PDF(Pubmed)
Abstract：
Adenosine triphosphate (ATP)-producing modules energized by light-driven proton pumps are powerful tools for the bottom-up assembly of artificial cell-like systems. However, the maximum efficiency of such modules is prohibited by the random orientation of the proton pumps during the reconstitution process into lipid-surrounded nanocontainers. Here, we overcome this limitation using a versatile approach to uniformly orient the light-driven proton pump proteorhodopsin (pR) in liposomes. pR is post-translationally either covalently or noncovalently coupled to a membrane-impermeable protein domain guiding orientation during insertion into preformed liposomes. In the second scenario, we developed a novel bifunctional linker, trisNTA-SpyTag, that allows for the reversible connection of any SpyCatcher-containing protein and a HisTag-carrying protein. The desired protein orientations are verified by monitoring vectorial proton pumping and membrane potential generation. In conjunction with ATP synthase, highly efficient ATP production is energized by the inwardly pumping population. In comparison to other light-driven ATP-producing modules, the uniform orientation allows for maximal rates at economical protein concentrations. The presented technology is highly customizable and not limited to light-driven proton pumps but applicable to many membrane proteins and offers a general approach to overcome orientation mismatch during membrane reconstitution, requiring little to no genetic modification of the protein of interest.
摘要：
由光驱动质子泵供电的三磷酸腺苷（ATP）生产模块是自下而上组装人造细胞样系统的强大工具。然而,这样的模块的最大效率被质子泵在重构过程中随机定向到脂质包围的纳米容器中所禁止。这里,我们使用一种通用的方法克服了这一限制，使光驱动质子泵蛋白视紫红质（pR）在脂质体中均匀定向。在插入预先形成的脂质体期间,pR在翻译后共价或非共价偶联至膜不可渗透的蛋白质结构域引导取向。在第二种情况下，我们开发了一种新的双功能接头，trisNTA-SpyTag,这允许任何含SpyCatcher的蛋白质和携带HisTag的蛋白质的可逆连接。通过监测矢量质子泵和膜电位的产生来验证所需的蛋白质取向。与ATP合酶结合，高效的ATP生产是由向内的抽水人口激励的。与其他光驱动ATP产生模块相比，均匀的方向允许在经济的蛋白质浓度下的最大速率。所提出的技术是高度可定制的，不限于光驱动质子泵，但适用于许多膜蛋白，并提供了一种克服膜重建过程中方向不匹配的通用方法。几乎不需要对感兴趣的蛋白质进行遗传修饰。
