STRUCTURE - ACTIVITY STUDIES OF COMPLEMENT PROTEIN C9

补体的第九组分(C9)负责细胞毒 补体的作用。因此，它是 免疫系统。C9是一种70kDa的糖蛋白，为了成为 有效的必须从稳定折叠的水溶性蛋白质转变为 一种固有的膜蛋白。这种转变的性质必须是 了解这种蛋白质是如何发挥作用的。为了获得这样的成绩 对C9原生结构的深入研究 将以水溶形式和膜结合形式启动。 信息静态结构将通过表面测绘获得 以及荧光光谱、电子显微镜和三维图像 重建和小角中子散射。反应的动力学 当蛋白质进入细胞膜时发生的结构变化将 其次是电子顺磁共振和荧光光谱分析。所有技术都将 从用特定探针标记蛋白质中受益。在过去 具体的标注受到可用站点数量较少的限制 一种蛋白质。然而，这个问题现在可以在一定程度上绕过。 通过定点突变和使用较新的光谱技术 具有足够灵敏度以检测少量突变的技术 蛋白质。方法是使用基因工程标记。 C9序列中的位点，用于标记的附着。 下一批资助期的具体目标是： 1.通过以下方法鉴定溶血功能所必需的多肽区域 用游离型半胱氨酸构建位点特异性C9突变体 标记和由以下片段组成的杂化分子的附着 裂解的(人)和非裂解的(马)C9分子。 2.确定细胞内是否存在跨膜区 通过映射天然C9的表面和膜结合的C9- 用免疫学、酶法和光谱学方法结合C9。 3.确定C8和C9之间的接触点，并测量 C9折叠和插入膜的动力学。 4.通过3-生成膜结合C9的低分辨结构 三维图像重建与小角中子散射 结合对比度变化程序。

The ninth component of complement (C9) is responsible for the cytotoxic action of complement. It is, therefore, an important component of the immune system. C9 is a 70 Kda glycoproteins which in order to be effective must change from a stably folded, water soluble protein into an intrinsic membrane protein. The nature of this transformation must be understood to appreciate how this protein functions. To gain such information a thorough investigation of the structure C9 in its native water soluble form and in its membrane bound form will be initiated. Information static structures will be obtained by surface mapping, EPR and fluorescence spectroscopy, electron microscopy and 3-D image reconstruction, and small angle neutron scattering. The kinetics of structural changes as they occur when the protein enters a membrane will be followed by EPR and fluorescence spectroscopy. All techniques will benefit from labeling of the protein with specific probes. In the past specific labeling was limited by the small number of sites available on a protein. However, this problem can now be circumvented to some extend by site-directed mutagenesis and the use of newer spectroscopic techniques of sufficient sensitivity to examine small amounts of mutant proteins. The approach is to employ genetically engineered labeling sites in the C9 sequence for the attachment of labels. The specific aims for the next grant period are: 1. To identify the peptide region necessary for hemolytic function by generation of C9 mutants with free cysteines for site-specific attachment of labels, and of hybrid molecules composed of segments from lytic (human) and non-lytic (horse) C9 molecules. 2. To determine the presence (or absence) of transmembrane regions in membrane-bound C9 by mapping of the surfaces of native C9 and membrane- bound C9 with immunologic, enzymatic and spectroscopic methods. 3. To identify contact sites between C8 and C9 and to measure the kinetics of C9 refolding and insertion into membranes. 4. To generate a low resolution structure of membrane-bound C9 by 3- dimensional image reconstruction and by small angle neutron scattering combined with contrast variation procedures.