A modular platform for one-step assembly of multi-component membrane systems by fusion of charged proteoliposomes.

Robert R Ishmukhametov,Richard M Berry,Aidan N Russell

doi:10.1038/ncomms13025

Robert R Ishmukhametov, Richard M Berry + Show 1 more

Open Access

https://doi.org/10.1038/ncomms13025

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Journal: Nature Communications	Publication Date: Oct 6, 2016
Citations: 31	License type: CC BY 4.0

Affiliation: University of Oxford

Abstract

An important goal in synthetic biology is the assembly of biomimetic cell-like structures, which combine multiple biological components in synthetic lipid vesicles. A key limiting assembly step is the incorporation of membrane proteins into the lipid bilayer of the vesicles. Here we present a simple method for delivery of membrane proteins into a lipid bilayer within 5 min. Fusogenic proteoliposomes, containing charged lipids and membrane proteins, fuse with oppositely charged bilayers, with no requirement for detergent or fusion-promoting proteins, and deliver large, fragile membrane protein complexes into the target bilayers. We demonstrate the feasibility of our method by assembling a minimal electron transport chain capable of adenosine triphosphate (ATP) synthesis, combining Escherichia coli F1Fo ATP-synthase and the primary proton pump bo3-oxidase, into synthetic lipid vesicles with sizes ranging from 100 nm to ∼10 μm. This provides a platform for the combination of multiple sets of membrane protein complexes into cell-like artificial structures.

Highlights

An important goal in synthetic biology is the assembly of biomimetic cell-like structures, which combine multiple biological components in synthetic lipid vesicles
In vivo vesicle fusion is driven by a large variety of fusion-promoting complementary membrane proteins found in viruses[10] and intracellular organelles[11,12,13,14], which must be present in both interacting membranes and which pull the bilayers toward each other in a fusion complex[15,16]
It can be overcome by using complementary DNA oligonucleotides[20,21], which are designed to insert themselves into the lipid bilayer and drive vesicle fusion as they hybridize and pull the membranes towards each other

Summary

Introduction

An important goal in synthetic biology is the assembly of biomimetic cell-like structures, which combine multiple biological components in synthetic lipid vesicles. We demonstrate the feasibility of our method by assembling a minimal electron transport chain capable of adenosine triphosphate (ATP) synthesis, combining Escherichia coli F1Fo ATP-synthase and the primary proton pump bo3-oxidase, into synthetic lipid vesicles with sizes ranging from 100 nm to B10 mm This provides a platform for the combination of multiple sets of membrane protein complexes into cell-like artificial structures. In vivo vesicle fusion is driven by a large variety of fusion-promoting complementary membrane proteins found in viruses[10] and intracellular organelles[11,12,13,14], which must be present in both interacting membranes and which pull the bilayers toward each other in a fusion complex[15,16] Some of these proteins[17,18,19] have been used for vesicle fusion in vitro but a limitation of this approach is that the accepting bilayer a priori must have the complementary protein in the membrane. Despite its simplicity, complementary charged lipids have not previously been used as a method for the fast delivery of transmembrane proteins from one lipid object to another

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