MEMBRANE PROTEIN TOPOLOGY--CYTOCHROME C

细胞膜的结构和功能的阐明 今天，组件仍然是科学公司的重大挑战之一。这个 当我们试图了解分子基础时，挑战变得迫在眉睫 常见的致命性遗传病，如囊性纤维化 缺陷归因于跨膜氯通道功能障碍。 通道是如何构建到膜中的？膜是否会影响 频道活动？它与哪些关键组件交互 膜旁边呢？这些都是关于膜的关键问题 因缺乏适当方法而未得到解答的蛋白质拓扑学 和模型系统。考虑到这些长期目标，我们建议 要解决膜拓扑挑战的特定方面，请执行以下操作 介绍了一种新的实验方法。这将被用来确定 细胞色素c是电子的一种成分，它的精确配置 运输系统，在膜表面。而晶体结构 已知，细胞色素c停靠在 膜表面以及它如何与其合作伙伴在功能上相互作用 电子传输系统仍然存在疑问。X射线驻波 (Xsw)将首次用来确定，具有亚角 分辨率，本征重原子，铁，在一个 细胞色素c单分子膜与朗缪尔-布洛杰特膜的静电结合 沉积在固体载体上的脂膜。后者将被用来 生成XSWs。蛋白质定向将通过执行以下操作来确认 额外的XSW测量以确定i)中的硒原子位置 硒蛋氨酸修饰三个或三个以上位点的细胞色素c 取代天然蛋氨酸和II)Ru原子在 细胞色素c在蛋白质表面的Cysl02处发生单一衍生化反应。在……里面 这样，至少五个已知参考点在 膜表面以上的蛋白质将被测定。这一次，一起 利用已知的蛋白质结构，将被用来建立 明确细胞色素c在膜表面的取向。 将确定还原和氧化时的取向变化 并与晶体结构中的构象变化相关联。 此外，蛋白质在水相中的分布 以及膜脂是如何调节这一点的 膜浴的特点及离子组成 解决方案将被确定并用于检验假设，即至少 线粒体中存在两个细胞色素c池。分开测量 将利用凋亡素c和细胞色素c共价结合到 膜表面。膜对接的成功确定 细胞色素c的作用机制和蛋白质 在膜结合形式和可溶形式之间的分布为 用X射线光电子能谱技术阐明黄曲霉毒素的结构和配置 一系列整合型和周围型蛋白质，其膜特性 由于缺乏适当的方法，仍然难以捉摸。

The elucidation of the structure and function of cellular membrane components remains today one of Science's grand challenges. The challenge becomes immediate as we seek to understand the molecular basis of such common lethal genetic diseases as cystic fibrosis where the defect is attributed to a malfunctioning transmembranal chloride channel. How is the channel built into the membrane? Does the membrane influence channel activity? What critical components does it interact with in, on, and next to the membrane? These are crucial questions concerning membrane protein topology that remain unanswered for want of appropriate methods and model systems. With these long term objectives in mind, we propose to tackle particular aspects of the membrane topology challenge by introducing a new experimental approach. This will be used to determine the precise disposition of cytochrome c, a component of the electron transport system, at the membrane surface. While the crystal structure of the protein is known, the manner in which cytochrome c docks on the membrane surface and how it interacts functionally with its partners in the electron transport system is still in question. X-ray standing waves (XSW) will be used for the first time to determine, with subangstrom resolution, the position of the intrinsic heavy atom, iron, In a cytochrome c monolayer electrostatically bound to a Langmuir-Blodgett lipid film deposited on a solid support. The latter will be used to generate the XSWs. Protein orientation will be confirmed by performing additional XSW measurements to determine i) selenium atom location in cytochrome c modified at three or more sites with selenomethionine substituting for the native methionine and ii) ruthenium atom location in cytochrome c derivatized singly at Cysl02 on the protein surface. In this way, the location of at least five known reference points in the protein above the membrane surface will be determined. This, together with the known structure of the protein, will be used to establish unequivocally the orientation of cytochrome c at the membrane surface. Changes in orientation upon reduction and oxidation will be determined and correlated with conformational changes seen in the crystal structure. Additionally, the distribution of the protein in the aqueous phase next to the membrane and how this is modulated by the membrane lipid characteristics as well as the ionic composition of the membrane bathing solution will be determined and used to test the hypothesis that at least two pools of cytochrome c exist in mitochondria. Separate measurements will make use of the apocytochrome c and cytochrome c covalently bound to the membrane surface. A successful determination of the membrane docking mechanism for cytochrome c and the manner in which the protein distributes between membrane-bound and soluble forms paves the way for the elucidation by the XSW technique of the structure and disposition of a host of integral and peripheral proteins whose membrane properties remain elusive for lack of appropriate methodologies.