Structure of Circadian Clock Complexes from Cyanobacteria by Three Dimensional EM

通过三维电镜研究蓝藻生物钟复合物的结构

• 批准号: 7924201
• 负责人: MARTIN EGLI
• 金额: $ 29.48万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7924201
• 关键词: Apical; Architecture; Binding; Binding Sites; Biochemical; Biochemical Reaction; Biological Clocks; Biological Process; C-terminal; Cells; Chromosomes; Chronotherapy; Circadian Rhythms; Cleaved cell; Clock protein; Complex; Cryoelectron Microscopy; Cyanobacterium; Data; Docking; Electron Microscopy; Event; Exhibits; Financial compensation; Gene Expression; Gene Expression Regulation; Genetic; Genetic Transcription; Genome; Goals; Health; Hormones; Hour; Human; Hybrids; In Vitro; Lead; Lipids; Macromolecular Complexes; Mental Health; Mental disorders; Metabolic; Methods; Modeling; Molecular; Mutation; Organism; Output; Peptides; Performance; Periodicity; Phosphorylation; Physiology; Property; Protein Dephosphorylation; Proteins; Resolution; Running; Signal Transduction; Site; Sleep; Sleeplessness; Solutions; Structure; Synechococcus; System; Systems Analysis; Techniques; Temperature; Time; Tissues; alertness; circadian pacemaker; depressive symptoms; flexibility; in vivo; insight; particle; promoter; protein complex; public health relevance; reconstitution; reconstruction; residence; seal; stoichiometry

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.

昼夜节律是自我维持的生化振荡器，是每日节奏的基础 睡眠/醒来，代谢活性，基因表达和许多其他生物学过程。 特性包括温度补偿，大约24小时的时间常数，以及 高精度。这些特性很难通过已知的生化反应来解释。这 这些异常振荡器机制的最终解释将需要表征 昼夜节律时钟分子成分的结构，功能和相互作用。这 已知表现出昼夜节律现象的最简单细胞是原核细菌。 遗传和生化研究已经鉴定出三种关键时钟蛋白，Kaia，Kaib和Kaic 在蓝细菌中，弹性菌。这三种蛋白质加ATP具有 在体外重建一个磷酸化 /去磷酸化周期，与24小时周期相似 观察到体内全球基因调节。这个体外昼夜节振荡器是最好的 昼夜节律发条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 KAIA-KAIB-KAIC分子振荡器，因此三维EM非常适合结构 该系统的分析。该提案的具体目的是1）对 KAIB/KAIC复合物具有突变形式的KAIC，2）确定A 选定的KAIB/KAIC复合物的次纳米分辨率（<10？）的冷冻结构。 表征允许昼夜节律发条以24振荡的分子机制 小时周期对于理解蓝细菌对人类的昼夜节律至关重要。详细的 来自蓝细菌的核心振荡器的结构分析将提供机制 在似乎驱动这种独特发条的受控蛋白质蛋白关联的基础上。 该提案的长期目标是应用包括三维EM在内的混合方法 表征在此期间形成的超分子蛋白复合物的结构和功能 蓝细菌昼夜节律振荡周期。