Project Summary/Abstract Circadian clocks are intracellular enzymatic systems that provide a reliable biochemical representation of local time with profound consequences to health across diverse organisms. Unlike most enzymes, biological clocks are insensitive to a range of physiological temperatures and cellular energy levels so that organisms can anticipate dawn and dusk reliably. Another criterion of circadian clocks is that they can be entrained through environmental cues and thereby stay synchronized to local time. However, the mechanisms of these defining hallmarks remain incompletely resolved in any organism, and thus represent outstanding gaps in knowledge across the field of biological timekeeping. Therefore, the overall vision for the next five years is to build upon our prior work to elucidate the mechanisms of temperature and metabolic compensation and entrainment in the circadian clock of cyanobacteria, a system widely valued by the circadian clocks community. Because proteins and their interactions underpin clock mechanisms regardless of organism, lessons learned here are expected to serve as important points of reference for the scientific community working on diverse circadian clocks. Over the past 20 years, we have made many impactful discoveries on mechanism of the cyanobacterial clock and developed innovative methodologies and tools along the way. Our lab recently reconstituted the intact cyanobacterial clock in vitro such that each component can be monitored in real time over several days, and our refined and predictive model is the framework for our hypotheses regarding mechanisms of temperature compensation, metabolic compensation, and entrainment. Thus, we are very well positioned to succeed at filling these critical gaps in knowledge. A major expected outcome of the work proposed here is a detailed cause-and-effect model linking clock protein behavior and interactions to clock phenotypes in vivo.