PROJECT SUMMARY This project examimes the structure, spectroscopy, function, and mechanism of metallproteins that regulate cell cycle progresion in response to programmatic fluctuations in intracellular zinc concentration. Zinc fluxes have recently been discovered to be key regulatory events in human physiology, including cortical neuron function, immune response, fertilization, and insulin secretion. Despite its apparent roles in these profound physiologies, the biochemistry of zinc action is not well understood. Little is known regarding the inorganic chemistry of metalloproteins involved in the instructive mechanisms that mediate cellular responses to zinc fluxes, zinc trafficking pathways, or the role of specific metalloprotein receptors in signaling events. To interpret and eventually intervene in diseases caused by disruption of such pathways, we must first elucidate the fundamental molecular mechanisms of zinc-dependent switching events. While zinc has traditionally been viewed as a static cofactor involved in protein structure and enzyme catalysis, recent studies support the idea that zinc binding sites in regulatory proteins respond to transient fluctuations in zinc availability and are switched on and off in ways that regulate key cellular events. We will test the hypothesis that regulatory zinc fluxes exert instructive control over the mammalian cell cycle through specific, receptor- mediated processes. This hypothesis is based on multiple lines of evidence, including: (1) data showing fluctuations zinc distribution at various points in the cell cycle for single cells; (2) live cell imaging demonstrating the movement of waves of zinc; and (3) physicochemical approaches showing colocalization of zinc with specific factors. The overarching objective of this proposal is to use biochemical and spectroscopic approaches to understand how the influx and efflux of zinc exert control over the cell cycle. Chemicals tools to probe and control the concentration of zinc will be created to further our understanding of zinc physiology. In addition, we will leverage emerging techniques in X-ray absorption and emission spectroscopy, NMR, and mass spectrometry to understand the chemical environment of zinc and probe the zinc occupancy of zinc-containing proteins that regulate cell cycle progression. Taken together, the results from these studies will elucidate a robust role for zinc signaling fluxes during the cell cycle.