This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor 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.
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