Eukaryotic circadian clocks are cell-autonomous timing devices that pace metabolism to the day-night cycle. These clocks are composed of transcriptional-translational feedback loops (TTLs) within which repressor proteins inhibit the transcriptional activators of their own genes. Light entrains the clock phase by stimulating photosensors that impinge directly on the components of the central TTFs. Natural variation in clock genes directly affects behavior. In humans, aberrant clock function causes mental illness (sleep disorders, depression, mania), cell growth deregulation (cancer) and metabolic defects (diabetes and obesity). This project proposes structural and mechanistic investigations of the key repressor and light-setting activities common to clocks in higher organisms. Biophysical studies will be conducted on model circadian systems- those of filamentous fungi (Neurospora crassa) and fruit flies (Drosophila melanogaster)-that are tractable for testing the biological relevance of mechanistic insights. These fungal and fly systems also exemplify the two general strategies used by higher organisms for light setting: transcription factor activation and repressor destruction. Specifically, the light activation mechanism of the Whitecollar (WC) fungal transcriptional activators WC1 and WC2 will be determined. Questions will be answered about how the interchange of light- oxygen- and voltage (LOV) sensing domains allows for light adaptation and fine-tuning of the activation response. The crystallographic structure of the FRQ-interacting RNA-helicase, the fungal scaffold protein will be determined and its interaction with the central repressor protein Frequency (FRQ) elucidated. Photophysical studies of the fly light sensor cryptochrome (dCRY) will be coupled with cellular and behavioral assays to determine the biologically relevant photocycle for dCRY. A combination of crystallographic and spectroscopic approaches will be employed to understand the activation mechanism of dCRY and its engagement of downstream targets. The effects of post-translational modifications on the properties of the central fly repressor protein period (PER) will be correlated with the capability of PER to target the transcriptional activator complex Clock:Cycle (dCLK:CYC). Structures will be determined of CYC and its interaction with PER defined. To accomplish these goals a complimentary set of structural techniques including X-ray crystallography, small-angle X-ray scattering, optical spectroscopy and pulsed-dipolar ESR spectroscopy (PDS) will be applied. For PDS, new methods for incorporating spin probes based on nitroxides, flavins, nucleotides and Cu(II) ions will be developed and deployed. Genetically encoded Cu(II) probes will report on dynamic conformations within and associations among clock components. Overall, the project aims to understand behavioral patterns controlled by clocks in terms of the key protein complexes involved in gene repression and light setting.
Circadian clocks impact nearly every aspect of behavior and their dysfunction contributes to mental illness (sleep disorders, depression, mania, schizophrenia), cancer, and metabolic disease (diabetes and obesity). Defining conserved molecular mechanisms underlying cellular timing will rationalize how genetic and environmental diversity affects metabolic cycles and reveal new strategies for therapeutic intervention.
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