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.

Homogeneous hybrid vesicles where lipid diffusion decreases with increasing polymer content but polymer diffusion is minimally affected by lipid composition.

Hybrid vesicles consisting of phospholipids and block‐copolymers are increasingly finding applications in science and technology. Herein, small angle X‐ray scattering (SAXS) and cryo‐electron tomography (cryo‐ET) are used to obtain detailed structural information about hybrid vesicles with different ratios of 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphocholine (POPC) and poly(1,2‐butadiene‐block‐ethylene oxide) (PBd22‐PEO14, Ms = 1800 g mol−1). Using single particle analysis (SPA) the authors are able to further interpret the information gained from SAXS and cryo‐ET experiments, showing that increasing PBd22‐PEO14 mole fraction increases the membrane thickness from 52 Å for a pure lipid system to 97 Å for pure PBd22‐PEO14 vesicles. Two vesicle populations with different membrane thicknesses in hybrid vesicle samples are found. As these lipids and polymers are reported to homogeneously mix, bistability is inferred between weak and strong interdigitation regimes of PBd22‐PEO14 within the hybrid membranes. It is hypothesized that membranes of intermediate structure are not energetically favorable. Therefore, each vesicle exists in one of these two membrane structures, which are assumed to have comparable free energies. The authors conclude that, by combining biophysical methods, accurate determination of the influence of composition on the structural properties of hybrid membranes is achieved, revealing that two distinct membranes structures can coexist in homogeneously mixed lipid‐polymer hybrid vesicles.

Hybrid vesicles consisting of phospholipids and block-copolymers are increasingly finding applications in science and technology. Herein, small angle X-ray scattering (SAXS) and cryo-electron tomography (cryo-ET) were used to obtain detailed structural information about hybrid vesicles with different ratios of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and poly(1,2-butadiene-block-ethylene oxide) (PBd22-PEO14, Ms = 1800 gmol-1) . Using single particle analysis (SPA) we were able to further interpret the information gained from SAXS and cryo-ET experiments, showing that increasing PBd22-PEO14 mole fraction increases the membrane thickness from 52 Å for a pure lipid system to 97 Å for pure PBd22-PEO14 vesicles. We find two vesicle populations with different membrane thicknesses in hybrid vesicle samples. As these lipids and polymers are known to homogeneously mix, bistability is inferred between weak and strong interdigitation regimes of PBd22-PEO14 within the hybrid membranes. It is hypothesized that membranes of intermediate structure are not energetically favourable. Therefore, upon formation, each vesicle selects one of these two membrane structures, which are assumed to have comparable free energies. We conclude that, by combining biophysical methods, an accurate determination of the influence of composition on the structural properties of hybrid membranes is achieved, revealing that two distinct membranes structures can coexist in homogeneously mixed lipid-polymer hybrid vesicles.