Tailoring Vibrio-Type Secretin Channel Protein GspD Toward "One-Take" Dual-Constriction Nanopore Sensors

Area of Science:

• Biophysics
• Structural Biology
• Nanotechnology

Background:

• Dual-constriction nanopores enhance single-molecule analyte detection.
• Existing biological nanopore complexes like CsgG-CsgF are unstable under high voltages, necessitating robust alternatives.

Purpose of the Study:

• To engineer a stable dual-constriction nanopore platform using the Type II general secretin VcGspD.
• To characterize the structure and function of the engineered VcGspD for advanced sensing applications.

Main Methods:

• Biochemical analysis and truncation of VcGspD domains.
• Cryo-electron microscopy (Cryo-EM) for structural determination.
• Electrophysiological characterization and site-directed mutagenesis (S346C, F472A).

Main Results:

• Truncated VcGspD (N0-N2) forms a stable 15-mer architecture.
• Engineered VcGspD exhibits a dual-constriction structure (≈2 nm diameters) stable in lipid bilayers.
• Mutations conferred high-voltage stability and enabled sensing of single-stranded DNA and polypeptides.

Conclusions:

• VcGspD is a viable and robust scaffold for dual-constriction nanopore sensing.
• The engineered channel overcomes stability issues of previous biological nanopores.
• This platform advances single-molecule analysis and secretin channel engineering.