Molecular mechanism of membrane transport in the bacterial multidrug resistance proteins

本工作的长期目标是了解细菌多药耐药的分子机制，并推进核磁共振光谱（NMR）研究膜蛋白结构的方法。多药耐药是指癌症和细菌细胞将各种化学上不同的有毒化合物从细胞中排出的能力，从而破坏化疗和抗菌化学物质的有效性。药物挤压是由称为多药物转运体的膜蛋白促进的。大肠杆菌多药耐药蛋白MdfA将作为研究药物挤压分子机制的模型。我们计划分析MdfA的全局折叠或整体结构，并使用多维核磁共振绘制蛋白质中的药物结合位点，这是一种特别适合研究小分子与蛋白质结合以及研究溶液中蛋白质动力学的方法。MdfA和相关的转运体利用跨膜离子梯度的能量将药物泵出细胞。我们将试图确定蛋白质结构中的离子结合位点，并研究蛋白质折叠的变化，这可能涉及药物和离子结合以及跨细胞膜的易位。为了在未来的MdfA研究中达到原子分辨率，我们将尝试用x射线晶体学对蛋白质进行结晶，以求解MdfA的结构。这项工作将促进我们对致病菌和癌细胞多重耐药现象的认识，并有望为未来癌症化疗新方法的开发和新的抗菌治疗方法的开发提供新的思路和贡献。在这个项目过程中开发的实验方法应该引起膜蛋白生物化学领域广泛的研究界的兴趣。

The long-term goal of this work is to understand the molecular mechanism of multidrug resistance in bacteria and to advance the methods of investigating the structure of membrane proteins by nuclear magnetic resonance spectroscopy (NMR).  Multidrug resistance refers to the ability of cancer and bacterial cells to expel various chemically different toxic compounds from the cell, thus undermining the effectiveness of chemo-therapy and antibacterial chemicals. Drug extrusion is facilitated by membrane proteins called multidrug transporters. The multidrug resistance protein MdfA from E.coli will be used as a model for studying the molecular mechanism of drug extrusion. We plan to analyze the global fold, or overall architecture, of MdfA, and to map the drug-binding site in the protein, using multi-dimensional NMR, a method uniquely suited for investigating small molecule bind-ing to proteins and for studying protein dynamics in solution.  The MdfA and related transporters use the energy of transmembrane ion gradients to pump drugs out of the cells. We will attempt to pinpoint the ion-binding site in the structure of the protein and to investigate changes in the fold of the protein, which may be involved in drug and ion binding and translocation across the cell membrane. To reach an atomic resolution in the future work on MdfA, we will try to crystallize the protein for solving the structure of MdfA by X-ray crystallography. This work will advance our understanding of the multiple drug resistance phe-nomenon in pathogenic bacteria and cancer cells, and, hopefully, will provide new ideas and contribute to the future development of new approaches to the chemotherapy of can-cer and to the development of new antibacterial treatments. The experimental approaches developed in the course of this project should be of interest to the broad research com-munity in the area of membrane protein biochemistry.