School of Chemistry and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK.
School of Food Science and Nutrition, University of Leeds, Leeds, UK.
Methods. 2018 Sep 1;147:142-149. doi: 10.1016/j.ymeth.2018.01.021. Epub 2018 Feb 2.
Hybrid vesicles composed of lipids and block copolymers hold promise for increasing liposome stability and providing a stable environment for membrane proteins. Recently we reported the successful functional reconstitution of the integral membrane protein cytochrome bo (ubiquinol oxidase) into hybrid vesicles composed of a blend of phospholipids and a block copolymer (PBd-PEO). We demonstrated that these novel membrane environments stabilise the enzymes' activity, prolonging their functional lifetime [Chem. Commun. 52 (2016) 11020-11023]. This approach holds great promise for applications of membrane proteins where enhanced durability, stability and shelf-life will be essential to creating a viable technology. Here we present a detailed account of our methods for membrane protein reconstitution into hybrid vesicles and discuss tips and challenges when using block copolymers compared to pure phospholipid systems that are more common materials for this purpose. We also extend the characterisation of these hybrid vesicles beyond what we have previously reported and show: (i) hybrid membranes are less permeable to protons than phospholipid bilayers; (ii) extended enzyme activity data is presented over a period of 500 days, which fully reveals the truly remarkable enhancement in functional lifetime that hybrid vesicles facilitate.
由脂质和嵌段共聚物组成的杂交囊泡有望提高脂质体的稳定性，并为膜蛋白提供稳定的环境。最近，我们成功地将整合膜蛋白细胞色素 bo（泛醇氧化酶）重组到由磷脂和嵌段共聚物（PBd-PEO）混合物组成的杂交囊泡中。我们证明这些新型膜环境稳定了酶的活性，延长了它们的功能寿命[Chem. Commun. 52 (2016) 11020-11023]。这种方法在膜蛋白的应用中具有很大的前景，在这些应用中，增强耐用性、稳定性和保质期对于创造可行的技术至关重要。在这里，我们详细介绍了将膜蛋白重组到杂交囊泡中的方法，并讨论了与更常见的磷脂体系相比，使用嵌段共聚物时的技巧和挑战，这些磷脂体系通常更适用于此目的。我们还扩展了对这些杂交囊泡的特性描述，超出了我们之前的报道，并展示了：（i）与磷脂双层相比，混合膜对质子的渗透性更低；（ii）提出了长达 500 天的酶活性扩展数据，充分揭示了杂交囊泡所带来的真正显著的功能寿命延长。
