The porinACj is an α-helical porin that spans the mycolic acid outer membrane of Gram-positive mycolate, Corynebacterium jeikeium. Here, we report that a 40-amino acid, synthetic peptide, pPorA corresponding to porin PorACj, inserts into the lipid bilayers and forms well-defined pores. By electrical recordings, we measured the single-channel properties that revealed the autonomous assembly of large conductance ion-selective synthetic pores. Further, we characterized the functional properties by blocking the peptide pores by cyclodextrins of different charge and symmetry. We deduced the subunit stoichiometry and putative structure of the pore by site-specific chemical modification in single-channel electrical recordings and gel electrophoresis. On the basis of these findings, we suggest that this is a large functional uniform transmembrane pore built entirely from short synthetic α-helical peptides. Accordingly, we propose a model demonstrating structural assembly of large α-helix-based peptide pores for understanding the action of antimicrobial peptides and for the design of pores with applications in biotechnology.

Tailored transmembrane alpha-helical pores with desired structural and functional versatility have promising applications in nanobiotechnology. Herein, we present a transmembrane pore DpPorA, based on the natural pore PorACj, built from D-amino acid α-helical peptides. Using single-channel current recordings, we show that DpPorA peptides self-assemble into uniform cation-selective pores in lipid membranes and exhibit properties distinct from their L-amino acid counterparts. DpPorA shows resistance to protease and acts as a functional nanopore sensor to detect cyclic sugars, polypeptides, and polymers. Fluorescence imaging reveals that DpPorA forms well-defined pores in giant unilamellar vesicles facilitating the transport of hydrophilic molecules. A second D-amino acid peptide based on the polysaccharide transporter Wza forms transient pores confirming sequence specificity in stable, functional pore formation. Finally, molecular dynamics simulations reveal the specific alpha-helical packing and surface charge conformation of the D-pores consistent with experimental observations. Our findings will aid the design of sophisticated pores for single-molecule sensing related technologies.

The selective translocation of molecules through membrane pores is an integral process in cells. We present a bacterial sugar transporter, CymA of unusual structural conformation due to a dynamic N terminus segment in the pore, reducing its diameter. We quantified the translocation kinetics of various cyclic sugars of different charge, size, and symmetry across native and truncated CymA devoid of the N terminus using singlechannel recordings. The chemically divergent cyclic hexasaccharides bind to the native and truncated pore with high affinity and translocate effectively. Specifically, these sugars bind and translocate rapidly through truncated CymA compared to native CymA. In contrast, larger cyclic heptasaccharides and octasaccharides do not translocate but bind to native and truncated CymA with distinct binding kinetics highlighting the importance of molecular charge, size and symmetry in translocation consistent with liposome assays. Based on the sugar-binding kinetics, we suggest that the N terminus most likely resides inside the native CymA barrel, regulating the transport rate of cyclic sugars. Finally, we present native CymA as a large nanopore sensor for the simultaneous single-molecule detection of various sugars at high resolution, establishing its functional versatility. This natural pore is expected to have several applications in nanobiotechnology and will help further our understanding of the fundamental mechanism of molecular transport.