ROLE OF ORDERED HELICAL SEGMENTS IN MEMBRANE PROTEINS

明确定义的蛋白质螺旋片段在几个方面起着关键作用。 细胞生物学过程，包括蛋白质易位进入或穿过 膜和跨脂质双层的离子流。 拟议 研究将调查有序α螺旋的贡献， 前导序列和离子通道。 计划的研究将描述， 第一次，领导序列的作用，在影响 蛋白质的结构和加工，以及最终定义 两种不同离子的膜孔结构和化学计量比 渠道 这项工作将通过结合两个强大的 技术：2D-NMR（溶液和固相）和固相肽 合成. 固相肽合成是必不可少的，以制备 否则无法获得的蛋白质/肽物质，用于物理研究 方法，以及允许在设计的两个巨大的灵活性， 研究领域和实验技术，以利用 的分析。 在蛋白质转运领域，固相合成将使我们能够 目的：1）阐明前体形式的三维结构， 蛋白质; 2）评估特定氨基酸残基在 影响前蛋白的构象; 3）动力学表征 加工酶-前导肽酶;和4）设计抑制剂 前导肽酶 在离子通道领域，固相肽 合成将允许我们1）定义的三维结构的 两亲性螺旋形成的离子通道， 乙酰胆碱受体和哺乳动物脑钠通道;和2） 观察打开和关闭这些通道的动态。 固相 将采用模板合成来确定螺旋的数目 包括通道。

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.