The cyanobacterium Synechococcus elongatus has been shown to keep nearly accurate time in the absence of environmental stimuli, maintaining, for instance, the daily biochemical rhythms of nitrogen fixation and photosynthesis that allow it to thrive on 24-hour cycles of sunlight and darkness. Research has narrowed the centrally responsible components of this biological clock down to three proteins, KaiA, KaiB, and KaiC, and it is now possible to reconstitute this clock in vitro, using only the three proteins and a supply of ATP (Nakajima et al., 2005).
The most complex of the three proteins, KaiC, exists as a hexamer in its native state. Two of the protein's amino acid residues, S431 and T432, demonstrate daily oscillations in their phosphorylation states. Immunoprecipitation assays reveal that changes in the degree of interaction among the three proteins are correlated with the periods of kinase and phosphatase activity of KaiC throughout the cycle (Nishiwaki et al., 2007). Other experiments suggest that synchronization of the clock is dependent upon monomer exchange among the KaiC hexamers, and that such exchanges are possible only while KaiC exists in certain phosphorylation states (Hiroshi et al., 2007).
In my research, I hope to learn more about the mechanism by which the clock maintains its rhythm. The main subjects of my study will be several phosphomimetic mutants, in which one or both of the two key amino acids are replaced by either glutamic acid or alanine. These substitutions are intended to simulate permanently phosphorylated or permanently dephosphorylated residues, respectively. After expressing and purifying each mutant, I will then use SDS-PAGE analysis to observe their behavior in the oscillator by monitoring the changes in phosphoform concentrations. The results will show the effect that phosphorylating each residue individually has on the interactions among the proteins. I will also perform immunoprecipitation assays upon mixtures of the mutants to determine what impact the phosphorylation state has upon the ability of the hexamers to undergo monomer exchange. Should these studies prove conclusive, I hope to use the data to help construct a more concrete mathematical model
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