Tailoring <i>Vibrio</i> ‐Type Secretin Channel Protein GspD Toward “One‐Take” Dual‐Constriction Nanopore Sensors

Dual-constriction nanopores offer a second sensing region that enhances the interactions with analytes at the single-molecule level. However, existing biological nanopore complexes, i.e., CsgG-CsgF, are prone to dissociation upon high voltages, enforcing the development of robust "one-take" platforms. Here, Type II general secretin protein D from Vibrio cholerae (VcGspD) as a promising scaffold with dual-constrictions is proposed and engineered. Biochemical analysis reveals that truncation of the N0-N2 domains yields stable multimerization, with the N3 domain being essential. Cryo-electron microscopy (Cryo-EM) resolves the truncated VcGspD (N0-N2) as a 15-mer architecture, confirming its structural integrity and determining localizations of P471 and F472. By introducing a point mutation at position 346 (S346C) and conjugating cholesterol-maleimide, stable channel insertion in lipid bilayers is achieved. Electrophysiological characterization demonstrates a predominantly low-conductance dual-constriction architecture with constriction diameters of ≈2 nm both at the cap and central constriction sites. The F472A mutation, together with the mutations on both constrictions, gives rise to convergent open-channel current and confers high-voltage stability, thus enabling efficient sensing of both single-stranded DNA and polypeptides. The findings establish VcGspD as a promising platform toward dual-constriction nanopore sensing, paving the way for advancements in the development and engineering of secretin channels.