Daily biological clocks are important to human physiology. For example, psychiatric and medical studies have shown that circadian rhythmicity is involved in some forms of depressive illness, """"""""jet lag,"""""""" drug tolerance/efficacy, memory, alertness, and insomnia. Therefore, understanding the biochemical mechanism of circadian clocks may lead to procedures which will be useful in the diagnosis and treatment of disorders that are relevant to sleep, mental health, and pharmacology. Despite the importance of clocked phenomena, however, clues to the nature of the underlying biochemical mechanism are only just beginning to emerge. Recent investigations report that protein-to-protein interactions play key roles in circadian clock mechanisms in prokaryotes and eukaryotes. We will investigate the temporal and spatial control of clock protein interaction, primarily using a new method that we developed that is optimal for studies of clock protein interactions. This method, which we call Bioluminescence Resonance Energy Transfer (BRET), uses a bioluminescent luciferase that is genetically fused to one candidate protein, and an acceptor fluorophore fused to another protein of interest. If two candidate clock proteins interact so as to bring the luciferase and fluorophore into proximity, resonance energy transfer occurs that can be measured as a shift in the color of the bioluminescence emission. BRET will be particularly useful for testing clock protein interactions and spatial distributions within native cells that exhibit daily rhythms in culture. Using proteins encoded by clock genes for which evidence exists of interaction, we will use the BRET system (and other confirmatory techniques) to assay clock protein interactions in situ, both in short term experiments and also over the daily cycle to appraise temporal control of protein interaction of clock proteins in prokaryotes, Drosophila, and mammals. Our hypothesis is that clock protein interaction will not be simply a function of the protein abundance, but that there will be phase-specific interaction regulated by other factors than merely the proteins' abundance.
