title: Detecting KaiC phosphorylation rhythms of the cyanobacterial circadian oscillator in vitro and in vivo.
abstract: The central oscillator of the cyanobacterial circadian clock is unique in the biochemical simplicity of its components and the robustness of the oscillation. The oscillator is composed of three cyanobacterial proteins: KaiA, KaiB, and KaiC. If very pure preparations of these three proteins are mixed in a test tube in the right proportions and with ATP and MgCl2, the phosphorylation states of KaiC will oscillate with a circadian period, and these states can be analyzed simply by SDS-PAGE. The purity of the proteins is critical for obtaining robust oscillation. Contaminating proteases will destroy oscillation by degradation of Kai proteins, and ATPases will attenuate robustness by consumption of ATP. Here, we provide a detailed protocol to obtain pure recombinant proteins from Escherichia coli to construct a robust cyanobacterial circadian oscillator in vitro. In addition, we present a protocol that facilitates analysis of phosphorylation states of KaiC and other phosphorylated proteins from in vivo samples.
authors: Center for Circadian Biology, University of California, San Diego, La Jolla, California, USA.
authors: Yong-Ick
authors: YI
authors: Kim
authors: Center for Circadian Biology, University of California, San Diego, La Jolla, California, USA.
authors: Joseph S
authors: JS
authors: Boyd
authors: Division of Genetics, University of Alicante, Alicante, Spain.
authors: Javier
authors: J
authors: Espinosa
authors: Center for Circadian Biology, University of California, San Diego, La Jolla, California, USA; Division of Biological Sciences, University of California, San Diego, La Jolla, California, USA. Electronic address: sgolden@ucsd.edu.
authors: Susan S
authors: SS
authors: Golden
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D002846:Chromatography, Affinity; D057906:Circadian Clocks; D056925:Circadian Rhythm Signaling Peptides and Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D057075:Enzyme Assays; D004926:Escherichia coli; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D046940:Synechococcus / Q000502:physiology*
chemical_list: D001426:Bacterial Proteins; D056925:Circadian Rhythm Signaling Peptides and Proteins; C114271:KaiC protein, cyanobacteria; D011994:Recombinant Proteins
keywords: Biological clock; Circadian rhythms; Cyanobacteria; FPLC; KaiC; Oscillatory reaction; Phos-tag; Phosphorylation; Protein purification; Synechococcus elongatus