A protocol for preparing nucleotide-free KaiC monomer

DOI Web Site 3 Citations 25 References Open Access
  • Mukaiyama Atsushi
    Research Center of Integrative Molecular Systems (CIMoS), Institute for Molecular Science Department of Functional Molecular Science, SOKENDAI (The Graduate University for Advanced Studies) RIKEN SPring-8 Center, Harima Institute
  • Osako Masato
    Division of Biological Science, Graduate School of Science, Nagoya University
  • Hikima Takaaki
    RIKEN SPring-8 Center, Harima Institute
  • Kondo Takao
    Division of Biological Science, Graduate School of Science, Nagoya University
  • Akiyama Shuji
    Research Center of Integrative Molecular Systems (CIMoS), Institute for Molecular Science Department of Functional Molecular Science, SOKENDAI (The Graduate University for Advanced Studies) RIKEN SPring-8 Center, Harima Institute

Abstract

The hexameric form of the KaiC protein is a core of the cyanobacterial biological clock, and its enzymatic activities exhibit circadian periodicity. The instability of the monomeric form of nucleotide-free KaiC has precluded its storage and detailed analyses of the activities of the reassembled hexamer. Here, we provide a protocol for preparing nucleotidefree KaiC monomer that is stable in solution and for triggering its reassembly into intact KaiC hexamer by the addition of ATP. A phosphate buffer containing glutamic acid and arginine enhanced the stability of KaiC monomer considerably. In addition, we found that reassembled KaiC hexamer was functionally active as the intact hexamer. This protocol provides a methodological basis for further analyses of first-turnover events of the ATPase/autokinase/autophosphatase activities of the KaiC hexamer.

Journal

  • BIOPHYSICS

    BIOPHYSICS 11 (0), 79-84, 2015

    The Biophysical Society of Japan

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