Molecular Mechanisms of Membrane Transport

膜运输的分子机制

• 批准号: 10652857
• 负责人: DAVID S CAFISO
• 金额: $ 34.93万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-09-06 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10652857
• 关键词: ATP Synthesis Pathway; Active Biological Transport; Affinity; Antibiotics; Apoptosis; Automobile Driving; Bacteria; Bacterial Infections; Behavior; Binding; Carbohydrates; Carrier Proteins; Cell membrane; Cell physiology; Cells; Cholera; Coupling; Crystallography; Data; Development; Dysentery; Electron Spin Resonance Spectroscopy; Electrons; Environment; Equilibrium; Escherichia coli; Funding; Gram-Negative Bacteria; Human Microbiome; Hydrophobicity; In Vitro; Ions; Iron; Iron Chelating Agents; Iron Chelation; Island; Knowledge; Length; Lipids; Lipopolysaccharides; Measurement; Mediating; Membrane; Membrane Proteins; Meningitis; Modeling; Molecular; Molecular Conformation; Motion; Movement; Nickel; Nutrient; Osmosis; Pertussis; Phospholipids; Physiologic pulse; Play; Preparation; Procedures; Proteins; Proton-Motive Force; Regulation; Research; Resolution; Role; Sampling; Site; Spin Labels; Structure; System; Testing; Transmembrane Transport; Vitamin B 12; Work; biophysical analysis; biophysical properties; experimental study; extracellular; in vivo; intermolecular interaction; molecular modeling; novel antibiotic class; novel strategies; pathogen; pathogenic bacteria; protein function; protein oligomer; protein protein interaction; protein structure; reconstitution; restraint; success; uptake

Active membrane transport is central to many cell processes, including the acquisition of nutrients, the establishment of ion-gradients, the regulation of osmotic balance, ATP synthesis, and apoptosis. The proposed work will address key questions regarding the mechanisms of nutrient uptake in Escherichia coli, and it will address questions regarding the structure and organization of these proteins in the bacterial outer-membrane. In E. coli, a range of nutrients are transported by specific outer-membrane proteins that derive energy by coupling to the inner- membrane protein TonB. These TonB-dependent transporters include BtuB, which is responsible for vitamin B12 transport, and FhuA, FecA and FepA, which are responsible for the transport of various forms of chelated iron. TonB-dependent transporters are abundant in Gram negative bacteria, and they are critical to the proper functioning of the human microbiome where they are responsible for the acquisition and initial processing of some carbohydrates. They are also essential for the success of many bacterial pathogens, such as those that result in meningitis, cholera, pertussis and dysentery; and because TonB-dependent transport is unique to bacteria, it is thought to be a target for the development of new classes of antibiotics. High-resolution crystallographic models have been obtained for approximately two dozen TonB- dependent transporters; however, the mechanism by which transport takes place is unclear. One difficulty is that structural and biophysical studies in-vitro have never been made on TonB- dependent transport proteins that are known to be active. The proposed work will test models for the molecular mechanisms of transport and examine structural states that are only observed in cells. The role of lipopolysaccharide in altering transport protein structure will also be examined. The proposed work will employ site-directed spin labeling and EPR spectroscopy where novel approaches have been developed to perform pulse experiments, such as double electron-electron resonance, in intact E. coli. In the outer-membrane, proteins are sequestered into domains or islands, which are thought to drive the turnover of outer-membrane proteins in bacteria. EPR will be used in E. coli to characterize the protein-protein interactions that drive domain formation and define the supramolecular structure of the outer-membrane.

主动膜运输是许多细胞过程的核心，包括获取 营养，建立离子梯度，渗透平衡的调节，ATP合成， 和凋亡。拟议的工作将解决有关机制的关键问题 大肠杆菌中的营养摄取，它将解决有关结构和结构的问题 这些蛋白质在细菌外膜中的组织。在大肠杆菌中，一系列营养 通过特定的外膜蛋白来运输，该蛋白通过耦合到内部 - 膜蛋白量。这些依赖TONB的转运蛋白包括BTUB， 负责维生素B12运输，以及FHUA，FECA和FEPA，这些负责 各种形式的螯合铁的运输。 tonb依赖性转运蛋白的克很丰富 阴性细菌，它们对于人类微生物组的正常功能至关重要 他们负责某些碳水化合物的获取和初步处理。他们是 对于许多细菌病原体的成功也至关重要，例如 脑膜炎，霍乱，百日咳和痢疾；而且因为依赖TONB的运输是独特的 对于细菌而言，它被认为是开发新类抗生素的靶标。 大约二十吨的高分辨率晶体学模型已获得 依赖转运蛋白；但是，运输发生的机制尚不清楚。 一个困难是，从未对Tonb-进行结构和生物物理研究。 已知活性的依赖转运蛋白。拟议的工作将测试模型 对于运输的分子机制，并检查仅观察到的结构状态 在细胞中。脂多糖在改变转运蛋白结构中的作用也将是 检查。拟议的工作将采用定向定向的自旋标签和EPR光谱法 已经开发出新的方法来执行脉冲实验的地方，例如双 电子 - 电子共振，完整的大肠杆菌。在外膜中，蛋白质被隔离 进入域或岛屿，被认为可以推动外膜蛋白的营业额 细菌。 EPR将用于大肠杆菌中，以表征驱动蛋白质蛋白质的相互作用 域的形成并定义外膜的超分子结构。