The cyanobacterial circadian oscillator is a system of three proteins, KaiA, KaiB, and KaiC, that maintain a 24-hour cycle in the phosphorylation state of KaiC. As demonstrated in 2005 by Nakajima et al., this oscillator is entirely post-translational and can be reconstructed in vitro from the three purified proteins and ATP.
I am studying mutants of KaiA, the homodimeric protein that promotes KaiC autophosphorylation. KaiA stimulates KaiC autophosphorylation at low relative concentrations, and its activity is inhibited by KaiB. The C-terminus of KaiA is known to interact with KaiC to promote phosphorylation, while the N-terminus is implicated in receiving inhibitory and environmental signals. Analyzing the functional properties of different KaiA mutants may lead to a more detailed mechanistic understanding of these roles.
Using the in vitro clock reaction, I am comparing the period of KaiA mutants to in vivo results (Nishimura et al., 2002), in which the period is extended. I am also characterizing the kinetics of these mutants, including the rate of initial KaiC phosphorylation and inhibition by KaiB. These altered kinetic parameters will be used to compare experimental results with predictions from a mathematical model of the oscillator. By quantifying the effects of KaiA mutants on the in vitro clock, I hope to describe the protein-protein interactions of the oscillator at a structural level.
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