Structure of Circadian Clock Complexes from Cyanobacteria by Three Dimensional EM

昼夜节律钟是自我维持的生化振荡器，其是生物钟的每日节律的基础。 睡眠/清醒、代谢活动、基因表达和许多其他生物过程。他们的 性能包括温度补偿，约24小时的时间常数， 高精度这些性质很难用已知的生物化学反应来解释。的 对这些不寻常的振荡器的机制的最终解释将需要描述 生物钟的分子组成部分的结构、功能和相互作用。的 已知表现出昼夜节律现象的最简单的细胞是原核蓝细菌。 遗传和生物化学研究已经确定了三个关键的时钟蛋白，KaiA，KaiB和KaiC 在蓝细菌细长聚球藻中。这三种蛋白质加上ATP能够 在体外重建与24小时循环平行的磷酸化/去磷酸化循环 在体内观察到全局基因调控。这种体外昼夜节律振荡器是目前最好的 用于昼夜节律时钟装置的结构和生物物理分析的系统。初级电子 显微镜（EM）数据表明，许多和大的构象变化发生在 KaiA-KaiB-KaiC分子振荡器，因此三维EM非常适合于结构 分析这个系统。该提案的具体目标是：1）对以下各项进行cryoEM评价： 三种形式的KaiB/KaiC复合物与突变形式的KaiC，和2）确定一个 所选KaiB/KaiC复合物的亚纳米分辨率（<10）cryoEM结构。 描述了允许昼夜节律时钟以24秒振荡的分子机制 小时周期对于理解蓝藻对人类的昼夜节律至关重要。详细 从蓝藻的核心振荡器的结构分析将提供的机制 这是控制蛋白质-蛋白质关联的基础，似乎是驱动这种独特的发条装置的基础。 该提案的长期目标是应用包括三维EM在内的混合方法 以表征超分子蛋白质复合物的结构和功能， 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.