Light-Driven ATP Regeneration in Diblock/Grafted Hybrid Vesicles

Abstract

Light-driven ATP regeneration systems combining ATP synthase and bacteriorhodopsin have been proposed as an energy supply in the field of synthetic biology. Energy is required to power biochemical reactions within artificially created reaction compartments like protocells, which are typically based on either lipid or polymer membranes. The insertion of membrane proteins into different hybrid membranes is delicate, and studies comparing these systems with liposomes are needed. Here we present a detailed study of membrane protein functionality in different hybrid compartments made of graft polymer PDMS-g-PEO and diblock copolymer PBd-PEO. Activity of more than 90 % in lipid/polymer-based hybrid vesicles could prove an excellent biocompatibility. A significant enhancement of long-term stability (80 % remaining activity after 42 days) could be demonstrated in polymer/polymer-based hybrids.

Keywords: ATP synthase; bacteriorhodopsin; diblock polymers; energy conversion; graft polymers; permeability.

Figure 1

Light‐driven ATP synthesis in lipid vesicles (100‐150 nm). A) Schematic representation of the ATP‐generating system reconstituted with bacteriorhodopsin (bR) and F_OF₁‐ATP synthase. F_OF₁‐ATP synthase uses the electrochemical gradient generated by bR to synthesize ATP. B) Photoinduced ATP synthesis in bR‐EF₀F₁ liposomes through on‐off cycles of light, in the absence of light (dark incubated liposomes), and in liposomes containing only bR (bR liposomes).

Figure 2

A) Schematic presentation of different membrane compositions: Pure PC vesicles (100/0 PC), hybrid vesicles made of 70 mol % PDMS‐g‐PEO (70/30 PDMS/PC), 50 mol % PBd‐PEO (50/50 PBd/PC) as well as a mixture of 50 mol % PDMS‐g‐PEO and 50 mol % PBd‐PEO (50/50 PDMS/PBd). Schematic representation of the nanoscale structures of B) phosphatidylcholine, C) diblock polymer PBd‐PEO and D) grafted polymer PDMS‐g‐PEO.

Figure 3

Size distribution of vesicles made of pure 100/0 PC, 70/30 PDMS/PC, 50/50 PBd/PC and 50/50 PDMS/PBd. A) Sizes of vesicles after extrusion through 100 nm pores. B) Size distribution of detergent‐treated vesicles prior to reconstitution. C) Sizes of vesicles after reconstitution and removal of detergent using bio beads.

Figure 4

Light‐driven ATP synthesis in lipid and hybrid vesicles. The activity is determined by linear regression. The inset bars show the activity normalized to pure PC liposomes. All error bars represent the standard error of three independent measurements.

Figure 5

A) Proton pump activity of bR in liposomes and different hybrid vesicles as measured by pH change upon irradiation with green light. pH change is detected by encapsulated pyranine. Results show the middle value of at least three separate measurements. B) Reconstitution efficiency of bR in different compartments. *P≤0.05, not significant (ns) P>0.05 (P values are generated by unequal variance t‐test (Welch's test) for comparison of each hybrid membrane composition to the lipid vesicle sample). n=3; errors represent the standard error of the mean (SEM).

Figure 6

Proteolytic cleavage of reconstituted bR with proteinase K (ProtK) shows mixed orientation in all lipid and hybrid vesicles. A) Expected sizes of proteolytic fragments for ProtK digestion of bR when the N‐terminal (red values) or C‐terminal (violet values) is exposed to the bulk solution. B) SDS‐PAGE gel analysis of the digestion products. Lane 1: band specific for ProtK enzyme only; lane 2: digestion product of not reconstituted bR; lanes 3–6: digest patterns for different lipid/hybrid vesicles containing solubilized bR; lane 7: undigested bR in lipid vesicles.

Figure 7

Proton permeability of vesicles as measured by pH change after addition of A) 2.4 mM HCl and B) 1.6 mM NaOH to the outer solution of vesicles. pH change is detected by encapsulated HPTS. The solid lines represent the simulations as predicted by the model described in the Supporting Information.

Figure 8

Hybrid vesicles improve long‐term stability of the ATP regeneration module. The activity is normalized to the activity at day 1. *P≤0.05, not significant (ns) P >0.05 (P values are generated by unequal variance t‐test (Welch's test) for comparison of each hybrid membrane composition to the lipid vesicle sample). n≥4; error bars represent standard error of the mean (SEM). Needs to be exchanged