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.