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.

Membrane protein pores have demonstrated applications in nanobiotechnology and single-molecule chemistry for effective detection of biomolecules. Here, we define the molecular basis of carbohydrate polymers translocation through a substratespecific bacterial nanopore, CymA, which has a 15-residue N terminus segment inside the pore, restricting its diameter. Using single-channel recordings, we determined the kinetics of cationic cyclic oligosaccharide binding and elucidated the translocation mechanism across the pore in real-time. The cationic cyclic hexasaccharide binds to the densely packed negatively charged residues at the extracellular side of the pore with high affinity, facilitating its entry into the pore driven by the applied voltage. Further, the dissociation rate constant increased with increasing voltages, indicating unidirectional translocation toward the pore exit. Specifically, a larger cationic cyclic octasaccharide rapidly blocked the pore more effectively, resulting in the complete closure of the pore with increasing voltage, implying only strong binding. Further, we show that uncharged oligosaccharides exclusively bind to the extracellular side of the pore and the electroosmotic flow most likely drives their translocation. We propose that CymA favors selective translocation of cyclic hexasaccharide and linear maltooligosaccharides due to an asymmetrical charge pattern and the N terminus that regulates the substrate transport. We suggest that this substrate-specific nanopore with sophisticated geometry will be useful for complex biopolymer characterization.

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.