SAXS DATA COLLECTION: CYANOBACTERIAL KAI ABC CIRCADIAN CLOCK

SAXS 数据收集：蓝细菌 Kai ABC 昼夜节律时钟

• 批准号: 7601751
• 负责人: MARTIN EGLI
• 金额: $ 1.18万
• 依托单位: ILLINOIS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7601751
• 关键词: Biochemical; Complex; Computer Retrieval of Information on Scientific Projects Database; Data Collection; Electron Microscopy; Feedback; Funding; Grant; Homo; Institution; Modeling; Molecular; Negative Staining; Phosphorylation; Phosphorylation Site; Proteins; Recombinant Proteins; Research; Research Personnel; Resources; Source; Structure; System; Techniques; Testing; United States National Institutes of Health; Work; X-Ray Crystallography; base; beamline; behavior test; circadian pacemaker; dimer; gel electrophoresis; reconstitution; research study; three-dimensional modeling

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The recent demonstrations that the cyanobacterial KaiABC circadian clock works independently from a transcriptional/translational oscillatory feedback loop and that it can be reconstituted by mixing the three recombinant proteins in the presence of ATP and Mg2+ renders this system an ideal candidate for a biochemical, biophysical and structural characterization of a molecular clock. The system involves three protein components, KaiA, KaiB and KaiC. KaiC is an autokinase and autophosphatase and constitutes the central cog of the clock. KaiA enhances phosphorylation of the KaiC homo-hexamer and KaiB antagonizes KaiAs action. The structural basis for their ability to regulate KaiC remains to be worked out. We have determined the only crystal structure of KaiC to date (Pattanayek et al., 2004), determined the phosphorylation sites (Xu et al., 2004), and have used a combination of X-ray crystallography, negative-stain electron microscopy (EM), gel electrophoresis (PAGE) and modeling to develop a three-dimensional model of a 1:1 complex between the KaiA dimer and KaiC hexamer from S. elongatus (Pattanayek et al., 2006). Recent progress in the structural characterization of Kai proteins and the binary KaiA-KaiC complex has been reviewed in Egli et al., 2007. To date we have conducted several preliminary experiments at the Bio-CAT beamline with the three Kai proteins from either S. elongatus or T. elongatus separately, or by mixing KaiA and KaiC or KaiB and KaiC together to test the behavior of binary complexes. Although problems with aggregation need to be overcome these initial tests indicate that SAXS may a useful technique for analyzing the interactions between Kai proteins.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 最近的证明表明，蓝细菌昼夜节律时钟独立于转录/翻译振荡反馈回路，并且可以通过在存在ATP和MG2+的情况下混合三个重组蛋白来重组，这是该系统的理想候选生物化，生物学，结构型的候选。该系统涉及三个蛋白质成分，即Kaia，Kaib和Kaic。 KAIC是一种自动动酶和自磷酸酶，构成了时钟的中心齿轮。 KAIA增强了KAIC均己糖的磷酸化，而Kaib会拮抗Kaia的作用。他们调节KAIC能力的结构基础仍有待解决。我们已经确定了迄今为止KAIC的唯一晶体结构（Pattanayek等，2004），确定了磷酸化位点（Xu等，2004），并使用了X射线晶体学，负膜晶体学，负电子显微镜（EM），凝胶电泳（PAGE）和模型中的X射线晶体学和KAIC的kaia S. 1：1：1：1：1：1：1：1：1：1：1：1：1。 Elongatus（Pattanayek等，2006）。 Egli等人，2007年已经回顾了KAI蛋白结构表征和二元KAIA-KAIC复合体的结构表征的最新进展。迄今为止，我们已经在Biio-Cat Beamine上进行了几项初步实验，并通过三个KAI蛋白从S. elongatus或T. eyongatus或T. eyongatus和Kaia和Kaia和Kaia组合的三个KAI蛋白进行了三个KAI蛋白，并进行了KAIA和KAIA的kaia和KAIA的kaia和KAIAC，复合物。尽管需要克服聚集问题的问题，这些初始测试表明SAXS可能是分析KAI蛋白之间相互作用的有用技术。