Two-component signalling is a widespread biological phenomenon found primarily in procaryotes but also in eucaryotes. Its abundant representation makes it clear that virtually all phenomena studied in bacteria are either directly or indirectly regulated by at least one two- component system. Furthermore, since it appears that all members within each component family share structural and function homology, there is a unifying theme which has allowed previously unrelated biological phenomena to share findings. This type of informational networking is rare and has accelerated the progress in many two-component systems. For example, results from seemingly diverse areas as osmoregulation (OmpR), sporulation (SpoOA/F) or chemotaxis (CheY) have been compared and used to gain insight into each particular system. Also, new members in the 100+ family of CheY homologs can immediately benefit from the cumulative database. Clearly, there are differences and variations but the basic motif appears to be retained. This proposal is focused primarily on the structure and function of CheY. CheY is in the fortunate position of being the only response regulator for which there is high resolution structural information to combine with extensive genetic and biochemical data. We have both solid-state crystallographic and solution NMR structures of CheY. In collaboration with Dr. F.W. Dahlquist, we propose to continue our efforts in solving the solution NMR structure of the phosphorylated form of CheY and defining the molecular mechanism of activation. In addition, we will further elucidate the sites of protein-protein interaction and the conversion of kinase binding domain to the activated flagellar motor binding domain of CheY. We will also study the enhancement of the phosphatase activity of CheZ when it is bound to CheAS. These ambitious experiments represent a fortuitous combination of events; the development of a biologically important signal transduction system, an overproduced and well characterized response regulator protein, and the newly emerging technical advances in crystallography and NMR spectroscopy.