Project Summary: Circadian clocks have evolved to synchronize organism physiology with environmental conditions. Central to their function is the ability to adapt to unique environmental niches to maintain a 24-hour daily cycle to optimize organism fitness. Although circadian networks differ in various organisms, two hallmarks of circadian networks are conserved across phyla: 1) A central oscillator composed of a transcription-translation feedback loop (TTFL), and 2) Sensory elements that entrain the TTFL to endogenous and environmental cues. Examination of circadian network topology across diverse organisms reveals two protein domain families are widely employed to integrate environmental stimuli into circadian networks. These are members of the Light-Oxygen-Voltage (LOV: fungi and plants) and Cryptochromes (CRY: plants and animals). Results in diverse organisms demonstrate that despite conservation, the mechanisms and roles of these proteins can differ considerably. How LOV and CRY proteins are able to adapt the magnitude and mode of signal propagation to adapt to environmental conditions is unknown. Based on recent computational and biophysical studies of LOV and CRY systems in plants, fungi, and animals, we hypothesize that LOV/CRY systems leverage a dynamic conformational landscape to enable integration of environmental variables that are species and/or environment specific. By understanding these complex landscapes, we can: 1) Predict how closely related organisms tune circadian responses to maximize fitness. 2) Develop strategies to manipulate organism physiology to rectify either deleterious (disease causing) errors or encourage beneficial adaptations. Herein, we develop a platform, amendable to undergraduate researchers, that integrates biological, biophysical, and computational approaches. We focus on two aims to verify plasticity in circadian networks. We specifically focus on systems that retain allosteric mechanisms analogous to those found in mammals, thereby allowing us to develop new strategies to impact human disease. Aim 1: Leveraging recent computational and structural data we will directly evaluate allosteric switch residues that alter the conformational landscape of LOV proteins. Specific focus will be on residues allowing closely related plant and fungal species to alter signaling dynamics in an environmentally specific manner. We will demonstrate tunability of these signaling networks, thereby developing new methodologies to manipulate organism physiology. Aim 2: Leveraging recent chemical and structural studies of a photoactive vertebrate CRY, we will demonstrate that analogous structural plasticity exists in CRY-based systems allowing organisms to alter the magnitude and direction of conformational responses. Using structural and biochemical approaches we will develop an optogenetic tool capable of manipulating mammalian circadian networks. The combined approach enables us to identify natural mechanisms employed to tune c...