者：
Schreiter, Eric R.
中科院分区：
文献类型：
--
作者：
Deo, Claire;Abdelfattah, Ahmed S.;Schreiter, Eric R.
使用荧光指示剂的功能成像彻底改变了生物学，但需要额外的传感器支架才能获得明亮的远红发射等特性。在这里，我们引入了一个新的“化学遗传学”荧光指示剂平台，利用与环境敏感的合成荧光团缀合的自标记 HaloTag 蛋白。我们解析了与罗丹明染料配体结合的 HaloTag 的晶体结构，以指导调节染料环境的工程工作。我们表明，HaloTag 与蛋白质传感器域的融合会在结合染料附近发生构象变化，从而导致荧光输出发生大而快速的变化。这种通用方法提供了明亮的远红钙和电压传感器，具有高度可调的光物理和化学特性，可以可靠地检测培养神经元中的单个动作电位。
Functional imaging using fluorescent indicators has revolutionized biology, but additional sensor scaffolds are needed to access properties such as bright, far-red emission. Here, we introduce a new platform for 'chemigenetic' fluorescent indicators, utilizing the self-labeling HaloTag protein conjugated to environmentally sensitive synthetic fluorophores. We solve a crystal structure of HaloTag bound to a rhodamine dye ligand to guide engineering efforts to modulate the dye environment. We show that fusion of HaloTag with protein sensor domains that undergo conformational changes near the bound dye results in large and rapid changes in fluorescence output. This generalizable approach affords bright, far-red calcium and voltage sensors with highly tunable photophysical and chemical properties, which can reliably detect single action potentials in cultured neurons.