title: Conversion between two conformational states of KaiC is induced by ATP hydrolysis as a trigger for cyanobacterial circadian oscillation.
abstract: The cyanobacterial circadian oscillator can be reconstituted in vitro by mixing three clock proteins, KaiA, KaiB and KaiC, with ATP. KaiC is the only protein with circadian rhythmic activities. In the present study, we tracked the complex formation of the three Kai proteins over time using blue native (BN) polyacrylamide gel electrophoresis (PAGE), in which proteins are charged with the anionic dye Coomassie brilliant blue (CBB). KaiC was separated as three bands: the KaiABC complex, KaiC hexamer and KaiC monomer. However, no KaiC monomer was observed using gel filtration chromatography and CBB-free native PAGE. These data indicate two conformational states of KaiC hexamer and show that the ground-state KaiC (gs-KaiC) is stable and competent-state KaiC (cs-KaiC) is labile and degraded into monomers by the binding of CBB. Repeated conversions from gs-KaiC to cs-KaiC were observed over 24 h using an in vitro reconstitution system. Phosphorylation of KaiC promoted the conversion from gs-KaiC to cs-KaiC. KaiA sustained the gs-KaiC state, and KaiB bound only cs-KaiC. An E77Q/E78Q-KaiC variant that lacked N-terminal ATPase activity remained in the gs-KaiC state. Taken together, ATP hydrolysis induces the formation of cs-KaiC and promotes the binding of KaiB, which is a trigger for circadian oscillations.
authors: Graduate School of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan.
authors: Katsuaki
authors: K
authors: Oyama
authors: College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan.
authors: Chihiro
authors: C
authors: Azai
authors: College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan.
authors: Kaori
authors: K
authors: Nakamura
authors: College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan.
authors: Syun
authors: S
authors: Tanaka
authors: Graduate School of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan.|College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan.
authors: Kazuki
authors: K
authors: Terauchi
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002940:Circadian Rhythm / Q000235:genetics*; D056925:Circadian Rhythm Signaling Peptides and Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D006868:Hydrolysis; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012394:Rosaniline Dyes / Q000737:chemistry; D046940:Synechococcus / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D056925:Circadian Rhythm Signaling Peptides and Proteins; C114269:KaiA protein, cyanobacteria; C114270:KaiB protein, cyanobacteria; C114271:KaiC protein, cyanobacteria; D011994:Recombinant Proteins; D012394:Rosaniline Dyes; D000255:Adenosine Triphosphate; C004692:coomassie Brilliant Blue