Autonomously Assembled Synthetic Transmembrane Peptide Pore

Abstract

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.

Designed alpha-helical barrels for charge-selective peptide translocation

“…S15 † ). 43 This particular peptide variant did not form self-assembled oligomers in SDS PAGE and the pores are formed by direct pPorA addition into the lipid bilayer (Fig. S15 † ).…”

“… 22,27,39 More recently, we engineered and constructed large synthetic alpha-helical transmembrane pores, pPorA based on natural porin PorACj of Corynebacterium jeikeium . 43,44 ( Fig. 1a ).…”

The Functional Nanopore Screen: A Versatile High-throughput Assay to Study and Engineer Protein Nanopores inEscherichia coli

“…Recent years also witnessed the development of nanopores for different biotechnological applications 3 , most prominently in the context of biosensing 4 , DNA sequencing 4 and single molecule studies 5 . Notable nanopore scaffolds include oligomeric toxins such as αHLA [6][7][8] , MspA 9 , FraC 10,11 and ClyA 12 , bacterial outer membrane proteins such as FhuA [13][14][15] and OmpG 16,17 and several self-assembling membrane peptides [18][19][20][21] .…”

De Novo Design of a Nanopore for DNA Detection that Incorporates a β-Hairpin Peptide

“…For example, an α-helical barreled peptide (35 a.a.) based on the Wza protein, an outer-membrane auxiliary protein associated with exopolysaccharide assembly systems, was redesigned from the wild type and assembled to form monodisperse nanopores in lipid membranes. 38,39 However although the Wza-based nanopore was successfully assembled to form the nanopore, tailoring its size and function remains challenging in terms of the design strategy.…”