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: 7048609
• 负责人: Andy LiWang
• 金额: $ 21.3万
• 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 2008-06-05
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7048609
• 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）的生理和行为的虚拟 所有生物体都以大约24小时的周期振荡，现在 这是由一种叫做生物钟的内源性振荡器引起的。 迄今为止研究的所有生物钟都是基于一个共同的设计， 时钟基因和时钟蛋白的转录/翻译反馈环。 生物钟蛋白的活性是由它与其他生物钟蛋白的相互作用来调节的。 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。由于没有高分辨率的结构， 生物钟蛋白已经解决，没有确切的结构预测可以 生物钟蛋白如何相互作用， 帮助实现振荡器的正常（或异常）昼夜节律周期性。作为 因此，昼夜节律起搏器在结构水平上的机制是 目前仍不清楚. 这里提出，在理解结构方面的重大进展 昼夜节律时间保持的基础可以通过调查 生物钟蛋白的三维结构和动力学 细长聚球藻、SasA、KaiA、KaiB和KaiC的核磁共振分析 共振光谱学（NMR）。具体的目标是解决的结构， 这些时钟蛋白的核磁共振，然后阐明蛋白质-蛋白质相互作用 是设定昼夜节律的核心。遗传学数据库 和生化信息的功能的昼夜节律钟的S。 elongatus是相当广泛的，如果映射到三维空间， 生物钟的结构，将使人们能够深入了解 生理节律起搏器的机制。例如，残基E103、R249 KaiA的E274突变改变了昼夜节律， 在表面上结合在一起，或者在核心中形成重要的氢键， 蛋白质，是这里提出的研究的重要问题。如果残留物 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。如果残留物 被埋在蛋白质的核心，E1 O3 K，R249 H和 具有野生型KaiA的E274 H突变体将揭示KaiA的结构和功能的方面。 对KaiA的功能很重要的动力学。 长期目标是最终研究时钟蛋白复合物 通过核磁共振来了解特定的蛋白质-蛋白质相互作用 调节昼夜节律。我们的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，紧密连接的 结合水分子或关键氢键的重排，以及它们如何 在设定昼夜节律的速度方面起着重要的作用。尽管一些 从这里提出的工作中得出的结论将具体到S。 elongatus，其他的，应该对所有的生物体都有普遍的影响， 包括人类