Crystal structure analysis of MFS transporter proteins

我们尝试构建了多药输出系统MFS、SMR和MATE中29个蛋白的表达系统。最后，分别建立了金黄色葡萄球菌QacA和NorA以及谷氨酸棒状杆菌CGL2611的表达纯化体系。我们也尝试使用Mistic融合表达系统，并证实了EbrA、EbrB、TetB（13个跨膜螺旋）和LmrB（14个跨膜螺旋）的表达。考察了QacA、NorA和CGL2611结晶样品制备的详细条件。通过透射电镜、CD测量和差示扫描量热法证实了添加假定底物的效果。对于QacA，假设底物罗丹明6G可以避免其聚集，并且三维结构也稳定。然而，在CGL2611的情况下，洗涤剂DDM在浓缩过程中存在问题，并且没有发现有效的制备底物。观察了5种去污剂和PEG400、PEG3350两种沉淀剂溶液的相图，建立了初始结晶条件。在此基础上，利用蒸汽扩散法和脂质立方相法在不同温度下筛选了QacA和NorA的初始结晶条件。观察到一些结晶物体，但没有得到衍射晶体

We have tried to construct expression systems for 29 proteins of multidrug export system MFS, SMR and MATE. Finally, expression and purification systems were established for three of them, QacA and NorA from Staphylococcus aureus, and CGL2611 from Corynebacterium glutamicum. We have also tried to use a Mistic fusion expression system, and have confirmed expression of EbrA, EbrB, TetB (13 transmembrane helices) and LmrB (14 transmembrane helices) .A detailed condition of preparing crystallization sample was examined for QacA, NorA and CGL2611. Effect of adding putative substrate was confirmed using Transmission Electron Microscope, CD measurement and Differential Scanning Calorimetry. For QacA, it was suggested that putative substrate Rhodamine 6G avoid its aggregation and three-dimensional structure was also stabilized. In the case of CGL2611, however, detergent DDM was troubling in a concentration process, and no putative substrate effective for its preparation was found.Phase diagram for solutions consist of 5 detergents and two precipitants, PEG400 and PEG3350, was observed, and initial crystallization conditions were established. With such informations, screening for initial crystallization conditions was done for QacA and NorA using a vapor diffusion method and a lipidic-cubic phase method at several temperatures. Some crystalline objects were observed, but no diffracting crystal was obtained