Optimized reconstitution of membrane proteins into synthetic membranes

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.