Ritzmann Noah, Thoma Johannes, Hirschi Stephan, Kalbermatter David, Fotiadis Dimitrios, Müller Daniel J
Department of Biosystems Science and Engineering, ETH Zürich, Basel, Switzerland.
Institute of Biochemistry and Molecular Medicine, University of Bern, Bern, Switzerland.
Biophys J. 2017 Sep 19;113(6):1181-1186. doi: 10.1016/j.bpj.2017.06.022. Epub 2017 Jul 8.
One major objective of synthetic biology is the bottom-up assembly of minimalistic nanocells consisting of lipid or polymer vesicles as architectural scaffolds and of membrane and soluble proteins as functional elements. However, there is no reliable method to orient membrane proteins reconstituted into vesicles. Here, we introduce a simple approach to orient the insertion of the light-driven proton pump proteorhodopsin (PR) into liposomes. To this end, we engineered red or green fluorescent proteins to the N- or C-terminus of PR, respectively. The fluorescent proteins optically identified the PR constructs and guided the insertion of PR into liposomes with the unoccupied terminal end facing inward. Using the PR constructs, we generated proton gradients across the vesicle membrane along predefined directions such as are required to power (bio)chemical processes in nanocells. Our approach may be adapted to direct the insertion of other membrane proteins into vesicles.
合成生物学的一个主要目标是自下而上组装简约型纳米细胞，其由脂质或聚合物囊泡作为结构支架，以及膜蛋白和可溶性蛋白作为功能元件组成。然而，目前尚无可靠方法来确定重构到囊泡中的膜蛋白的方向。在此，我们介绍一种简单方法，可使光驱动质子泵视紫质（PR）插入脂质体时确定其方向。为此，我们分别在PR的N端或C端设计了红色或绿色荧光蛋白。荧光蛋白通过光学方法识别PR构建体，并引导PR插入脂质体，使未占据的末端朝向脂质体内部。利用这些PR构建体，我们沿着预定方向在囊泡膜上产生了质子梯度，这是为纳米细胞中的（生物）化学过程提供能量所必需的。我们的方法可能适用于指导其他膜蛋白插入囊泡。
