Fluorescence Correlation Spectroscopy to Monitor Kai Protein-based Circadian Oscillations in Real Time
Fluorescence Correlation Spectroscopy to Monitor Kai Protein-based Circadian Oscillations in Real Time
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DOI：
10.1074/jbc.m111.265777
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发表时间：
2012-01-27
影响因子：
4.8
通讯作者：
Oyama, Tokitaka
中科院分区：
文献类型：
--
作者：
Goda, Kazuhito;Ito, Hiroshi;Oyama, Tokitaka
动态蛋白-蛋白相互作用在细胞调控系统中起着重要作用。蓝藻生物钟是一个振荡系统，可以在体外通过混合ATP和三种时钟蛋白：KaiA， KaiB和KaiC来重建。KaiB与含KaiC复合物的结合和解离对KaiC的昼夜磷酸化和去磷酸化至关重要。我们开发了一种自动化、无创的方法，利用共聚焦荧光相关光谱（FCS）实时监测动态复合物的形成，并均匀标记KaiB作为探针。用于FCS测量的标记的KaiB的纳摩尔浓度不会干扰振荡系统，但在测量期间（50 - 5天）表现与野生型相似。荧光探针在反复激光照射下稳定。作为一项应用，我们表明该检测系统允许分析同一样品中长期昼夜节律振荡和对温度变化（类似于10分钟）的短期响应的动态。这表明，在高温脉冲刺激后，由于KaiB与KaiC复合物的解离，时钟发生了相移。这种监测方法将提高我们对这种细胞昼夜节律振荡器的机制的理解，并提供一种评估生物系统中动态蛋白质相互作用的方法，其特征是与Kai蛋白观察到的相似。
Dynamic protein-protein interactions play an essential role in cellular regulatory systems. The cyanobacterial circadian clock is an oscillatory system that can be reconstituted in vitro by mixing ATP and three clock proteins: KaiA, KaiB, and KaiC. Association and dissociation of KaiB from KaiC-containing complexes are critical to circadian phosphorylation and dephosphorylation of KaiC. We developed an automated and noninvasive method to monitor dynamic complex formation in real time using confocal fluorescence correlation spectroscopy (FCS) and uniformly labeled KaiB as a probe. A nanomolar concentration of the labeled KaiB for FCS measurement did not interfere with the oscillatory system but behaved similarly to the wild-type one during the measurement period (>5 days). The fluorescent probe was stable against repeated laser exposure. As an application, we show that this detection system allowed analysis of the dynamics of both long term circadian oscillations and short term responses to temperature changes (similar to 10 min) in the same sample. This suggested that a phase shift of the clock with a high temperature pulse occurred just after the stimulus through dissociation of KaiB from the KaiC complex. This monitoring method should improve our understanding of the mechanisms underlying this cellular circadian oscillator and provide a means to assess dynamic protein interactions in biological systems characterized by rates similar to those observed with the Kai proteins.