Abstract
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.
Highlights
Designing, creating and manipulating existing or novel artificial biological systems is the core concept of synthetic biology[1]
In recent work we demonstrated the extended functionality of the PR-N221C mutant (N220C in the reference) that can be repeatedly switched off and on by chemical modification with the sulfhydryl-modifying agent sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES) and reduction with β-mercaptoethanol (β-ME)[10]
We recently demonstrated the possibility of chemically switching off and on the proton pumping activity of the engineered PR-N221C mutant based on the chemical modification of the site- introduced cysteine[10]
Summary
Designing, creating and manipulating existing or novel artificial biological systems is the core concept of synthetic biology[1]. The membrane protein proteorhodopsin (PR) is a well-characterized light-driven proton pump[9] that can be genetically engineered and overexpressed in Escherichia coli[10,11] These features make PR an ideal energizing module candidate for biomolecular nanoreactors. In recent work we demonstrated the extended functionality of the PR-N221C mutant (N220C in the reference) that can be repeatedly switched off and on by chemical modification with the sulfhydryl-modifying agent sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES) and reduction with β-mercaptoethanol (β-ME)[10] This is a valuable approach to prevent unintended PR activation, e.g., during imaging of PR containing samples with a light or confocal laser scanning microscope, or to control its activity in situations without possibility to regulate the source of illumination, e.g. when using sunlight. These properties established the presented proteoliposomes as a solid foundation for complex synthetic systems
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