Architecture and function of membrane proteins in planar supported bilayers: A study with photosynthetic reaction centers
Architecture and function of membrane proteins in planar supported bilayers: A study with photosynthetic reaction centers
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DOI：
10.1021/bi961432i
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发表时间：
1996-11-26
期刊：
影响因子：
2.9
通讯作者：
Boxer, SG
中科院分区：
文献类型：
--
作者：
Salafsky, J;Groves, JT;Boxer, SG
我们提出了一种简单方便的方法来创建包含定向和功能性光合反应中心（RC）的流体支撑双层。（1）支撑双层是通过蛋白脂质体与玻璃表面融合而制备的。蛋白脂质体是通过将 RC 自发插入预先形成的小单层囊泡中来制备的。这些囊泡中的 RC 的方向是细胞色素 c 结合表面位于外侧，H 亚基朝向内侧。与玻璃表面融合后，RC 保持功能性且高度定向，细胞色素 c 结合表面暴露于本体溶液中。支撑双层中的 RC 的表面密度为 10(11) RCs/cm(2) 量级。支持的脂质双层的质量通过落射荧光显微镜和光漂白后的荧光恢复来表征脂质的长距离横向移动性。我们证明可以在大面积（例如 1 cm(2)）的干净玻璃表面上制备均匀的流体双层。这些支撑双层中的脂质可以横向移动，并且它们的扩散系数与其他流体双层系统中获得的值一致。这种流动性不受 RC 存在的影响；然而，带有位点特异性荧光标记的 RC 是固定的，尽管保留了电荷分离和细胞色素 c 结合特性。我们推测这是 H 亚基的球状结构域与玻璃基板之间相互作用的结果。由于与完整 RC 相关的独特光谱和功能特征，该系统是非生物界面支持双层中跨膜蛋白的最佳表征示例之一。
We present a simple and convenient method for creating fluid supported bilayers which contain oriented and functional photosynthetic reaction centers (RCs).(1) The supported bilayers are prepared by fusion of proteoliposomes with a glass surface. The proteoliposomes are prepared by spontaneous insertion of RCs into preformed small, unilamellar vesicles. The RCs in these vesicles are shown to be oriented with the cytochrome c binding surface on the outside and the H-subunit facing inside. Upon fusion to glass surfaces, the RCs remain functional and highly oriented, with the cytochrome c binding surface exposed to the bulk solution. The RCs in the supported bilayers are at a surface density of order 10(11) RCs/cm(2). The quality of the supported lipid bilayer is characterized by epifluorescence microscopy and the long-range lateral mobility of the lipids by fluorescence recovery after photobleaching. We demonstrate that homogeneous, fluid bilayers can be prepared over large areas (e.g., 1 cm(2)) of clean glass surfaces. The lipids in these supported bilayers are laterally mobile, and their diffusion coefficient agrees with values obtained in other fluid bilayer systems. This fluidity is unaffected by the presence of RCs; however, the RCs bearing a site-specific fluorescent label are immobile, despite retaining their charge separation and cytochrome c binding properties. We speculate that this results from interactions between the globular domain of the H-subunit and the glass substrate. Because of the unique spectroscopic and functional signatures associated with intact RCs, this system is one of the best characterized examples of a transmembrane protein in a supported bilayer at a nonbiological interface.