This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The Chivers lab is interested in how cells control intracellular levels of essential trace metals. A balance must be achieved between levels that are sufficient for growth and those that are toxic to the cell. They focus on the regulation and intracellular trafficking of nickel in microbes, in particular Escherichia coli and Helicobacter pylori. In these bacteria, nickel-enzymes are synthesized with the help of specific nickel-binding chaperones. The nickel-dependent transcriptional regulator NikR binds free nickel ions and represses expression of nickel uptake genes. NikR has also been shown to postively regulate gene expression of at least one operon (urrease) in H. pylori. The function of NikR indicates that free nickel ions accumulate only after sufficient nickel has been acquired to synthesize nickel-dependent enzymes. The high-affinity of NikR for nickel ions, however, suggests that nickel-trafficking pathways must actively compete with NikR for newly acquired nickel ions. A goal is to study the mechanism by which this partitioning is achieved. Our approach combines biochemical and biophysical studies of purified proteins (for example, fluorescence and UV-visible spectroscopy) with in vivo studies of protein function (molecular genetics, reporter assays) to correlate molecular properties, such as ligand-binding affinity, with biological function. A new direction is to use mass spectrometry, especially some of the biophysical approaches developed in the Michael Gross lab.