Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA.
Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.
Nat Chem Biol. 2021 Jun;17(6):718-723. doi: 10.1038/s41589-021-00775-w. Epub 2021 Apr 1.
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.
利用荧光指示剂的功能成像技术已经彻底改变了生物学，但还需要额外的传感器支架来获取诸如明亮的远红发射等特性。在这里，我们引入了一种新的“化学生物学”荧光指示剂平台，该平台利用与环境敏感的合成荧光团偶联的自标记 HaloTag 蛋白。我们解决了 HaloTag 与罗丹明染料配体结合的晶体结构，以指导工程努力来调节染料环境。我们表明，HaloTag 与在结合染料附近发生构象变化的蛋白质传感器结构域融合会导致荧光输出的大幅快速变化。这种可推广的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性质，可可靠地检测培养神经元中的单个动作电位。
