Abstract of the Funded Project (R35 GM141849) Circadian rhythms arise from genetically encoded clocks that are intimately linked to external cues like light to synchronize physiology and behavior with the 24-hour solar cycle. Although the genetic networks that give rise to circadian rhythms are now relatively well established, we still don’t understand many of the fundamental, molecular steps that determine the ~24-hour basis of these clocks and how they respond to external time-setting cues. By integrating structural biology and solution biophysical methods with biochemistry and cell biology, we aim to determine the underlying biochemical principles that lead to the day-long timescale of circadian signaling and uncover the mechanisms that allow biological clocks to faithfully maintain intrinsic timing and respond robustly to external cues. With prior NIGMS funding, we studied clock systems from mammals and cyanobacteria to discover how different clock proteins assemble into regulatory complexes and identified how protein dynamics, enzyme activity and/or post-translational modifications impact clock timing. Our comparative biochemical approach highlighted surprising commonalities, such as the competition for mutually exclusive binding sites, between these clocks despite their different molecular architectures. Here, we will continue to pursue the structural basis of protein assemblies from diverse biological clocks, determine the consequences of post- translational modifications on clock protein function, study the molecular basis for entrainment of clocks to external cues, and seek out new inroads for pharmacological intervention. Funding from the MIRA program would provide us with the resources and flexibility to explore commonalities in mechanisms of biological timekeeping across a diverse array of species from cyanobacteria to humans.