Salafsky J, Groves J T, Boxer S G
Department of Chemistry, Stanford University 94305-5080, USA.
Biochemistry. 1996 Nov 26;35(47):14773-81. doi: 10.1021/bi961432i.
We present a simple and convenient method for creating fluid supported bilayers which contain oriented and functional photosynthetic reaction centers (RCs). 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/cm2. 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 cm2) 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.
我们提出了一种简单便捷的方法来制备包含定向且具有功能的光合反应中心（RCs）的流体支撑双层膜。支撑双层膜通过蛋白脂质体与玻璃表面融合来制备。蛋白脂质体通过将RCs自发插入预先形成的小单层囊泡中制备而成。这些囊泡中的RCs显示为细胞色素c结合表面向外，H亚基向内定向排列。与玻璃表面融合后，RCs仍保持功能且高度定向，细胞色素c结合表面暴露于本体溶液中。支撑双层膜中的RCs表面密度约为10(11)个RCs/cm2。通过落射荧光显微镜表征支撑脂质双层膜的质量，通过光漂白后荧光恢复来表征脂质的长程横向流动性。我们证明可以在大面积（例如1 cm2）的清洁玻璃表面上制备均匀的流体双层膜。这些支撑双层膜中的脂质具有横向流动性，其扩散系数与在其他流体双层膜系统中获得的值一致。这种流动性不受RCs存在的影响；然而，带有位点特异性荧光标记的RCs尽管保留了电荷分离和细胞色素c结合特性，但却是固定不动的。我们推测这是由于H亚基的球状结构域与玻璃底物之间的相互作用所致。由于与完整RCs相关的独特光谱和功能特征，该系统是在非生物界面的支撑双层膜中跨膜蛋白特征最明确的例子之一。
