TRANSPORT MECHANISM OF THE MULTIDRUG RESISTANCE EFFLUX PROTEIN, EMRE

描述（由申请人提供）：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将质子输入耦合到E中的聚芳族阳离子输出。大肠杆菌，从而赋予耐药的广泛范围内的这种类型的药物。虽然细节尚不清楚，但蛋白质构象变化必须发生在运输过程中，允许响应底物结合而交替进入膜的任一侧。该项目使用溶液NMR光谱研究EmrE的运输机制，以确定运输循环中多个状态的结构沿着这些状态之间的构象交换动力学。NMR提供了一种独特的工具来获得这些信息，因为动力学和结构数据是在蛋白质的多个位点上以原子分辨率同时测量的。将使用现代解决方案NMR和单分子FRET的EmrE溶解在快速翻滚的bicelles的动力学的定量测量。单分子FRET提供了一种互补的方法，可以检测被群体平均化所掩盖的细节，并且可以在双胞和脂质体中使用。将该数据与标准结合和转运试验进行比较，以实验性检验与多药外排相关的偶联反向转运的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运输的限速步骤，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，从而导致不同的构象转换率。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MDR外排引起的细菌抗生素耐药性。

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.