A protocol for preparing nucleotide-free KaiC monomer

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 hexa...

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Bibliographic Details
Published in:BIOPHYSICS 2015, Vol.11, pp.79-84
Main Authors: Mukaiyama, Atsushi, Osako, Masato, Hikima, Takaaki, Kondo, Takao, Akiyama, Shuji
Format: Article
Language:English
Subjects:
circadian clock
Experimental Methods and Protocols
KaiC
solution structure
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Summary: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.
ISSN:1349-2942
1349-2942
DOI:10.2142/biophysics.11.79