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A protocol for preparing nucleotide-free KaiC monomer.

Atsushi Mukaiyama¹, Masato Osako², Takaaki Hikima³

¹Research Center of Integrative Molecular Systems (CIMoS), Institute for Molecular Science, 38 Nishigo-Naka, Myodaiji, Okazaki 444-8585, Japan; Department of Functional Molecular Science, SOKENDAI (The Graduate University for Advanced Studies), 38 Nishigo-Naka, Myodaiji, Okazaki 444-8585, Japan; RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan.

Biophysics (Nagoya-Shi, Japan)

|August 6, 2016

Summary

Researchers developed a protocol to stabilize the KaiC protein monomer, enabling its reassembly into a functional hexamer. This breakthrough facilitates detailed studies of the cyanobacterial biological clock

Keywords:

KaiC circadian clock solution structure

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Area of Science:

Biochemistry
Molecular Biology
Chronobiology

Background:

The KaiC protein hexamer is central to the cyanobacterial biological clock.
Instability of nucleotide-free KaiC monomer hinders research into hexameric activities.

Purpose of the Study:

To develop a protocol for stabilizing nucleotide-free KaiC monomer.
To enable reassembly of stable KaiC monomer into a functional hexamer.
To facilitate detailed analysis of KaiC hexamer's enzymatic activities.

Main Methods:

Preparation of stable nucleotide-free KaiC monomer using a specific phosphate buffer with glutamic acid and arginine.
Triggering reassembly of KaiC monomer into hexamer via ATP addition.
Functional assessment of the reassembled KaiC hexamer.

Main Results:

A stable, nucleotide-free KaiC monomer solution was successfully prepared.
The addition of ATP induced reassembly into a functional KaiC hexamer.
The reassembled KaiC hexamer exhibited activity comparable to the intact hexamer.

Conclusions:

The developed protocol overcomes the instability of KaiC monomer.
This method allows for detailed investigation of KaiC hexamer's ATPase/autokinase/autophosphatase activities.
Provides a foundation for studying circadian clock mechanisms in cyanobacteria.