ENERGY COUPLING BY THE YEAST PLASMA MEMBRANE ATPASE

酵母质膜ATP酶的能量耦合

• 批准号: 2179220
• 负责人: David S Perlin
• 金额: $ 31.96万
• 依托单位: PUBLIC HEALTH RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-04-01 至 1996-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2179220
• 关键词: Escherichia coli; SDS polyacrylamide gel electrophoresis; Saccharomyces; adenosinetriphosphatase; bioenergetics; cell membrane; drug resistance; enzyme structure; gene expression; high performance liquid chromatography; hydrogen channel; hydrogen transport; membrane model; membrane transport proteins; molecular cloning; mutant; nucleic acid sequence; polymerase chain reaction; protein sequence; protein structure function; site directed mutagenesis

The long term objective of this project is to understand the structural basis for coupling ATP hydrolysis to proton transport by the plasma membrane H+-ATPase of yeast. Energy coupling is a fundamental process in biology that frequently involves the conversion of chemical energy to mechanical work. The mechanics underlying coupling in transport enzymes are poorly understood. the yeast H+-ATPase is a proton pump that has been chosen for these studies because it is amenable to biochemical and genetic analyses that are essential for probing coupling. In addition, the H+- ATPase shares significant sequence homology with, and closely resembles in structure and function, important P-type ATPases from animal cells including the Na+,K+-ATPase, H+, K+-ATPase and Ca2+- ATPase. This project will focus on a localized region of protein structure that was implicated from genetic studies, completed in the prior project period, to play an important role in coupling. This region consists of a cytoplasmic hydrophilic loop domain termed the 'phosphatase' domain and includes transmembrane segments 2 and 3. The operating premise in this proposal is that this region provides a structural linkage between proton translocation and ATP hydrolysis domains. This proposal seeks to probe this region genetically in an effort to provide detailed evidence for the role of specific amino acids and/or localized regions of protein structure in coupling. Localized random mutagenesis will be used to generate mutations within the target region and potential pmal coupling mutants will be selected on the basis of hygromycin B resistance and low pH sensitivity. The mutations will be genetically identified and mutant enzymes characterized for assembly and stability properties, the kinetics of ATP hydrolysis and proton transport, and the stoichiometry of H+ transported to ATP hydrolyzed (coupling ratio). Primary site mutations inducing prominent cellular and biochemical phenotypes will be used in revertant analyses to identify local and long-range protein structure interactions. Site-directed mutagenesis will be used to modify residues identified from initial screening routines to be important to function and amino acid residues flanking important primary sites will be extensively modified by saturation mutagenesis to examine effects of localized structure on coupling. Finally, molecular modeling will be used as a visualization and prediction tool to model local regions of protein structure, as well as interactions between closely apposed protein structure elements. The methodological approaches include random, saturation and site- directed mutagenesis, DNA sequence analysis, PCR cloning and mutagenesis, revertant analysis, ATP hydrolysis and H+-transport kinetics, SDS-PAGE, Western blot analysis, H+/ATP stoichiometry and molecular modeling.

该项目的长期目标是了解 耦合ATP水解与质子传输的结构基础 酵母的质膜H+-ATPase。 能量耦合是 生物学的基本过程，经常涉及 化学能量转换为机械工作。 力学 传输酶的基本耦合知之甚少。 酵母H+-ATPase是已为此选择的质子泵 研究是因为它可以接受生化和遗传分析 这对于探测耦合至关重要。 另外，H+ - ATPASE与并与 与结构和功能相似，重要的P型ATPase 动物细胞包括Na+，K+-ATPase，H+，K+-ATPase和Ca2+ - ATPase。 该项目将集中于蛋白质结构的局部区域 这与遗传研究有关，在先前完成 项目时期，在耦合中发挥重要作用。 这个区域 由称为的细胞质亲水环域组成 “磷酸酶”结构域，包括跨膜段2和3。 该提案的运营前提是该地区提供 质子易位和ATP之间的结构联系 水解结构域。 该建议旨在调查该地区 从基因上讲，以提供有关该角色的详细证据 特定氨基酸和/或蛋白质的局部区域 耦合中的结构。 局部随机诱变将用于产生突变 在目标区域和潜在的PMAL耦合突变体中将 根据湿霉素B耐药性和低pH值选择 灵敏度。 突变将被遗传鉴定，并且 用于组装和稳定性特征的突变酶， ATP水解和质子转运的动力学，以及 H+的化学计量法转运为ATP水解（耦合比）。 诱导突出的细胞和生化的主要位点突变 表型将用于重新分析，以识别局部和 远程蛋白质结构相互作用。 站点定向 诱变将用于修饰从初始鉴定的残基 筛选程序对功能和氨基酸很重要 重要的主要原理侧面的残留物将是广泛的 通过饱和诱变进行修改以检查局部的影响 耦合的结构。 最后，分子建模将被用作 可视化和预测工具，以建模 蛋白质结构以及紧密相关的相互作用 蛋白质结构元素。 方法学方法包括随机，饱和和位点 定向诱变，DNA序列分析，PCR克隆和 诱变，恢复分析，ATP水解和H+转移 动力学，SDS-PAGE，WESTERT印迹分析，H+/ATP化学计量学和 分子建模。