Protein-Lipid Interactions

蛋白质-脂质相互作用

• 批准号: 7743374
• 负责人: ROBERT B GENNIS
• 金额: $ 54.25万
• 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 2011-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7743374
• 关键词: Active Sites; Aerobic; Cell Respiration; Charge; Chemistry; Copper; Coupled; Dioxygen; Electron Transport; Enzymes; Event; Family; Free Energy; Funding Applicant; Grant; Heme; Human; Individual; Kinetics; Lesion; Lipids; Measurement; Measures; Medical; Membrane; Mitochondria; Molecular; Monitor; Movement; Mutation; Nature; Organism; Oxidases; Oxygen; Pathway interactions; Planets; Proteins; Proton Pump; Protons; Reaction; Research; Respiration; Roentgen Rays; Side; Site-Directed Mutagenesis; Solutions; Spectroscopy, Fourier Transform Infrared; Spectrum Analysis; Structure; Symptoms; Techniques; Time; Water; cytochrome c oxidase; driving force; interest; member; mutant; pathogen; pathogenic bacteria; protein structure; protonation; respiratory; uptake; voltage

DESCRIPTION (provided by applicant): Funds are being requested to study the molecular mechanism of respiratory oxidases. Virtually all aerobic organisms on the planet utilize a respiratory oxidase from either the heme-copper superfamily or from the bd-family of enzymes. We are studying both groups of enzymes as part of the proposed research. All of the oxidases reduce dioxygen to water, and use the free energy to generate a voltage across the membrane. Most of the charge separation that is driven by these enzymes comes from protons moving across the membrane within the oxidase protein, either to the enzyme active site, to be consumed to form water, or to the bulk solution on the opposite side of the membrane from which protons are taken up, i.e., proton pumping. We are primarily interested in the pathways through which protons move within the proteins, and in the driving forces that result in proton translocation. Which groups are protonated and when? The catalytic cycle consists of a sequence of proton-coupled electron transfer reactions, and we are interested in how the electron transfer is coupled to moving protons, including the proton pump. We use rapid kinetics techniques to examine individual steps in the reaction, monitoring charge movements across the membrane by time- resolved voltage measurements, and correlating this to the electron transfer events, monitored by UV/vis spectroscopy. FTIR spectroscopy is also utilized to monitor changes in protein structure and the protonation of individual residues. Several X-ray structures serve as guides for site-directed mutagenesis, and we can characterize each mutant by measuring the uptake and release of protons from the enzymes, as well as intra-protein proton translocation. In the next grant period, the emphasis will be on trying to characterize intermediates in the reaction chemistry, and to expand our studies to include "non-canonical" oxidases, which lack what we have assumed to be essential residues, although these "non-canonical oxidases function perfectly well. The human mitochondrial aa3-type cytochrome c oxidase is a member of the heme-copper superfamily. One aspect of medical interest concerns the many mitochondrial genetic defects which influence the function of the mitochondrial oxidase, leading to insufficient ability to make ATP and a variety of symptoms depending on the nature of the lesion. A second point of medical relevance concerns the possible vulnerability of pathogenic bacteria to agents that compromise their aerobic respiration. The need for pathogens to survive under conditions of low oxygen, as in intracellular regions, often requires specialized oxidases, such as the bd-type of cbb3-type enzymes.

描述（由申请人提供）：要求资金研究呼吸氧化酶的分子机制。实际上，地球上的所有有氧生物都使用血红素超级家族或酶的BD家庭的呼吸道氧化酶。作为拟议研究的一部分，我们正在研究两组酶。所有氧化酶都将二氧化物降低到水中，并使用自由能在整个膜上产生电压。这些酶驱动的大部分电荷分离来自质子在氧化酶蛋白内移动到氧化酶蛋白中的质子，要么被消耗到酶的活性位点，要么被消耗以形成水，要么在膜的另一侧散装质子，从而从中取走了质子的质子泵，即蛋白质泵。我们主要对质子在蛋白质中移动的途径以及导致质子易位的驱动力感兴趣。哪些组是质子化的？何时？该催化循环由一系列质子耦合的电子转移反应组成，我们对电子转移如何与包括质子泵在内的移动质子耦合感兴趣。我们使用快速的动力学技术来检查反应中的各个步骤，通过时间分辨的电压测量来监测跨膜的电荷运动，并将其与通过UV/VIS光谱监测的电子传输事件相关联。 FTIR光谱也用于监测蛋白质结构的变化和单个残基的质子化。几种X射线结构是定向诱变的指南，我们可以通过测量酶从酶的摄取和释放以及蛋白质内质子易位来表征每个突变体。在下一个批准期间，重点将是试图在反应化学中表征中间体，并将我们的研究扩展到包括“非人道主义”氧化酶，尽管这些氧化酶缺乏我们认为是必不可少的残留物，尽管这些“这些非经典氧化酶都很好地发挥作用。关注许多线粒体遗传缺陷，这些缺陷会影响线粒体氧化酶的功能，从而导致不足以使ATP和各种症状，具体取决于病变的性质，这是医疗相关性的第二点。区域通常需要专门的氧化酶，例如CBB3型酶的BD型。