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: 6719590
• 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/6719590
• 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：(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 已知是一种名为生物钟的内源振荡器的结果。 到目前为止研究的所有生物钟都是基于一个共同的设计，该设计包括 时钟基因和时钟蛋白的转录/翻译反馈环。 时钟蛋白的活性受其与其他蛋白质的相互作用的调节 时钟蛋白和这些蛋白质之间的相互作用在 昼夜节律的周期性。由于没有高分辨率结构的 生物钟蛋白质已经被解决了，没有确切的结构预测可以 关于时钟蛋白如何相互作用以及它们之间的相互作用 帮助实现振荡器的正常(或异常)昼夜周期。AS 因此，昼夜节律起搏器在结构水平上的机制是 仍不清楚。 这里提出，在理解结构方面的重大进展 昼夜节律守时的依据可以通过研究 中华绒毛虫时钟蛋白的三维结构和动力学 长聚球藻、Sasa、KaIA、Kaib和KaiC的核磁检测 共振谱(核磁共振)。其具体目标是解决结构问题 对这些时钟蛋白进行核磁共振分析，从而阐明蛋白质之间的相互作用 对设定昼夜节律的节奏至关重要。《基因数据库》 以及关于苏云金杆菌生物钟功能的生化信息。 拉长是相当广泛的，如果映射到三维空间 生物钟的结构，将允许对分子的洞察 昼夜节律起搏器的机制。例如，残基E103、R249 突变改变昼夜节律的Kaia的E274基因被聚为一类 或在核心处形成重要的氢键 蛋白质是本文提出的研究的重要问题。如果残留物 在表面聚集在一起，核磁共振研究将确定它们是否 形成与Kaib、Kaic和/或SASA的二聚体接口的一部分。如果残留物 都埋在蛋白质的核心，比较E103K、R249H和 带有野生型KaIA的E274H突变体将揭示结构和 对Kaia的功能很重要的动态。 长期目标是最终研究时钟蛋白复合体。 通过核磁共振来努力了解特定的蛋白质-蛋白质相互作用 调节昼夜节律。我们远景目标的关键问题是 KaIA、Kaib、SASA和KaiC之间的相互作用是否涉及变构， 主干或侧链动力学的局部性变化，紧密的位移 结合水分子或关键氢键的重排，以及它们是如何 在设定昼夜节律的节奏方面起着重要作用。尽管有些人 本文提出的工作所得出的结论将是S。 其他的，应该对所有的生物体都有普遍的影响， 包括人类。