PROTEIN/LIPID INTERACTIONS

蛋白质/脂质相互作用

• 批准号: 6330000
• 负责人: ROBERT B GENNIS
• 金额: $ 48.16万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-01-01 至 2002-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6330000
• 关键词: Escherichia coli; Raman spectrometry; Rhodospirillales; active sites; bacterial proteins; chemical binding; copper; crystallization; cytochrome oxidase; electron spin resonance spectroscopy; electron transport; enzyme mechanism; enzyme structure; heme; hydrogen transport; infrared spectrometry; interferometry; intermolecular interaction; laboratory mouse; ligands; membrane lipids; membrane proteins; microorganism metabolism; oxidation reduction reaction; protein degradation; protein sequence; respiratory enzyme; site directed mutagenesis; ubiquinone

This proposal is focussed on the study of two bacterial respiratory oxidases. A combination of biochemical, biophysical and genetics techniques will be employed to explore how these two membrane-bound enzymes function to reducer molecular oxygen to water and, concomitantly, to translocate protons across the bacterial membrane to generate a proton motive force. The bd-type ubiquinol oxidase of E. coli contain two subunits and three heme prosthetic groups. Several oxygenated forms of this enzyme have been spectroscopically characterized under equilibrium conditions. One of our goals is to monitor the time evolution of these forms during a single turnover of the oxidase using flow-flash transient kinetics techniques. Studies such as this with the native oxidase are absolutely essential to provide the background information and the spectroscopic techniques which are essential in order to properly utilize mutagenesis as a tool for addressing questions of structure and function. Genetics methods along with biochemical techniques have defined regions of the polypeptides that are involved in heme binding, ubiquinol binding, and possibly, subunit interaction. These studies will be extended to define additional residues that may be critically involved in the active sites. A variety of techniques will be utilized in the characterization of mutants, including resonance Raman, electron spin resonance, and Fourier transform infrared spectroscopies. Our working model places the two active sites of this enzyme, for quinol oxidation and for the reduction of oxygen to water, on opposite sides of the membrane. The net proton translocation across the membrane which is observed during enzyme turnover can be explained by the chemistry occurring at these two separated active sites, without the need to postulate a proton-conducting channel. This model will be further tested. A second project is to examine the aa(3)-type cytochrome c oxidase of Rb. sphaeroides. This enzyme is closely related to the eukaryotic cytochrome c oxidase. We have developed the techniques required to evaluate structural perturbations on each of the metal redox centers within subunit I (heme a, heme a(3) and Cu(B)) caused by specific amino acid substitutions. The consequences of specific mutations on both proton pumping and electron transfer can also be quantified. Mutations will be made in selected residues that are highly conserved in this family of oxidases in order to identify residues critical for heme and Cu binding and also for proton movements during turnover. The aa(3)-type oxidase must have a proton-conducting channel spanning the membrane. Among our targets will residues which are components of the channel.

该提案重点研究两种呼吸道细菌 氧化酶。 生物化学、生物物理和遗传学的结合 将采用技术来探索这两种膜结合的方式 酶的功能是将分子氧还原成水，同时， 使质子穿过细菌膜产生质子 动力。 大肠杆菌的bd型泛醇氧化酶含有两个 亚基和三个血红素假体组。 几种含氧形式 该酶已在平衡状态下进行了光谱表征 状况。 我们的目标之一是监控这些的时间演变 使用流动闪蒸瞬态在氧化酶的单次周转期间形成 动力学技术。 诸如此类的关于天然氧化酶的研究是 提供背景信息和 为了正确利用光谱技术所必需的 诱变作为解决结构和功能问题的工具。 遗传学方法和生化技术已经确定了区域 参与血红素结合、泛醇结合的多肽， 以及可能的亚基相互作用。 这些研究将延伸至 定义可能与活性密切相关的其他残基 网站。 表征过程中将使用多种技术 突变体，包括共振拉曼、电子自旋共振和 傅里叶变换红外光谱。 我们的工作模型将 该酶的两个活性位点，用于醌氧化和 在膜的两侧，氧气还原成水。 网 在酶过程中观察到质子跨膜易位 营业额可以通过这两个地方发生的化学反应来解释 分离的活性位点，无需假设质子传导 渠道。该模型将被进一步测试。 第二个项目是检查 Rb 的 aa(3) 型细胞色素 c 氧化酶。 球状体。该酶与真核细胞色素密切相关 c氧化酶。 我们已经开发了评估所需的技术 每个金属氧化还原中心的结构扰动 由特定氨基酸引起的亚基 I（血红素 a、血红素 a(3) 和 Cu(B)） 替换。 两个质子的特定突变的后果 泵浦和电子转移也可以量化。 突变将是 由该家族中高度保守的选定残基制成 氧化酶，以确定对血红素和铜结合至关重要的残基 以及周转过程中的质子运动。 aa(3)型氧化酶 必须有一个跨越膜的质子传导通道。 我们当中 目标是作为通道组成部分的残留物。