The circadian clock orchestrates metabolism and cell division and imposes important consequences to health and diseases, such as obesity, diabetes, and cancer. It is well established that phosphorylation-dependent circadian rhythms are the result of circadian clock protein interactions, which regulate many intercellular processes according to time of day. The phosphorylation-dependent circadian rhythm undergoes a succession of phases: Phosphorylation Phase → Transition Phase → Dephosphorylation Phase. Each phase induces the next phase. However, the mechanism of each phase and how the phosphorylation and dephosphorylation phases are prevented from interfering with each other remain elusive. In this research, we used a newly developed isotopic labeling strategy in combination with a new type of nuclear magnetic resornance (NMR) experiment to obtain the structural and dynamic information of the cyanobacterial KaiABC oscillator system. This system is uniquely suited for the mechanistic studies: mixing KaiA, KaiB KaiC, and ATP generates a self-sustained ~24 h rhythm of KaiC phosphorylation in vitro. Our data strongly suggest that the dynamic states of KaiC underpin the timing mechanism of cyanobacterial oscillator.

The cyanobacterium Synechococcus elongatus builds a circadian clock on an oscillator comprised of three proteins, KaiA, KaiB, and KaiC, which can recapitulate a circadian rhythm of KaiC phosphorylation in vitro. The molecular structures of all three proteins are known, and the phosphorylation steps of KaiC, the interaction dynamics among the three Kai proteins, and a weak ATPase activity of KaiC have all been characterized. A mutant of a clock gene in the input pathway, cikA, has a cell division defect, and the circadian clock inhibits the cell cycle for a short period of time during each cycle. However, the interaction between the circadian cycle and the cell cycle and the molecular mechanisms underlying it have been poorly understood. In addition, the subcellular localization of clock proteins and possible localization dynamics, which are critical in the timing circuit of eukaryotic clock systems and might also shed light on the interaction between circadian cycle and cell cycle, have remained largely unknown. A combination of genetics, cell biology, and microscopy techniques has been employed to investigate both questions. This work showed that the cell division defect of a cikA mutant is a function of the circadian clock. High ATPase activity of KaiC coincides with the inhibition of cytokinesis by the circadian clock. CikA likely represses KaiC's ATPase activity through an unknown protein, which in cikA's absence stimulates both the ATPase and autokinase activities independently of KaiA or KaiB. SasA-RpaA acts as an output in the control of cell division, and the localization of FtsZ is the target, although it still remains to be seen how RpaA, directly or indirectly, inhibits FtsZ localization. The project also showed that clock proteins are localized to the cell poles. KaiC is targeted to the cell pole in a phosphorylation-dependent manner. KaiB and CikA are also found at the poles independently of KaiC. KaiA likely only localizes to the cell pole during the dephosphorylation phase, which is dependent on both KaiB and KaiC, specifically on the phosphorylation of KaiC at S431. Overall, significant progress was made in both areas and this project sheds light on how the circadian oscillator operates in cyanobacterial cells and interacts with another fundamental cellular function.

Thesis (M.A.)--Hartford Seminary, 1944. / Includes bibliographical references (p. 180-181).

Circadian oscillators or clocks are a widespread, endogenous class of oscillatory mechanisms that control the ~24h temporal pattern of diverse organism functions. In cyanobacteria this mechanism is formed by three proteins, KaiA, KaiB and KaiC. KaiA is shown here to be a two domain protein that directly interacts with KaiC and enhances the KaiC autokinase activity. The amino-terminal domain of KaiA can be structurally categorized as a pseudo-receiver, a class of proteins used in signaling cascades and activated by direct protein??protein interactions. The carboxy-terminal domain interacts directly with KaiC, is sufficient to enhance the KaiC autokinase activity in a manner similar to full-length KaiA, and adopts a unique, all α-helical dimeric fold. The structure of this domain raises interesting probabilities regarding the mode of KaiA??KaiC interaction. The two KaiA domains are shown to directly interact with each other, which suggests a possible mechanism of signal transfer from the amino to carboxy-terminal domain. Hydrogen bonds are of paramount importance in nucleic acid structure and function. Here we show that changes in the width and anharmonicity of vibrational potential energy wells of hydrogen bonded groups can be measured in nucleic acids and can possibly be correlated to structural properties, such as length. Deuterium/protium fractionation factors, which are sensitive to the vibrational potential well width, were measured for the imino sites of thymidine residues involved in A:T base pairs or free in solution, and a correlation was established between decreasing fractionation factors and increasing imino proton chemical shift, δH3. Similarly, a correlation was observed between δH3and deuterium isotope effects (DIE) on chemical shift of thymidine carbon atoms. Combined these results indicate that as hydrogen-bond strength increases the vibrational potential wells of imino protons widen with a corresponding increase in anharmonicity. However, trans-hydrogen bond DIE on carbon chemical shifts of A:T base-paired adenosine residues do not correlate with those measured on thymidine residues. We propose that this lack of correlation is due to DIE dependence on base-pair geometry, which is not easily measured by traditional NMR experiments.

Circadian oscillators or clocks are a widespread, endogenous class of oscillatory mechanisms that control the ~24h temporal pattern of diverse organism functions. In cyanobacteria this mechanism is formed by three proteins, KaiA, KaiB and KaiC. KaiA is shown here to be a two domain protein that directly interacts with KaiC and enhances the KaiC autokinase activity. The amino-terminal domain of KaiA can be structurally categorized as a pseudo-receiver, a class of proteins used in signaling cascades and activated by direct protein??protein interactions. The carboxy-terminal domain interacts directly with KaiC, is sufficient to enhance the KaiC autokinase activity in a manner similar to full-length KaiA, and adopts a unique, all α-helical dimeric fold. The structure of this domain raises interesting probabilities regarding the mode of KaiA??KaiC interaction. The two KaiA domains are shown to directly interact with each other, which suggests a possible mechanism of signal transfer from the amino to carboxy-terminal domain. Hydrogen bonds are of paramount importance in nucleic acid structure and function. Here we show that changes in the width and anharmonicity of vibrational potential energy wells of hydrogen bonded groups can be measured in nucleic acids and can possibly be correlated to structural properties, such as length. Deuterium/protium fractionation factors, which are sensitive to the vibrational potential well width, were measured for the imino sites of thymidine residues involved in A:T base pairs or free in solution, and a correlation was established between decreasing fractionation factors and increasing imino proton chemical shift, δH3. Similarly, a correlation was observed between δH3and deuterium isotope effects (DIE) on chemical shift of thymidine carbon atoms. Combined these results indicate that as hydrogen-bond strength increases the vibrational potential wells of imino protons widen with a corresponding increase in anharmonicity. However, trans-hydrogen bond DIE on carbon chemical shifts of A:T base-paired adenosine residues do not correlate with those measured on thymidine residues. We propose that this lack of correlation is due to DIE dependence on base-pair geometry, which is not easily measured by traditional NMR experiments.