Structure of Circadian Clock Complexes from Cyanobacteria by Three Dimensional EM

生物钟是一种自我维持的生物化学振荡器，它构成了 睡眠/清醒、代谢活动、基因表达和许多其他生物过程。他们的 特性包括温度补偿、大约24小时的时间常数以及 精确度高。这些特性很难用已知的生化反应来解释。这个 对这些不寻常的振荡器的机制的最终解释将需要描述 生物钟分子组成的结构、功能和相互作用。这个 已知的最简单的呈现昼夜节律现象的细胞是原核蓝藻。 遗传和生化研究已经确定了三种关键的时钟蛋白，KaiA，Kaib和KaiC 在蓝藻细长聚球藻中。这三种蛋白质加上三磷酸腺苷都有能力 在体外重建与24小时周期平行的磷酸化/去磷酸化循环 观察活体内的全球基因调控。这种体外生物钟振荡器是目前最好的。 生物钟的结构和生物物理分析系统。初步电子 显微镜(EM)数据表明，在细胞内发生了大量的构象变化 Kaia-Kaib-KaiC分子振荡器，因此三维电磁非常适合于结构 对这一制度进行了分析。该提案的具体目标是1)对以下各项进行低温EM评估 Kaib/KaiC复合体的三种形式与KaiC的突变形式，以及2)确定 选定的Kaib/KaiC络合物的亚纳米分辨率(&lt；10？)低温EM结构。 描述了允许昼夜节律的钟表随着24小时振荡的分子机制 小时周期对于人类理解蓝藻的昼夜节律是必不可少的。详细 蓝藻核心振荡器的结构分析将提供机制 在受控制的蛋白质-蛋白质关联之下，似乎驱动着这种独特的发条。 这项提议的长期目标是应用混合方法，包括三维电磁 以表征在生物化学过程中形成的超分子蛋白质复合体的结构和功能 蓝藻昼夜振荡周期。

Circadian clocks are self-sustained biochemical oscillators that underlie daily rhythms of sleep/waking, metabolic activity, gene expression, and many other biological processes. Their properties include temperature compensation, a time constant of approximately 24 hours, and high precision. These properties are difficult to explain by known biochemical reactions. The ultimate explanation for the mechanism of these unusual oscillators will require characterizing the structures, functions, and interactions of the molecular components of circadian clocks. The simplest cells that are known to exhibit circadian phenomena are the prokaryotic cyanobacteria. Genetic and biochemical studies have identified three key clock proteins, KaiA, KaiB, and KaiC in the cyanobacterium Synechococcus elongatus. These three proteins plus ATP are competent to reconstitute a phosphorylation / dephosphorylation cycle in vitro that parallels the 24 hour cycles observed for global gene regulation in vivo. This in vitro circadian oscillator is the best available system for structural and biophysical analyses of a circadian clockwork. Preliminary electron microscopy (EM) data suggests that numerous and large conformational changes occur within the KaiA-KaiB-KaiC molecular oscillator, thus three-dimensional EM is well suited for structural analysis of this system. The specific aims of this proposal are 1) perform a cryoEM evaluation of three forms of the KaiB/KaiC complex with mutated forms of KaiC, and 2) determine a subnanometer resolution (<10¿) cryoEM structure of the chosen KaiB/KaiC complex. Characterizing the molecular mechanisms that allow a circadian clockwork to oscillate with a 24 hour cycle is essential for understanding circadian rhythms in cyanobacteria to humans. Detailed structural analysis of the core oscillator from cyanobacteria will provide the mechanisms underlying the controlled protein-protein associations that appear to drive this unique clockwork. The long-term goal of this proposal is to apply hybrid methods including three-dimensional EM to characterize the structure and function of supramolecular protein complexes formed during the cyanobacterial circadian oscillation cycle.