Light- and Bioluminescence-Controlled Reversible Protein Conjugation on the Inner Leaflet of Asymmetric...

Living cells maintain complex nonequilibrium functions through the spatiotemporal regulation of membrane-associated protein localization, utilizing energy and external stimuli to orchestrate sequential signaling and biochemical reactions. Bottom-up synthetic cells composed of phospholipid bilayers, such as lipid vesicles and liposomes, have provided valuable insights into cellular organization and enabled various applications in biomolecular robotics and drug delivery. Reversible, light-, or bioluminescence-dependent protein interactions-such as those mediated by the LOV2-Zdk pair-enable precise spatiotemporal control over protein localization. To build more complex cell-mimicking systems, protein- or polypeptide-based vesicles have been developed, offering advantages such as genetic programmability and functional diversity. However, reversible protein accumulation on protein-based inner leaflets has not yet been achieved. Here, we report light- and bioluminescence-controlled protein accumulation systems on protein-based leaflets of asymmetric hybrid vesicles composed of phospholipids, LOV2, and FKBP-fused amphiphilic proteins (oleosin). We achieved multiple reversible and spatially controlled cycles of protein conjugation and dissociation, and enabled internal LOV2 activation via Renilla luciferase bioluminescence without external illumination. Our asymmetric phospholipid-oleosin hybrid vesicles provide a versatile platform for constructing stimuli-responsive synthetic cells and designing autonomous functional biomolecular systems.