Towards synthetic respiration – methods for the co-reconstitution of respiratory chain enzymes

During oxidative phosphorylation, membrane embedded complexes I to V functionally interact to form ATP from cellular reducing equivalents. Normally, these enzymes are investigated individually and therefore, their functional interplay is not yet understood in detail. To get further insights on interactions of respiratory chain enzymes on a molecular basis such as the formation of supercomplexes, they have to be extracted from their complex native environment and co-reconstituted into a well-defined membrane mimicking system such as liposomes. To obtain co-reconstitutions of several membrane proteins (MP), we developed a strategy where we first reconstitute each purified MP into liposomes and in a second step, fuse the proteoliposomes. We recently successfully applied SNARE-mediated fusion of proteoliposomes, but this method is limited to one round of fusion and therefore only interactions of two MPs can be studied. To investigate more complex systems, more than one round of liposome fusion are necessary. We recently developed proteoliposome fusion by oppositely charged lipids as an alternative to SNARE-dependent fusion [1]. Using fluorescent lipid mixing assays and tunable resistive pulse sensing, we established protocols for charge mediated liposome fusion in our hands and applied the optimised conditions to fuse liposomes containing respiratory chain enzymes. Using this strategy, it was possible to co-reconstitute different terminal oxidases and the E. coli ATP synthase, imitating the last step of oxidative phosphorylation. The oxidase thereby creates an electrochemical proton gradient that energizes the ATP synthase, requiring an intact (proton tight) lipid bilayer after the fusion process. To obtain even more complex systems, we fused small vesicles (~40 nm) with giant vesicles (~50 µm) using charge-mediated fusions. In this system several fusion events can happen and we could show the co-reconstitution of up to three membrane proteins