TRANSPORT MECHANISM OF THE MULTIDRUG RESISTANCE EFFLUX PROTEIN, EMRE

描述(申请人提供)：通过多药耐药(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是已知的最小的MDR转运体之一，使其成为研究MDR外流的最低要求的理想系统。EmRE在大肠杆菌中将质子输入与多芳基阳离子输出结合在一起，从而对这类药物的广泛范围产生抗药性。虽然细节尚不清楚，但蛋白质的构象变化必须发生在运输过程中，允许交替进入膜的两侧以响应底物的结合。本项目利用溶液核磁共振波谱研究了EMRE的输运机制，以确定输运循环中多个态的结构以及这些态之间的构象交换动力学。核磁共振提供了一个独特的工具来获得这种信息，因为动力学和结构数据是以原子分辨率在蛋白质的多个位置同时测量的。将使用现代溶液核磁共振和单分子FRET定量测量EmRE在快速翻滚的双柱中的增溶动力学。单分子FRET提供了一种补充方法，可以检测被群体平均遮盖的细节，并可用于双分子和脂质体。这些数据将与标准的结合和转运分析相比较，以实验检验与多药外流相关的偶联反向端口单位交替进入模型中的两个重要假设：(I)向内和向外的状态之间的构象相互转换是运输的限速步骤，(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相互转换速率。这些知识将提高我们对二次主动转运的理解，并有助于努力对抗由于MDR外排而产生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.