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Effects of different reconstitution procedures on membrane protein activities in proteopolymersomes

The development of membrane protein reconstitution methods in polymersomes is regarded as a major challenge in replicating cellular functions in engineered cellular mimetic systems. We present a solvent-free membrane protein reconstitution method which can be used in polymersomes. To test our method, we reconstructed in vitro proton-powered ATP synthesis using engineered artificial organelles (BR/F0F1-ATP synthase reconstituted proteopolymersomes). We compared the functionality of BR and ATP synthase between two preparation methods. From the difference in the direction of proton pumping and the ATP production profile, it is evident that the relative orientation of BR can be determined by the condition of the proteins (BR monomer, purple membrane), together with the incorporation techniques. As the new procedure eliminates the problem of protein denaturation during incorporation, this research is expected to enhance the potential applications of synthetic membranes in the future fabrication of hybrid protein/polymer systems.