Biological nanopores have been used for single-molecule detection and commercialized as detectors for nanopore DNA sequencers. While biological nanopores also show potential for peptide sequencing, their resolution in distinguishing amino acids remains limited. In this study, we explored de novo designed nanopores as alternative sequencing platforms. We previously reported a de novo designed peptide, SVG28, which assembles into a β-barrel nanopore in lipid bilayer membrane and can detect polypeptides. However, SVG28 mainly has three oligomeric states, resulting in nanopores varying sizes. Here, we used the extracellular domain of α-hemolysin as a scaffold for SVG28, enabling assembly with an exact heptameric stoichiometry. This chimeric nanopore was expressed, purified, and evaluated both structurally and functionally including cryo-electron microscopy, electrophysiological measurement, and molecular dynamics simulations. This chimeric approach successfully controlled the oligomeric state of SVG28 to a heptamer and revealed unique electrophysiological properties. We further developed the chimeric nanopore by introducing mutations—one designed to enhance structural stability and another to promote interaction with amino acids. Furthermore, we demonstrated peptide sequencing using this chimeric nanopore employing methodologies established in previous studies, on biological nanopores. Our results demonstrate the potential of peptide sequencing with de novo designed nanopores.
Nakada et al. (Sun,) studied this question.