Real-Time In Vitro Fluorescence Anisotropy of the Cyanobacterial Circadian Clock
Real-Time In Vitro Fluorescence Anisotropy of the Cyanobacterial Circadian Clock
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DOI：
10.3390/mps2020042
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发表时间：
2019-06-01
影响因子：
2.4
通讯作者：
LiWang, Andy
中科院分区：
文献类型：
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作者：
Heisler, Joel;Chavan, Archana;LiWang, Andy
独一无二的是，蓝藻的生物钟可以在活细胞的复杂环境之外重建，大大简化了对正常工作的生物计时器的研究。核心振荡器成分只由三种蛋白质组成，KaIA、Kaib和KaiC，它们与ATP一起经历组装和拆解波，驱动KaiC的磷酸化节奏。通常，这些反应的时间点是事后通过变性聚丙烯酰胺凝胶电泳法分析的，因为这项技术解决了KaiC磷酸化的不同状态。在这里，我们描述了一种更灵敏的方法，它允许实时监测时钟反应。通过用荧光团标记其中一个时钟蛋白，在这种情况下是Kaib，体外时钟反应可以通过几周的分钟时间尺度上的荧光各向异性来监测。
Uniquely, the circadian clock of cyanobacteria can be reconstructed outside the complex milieu of live cells, greatly simplifying the investigation of a functioning biological chronometer. The core oscillator component is composed of only three proteins, KaiA, KaiB, and KaiC, and together with ATP they undergo waves of assembly and disassembly that drive phosphorylation rhythms in KaiC. Typically, the time points of these reactions are analyzed ex post facto by denaturing polyacrylamide gel electrophoresis, because this technique resolves the different states of phosphorylation of KaiC. Here, we describe a more sensitive method that allows real-time monitoring of the clock reaction. By labeling one of the clock proteins with a fluorophore, in this case KaiB, the in vitro clock reaction can be monitored by fluorescence anisotropy on the minutes time scale for weeks.