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

During oxidative phosphorylation in bacteria and mitochondria, 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. We recently described the functional co-reconstitution of terminal oxidases and the ATP synthase by a detergent-mediated process and investigated the phenomenon of “mild uncoupling” using low amounts of different ionophores as well as recoupling mediated by 6-ketocholestanol [1]. To obtain even more complex systems containing three or more membrane proteins, a different approach was developed. First, membrane proteins were reconstituted individually into liposomes and subsequently fused to form larger liposomes containing all protein populations. Different methods were described to fuse liposomes such as the use of SNARE proteins, SNARE-mimicking peptides and DNA oligomers or oppositely charged lipids. Here, we used oppositely-charged lipids as they are easy to handle and no synthesis of SNARE proteins or SNARE-mimicking molecules is necessary. Successful fusion was demonstrated by functional coupling of the reconstituted proteins, and it was possible to functionally co-reconstitute E. coli ATP synthase and bo3 oxidase into unilamellar liposomes ranging from 100 nm to 50 µm in size [2]. The same approach was used to demonstrate the successful co-reconstitution of three different membrane proteins into giant unilamellar vesicles. The described method is thus a versatile tool for the bottom up synthesis of minimal cell systems.