The broad long term objective of this proposal is to understand the molecular basis of superoxide production in human neutrophils. The proposed investigation will focus on the structural changes of human neutrophil flavocytochrome b (Cytb) upon activation of the NADPH oxidase. The fundamental assumption made in this proposal is that alterations in the structure of this electron transferase regulate the flow of electrons across the membrane it spans. Elucidation of the structural mechanism of regulation of this electron flow will provide crucial information necessary to understand the molecular basis of an essential process in phagocyte-mediated microbicidal killing and tissue injury. Examination of the structure/function relationships existing in this integral membrane glycoprotein may lead to the development of rationally designed drugs that could ameliorate neutrophil mediated tissue damage and enhance neutrophil mediated microbicidal killing. More specifically, this proposal outlines strategies for exploiting 9 monoclonal and recombinant antibodies that recognize unique native flavocytochrome b epitopes, defining their placement on the surface of flavocytochrome b. It describes a plan to covalently modify these antibodies with fluorescent probes, to triangulate heme sites using fluorescence resonance energy transfer, measure the distance between antibody sites, and determine how these distances change upon activation of the oxidase. The proposal also outlines a strategy to determine surface topology and intramolecular proximities. This strategy includes selective crosslinking of purified flavocytochrome followed limited proteolysis and HPLC/mass spectrometry analysis. It also outlines a plan to analyze Cytb molecular shape by single molecule conventional and cryoelectron microscopy. Lastly, the proposal exploits recent progress made in antibody imprinting, an application of phage display analysis developed by the Principal Investigator, which identifies structural elements of antibody binding sites. This information will be used to help produce a nearest neighbor map of the antigen epitope to aid in design of peptide inhibitors of the antibody-antigen interactions and produce raw material for making antibody-peptide complexes for cocrystallization and structure determination under other support. Successful completion of this work will initiate the development of a structural model of the flavocytochrome that will be able to incorporate its known sequence, transmembrane topology, gross molecular shape, and antibody imprint structure of discrete surface sites and be essential to the interpretation of any fully solved x-ray crystal structure.
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