MODULATION OF MEMBRANE FUNCTION BY COMPLEMENT

拟议研究的长期目标是 的性质和程度的相互作用， 末端补体蛋白（C5、C6、C7、C8、C9）， 一起形成膜攻击复合物（MAC）， 补体，以及人工和天然的脂质和蛋白质 靶向膜。 我们想了解 在细胞表面组装这种复合物，并阐明 MAC可以引起免疫的机制 细胞溶解和细胞死亡或保护宿主细胞免于自噬， 杀伤性 我们希望通过使用一种组合来实现我们的长期目标。 生物物理、生物化学和免疫化学技术， 研究MAC的组装及其对目标的影响 膜的 我们的研究将主要集中在C9，因为 这种蛋白质提供了细胞的主要溶解和细胞毒性功能， Mac. 我们将使用合成肽的方法来产生一个 天然C9和C9与靶结合时的表位图 膜的 圆二色性和电子自旋共振（ESR） 光谱学将用于分析构象变化， 蛋白质在细胞膜上组装并穿透细胞膜。 脂质双层结构的相应变化将是 通过ESR和差示扫描量热法监测。 的 所得MAC-膜复合物将通过冷冻可视化， 断口电子显微镜 活性中心定位 将尝试通过产生蛋白水解片段， 溶解或细胞毒活性。 这些活动将由 测量标记物从脂质体的释放，或 电导跨越平面脂质双层，并通过测试肽 直接影响血细胞和细菌的存活。

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.