活细胞中探究ATP合酶生成过程中的组装机制及质量控制因子

ATP合酶是一种广泛存在、负责能量转换的多亚基膜蛋白复合体。其典型亚基组成模式为：α3β3γεδab2c8-15。可分为插入细胞膜内的Fo部分(ab2c8-15)和膜外的F1（α3β3γεδ）两部分。其完成能量转换是通过一种独特的旋转催化机制实现。尽管人们一直试图知道这一精巧蛋白质机器是如何组装而成的，但过往研究大多基于通过遗传学手段以及体外生物化学手段开展。.本申请主要拟通过非天然氨基酸介导的活细胞中的蛋白质光交联方法体系来探究其在细菌中的组装机制。非天然氨基酸将通过遗传学方法被特异地或非特异地，通过核糖体上的翻译过程引入到亚基蛋白质中。以此可跟踪活细胞中其新生肽链的包括转运，折叠和组装等在内的分子过程。结合免疫印迹和质谱鉴定等蛋白质检测方法，有望揭示ATP合酶这一蛋白质机器的生成组装过程，以及参与其中的质量控制因子，进而认识该重要蛋白质机器的功能机制和调控机制。

ATP synthase is a multisubunit protein complex that acts as the major energy transducer in all organisms. It has the typical composition as α3β3γεδab2c8-15, and can be divided into two parts: the membrane-integrated FO part (ab2c8-15) and the membrane-extrinsic F1 part (α3β3γεδ）. It functions via a unique rotational catalytic mechanism. Although how such an amazing complex are formed has attracted much attention, most of the researches have been conducted via genetic or in vitro biochemical approaches. . Here we plan to explore this by performing unnatural amino acid-mediated in vivo protein photo-crosllinking analysis. In this regard, we will introduce the photo-reactive unnatural amino acid into each of its subunits, specifically at particular residue positions or non-specifically at certain residue positions during the ribosomal translational of the polypeptides. It follows that we can follow how each subunit is translocated and assembled during its biogenesis in living cells, by analyzing the interacting proteins of each nascent subunit, via immunoblotting and mass spectrometry analysis. We are confident that our research will unveil the assembly and quality control mechanisms of this unusual protein complex during its biogenesis, which in turn will greatly help us to further understand its functional and regulatory mechanisms in living cells.