ORDERED HELICAL SEGMENTS AND MEMBRANE PROTEINS

明确定义的蛋白质螺旋片段在以下几个方面发挥着关键作用 包括蛋白质转运在内的细胞生物学过程 膜和离子在脂质双层上的流动。建议数 研究将调查有序的α螺旋对AS的贡献 前导序列和离子通道。计划中的研究将描述， 第一次，领导序列在影响 蛋白质的结构和加工，以及最终定义 两种不同离子膜孔的结构和化学计量比 频道。这项工作将通过将两个强大的 技术：2D-核磁共振(溶液和固相)和固相肽 综合。固相多肽合成是制备多肽的关键 否则无法获得用于物理研究的蛋白质/多肽种类 方法，并允许在设计时具有极大的灵活性 要研究的领域和要利用的实验技术 分析。 在蛋白质转运领域，固相合成将使我们能够 目的：1)阐明一种前驱形式的三维结构 蛋白质；2)评估特定氨基酸残基在 对前蛋白构象的影响；3)动力学表征 4)酶前体肽酶抑制剂的设计 领头多肽酶。在离子通道方面，固相肽 合成将允许我们1)定义三维结构 两种鱼雷的两亲性螺旋形成的离子通道 乙酰胆碱受体与哺乳动物脑钠通道； 观察打开和关闭这些通道的动态。固相 将使用模板合成来定义螺旋的数量 包括通道的通道。

Well defined helical segments of proteins play a critical role in several cell biological processes including protein translocation into or across membranes and the flow of ions across the lipid bilayer. The proposed research will investigate the contribution ordered alpha-helices have as both leader sequences and ion channels. The planned studies will describe, for the first time, the roles of the leader sequence in influencing the structure and processing of a protein as well as defining conclusively the structure and stoichiometry of the membrane pores for two different ion channels. This work will be accomplished by combining two powerful techniques: 2D-NMR (solution and solid-phase) and solid-phase peptide synthesis. Solid-phase peptide synthesis is essential in order to prepare otherwise unobtainable protein/peptide species for study by physical methods as well as permitting great flexibility in the design of both the domains to be studied and the experimental techniques to be utilized for the analysis. In the area of protein translocation, solid phase synthesis will enable us to: 1) elucidate the three-dimensional structure of a precursor form of a protein; 2) assess the role of particular amino acid residues in influencing the preprotein's conformations; 3) characterize kinetically the processing enzyme - leader peptidase; and 4) design of inhibitors for leader peptidase. IN the area of ion channels, solid-phase peptide synthesis will allow us 1) to define the three-dimensional structure of the ion channels formed by amphipathic helices for both the Torpedo acetylcholine receptor and the mammalian brain sodium channel; and 2) observe the dynamics that open and close these channels. Solid-phase template synthesis will be employed to define the number of helices comprising the channels.