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Crystal structure analysis of MFS transporter proteins

MFS转运蛋白的晶体结构分析

基本信息

批准号：
18570101
负责人：
WATANABE Nobuhisa
金额：
$ 2.55万
依托单位：
Nagoya University (2007)Hokkaido University (2006)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18570101/
关键词：
Structural Biology Membrane Proteins Multidrug Efflux Systems Secondary Transporters X-rav crystallography

项目摘要

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

我们尝试构建了多药输出系统MFS、SMR和Mate的29个蛋白的表达系统。最后，建立了金黄色葡萄球菌的QacA和NorA和谷氨酸棒杆菌的CGL2611三个基因的表达和纯化体系。我们还尝试使用Mistic融合表达系统，证实了Ebra、EBRB、TetB(13个跨膜螺旋)和LmrB(14个跨膜螺旋)的表达。对QacA、Nora和CGL2611结晶样品的制备条件进行了详细的考察。用透射电子显微镜、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，推测底物罗丹明6G避免了其聚集，并且三维结构也稳定了。然而，对于CGL2611，洗涤剂DDM存在浓缩过程，没有找到制备它的有效底物。观察了由5种洗涤剂和两种沉淀剂PEG400和PEG3350组成的溶液的相图，并建立了初始结晶条件。利用这些信息，使用气相扩散法和脂质立方相法在几个温度下对QacA和NorA的初始结晶条件进行了筛选。观察到了一些晶态物体，但没有观察到衍射晶。