Membrane protein selectively oriented on solid support and reconstituted into a lipid membrane.
Membrane protein selectively oriented on solid support and reconstituted into a lipid membrane.
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膜蛋白选择性地定向在固体支持物上并重构为脂质膜。
DOI：
10.1021/la062227z
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发表时间：
2007
期刊：
影响因子：
--
通讯作者：
O. Lambert
中科院分区：
文献类型：
--
作者：
S. Trépout;S. Mornet;H. Benabdelhak;A. Ducruix;A. Brisson;O. Lambert
固体支持物上的模拟功能膜正在出现用于膜生物传感器的开发或用于膜介导过程的研究，并且应该对生物诊断产生重要影响。我们建立了一种方法来重建膜蛋白到脂质膜上的选择性取向的固体支持物。膜蛋白OprM，OprM-MexA-MexB多药外排泵的一个组成部分，溶解在洗涤剂中，通过其胞外结构域固定在氨基硅烷修饰的二氧化硅表面。通过去污剂去除将定向蛋白质重构成脂质膜。在二氧化硅纳米颗粒和平面二氧化硅表面上进行的膜蛋白重构过程分别通过低温电子显微镜（cryo-EM）和石英晶体微天平与耗散监测（QCM-D）进行。氨基硅烷改性的二氧化硅表面上的选择性蛋白质取向进行了评估，通过冷冻-EM和二氧化硅表面上的非特异性蛋白质沉积进行了比较。最后，结合MexA，周质组成部分的三方外排复合物，监测与QCM-D上的定向OprM蛋白单层。大的吸附质量给出了一个直接的证据，MexA与OprM的周质螺旋部分的高亲和力。
Mimetic functional membranes on solid support are now emerging for the development of membrane biosensor or for the study of membrane-mediated processes and should have an important impact on biodiagnostics. We established a method to reconstitute a membrane protein into a lipid membrane in a selective orientation on a solid support. Membrane protein OprM, a component of OprM-MexA-MexB multidrug efflux pump, solubilized in detergent was immobilized via its extracellular domain on aminosilane-modified silica surface. The oriented protein was reconstituted into a lipid membrane by detergent removal. The membrane protein reconstitution process carried out on silica nanoparticles and on planar silica surfaces was followed by cryo-electron microscopy (cryo-EM) and quartz crystal microbalance with dissipation monitoring (QCM-D) respectively. The selective protein orientation on aminosilane-modified silica surface was assessed by cryo-EM and was compared to the nonspecific protein deposition on silica surface. Finally, the binding of MexA, a periplasmic component of the tripartite efflux complex, was monitored with QCM-D on the oriented OprM protein monolayer. The large adsorbed mass gave a direct evidence of the high affinity of MexA with the periplasmic helical part of OprM.