期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2020
卷号：117
期号：26
页码：15006-15017
DOI：10.1073/pnas.1919306117
出版社：The National Academy of Sciences of the United States of America
摘要：Cytochrome bo 3 ubiquinol oxidase is a transmembrane protein, which oxidizes ubiquinone and reduces oxygen, while pumping protons. Apart from its combination with F 1 F o -ATPase to assemble a minimal ATP regeneration module, the utility of the proton pump can be extended to other applications in the context of synthetic cells such as transport, signaling, and control of enzymatic reactions. In parallel, polymers have been speculated to be phospholipid mimics with respect to their ability to self-assemble in compartments with increased stability. However, their usability as interfaces for complex membrane proteins has remained questionable. In the present work, we optimized a fusion/electroformation approach to reconstitute bo 3 oxidase in giant unilamellar vesicles made of PDMS- g -PEO and/or phosphatidylcholine (PC). This enabled optical access, while microfluidic trapping allowed for online analysis of individual vesicles. The tight polymer membranes and the inward oriented enzyme caused 1 pH unit difference in 30 min, with an initial rate of 0.35 pH·min −1 . To understand the interplay in these composite systems, we studied the relevant mechanical and rheological membrane properties. Remarkably, the proton permeability of polymer/lipid hybrids decreased after protein insertion, while the latter also led to a 20% increase of the polymer diffusion coefficient in polymersomes. In addition, PDMS- g -PEO increased the activity lifetime and the resistance to free radicals. These advantageous properties may open diverse applications, ranging from cell-free biotechnology to biomedicine. Furthermore, the presented study serves as a comprehensive road map for studying the interactions between membrane proteins and synthetic membranes, which will be fundamental for the successful engineering of such hybrid systems.
