Circadian clocks underlie the rhythmic physiological functions and behaviors of eukaryotic organisms in all kingdoms of life. The clock is a widespread cellular mechanism that adjusts to environmental cues like light and temperature and impacts many aspects of our health such as sleep patterns, time-perception, and aging, as well as the treatment and prevention of disease. A complex signaling network that integrates transcriptional feedback loops maintains the rhythm, and although genetics and cell biology studies have identified the functions of clock genes within the loops, the molecular mechanisms of signal processing and propagation are unknown. Two clock components that are important for light entrainment - light, oxygen, and voltage (LOV) domains and Cryptochromes (CRY) - will be structurally and computationally characterized in the model systems of Neurospora (fungi), Rhodobacter (bacteria), and Drosophila (flies) in order to understand how light signals are processes and propagated. The LOV and CRY components are both flavin-containing photosensors that exhibit conformational changes upon light-excitation, but vary in terms of their photochemistry and structure. To determine whether LOV domains exhibit analogous photochemistry and similar light-induced structural changes in different species despite significant differences in terminal residues, Rhodobacter LOV (RLOV) and a LOV protein in Neurospora, Vivid (VVD), will be investigated in both light- and dark-states by biochemical, structural and computational methods. The goal is to determine how protein conformational changes correlate to cofactor photochemistry, and how the changes induce signal propagation to LOV partners and downstream clock components. Similarities of RLOV to VVD would indicate a mechanism shared between protein families, and would be meaningful as a broad mechanism of LOV domain chemistry. Drosophila Cryptochrome (dCRY) is known to transduce light signals associated with circadian clocks. Uncertainties in dCRY photochemistry and signal transduction will also be examined by biochemical, structural, and computational methods. Significant conformational changes of dCRY have been associated with light- activation;thus, structural characterization of dCRY in light-induced and point mutation-designed alternative conformations will be completed using x-ray crystallography to elucidate the changes. Additionally, Timeless (TIM), a protein partner of CRY that impacts transcription of clock controlled genes, will be structurally characterized in combination with dCRY to provide knowledge of how the light signal is converted to activate downstream effects. Ultimately, structural characterization of clock components using x-ray crystallography, kinetic analyses from time-resolved spectroscopic methods, and quantum chemical calculations of the flavin- containing active centers, will provide insight into how flavin photochemistry generates protein conformational changes, and how structural these changes lead to signal propagation.

Public Health Relevance

The circadian clock is a mechanism responsible for coordinating cellular metabolism, the combination of chemical reactions that sustain body function, with a daily cycle. Circadian clocks are present in all kingdoms of life, but in humans the clock impacts a wide variety of physiological processes including sleep patterns, appetite, time perception, hormone production, and aging. This research proposal outlines an investigation of light-sensitive proteins that are critical components of circadian clocks to understand how they work, their structural components and features, and how they communicate with other proteins.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Postdoctoral Individual National Research Service Award (F32)
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Special Emphasis Panel (ZRG1-F04-A (20))
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Flicker, Paula F
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Cornell University
Schools of Arts and Sciences
United States
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Conrad, Karen S; Manahan, Craig C; Crane, Brian R (2014) Photochemistry of flavoprotein light sensors. Nat Chem Biol 10:801-9
Conrad, Karen S; Bilwes, Alexandrine M; Crane, Brian R (2013) Light-induced subunit dissociation by a light-oxygen-voltage domain photoreceptor from Rhodobacter sphaeroides. Biochemistry 52:378-91