TRANSPORT MECHANISM OF THE MULTIDRUG RESISTANCE EFFLUX PROTEIN, EMRE

多药耐药流出蛋白 EMRE 的转运机制

• 批准号: 8185047
• 负责人: Katherine Anne Henzler-Wildman
• 金额: $ 28.88万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8185047
• 关键词: Active Biological Transport; Address; Affinity; Antibiotic Resistance; Bacteria; Bacterial Antibiotic Resistance; Bacterial Drug Resistance; Binding; Biochemical; Biological Assay; Biological Models; Cartoons; Cations; Chemicals; Coupled; Couples; Coupling; Data; Detergents; Escherichia coli; Fluorescence Resonance Energy Transfer; Future; Kinetics; Knowledge; Link; Liposomes; Measurement; Measures; Membrane; Membrane Proteins; Methods; Micelles; Modeling; Molecular Conformation; Motion; Multi-Drug Resistance; Mutate; NMR Spectroscopy; Pharmaceutical Preparations; Population; Process; Protein Dynamics; Proteins; Protons; Relative (related person); Resistance; Resolution; Role; Side; Site; Solutions; Specificity; Structure; System; Technology; Testing; Thermodynamics; Titrations; Transport Process; Vertebral column; antiport; base; combat; dimer; functional outcomes; improved; insight; movie; research study; response; single-molecule FRET; solute; tool

DESCRIPTION (provided by applicant): Active export of drug molecules by multidrug resistance (MDR) efflux proteins is one important mechanism of bacterial drug resistance. EmrE is one of the smallest known MDR transporters, making it an ideal system to study the minimum requirements for MDR efflux. EmrE couples proton import to polyaromatic cation export in E. coli, thus conferring resistance to a broad range of drugs of this type. Although the details are not known, protein conformational change must occur during transport, allowing alternating access to either side of the membrane in response to substrate binding. This project investigates the transport mechanism of EmrE using solution NMR spectroscopy to determine the structures of the multiple states in the transport cycle along with the kinetics of conformational exchange between those states. NMR offers a unique tool to obtain this information, since kinetic and structural data are measured simultaneously at multiple sites across the protein with atomic resolution. Quantitative measurement of the dynamics of EmrE solubilized in fast-tumbling bicelles will be performed using modern solution NMR and single molecule FRET. Single molecule FRET provides a complementary method that can detect details obscured by population averaging and can be used both in bicelles and in liposomes. This data will be compared with standard binding and transport assays to experimentally test two important hypotheses in the single-site alternating access model of coupled antiport with relevance to multidrug efflux: (i) conformational inter- conversion between inward- and outward-facing states is the rate-limiting step for transport, and (ii) binding substrates with different affinities leads to a different energy landscape of the bound state, and thus different rates of conformational interconversion. This knowledge will improve our understanding of secondary active transport and aid efforts to combat bacterial antibiotic resistance due to MDR efflux. PUBLIC HEALTH RELEVANCE: Active export of drug molecules by multidrug resistance (MDR) efflux proteins is one way bacteria achieve antibiotic resistance. This project investigates the transport mechanism of the small multidrug resistance transporter, EmrE, using solution NMR spectroscopy and single molecule FRET. It will improve our understanding of multidrug recognition and secondary active transport mechanisms, aiding future efforts to combat bacterial antibiotic resistance due to MDR efflux.

描述（申请人提供）：多药耐药（MDR）外排蛋白主动输出药物分子是细菌耐药的重要机制之一。 EmrE 是已知最小的 MDR 转运蛋白之一，使其成为研究 MDR 外排最低要求的理想系统。 EmrE 将大肠杆菌中的质子输入与多芳香族阳离子输出耦合，从而赋予对此类药物的广泛耐药性。尽管细节尚不清楚，但蛋白质构象变化必须在运输过程中发生，从而允许响应底物结合而交替进入膜的任一侧。该项目利用溶液核磁共振波谱研究 EmrE 的转运机制，以确定转运循环中多个状态的结构以及这些状态之间构象交换的动力学。 NMR 提供了一种独特的工具来获取这些信息，因为动力学和结构数据是在蛋白质的多个位点以原子分辨率同时测量的。将使用现代溶液 NMR 和单分子 FRET 对溶解在快速翻滚 bicelles 中的 EmrE 动力学进行定量测量。单分子 FRET 提供了一种补充方法，可以检测群体平均所掩盖的细节，并且可用于 bicelles 和脂质体中。该数据将与标准结合和转运测定进行比较，以实验测试与多药物流出相关的偶联反端口单位点交替进入模型中的两个重要假设：（i）向内和向外状态之间的构象相互转换是转运的限速步骤，以及（ii）具有不同亲和力的结合底物导致结合态的不同能量景观，因此不同的转运速率 构象相互转换。这些知识将提高我们对次级主动转运的理解，并有助于对抗由于 MDR 外流引起的细菌抗生素耐药性。 公共卫生相关性：通过多药耐药性 (MDR) 外排蛋白主动输出药物分子是细菌实现抗生素耐药性的一种方式。该项目利用溶液核磁共振波谱和单分子 FRET 研究小型多药耐药转运蛋白 EmrE 的转运机制。它将提高我们对多药识别和次级主动转运机制的理解，有助于未来对抗由于多药耐药外流而导致的细菌抗生素耐药性。