An engineered dimeric protein pore that spans adjacent lipid bilayers

The bottom-up construction of artificial tissues is an underexplored area of synthetic biology. An important challenge is communication between constituent compartments of the engineered tissue and between the engineered tissue and additional compartments, including extracellular fluids, further engineered tissue and living cells. Here we present a dimeric transmembrane pore that can span two adjacent lipid bilayers and thereby allow aqueous compartments to communicate. Two heptameric staphylococcal α-hemolysin (αHL) pores were covalently linked in an aligned cap-to-cap orientation. The structure of the dimer, (α7)(2), was confirmed by biochemical analysis, transmission electron microscopy (TEM) and single-channel electrical recording. We show that one of two β barrels of (α7)(2) can insert into the lipid bilayer of a small unilamellar vesicle, while the other spans a planar lipid bilayer. (α7)(2) pores spanning two bilayers were also observed by TEM.