PROTEIN-LIPID INTERACTIONS

蛋白质-脂质相互作用

• 批准号: 3335140
• 负责人: ROBERT B GENNIS
• 金额: $ 20.94万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-12-01 至 1992-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3335140
• 关键词: Escherichia coli; Raman spectrometry; active transport; allosteric site; bacterial proteins; binding proteins; bioenergetics; chemical binding; circular dichroism; conformation; cytochrome oxidase; electron spin resonance spectroscopy; electron transport; enzyme complex; enzyme mechanism; enzyme structure; fluorescence; genetic manipulation; heme; immunochemistry; laboratory rabbit; ligands; lipid bilayer membrane; membrane lipids; membrane model; membrane permeability; membrane proteins; membrane structure; microorganism metabolism; molecular polarity; nonblood lipoprotein; operon; oxygen; protein engineering; protein sequence; proteins; proteolysis

Our laboratory has for a number of years been interested in the structure and function of membrane-bound enzymes. During the past several years we have purified the two cytochrome complexes which are the membrane-bound terminal oxidases of the aerobic respiratory chain of E. coli. One of these enzymes, the cytochrome d terminal oxidase complex, is the focus of all the work proposed in this grant. The cyto-chrome d complex is of particular importance in that it is representative of a class of membrane-enzymes which couples an electron transfer reaction to the electrogenic translocation of protons across the membrane bilayer. Our long term goal is to understand the mechanism by which this enzyme generates a proton motive force. Many diseases are characterized by bioenergetic deficiencies, and understanding the structure and mechanism of a protein which functions as a "coupling site" is of fundamental importance in elucidating the nature of some of these defects. In order to accomplish our goal, we are emphasizing the use of molecular genetics and immunological techniques directed at obtaining structural information about this protein. We will utilize several approaches to determine which portions of each polypeptide are periplasmic, cytoplasmic, or buried with the membrane bilayer. Proteolysis, antibodies binding to defined epitopes, and gene-fusion technology will all be used to define the protein topography within the cytoplasmic membrane. Specific amino acid residues involved in catalysis or heme-binding will be identified primarily through the use of genetics. In particular, site-directed mutagenesis will be used to define which of the total of 10 histidine residues act as heme axial ligands. Biophysical and biochemical approaches will also be used to obtain similar information and help clarify the specific role played by each of the heme centers in this enzyme. Finally, the genetic expression of this enzyme is regulated by oxygen, and we will identify the genes required for this regulation as well as the specific DNA sequence in the cytochrome operon (cyd) which is responsible for the response to oxygen.

我们的实验室多年来一直对 膜结合酶的结构和功能。 过去期间 几年来我们纯化了两种细胞色素复合物 是有氧呼吸的膜结合末端氧化酶 大肠杆菌链。 这些酶之一，细胞色素 d 末端 氧化酶复合物，是本文提出的所有工作的重点 授予。 细胞色素 d 复合物在以下方面特别重要： 它是一类膜酶的代表 将电子转移反应与生电耦合 质子穿过双层膜的易位。 我们的长 术语目标是了解这种酶的机制 产生质子动力。很多疾病都有其特点 通过生物能缺陷，并了解结构和 作为“偶联位点”的蛋白质的机制是 阐明其中一些问题的性质具有根本重要性 缺陷。 为了实现我们的目标，我们强调使用 分子遗传学和免疫学技术 获得有关该蛋白质的结构信息。 我们将 利用多种方法来确定每个方法的哪些部分 多肽是周质的、细胞质的或埋藏在 膜双层。 蛋白水解，抗体结合定义 表位和基因融合技术都将用于定义 细胞质膜内的蛋白质拓扑。 特定氨基 参与催化或血红素结合的酸残基将是 主要通过使用遗传学来识别。 尤其， 定点诱变将用于定义总数中的哪一个 10 个组氨酸残基充当血红素轴向配体。 生物物理学 生化方法也将用于获得类似的 信息并有助于阐明每个人所扮演的具体角色 血红素以这种酶为中心。 最后，该酶的基因表达受到调节 氧气，我们将确定这种调节所需的基因 以及细胞色素操纵子 (cyd) 中的特定 DNA 序列 它负责对氧气的反应。