Here we use an innovative, combined chemical and biophysical approach to decipher the molecular mechanism circadian clocks use to integrate complex environmental sensing pathways into regulation of metabolism and development. Synchronization of cellular physiology with diurnal changes in environmental variables is a central aspect of circadian clocks. Notably, desynchronization of master and peripheral clocks due to disruption in sleep cycles or altered metabolic function have been implicated in the onset and progression of diseases ranging from obesity, diabetes and heart disease. Notably, the molecular mechanisms gating reciprocal regulation of metabolism and the core circadian oscillator have been hampered by the complexity and number of entrainment pathways in vertebrates. In contrast, the principal environmental variable regulating circadian function in plants is blue-light, enabling precise spatial and temporal interrogation of protein:protein interaction networks integrating environmental factors into circadian regulation of metabolism and development. Herein, we focus on elucidating the role of flavin- binding photoreceptors in mediating circadian function in the model organism Arabidopsis thaliana. A complete understanding of how flavin chemistry dictates activation of protein degradation pathways in a circadian manner can facilitate analysis of similar environmentally sensitive pathways in higher organisms including humans. To map a reaction trajectory beginning from initial photon absorption to alteration of organism physiology we will focus on three primary factors. 1.) Define the chemical and photochemical activation mechanisms of A. thaliana circadian clock photoreceptors. The Zeitlupe (ZTL), Flavin-Kelch-Fbox-1 (FKF1) and LOV-Kelch-Protein (LKP2) family of photoreceptors couples activation of a flavin-binding domain to alteration in protein stability though regulation of light-activated F-Box domains. Using a combination of spectroscopic techniques we will unravel chemical mechanisms regulating ZTL/FKF1/LKP2 function. 2.) Elucidate structural dynamics regulating environmental sensing. Using a combination of NMR and X-ray crystallography, we will decipher atomic resolution detail of how alteration in flavin chemistry dictates alterations in protein structure to selectively excite multple signaling pathways. 3.) Regulation of protein:protein networks. Structural characterization of the photoactivation process will guide design of protein variants to selectively disrupt formation of signaling complexes. Mapping of interaction surfaces will facilitate interrogation of cellular signaling.
The proposed project focuses on elucidating the molecular mechanisms organisms use to entrain circadian rhythms to alterations in environmental variables. Keen understanding of sensory adaptation and the role of stress responses in mediating alteration of circadian function is of central importance to human physiology and disease. By working in the model organism, Arabidopsis thaliana, we can delineate sensory adaptation from the core circadian mechanism through a focus on photosensory receptors. Deciphering the molecular mechanism governing integration of blue-light and oxidative stress into the circadian machinery will facilitate our understanding of analogous processes in humans and other animals.