ROLE OF ORDERED HELICAL SEGMENTS IN MEMBRANE PROTEINS

有序螺旋片段在膜蛋白中的作用

• 批准号: 3468042
• 负责人: John M Tomich
• 金额: $ 11万
• 依托单位: CHILDREN'S HOSPITAL OF LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-12-01 至 1994-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3468042
• 关键词: brain; cholinergic receptors; conformation; electrophysiology; exopeptidase; gap junctions; laboratory rat; lipid bilayer membrane; membrane channels; membrane proteins; nuclear magnetic resonance spectroscopy; peptide chemical synthesis; potassium channel; protein signal sequence; protein structure function; protein transport; sodium channel

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.

蛋白质的明确螺旋片段在多个方面发挥着关键作用 细胞生物学过程，包括蛋白质易位进入或跨过 膜和离子穿过脂质双层的流动。 拟议的 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的结构和化学计量 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。 为了制备肽，固相合成是必不可少的 物理研究无法获得的蛋白质/肽种类 方法以及允许设计的极大灵活性 要研究的领域和要使用的实验技术 分析。 在蛋白质易位领域，固相合成将使我们能够 目的：1）阐明a的前体形式的三维结构 蛋白质; 2）评估特定氨基酸残基的作用 影响前蛋白的构象； 3) 动力学表征 加工酶——前导肽酶； 4）抑制剂的设计 前导肽酶。 在离子通道领域，固相肽 综合将使我们能够1）定义三维结构 鱼雷的两亲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 将采用模板合成来定义螺旋数 包括渠道。