MODULATION OF MEMBRANE FUNCTION BY COMPLEMENT

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The long range objectives of the proposed research are to characterize the nature and the extent of interaction between the terminal complement proteins (C5, C6, C7, C8, C9), which together form the membrane attack complex (MAC) of complement, and lipids and proteins in artificial and natural target membranes. We want to understand the mechanism of assembly of this complex on a cell's surface and elucidate the mechanisms through which the MAC can either cause immune cytolysis and cell death or protect host cells from auto- destruction. We expect to attain our long range goals by using a combination of biophysical, biochemical and immunochemical techniques to investigate the assembly of the MAC and its effect on the target membrane. Our studies will focus predominantly on C9 because this protein provides the main lytic and cytotoxic functions of the MAC. We will use the synthetic peptide approach to generate an epitope map of native C9 and of C9 when bound to target membrane. Circular dichroism and electron spin resonance (ESR) spectroscopy will be used to analyze conformational changes in the protein as they assemble on and penetrate into the membrane. The corresponding changes in lipid bilayer structure will be monitored by ESR and differential scanning calorimetry. The resulting MAC-membrane complex will visualized by freeze- fracture electron microscopy. Localization of the active center of C9 will be attempted by producing proteolytic fragments with lytic or cytotoxic activity. Such activities will be assayed by measuring release of markers from liposomes, or changes in conductance across planar lipid bilayers, and by testing peptides directly for their effects on survival of blood cells and bacteria.