Real-time in vitro fluorescence anisotropy of the cyanobacterial circadian clock

Joel Heisler, Archana Chavan, Yong Gang Chang, Andy Liwang

Research output: Contribution to journalArticleOtherpeer-review

3 Citations (Scopus)

Abstract

Uniquely, the circadian clock of cyanobacteria can be reconstructed outside the complex milieu of live cells, greatly simplifying the investigation of a functioning biological chronometer. The core oscillator component is composed of only three proteins, KaiA, KaiB, and KaiC, and together with ATP they undergo waves of assembly and disassembly that drive phosphorylation rhythms in KaiC. Typically, the time points of these reactions are analyzed ex post facto by denaturing polyacrylamide gel electrophoresis, because this technique resolves the different states of phosphorylation of KaiC. Here, we describe a more sensitive method that allows real-time monitoring of the clock reaction. By labeling one of the clock proteins with a fluorophore, in this case KaiB, the in vitro clock reaction can be monitored by fluorescence anisotropy on the minutes time scale for weeks.

Original languageEnglish
Article number42
Number of pages15
JournalMethods and Protocols
Volume2
Issue number2
DOIs
Publication statusPublished - 24 May 2019
Externally publishedYes

Keywords

  • Circadian clock
  • Cyanobacteria
  • Fluorescence
  • Phosphorylation
  • Protein

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