Circadian (daily) rhythms are a crucial component of human health that regulates sleep, alertness, hormones, metabolism, and many other biological processes. The ultimate explanation for the mechanism of circadian oscillators will require characterizing the structures, functions, and interactions of the molecular components of these clocks. The current project is to elucidate the basic principles of circadian clocks at a biophysical/molecular level in the cyanobacterial model system, where genetic/biochemical studies have identified three key clock proteins, KaiA, KaiB, and KaiC, that can reconstitute a circadian oscillator in vitro. This remarkable demonstration has led to a re-evaluation of our understanding of circadian clocks in all organisms, including mammals. Moreover, atomic resolution structures of KaiA, KaiB, and KaiC proteins have been determined that enable truly molecular analyses of clock mechanisms. This research project will focus on answering two fundamental questions in chronobiology: how do circadian enzymes work? and what is the adaptive advantage of circadian mechanisms? Regarding the first question of enzymatic mechanism, we will determine the basis of the central property of temperature compensation of the core clockwork by biochemical/biophysical, genetic, and structural approaches. Temperature-compensation mutations of the Kai proteins will be studied to generate specific hypotheses that will be tested by novel in vitro biochemical analyses (e.g., single-molecule dynamics) and targeted mutations. The biochemical data that result from the analyses of these mutants will be used to generate models that account for the temperature compensated, 24 h time constant of the circadian oscillator. Regarding the second overall question of adaptive advantage, differential expression of circadian rhythms under some conditions but not others is based on novel mechanisms of codon usage, and the mechanism of this adaptive phenomenon will be analyzed, as well as recruited to maximize cost-effective synthesis of bioproducts. Finally, a novel hypothesis with far-reaching implications will be evaluated, namely that accurate circadian timekeeping requires compensation for metabolic perturbations, of which temperature change is only one among many such perturbations. The answers to these questions will lead to wide-ranging general insights into the mechanisms and applications of biological timekeeping.
This MERIT project will clarify circadian mechanisms at molecular levels that were heretofore unreachable. Biological clocks have been found to be crucial for physical and mental health, and knowledge of circadian mechanisms along with the development of therapies to properly phase sleep will allow us to enhance health, performance, and well-being in addition to improving the quality of life for depressed subjects.
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