SAXS DATA COLLECTION: CYANOBACTERIAL KAI ABC CIRCADIAN CLOCK

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 最近的证据表明，蓝藻KaiABC生物钟的工作独立于转录/翻译振荡反馈回路，它可以通过混合三种重组蛋白在ATP和Mg 2+的存在下，使该系统的生化，生物物理和结构表征的分子时钟的理想候选人重建。该系统涉及三种蛋白质组分，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同源六聚体的磷酸化，KaiB拮抗KaiA的行动。它们监管KaiC能力的结构基础仍有待确定。我们已经确定了迄今为止KaiC的唯一晶体结构（Pattanayek等人，2004），确定磷酸化位点（Xu et al.，2004），并且已经使用X射线晶体学、负染电子显微镜（EM）、凝胶电泳（PAGE）和建模的组合来开发来自S. elongatus（Pattanayek等，2006年）。Kai蛋白和二元KaiA-KaiC复合物的结构表征的最新进展已经在Egli等人，2007.迄今为止，我们已经在Bio-CAT光束线上用来自S. elongatus或T.通过将KaiA和KaiC或KaiB和KaiC混合在一起来测试二元复合物的行为。虽然聚集的问题需要克服，这些初步的测试表明，SAXS可能是一个有用的技术，用于分析Kai蛋白之间的相互作用。

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.