Molecular Mechanisms of Membrane Transport

膜运输的分子机制

• 批准号: 10321589
• 负责人: DAVID S CAFISO
• 金额: $ 32.7万
• 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 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10321589
• 关键词: Action Potentials; Active Biological Transport; Address; Affinity; Aging; Anti-Bacterial Agents; Antibiotics; Apoptosis; Bacteria; Bacterial Infections; Binding; Carbohydrates; Carrier Proteins; Cell membrane; Cells; Cellular Metabolic Process; Cholera; Complex; Coupling; Data; Development; Disease; Dysentery; Electron Spin Resonance Spectroscopy; Electrons; Environment; Equilibrium; Escherichia coli; Funding; Gram-Negative Bacteria; Human Microbiome; Ions; Iron; Iron Chelating Agents; Island; Knowledge; Label; Lipopolysaccharides; Maintenance; Measurement; Measures; Membrane; Membrane Proteins; Meningitis; Modeling; Molecular; Molecular Conformation; Nickel; Normal Cell; Nutrient; Pertussis; Physiologic pulse; Preparation; Process; Proteins; Research; Resolution; Rotation; Site; Spin Labels; Structure; System; Testing; Transmembrane Transport; Vitamin B 12; Work; analog; bactericide; colicin; extracellular; flexibility; molecular modeling; novel; novel antibiotic class; novel strategies; nuclease; pathogen; pathogenic bacteria; periplasm; protein function; protein protein interaction; receptor; receptor binding; reconstitution; simulation; success; uptake

Project Summary Active membrane transport is a critical process for normal cell metabolism, including the maintenance of ion-gradients, osmotic balance, action potentials 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, rare nutrients are sequestered 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, such as BtuB, also acts as receptors for antibacterial proteins called colicins, which are produced by bacteria to eliminate other bacteria. TonB- dependent transporters are abundant in Gram negative bacteria and are critical to the proper functioning of the human microbiome as well as the success of many bacterial pathogens, such as those that result in meningitis, cholera, pertussis and dysentery. Because they are unique to bacteria, these transporters are a rational target for the development of new classes of antibiotics. High-resolution crystallographic models have been obtained for a number of TonB-dependent transporters; however, the mechanism by which transport takes place is unclear. The proposed work will test models for the molecular mechanisms of transport primarily through the use of site-directed spin labeling and EPR spectroscopy. New approaches have been developed to perform double electron-electron resonance in intact E. coli, and these approaches will be used to determine the conformation of TonB-dependent transporters in E. coli under conditions where transport takes place. 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. Finally, EPR on actively metabolizing E. coli will be used to test models for the import of colicin E3 into the bacterial cell.

项目摘要 主动膜转运是正常细胞代谢的关键过程，包括 维持离子梯度，渗透平衡，动作电位和凋亡。提议 工作将解决有关大肠含量营养摄取机制的关键问题 大肠杆菌将解决有关这些蛋白质的结构和组织的问题 细菌外膜。在大肠杆菌中，稀有营养因素被特定的外部隔离 膜蛋白通过与内膜蛋白TONB耦合来得出能量。这些 TONB依赖性转运蛋白包括负责维生素B12转运的BTUB，并且 FHUA，FECA和FEPA，负责各种形式的螯合 铁。 TONB依赖性转运蛋白（例如BTUB）也充当抗菌的受体 蛋白质称为结菌素，由细菌产生以消除其他细菌。 tonb- 依赖性转运蛋白在革兰氏阴性细菌中很丰富，对适当的 人类微生物组的功能以及许多细菌病原体的成功， 例如导致脑膜炎，霍乱，百日咳和痢疾的。因为他们是 这些转运蛋白是细菌独有的，是开发新类别的合理目标 抗生素。 已经获得了许多依赖TONB的高分辨率晶体学模型 转运蛋白；但是，运输发生的机制尚不清楚。提议 工作将主要通过使用来测试传输的分子机制 定向的自旋标记和EPR光谱。已经开发了新方法 在完整的大肠杆菌中执行双电子电子共振，这些方法将被使用 在条件下确定大肠杆菌中TONB依赖性转运蛋白的构象 运输发生。在外膜中，蛋白质被隔离到结构域或 岛屿被认为可以推动细菌中外膜蛋白的营业额。 EPR 将在大肠杆菌中用于表征驱动结构域形成的蛋白质蛋白质相互作用 并定义外膜的超分子结构。最后，EPR积极 代谢大肠杆菌将用于测试将结菌素E3进口到细菌细胞中的模型。