Circadian clocks synchronize cellular metabolism to the diurnal light cycle. In humans, our biological clocks impact many aspects of our physiology, including sleep, mental well-being, and the prevention and treatment of disease. Although genetics and cell biology have made great progress in understanding the function of clock genes, the molecular mechanisms that compose the underlying transcriptional feedback loops, and their light entrainment, are not well understood. Eukaryotic model systems such as Neurospora, (filamentous fungi) and Drosophila (flies) share similarities with higher metazoan clocks and importantly have well defined photoreceptors and molecular oscillators homologous to their mammalian counterparts. New structures of the flavin-containing photosensors in different states of activation have provided insight into their chemical reactivity and primary functions. For the fungal photoreceptors White-collar-1 (WC-1) and Vivid (VVD), photo-induced swapping of their light, oxygen and voltage (LOV) domains, will be investigated as a mechanism to explain light adaptation. Recent results on light signal propagation by Drosophila Cryptochrome (CRY) suggest novel relationships among flavin chemistry, conformational signaling and recognition of the Timeless protein. The effects of LOV domains and CRYs on their partners and downstream targets will be studied by x-ray crystallography, spectroscopy, biochemistry and cell biology. At the center of these studies is the question of how protein conformational change links cofactor photochemistry to the modulation of protein assembly. New methods developed for acquiring time-resolved spectroscopic and x-ray scattering data will be applied to reconcile cofactor electronic structure with protein conformational state. Breakthroughs in protein expression and purification of several key clock components provide accessibility to structural studies for the first time. The rational design and production of variant photosensors with perturbed photocycles, redox potentials, conformational coupling and recognition properties allow molecular mechanisms to be tested in vivo. Parallel investigations of related mammalian clock proteins, including new diffraction quality crystals, will spearhead comprehensive mechanistic studies in these highly complex systems. Ultimately, studies of clock proteins will provide a molecular rationale for behavioral responses and provide a basis for advancing the treatment of mental disorders and many other maladies. PUBLIC HEALTH RELEVANCE: These studies will reveal molecular mechanisms at the core of eukaryotic circadian clocks, which in humans impact many aspects of physiology, including sleep, mental well-being and metabolic regulation. An understanding of the reactivity and interactions of clock components provides a basis for molecular intervention in the treatment of sleep disorders, depression, obesity and cancer.