SAXS DATA COLLECTION: CYANOBACTERIAL KAI ABC CIRCADIAN CLOCK

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 最近的研究表明，蓝藻KaiABC生物钟独立于转录/翻译振荡反馈环工作，并且它可以在ATP和Mg2+存在的情况下通过混合三种重组蛋白而重组，这使得该系统成为分子钟的生化、生物物理和结构表征的理想候选者。该系统包括三个蛋白质组分，KaIA，Kaib和KaiC。KaiC是一种自激酶和自磷酸酶，构成了时钟的中心齿轮。Kaia增强KaiC高六聚体的磷酸化，Kaib拮抗Kaias的作用。它们监管KaiC的能力的结构基础仍有待研究。我们已经确定了到目前为止唯一的KaiC的晶体结构(Pattanayek等人，2004年)，确定了磷酸化位点(Xu等人，2004年)，并使用X射线结晶学、负染色电子显微镜(EM)、凝胶电泳(PAGE)和建模的组合来开发KaIA二聚体和来自长链球菌的KaiC六聚体之间1：1的复合体的三维模型(Pattanayek等人，2006年)。Egli等人在2007年综述了KAI蛋白和KaIA-KaiC二元复合体的结构表征的最新进展。到目前为止，我们已经在Bio-CAT光束线上分别使用来自长链球菌或长链球菌的三个KAI蛋白，或者通过将KaIA和KaiC或Kaib和KaiC混合在一起来测试二元复合体的行为，进行了几次初步实验。尽管需要克服聚集方面的问题，但这些初步测试表明，SAXS可能是一种分析KAI蛋白之间相互作用的有用技术。

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.