Optimized reconstitution of membrane proteins into synthetic membranes

Roland Goers,Daniel J Müller,Johannes Thoma,Claudio Alter,Gesine Gunkel-Grabole,Dimitrios Fotiadis,Alfredo Di Silvestro,Noah Ritzmann,Wolfgang Meier

doi:10.1038/s42004-018-0037-8

Roland Goers, Daniel J Müller + Show 7 more

Open Access

https://doi.org/10.1038/s42004-018-0037-8

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Abstract

Light-driven proton pumps, such as proteorhodopsin, have been proposed as an energy source in the field of synthetic biology. Energy is required to power biochemical reactions within artificially created reaction compartments like proto- or nanocells, which are typically based on either lipid or polymer membranes. The insertion of membrane proteins into these membranes is delicate and quantitative studies comparing these two systems are needed. Here we present a detailed analysis of the formation of proteoliposomes and proteopolymersomes and the requirements for a successful reconstitution of the membrane protein proteorhodopsin. To this end, we apply design of experiments to provide a mathematical framework for the reconstitution process. Mathematical optimization identifies suitable reconstitution conditions for lipid and polymer membranes and the obtained data fits well to the predictions. Altogether, our approach provides experimental and modeling evidence for different reconstitution mechanisms depending on the membrane type which resulted in a surprisingly similar performance.

Highlights

Light-driven proton pumps, such as proteorhodopsin, have been proposed as an energy source in the field of synthetic biology
Methodologies from biology and engineering are combined in the bottom-up approach in synthetic biology[1,2], aiming at building a biological system with a desired functionality from the bottom by using dedicated building blocks
Mainly robust membrane proteins have been used for reconstitution in polymer membranes and the goal to combine biological with synthetic parts from chemistry has only partly been achieved[3]

Summary

Introduction

Light-driven proton pumps, such as proteorhodopsin, have been proposed as an energy source in the field of synthetic biology. Block-poly(2-methyloxazoline) (PMOXA-PDMS-PMOXA) triblock copolymers are commonly used for self-assembly involving proteins or other biomolecules Their low glass transition temperature and resulting flexibility, as well as lateral diffusion properties make them good candidates for the reconstitution of membrane proteins[3]. The combination of polymer membranes (based on e.g. PDMSPMOXA, PB-PEO, or other polymer blocks3) and the efficiency and selectivity of biological components such as enzymes and membrane proteins combines the “best of both worlds” and can be exploited towards building synthetic nanoscale devices[3] Such molecular factories can be envisioned performing enzymatic production or degradation of specific compounds (antibiotics, etc.) or take over a desired functionality[17]. Mainly robust membrane proteins have been used for reconstitution in polymer membranes and the goal to combine biological with synthetic parts from chemistry has only partly been achieved[3]

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