Durable proteo-hybrid vesicles for the extended functional lifetime of membrane proteins in bionanotechnology† †Electronic supplementary information (ESI) available: Additional supporting data and experimental methods. See DOI: 10.1039/c6cc04207d Click here for additional data file.

Significant enhancement of membrane protein functional durability is demonstrated when reconstituted in hybrid lipid–block copolymer vesicles compared to conventional proteoliposomes.

. Aggregate size distributions by reconstitution method 1. Hydrodynamic diameter distributions for samples containing different mol% block copolymer after detergent extraction using Bio-Beads, as described in Method 1.   Fig. 2. The data shows Z-average particle diameter (d/nm), Polydispersity Index (PdI), Size peaks form the particle size distribution profile and corresponding % Volume for samples containing different polymer:lipid ratios (expressed in polymer mol%).  (see table S2) and variability in these separation experiments that are run on a benchtop gravity column. Figure S4: Enzymatic activity of cyt bo3 over time, before and after purification of vesicles in 50% and 75% PHV samples. Comparison of enzymatic activity of cyt bo3 in reconstituted PHVs prepared by method 2 and separated on a Sephadex G50 column, over a period of 44 days. The protein activity is calculated with respect to the initial activity after preparation on day 0 and error bars represent the standard error. Each measurement is the average of 2 independent PHV preparations.

Reconstitution Method 2
The second method was adapted from Geertsma, E.R. et al. [4] Lipid / block-copolymer films were prepared as described in method 1. Vesicles were formed by adding buffer (20 mM HEPES, 10 mM NaCl, pH 7.4), followed by incubation at 50°C for 5 min and vortex for 1 min, then incubate for further 5 min and vortex again. The resultant suspension was put through five freeze-thaw-vortex cycles and subsequently extruded 11 times through a 100 nm pore size polycarbonate membrane filter using an Avanti Mini-Extruder to form nanovesicles.

PHV Characterisation
To verify the formation of vesicular structures with both Method 1 and Method 2, the samples were analysed using dynamic light scattering (DLS) as well as using Carboxyfluorescein encapsulation and leakage experiments. DLS experiments were performed using a Malvern Zetasizer Nano ZS: the sample was measured at a fixed 173°scattering angle. Three measurements for each sample were performed at 25°C and the vesicle size was reported as the average of the three measurements.
For electron microscopy analysis Lacey grids (Agar Scientific) were glow discharged for 10 seconds using an EasiGlow system. Following this 3.0 µl of the PHV sample (6.5 mM amphiphile (lipid + block copolymer) concentration) was applied to the grid which was subsequently blotted for 4 s at blot force 2, using a Vitrobot mark IV. Grids were imaged on an FEI F20 microscope working at 200 KV and operating in low dose mode. Images were collected on a Gatan 4K x 4K CCD detector at a range of magnifications.
Unencapsulated CF was removed via gel filtration using a Sephadex G50 medium column, eluting with HEPES buffer without CF. To destabilise the vesicles and release encapsulated CF, 50μL of 10% Triton X-100 (w/v) was added to the sample and the fluorescence emission was recorded as counts per second (cps). The increase in fluorescence output was calculated by subtracting the background emission from the fluorescence emission recorded after the addition of Triton X-100.

Protein activity assay
The enzymatic activity assay was similar to that outlined in Rumbley, et al. [5] . First the substrate, Decylubiquinone (acquired form Sigma-Aldrich, CAS No: 55486-00-5), was solubilised in absolute ethanol. The concentration of the prepared decylubiquinone solution was confirmed spectroscopically at 275 nm (ε = 19 mM -1 cm -1 in absolute ethanol) to be 1.695 mM. For the assay, decylubiquinone (DUQ) was reduced to decylubiquinol (DUQH2) using sodium borohydride crystals in a method similar to that described in Trounce, I.A. et al. [6] To remove excess hydride ions (H -) remaining in the solution, 0.1 M HCl was added (1 µL per 10 µL of DUQ solution). For the assay, 70 µL of prepared PHV, and 20 µL of 1.695 mM DUQH2 was diluted in HEPES buffer (40 mM HEPES, 20 mM NaCl, pH 7.4) to a final volume of 700 µL and the oxidation of DUQH2 was monitored at 275 nm (ε = 12.25 mM -1 cm -1 in aqueous solution [7] ) for 5 min.
The activity of cyt bo3 is calculated by converting the absorbance reading at 275 nm into µmoles of Decylubiquinone (using ε = 12250 M -1 cm -1 ) and plotted against time. The initial slope of the curve in a plot of µmoles of Decylubiquinone against time (gradient of the initial 20 seconds) is calculated and converted to represent rate of Decylubiquinone turnover per ml of prepared sample. The resulting value thus represents the enzymatic activity of cyt bo3. The raw data had some noise due to light scattering from the vesicles and this contributes to the overall error in the calculated enzymatic activity.

Chromatographic separation of PHVs
PHV samples were run on a Sephadex G50 gravity column. 0.5 mol% 1,2-dioleoyl-sn-glycero-3phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt) (Rh-DOPE; Avanti Polar Lipids; CAS no. 384833-00-5) was included in the membrane of these PHVs during formation so they could be visually tracked on the column. 0.5 ml eluted fractions were collected and quantified by UVvis adsorption spectroscopy at 570 nm (to measure Rh-DOPE content) and dynamic light scattering for particle size distributions. These fractions were further analysed for their protein activity as described above.