The use of protein nanopores has shown great promise for the identification and characterization of molecules in nanopore sensing technologies. For detailed characterization of analytes, pores with uniquely suited characteristics are required. To expand the pool of available nanopores, we structurally and biochemically characterised two potential pore-forming proteins: the actinoporin-like protein MoFa from the coral Orbicella faveolata and the cytolysin Cyt2Aa from Bacillus thuringiensis. MoFa forms oligomeric pores on lipid membranes containing sphingomyelin. The membrane is penetrated by α-helices located at the N-terminus. The pore is a funnel-shaped protein-lipid complex, where at least 112 lipids are bound to the pore, consisting of eight MoFa protomers. We quantified protein-lipid interactions and by using molecular dynamics simulations showed that the pore limits the movement of surrounding membrane lipids. We found that, in addition to octameric pores MoFa also forms heptameric and nonameric pores, the three-dimensional structures of which we also determined by cryo electron microscopy. The stoichiometry of the pores depends in part on the composition of the lipid membranes. Based on the pore structures, we designed new constructs for sensing applications. In contrast to MoFa, Cyt2Aa does not form pores when exposed to a lipid membrane but forms filamentous oligomers. Our results show that Cyt2Aa does not act as a classical pore-forming protein and does not form ring-shaped pores, but instead disrupts the membrane integrity similar to detergents by solubilization. The formation of filamentous oligomers of Cyt2Aa is also triggered by the contact of the protein with detergents. Cyt2Aa filaments have reduced hemolytic activity and are not suitable for use in nanopore sensing. The main result of this work are different MoFa pore structures and newly designed versions, which provide an excellent starting point for the future development of sensing applications.
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