Supplement for AKTA pure 25 M FPLC System

This research program focuses on two areas of bioinorganic chemistry: metal transport by P1B-ATPases and biological methane oxidation by particulate methane monooxygenase (pMMO). Common themes of these two projects include understanding the structure and function of integral membrane metalloproteins, elucidating the atomic details of metal sites within these proteins, and establishing molecular mechanisms of metal ion transport or of catalysis by metal ions. These two processes are fundamentally different: transport requires a highly specific metal binding site with dynamically changing affinities whereas catalysis demands a specifically tailored site for chemical transformations. In both cases, the long term goal is to understand how the larger context of the protein scaffold confers these functional properties. The P1B-ATPases, integral membrane proteins that use the energy of ATP hydrolysis to transport metal ions across membranes, play a key role in metal homeostasis in all organisms. In particular, P1B-ATPases are linked to human diseases of metal metabolism and to the virulence of human pathogens. Despite their universal importance, fundamental issues related to P1B-ATPase structure and function remain unresolved, including the molecular basis of metal ion specificity and the mechanism of transport. These questions will be addressed by characterizing a range of P1B-ATPases that transport different metal substrates. Experimental approaches include biochemical characterization, metal binding studies, spectroscopy, in vitro activity assays, in vivo analysis, spectroscopy, and crystallography. Particulate methane monooxygenase (pMMO) is an oligomeric, integral membrane metalloenzyme that converts methane to methanol in methanotrophic bacteria, organisms that utilize methane as their sole source of carbon and energy. Methanotrophs are a potential means to mitigate the deleterious effects of global warming on human health. In addition, methanotrophs can oxidize substrates besides methane, including halogenated hydrocarbons, and have therefore been targeted for bioremediation applications. Major questions central to pMMO structure and function will be addressed, including the atomic details of the active site, the chemical mechanisms of oxygen and methane activation, the roles of the different protein subunits, and the molecular basis for substrate specificity. The experimental approach involves biochemical, spectroscopic, mechanistic, and crystallographic characterization of native pMMOs, recombinant variant pMMOs, and recombinant soluble pMMO domains.