Redesign of Translocon EXP2 Nanopore for Detecting Peptide Fragments

Nanopore sensing is a rapid, label-free technique that enables single-molecule detection and is successfully applied to nucleic acid sequencing. Extending this technology to the detection and sequencing of peptides and proteins is a key area of interest. However, the complex structures and diverse charge distributions of peptides and proteins present challenges for extensive detection using existing nanopores. In this study, the focus is on the EXP2 nanopore derived from the malaria parasite Plasmodium falciparum to address these challenges. Previously, it is characterized wild-type EXP2 (WT-EXP2) nanopores and demonstrated their ability to detect polypeptides, although intrinsic electrical noise from the pore posed difficulties for accurate detection. To overcome these limitations, several EXP2 nanopore mutants are designed, including EXP2_ΔD231, EXP2_NC, and EXP2_NC ^K42D/S46F, to reduce electrical noise and improve peptide detection accuracy. The EXP2_ΔD231 mutant reduced electrical noise by more than 50% compared to WT-EXP2 and improved the discrimination accuracy of oligoarginine peptides. In addition, the EXP2_ΔD231 detected and discriminated eight different peptides, ranging in molecular weight from small to large, that are previously challenging to detect using a single nanopore type. These results suggest that engineered EXP2 nanopores could serve as effective tools for peptide and protein detection and sequencing, contributing to the broader application of nanopore technology in biochemical and clinical research.