Design and assembly of a chemically switchable and fluorescently traceable light-driven proton pump system for bionanotechnological applications.

Energy-supplying modules are essential building blocks for the assembly of functional multicomponent nanoreactors in synthetic biology. Proteorhodopsin, a light-driven proton pump, is an ideal candidate to provide the required energy in form of an electrochemical proton gradient. Here we present an advanced proteoliposome system equipped with a chemically on-off switchable proteorhodopsin variant. The proton pump was engineered to optimize the specificity and efficiency of chemical deactivation and reactivation. To optically track and characterize the proteoliposome system using fluorescence microscopy and nanoparticle tracking analysis, fluorescenlty labelled lipids were implemented. Fluorescence is a highly valuable feature that enables detection and tracking of nanoreactors in complex media. Cryo-transmission electron microscopy, and correlative atomic force and confocal microscopy revealed that our procedure yields polylamellar proteoliposomes, which exhibit enhanced mechanical stability. The combination of these features makes the presented energizing system a promising foundation for the engineering of complex nanoreactors.