Abstract
Modules that switch protein-protein interactions on and off are essential to develop synthetic biology; for example, to construct orthogonal signaling pathways, to control artificial protein structures dynamically, and for protein localization in cells or protocells. In nature, the E. coli MinCDE system couples nucleotide-dependent switching of MinD dimerization to membrane targeting to trigger spatiotemporal pattern formation. Here we present a de novo peptide-based molecular switch that toggles reversibly between monomer and dimer in response to phosphorylation and dephosphorylation. In combination with other modules, we construct fusion proteins that couple switching to lipid-membrane targeting by: (i) tethering a ‘cargo’ molecule reversibly to a permanent membrane ‘anchor’; and (ii) creating a ‘membrane-avidity switch’ that mimics the MinD system but operates by reversible phosphorylation. These minimal, de novo molecular switches have potential applications for introducing dynamic processes into designed and engineered proteins to augment functions in living cells and add functionality to protocells.
Highlights
Modules that switch protein-protein interactions on and off are essential to develop synthetic biology; for example, to construct orthogonal signaling pathways, to control artificial protein structures dynamically, and for protein localization in cells or protocells
Such artificial molecular switches are important for the development of bioengineering and synthetic biology, as they would allow new regulatory mechanisms and circuits to be incorporated into host cells, or synthetic ‘protocells’, endowing their host compartments with new, dynamic functionality[4]
The recognition motif was placed such that the phospho-acceptor residue (Ser) was at the a position of the second heptad repeat of the four-heptad peptide sequence (Fig. 1a and Table 1)
Summary
Modules that switch protein-protein interactions on and off are essential to develop synthetic biology; for example, to construct orthogonal signaling pathways, to control artificial protein structures dynamically, and for protein localization in cells or protocells. In combination with other modules, we construct fusion proteins that couple switching to lipid-membrane targeting by: (i) tethering a ‘cargo’ molecule reversibly to a permanent membrane ‘anchor’; and (ii) creating a ‘membrane-avidity switch’ that mimics the MinD system but operates by reversible phosphorylation These minimal, de novo molecular switches have potential applications for introducing dynamic processes into designed and engineered proteins to augment functions in living cells and add functionality to protocells. There is a demand to create de novo switches that are orthogonal to existing cellular systems, that enable regulation by different forms of stimuli, and that operate in more-complex environments such as in cells and at membranes Such artificial molecular switches are important for the development of bioengineering and synthetic biology, as they would allow new regulatory mechanisms and circuits to be incorporated into host cells, or synthetic ‘protocells’, endowing their host compartments with new, dynamic functionality[4]. The ability to tether a molecular cargo to a cellular or artificial membrane reversibly has important applications in biotechnology and synthetic biology[13,14]
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