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

本研究旨在建立固相脂双层的实验平台，以揭示光合膜蛋白、捕光复合体2(LH2)和捕光MM复合体(LH1-RC)的组装结构与功能之间的关系。最近，原子力显微镜(AFM)揭示了细菌光合膜中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“蛋白质NaY”结构。我们尝试构建LH1-RC和LH2在支撑的脂质双层膜上的组装，成功地将LH1、反应中心(RC)和LH1-RC络合物组装到氨基修饰的ITO电极上。当RC与LHL组装时，RC的光电流产生增强，这表明LH1的光捕获和能量传递可以在电极上工作。我们建立了一种新的方法，将LH2掺入到新分离的脂膜…中膜中有更多的氮。简而言之，LH2通过囊泡融合和随后的侧向扩散选择性地排列在阴离子平面膜的流体区域上。通过全内反射荧光(TIRF)显微镜观察到LH2/LH1-RC的排列过程，通过将LH2和LH1-RC连续沉积在氨基修饰的玻片上，成功地构建了LH2/LH1-RC在支撑脂质双层中的共存阵列，LH1、反应中心(RC)和LH1-RC络合物成功地组装在氨基修饰的ITO电极上。当RC与LH1组装时，RC的光电流产生增强，这表明LH1的光捕获和能量转移可以在电极上工作。该结构在平面膜和衬底之间提供了薄水层。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。作为实验平台，该体系结构使得研究LH1-RC和LH2在平面膜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