STRUCTURE/ACTIVITY STUDIES OF COMPLEMENT PROTEIN C9

补体的第九组分（C9）负责细胞毒性的免疫调节。 补的作用。因此，它是 免疫系统C9是一种70 Kda的糖蛋白， 有效的必须从稳定折叠的水溶性蛋白质转变为 一种内在膜蛋白。这种转变的性质必须是 了解这种蛋白质的功能。获得这样 信息的结构C9在其原生的彻底调查 水溶性形式和其膜结合形式将被引发。 通过表面映射、电子顺磁共振（EPR）等方法获得信息静态结构 荧光光谱、电子显微镜和三维图像 重建和小角中子散射。的动力学 当蛋白质进入细胞膜时， 然后进行EPR和荧光光谱分析。所有技术将 受益于用特异性探针标记蛋白质。过去 特定的标签是有限的网站数量少， 蛋白质。然而，这个问题现在可以在一定程度上规避 通过定点诱变和使用新的光谱技术， 足够灵敏的技术来检查少量的突变体 proteins.方法是采用基因工程标记 C9序列中用于连接标记的位点。 下一个赠款期的具体目标是： 1.通过以下方法鉴定溶血功能所必需的肽区： 产生具有游离半胱氨酸的C9突变体用于位点特异性 标签的附着，以及由来自 裂解性（人）和非裂解性（马）C9分子。 2.为了确定跨膜区的存在（或不存在）， 膜结合的C9通过映射天然C9和膜结合的C9的表面， 结合C9的免疫学，酶和光谱方法。 3.确定C8和C9之间的接触部位，并测量 C9重折叠和插入膜的动力学。 4.为了通过3-氨基-2-甲基-N- 中子小角散射三维图像重建 结合对比度变化过程。

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.