Tertiary Structures of Circadian Clock Proteins by NMR

通过 NMR 观察昼夜节律钟蛋白的三级结构

• 批准号: 6419683
• 负责人: Andy LiWang
• 金额: $ 21.83万
• 依托单位: TEXAS A&M UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6419683
• 关键词: bacteria; bacterial genetics; bacterial proteins; chemical chain length; circadian rhythms; computer simulation; gene expression; genetic regulation; hydrogen bond; intermolecular interaction; model design /development; molecular assembly /self assembly; molecular dynamics; molecular shape; molecular site; nuclear magnetic resonance spectroscopy; physical model; point mutation; protein purification; protein sequence; protein structure function; radiotracer; site directed mutagenesis; structural biology; transcription factor

DESCRIPTION: (provided by applicant) The physiology and behavior of virtually all organisms oscillate with a periodicity of approximately 24 h, which is now known to be the result of an endogenous oscillator called the circadian clock. All circadian clocks studied thus far are based on a common design composed of a transcription/translation feedback loop of clock genes and clock proteins. The activity of a clock protein is modulated by its interactions with other clock proteins and these protein-protein interactions play an important role in the periodicity of the circadian rhythm. As no high-resolution structure of a circadian clock protein has been solved, no firm structural predictions can be made as to how clock proteins interact and, therefore, how their interactions help achieve a normal (or abnormal) circadian periodicity of the oscillator. As a result, the mechanism of the circadian pacemaker at the structural level is still unclear. It is proposed here that significant advances in understanding the structural basis of circadian time keeping can be achieved by investigating the three-dimensional structures and dynamics of the clock proteins of Synechococcus elongatus, SasA, KaiA, KaiB and KaiC using nuclear magnetic resonance spectroscopy (NMR). The specific aims are to solve the structures of these clock proteins by NMR and then elucidate the protein-protein interactions central to setting the pace of the circadian rhythm. The data base of genetic and biochemical information on the function of the circadian clock of S. elongatus is quite extensive and, if mapped onto the three dimensional architecture of the circadian clock, will allow insights into the molecular mechanism of a circadian pacemaker. For example, whether residues E103, R249 and E274 of KaiA, whose mutation alter the circadian rhythm, are clustered together on the surface or make important hydrogen bonds in the core of the protein, are important questions of the research proposed here. If the residues are clustered together on the surface, NMR studies will ascertain whether they form part of the dimer interface with KaiB, KaiC, and/or SasA. If the residues are buried in the core of the protein, comparisons of the E1O3K, R249H and E274H mutants with wild-type KaiA will reveal aspects of the structures and dynamics that are important to the function of KaiA. The long-range objective is to eventually investigate clock protein complexes by NMR in an effort to understand how specific protein-protein interactions modulate circadian rhythms. Key questions of our long-range objectives are whether interactions between KaiA, KaiB, SasA, and KaiC involve allostery, localized changes in backbone or side chain dynamics, displacement of tightly bound water molecules or rearrangement of crucial hydrogen bonds, and how they play important roles in setting the pace of the circadian rhythm. Although some of the conclusions resulting from the work proposed here will be specific to S. elongatus, others, should have universal ramifications for all organisms, including humans.

描述：（由申请人提供）虚拟的生理和行为 所有生物体都以大约 24 小时的周期振荡，现在是 已知是称为生物钟的内源性振荡器的结果。 迄今为止研究的所有生物钟均基于以下共同设计： 时钟基因和时钟蛋白的转录/翻译反馈环。 时钟蛋白的活性通过其与其他蛋白的相互作用来调节 时钟蛋白和这些蛋白质-蛋白质相互作用在 昼夜节律的周期性。由于没有高分辨率结构 生物钟蛋白已被解决，但无法做出确切的结构预测 关于时钟蛋白如何相互作用以及它们的相互作用如何 帮助实现振荡器的正常（或异常）昼夜节律周期性。作为 由此可见，昼夜节律起搏器在结构层面的作用机制是 仍不清楚。 这里建议在理解结构性方面取得重大进展 昼夜节律计时的基础可以通过研究来实现 时钟蛋白的三维结构和动力学 使用核磁分析细长聚球藻、SasA、KaiA、KaiB 和 KaiC 共振光谱（NMR）。具体目标是解决 通过 NMR 分析这些时钟蛋白，然后阐明蛋白质-蛋白质相互作用 对于设定昼夜节律的节奏至关重要。遗传数据库 以及有关 S 生物钟功能的生化信息。 elongatus 相当广泛，如果映射到三维 生物钟的结构，将有助于深入了解分子 昼夜节律起搏器的机制。例如，残基E103、R249是否 KaiA的E274和E274，其突变改变了昼夜节律，聚集在一起 在表面聚集或在核心形成重要的氢键 蛋白质，是这里提出的研究的重要问题。如果残留 在表面聚集在一起，核磁共振研究将确定它们是否 与 KaiB、KaiC 和/或 SasA 形成二聚体界面的一部分。如果残留 埋藏在蛋白质的核心，E1O3K、R249H 和 具有野生型 KaiA 的 E274H 突变体将揭示其结构和 对 KaiA 功能很重要的动态。 长期目标是最终研究时钟蛋白复合物 通过 NMR 来了解特定的蛋白质-蛋白质相互作用 调节昼夜节律。我们长期目标的关键问题是 KaiA、KaiB、SasA 和 KaiC 之间的相互作用是否涉及变构， 主链或侧链动力学的局部变化，紧密的位移 结合水分子或关键氢键的重排，以及它们如何 在设定昼夜节律的节奏方面发挥着重要作用。虽然有些 这里提出的工作得出的结论将针对 S. elongatus 等应该对所有生物体产生普遍影响， 包括人类。