Assembly of transmembrane pores from mirror-image peptides

Abstract

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.

Engineering Biological Nanopore Approaches toward Protein Sequencing

“…Such control of the nanopore’s morphology can be used to optimize its sensing ability, ensuring measurable changes in the ionic current amplitude above the noise level for individual peptides, proteins, and PTMs. This fine-tuning can be applied to artificial nanopores: solid-state nanopores, ,− nanopipettes, − chemosynthetic membrane channels, and hybrid nanopores, , as well as biological nanopores: DNA-based channels, ,− peptide-based transmembrane pores, helicase nanopores, ligand-gated pores, − transmembrane β barrels, voltage-dependent anion channels (VDAC) of the mitochondrion, , etc., as needed, depending on the experimental conditions and particular analytes of interest. Additionally, simultaneous sensing with a range of different nanopores could lead to a comprehensive understanding of proteome and protein isoform diversity .…”

Synthetic α-Helical Nanopore Reactor for Chemical Sensing

“…Recently, advanced computational designs , and the modulation of natural assemblies have been exploited to build functional transmembrane structures . We recently presented the first report of a large functional and ion-selective α-helical nanopore made from the self-assembly of synthetic peptides based on the natural PorACj assembly. , In addition to their facile synthesis and autonomous assembly, these pores could be exploited for the charge-selective sensing of charged cyclic sugars and polypeptides …”

“…27 We recently presented the first report of a large functional and ion-selective α-helical nanopore made from the self-assembly of synthetic peptides based on the natural PorACj assembly. 28,29 In addition to their facile synthesis and autonomous assembly, these pores could be exploited for the charge-selective sensing of charged cyclic sugars and polypeptides. 30 The nanopore approach has also extended beyond stochastic sensing and sequencing applications.…”

Sensing PEGylated Peptide Conformations Using a Protein Nanopore

“…Engineered protein pores have been used to sense and characterize various biomolecules such as nucleic acid fragments, oligosaccharides, peptides, and protein segments. − Membrane protein-based nanopores are widely exploited as they can be easily obtained from natural sources with high structural stability. ,,− Their unique geometry and permeability components have been used to build nanopore sensors. ,, Furthermore, they are compatible with studying many biological molecules due to their constriction zone and are flexible to protein engineering to enable high-resolution single-molecule detection. ,− Recently, synthetic DNA and α-helical pores have been introduced due to their sophisticated structure for sensing analytes. − Most nanopore studies have focused on simple small molecules and lengthened polymer chains. ,− Recently, nanopore technology has been used to sense complex biomacromolecules, including peptide and protein segments. − The unstructured peptides can be easily detected using nanopores, whereas larger protein fragments pose challenges due to their intricate folding pattern. ,− ,, In such cases, wide-diameter pores of defined geometry would expand the scope for detecting large folded proteins and complex biopolymers of stable structural conformations. ,, …”