Elucidating the ticking of an in vitro circadian clockwork.
Elucidating the ticking of an in vitro circadian clockwork.
复制标题
阐明体外昼夜节律发条的滴答作响。
DOI：
10.1371/journal.pbio.0050093
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发表时间：
2007-04
期刊：
影响因子：
9.8
通讯作者：
Johnson, Carl Hirschie
中科院分区：
文献类型：
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作者：
Mori, Tetsuya;Williams, Dewight R;Byrne, Mark O;Qin, Ximing;Egli, Martin;Mchaourab, Hassane S;Stewart, Phoebe L;Johnson, Carl Hirschie
生化振荡器可以在体外用三种纯化的蛋白质重组，表现出昼夜节律的显著特性，包括自我维持的24小时周期，这是温度补偿的。我们通过电子显微镜和自然凝胶电泳法定量三种蛋白(KaIA、Kaib和KaiC)之间的相互作用，分析了这种振荡器的生化基础，以阐明KAI蛋白之间形成复合体的时间。这些数据被用来推导出体外振荡器的动力学模型，该模型准确地再现了KaiABC复合体和KaiC磷酸化的节奏，并与对单个KAI蛋白相互作用的生物物理观察一致。我们用荧光共振能量转移(FRET)证实了KaiC六聚体之间发生了单体交换。该模型表明，这种单体交换的功能可能是在反应中保持KaiC六聚体之间的同步性，从而维持高幅度的振荡。最后，我们对一个体外振荡器进行了第一次微扰分析，利用温度脉冲来重置KaiABC振荡器的相位，从而测试了这种独特的昼夜节律振荡器的重置特性。这项研究分析了生物钟的分子细节，达到了前所未有的水平。生物钟存在于从细菌到人类的各种生物体中。生物钟的一个核心功能是控制对每日光暗周期的适应性反应。因此，改变时钟(例如，通过时差或轮班工作)会影响人类的心理和身体健康。人们普遍认为昼夜节律振荡的潜在分子机制是一个自我调节的转录/翻译反馈环。然而，在蓝藻中，只有三个纯化的Clock蛋白可以在试管中(体外)重建蛋白质磷酸化的昼夜节律。利用这个体外系统，我们发现这三种蛋白质在整个周期中相互作用，形成不同组成的复合体。我们为体外振荡器推导了一个动力学模型，该模型准确地再现了复合体和蛋白质磷酸化的节律。其中一种蛋白质经历了其单体的相变交换，该模型表明这种单体交换允许维持强劲的振荡。最后，我们用温度脉冲来扰动体外振荡器，以演示这种独特的昼夜节律振荡器的重置特性。我们的研究分析了生物钟的分子细节，达到了前所未有的水平。从蓝藻生物钟衍生的三启蛋白体外振荡器的相互作用动力学被测定到前所未有的分子细节水平。
A biochemical oscillator can be reconstituted in vitro with three purified proteins, that displays the salient properties of circadian (daily) rhythms, including self-sustained 24-h periodicity that is temperature compensated. We analyze the biochemical basis of this oscillator by quantifying the time-dependent interactions of the three proteins (KaiA, KaiB, and KaiC) by electron microscopy and native gel electrophoresis to elucidate the timing of the formation of complexes among the Kai proteins. The data are used to derive a dynamic model for the in vitro oscillator that accurately reproduces the rhythms of KaiABC complexes and of KaiC phosphorylation, and is consistent with biophysical observations of individual Kai protein interactions. We use fluorescence resonance energy transfer (FRET) to confirm that monomer exchange among KaiC hexamers occurs. The model demonstrates that the function of this monomer exchange may be to maintain synchrony among the KaiC hexamers in the reaction, thereby sustaining a high-amplitude oscillation. Finally, we apply the first perturbation analyses of an in vitro oscillator by using temperature pulses to reset the phase of the KaiABC oscillator, thereby testing the resetting characteristics of this unique circadian oscillator. This study analyzes a circadian clockwork to an unprecedented level of molecular detail. Circadian biological clocks are present in a diverse range of organisms, from bacteria to humans. A central function of circadian clocks is controlling the adaptive response to the daily cycle of light and darkness. As such, altering the clock (e.g., by jet lag or shiftwork) affects mental and physical health in humans. It has generally been thought that the underlying molecular mechanism of circadian oscillations is an autoregulatory transcriptional/translational feedback loop. However, in cyanobacteria, only three purified clock proteins can reconstitute a circadian rhythm of protein phosphorylation in a test tube (in vitro). Using this in vitro system we found that the three proteins interact to form complexes of different compositions throughout the cycle. We derived a dynamic model for the in vitro oscillator that accurately reproduces the rhythms of complexes and of protein phosphorylation. One of the proteins undergoes phase-dependent exchange of its monomers, and the model demonstrates that this monomer exchange allows the maintenance of robust oscillations. Finally, we perturbed the in vitro oscillator with temperature pulses to demonstrate the resetting characteristics of this unique circadian oscillator. Our study analyzes a circadian clockwork to an unprecedented level of molecular detail. The interaction dynamics of the three-Kai-protein in vitro oscillator derived from the cyanobacterial circadian clock were determined to an unprecedented level of molecular detail.