Direct observation of molecular assembly of photosynthetic membrane proteins in solid-supported lipid bilayers

为了揭示光合膜蛋白与捕光复合物2 （LH2）和捕光mm复合物（LH1-RC）的组装结构和功能之间的关系，本研究旨在建立固体负载脂双分子层的实验平台。最近，利用原子力显微镜（AFM）研究了细菌光合膜中LH2和LH1-RC的阵列结构。在膜中，六聚体LH1-RCs被LH2分子包围，形成“蛋白nay”结构。我们试图构建LH1-RC和LH2在支持的脂质双层膜中的组装。LH1、反应中心（RC）和LH1-RC配合物成功组装在氨基修饰的ITO电极上。当RC与LHl组装时，可以观察到RC产生的光电流增强，这表明LH1可以在电极上进行光收集和能量传递。我们已经建立了一种新的方法，将LH2结合到一个新分离的脂质结构域中。简而言之，通过囊泡融合和随后的横向扩散，将LH2结合到阳离子囊泡货物（蛋白脂质体）中，选择性地排列在阴离子平面膜的流体域上。利用全内反射荧光（TIRF）显微镜观察了LH2/LLH1-RC的排列过程，通过将含有LH2和LH1-RC的囊泡载体连续沉积在氨基修饰的盖层上，成功构建了LH2/LLH1-RC在负载的脂质双分子层中共存的阵列。LH1、反应中心（RC）和LH1-RC配合物成功组装在氨基修饰的ITO电极上。当RC与LH1组装时，可以观察到RC产生的光电流增强，这表明LH1可以在电极上进行光收集和能量传递。通过亲和素与生物素的相互作用，成功构建了由亲和素固定的底物拴住的平面脂质双分子层。该结构在平面膜和衬底之间提供薄水层。水层允许具有大的突出亲水结构域的LH1-RC在膜内横向扩散。该结构作为实验平台，使研究LH1-RC和LH2在平面膜Less中的动态组装成为可能

This research has aimed to establish experimental platforms using solid-supported lipid bilayer in order to reveal the relation between the assembling structure and function of photosynthetic membrane proteins, light-harvesting complex 2 (LH2) and light-harvesting mm complex (LH1-RC). Recently, the array structure of LH2 and LH1-RC in a bacterial photosynthetic membrane has been revealed by atomic force microscopy (AFM). In the membrane, hexameric LH1-RCs were surrounded by LH2 molecules, forming 'protein nay' structure. We attempted to construct assembly of LH1-RC and LH2 in a supported lipid bilayer membrane.LH1, reaction center(RC), and LH1-RC complexes were successfully assembled onto an amino-modified ITO electrode. Enhanced photocurrent generation by RC was observed when the RC was assembled with LHl, indicating that light harvesting and energy transfer by LH1 could work on the electrode.We have established a novel method for incorporation of LH2 into a phew-separated lipid domai … More n in the membrane. In brief, LH2 incorporated into a cationic vesicle cargo (proteoliposome) is selectively arrayed on the fluid domain of the anionic planar membrane via vesicle fusion and subsequent lateral diffusion. The prceess of arraying has been observed by total internal reflection fluorescence (TIRF) microscopy An LH2/LLH1-RC coexistent array in a supported lipid bilayer was successfully constructed by consecutive deposition of vesicle cargos incorporating LH2 and LH1-RC onto an amino-modified coverslip.LH1, reaction center (RC), and LH1-RC complexes were successfully assembled onto an amino-modified ITO electrode. Enhanced photocurrent generation by RC was observed when the RC was assembled with LH1, indicating that light harvesting and energy transfer by LH1 could work on the electrode.A planar lipid bilayer tethered by an avidin-immobilized substrate via avidin-biotin interaction was successfully constructed. The architecture provides thin water layer between the planar membrane and the substrate. The water layer allows the LH1-RC possessing large protrudent hydrophilic domain to diffuse laterally within the membrane. As the experimental platform, this architecture makes it possible to investigate dynamic assembling of LH1-RC and LH2 in the planar membrane Less