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: 8293037
• 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/8293037
• 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.

描述（由申请人提供）：通过多药耐药性（MDR）外排蛋白主动导出药物分子是细菌耐药性的一种重要机制。 EMRE是最小的MDR转运蛋白之一，它是研究MDR外排最低要求的理想系统。 EMRE夫妇质子在大肠杆菌中进口到多野阳离子出口，从而赋予对这种类型的广泛药物的耐药性。尽管细节尚不清楚，但在运输过程中必须发生蛋白质构象变化，从而可以响应底物结合而交替进入膜的两侧。该项目使用溶液NMR光谱研究EMRE的运输机制，以确定运输周期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提供了一个独特的工具来获取此信息，因为在具有原子分辨率的蛋白质的多个位点同时测量了动力学和结构数据。将使用现代溶液NMR和单分子fret进行定量测量EMRE溶解的EMRE动力学。单分子FRET提供了一种互补方法，可以检测被人口平均掩盖的细节，并且可以在双层和脂质体中使用。 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构象互连。这些知识将提高我们对二级主动运输和援助努力，以打击由于MDR外排构成的细菌抗生素耐药性。