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Single OmpG Nanopore Gating Regulated by Linker Length for Protease Detection
Koki Kamiya1, Haruka Suzuki1, Kazuha Endo1
1Graduate School of Science and Technology, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515, Japan.
ACS Applied Bio Materials
|March 16, 2026
Summary
N-terminal linkers fused to OmpG proteins enable stable nanopore sensing for protease detection. Protease cleavage of these linkers reverses gating dynamics, allowing highly accurate, specific identification of target proteases.
Area of Science:
- Biophysics
- Nanotechnology
- Molecular Biology
Background:
- Nanopore sensing offers advanced single-molecule analysis capabilities.
- The OmpG protein's gating properties are ideal for occlusion-based detection.
- Internal loop modifications in OmpG can destabilize its structure.
Purpose of the Study:
- To investigate N-terminal linker fusion as a method to stabilize OmpG for nanopore sensing.
- To introduce protease cleavage sites into OmpG via N-terminal linkers.
- To assess the feasibility of using modified OmpG for specific protease detection.
Main Methods:
- Constructing OmpG variants with N-terminal functional linkers.
- Utilizing a planar lipid bilayer system to monitor ionic currents.
- Employing molecular dynamics simulations to analyze linker interactions.
- Performing protease cleavage experiments and analyzing gating dynamics.
Main Results:
- N-terminal linker length significantly influenced OmpG gating dynamics.
- Linker-fused variants showed high gating frequencies (over 3.6 events/s).
- Protease treatment reversed gating, enabling up to 90.9% accurate linker presence discrimination.
- Molecular dynamics confirmed longer linkers occlude pore entrances more frequently.
Conclusions:
- N-terminal linker fusion preserves OmpG structural integrity for nanopore applications.
- Protease-mediated cleavage of linkers provides a reliable signal for detection.
- This modified OmpG platform demonstrates high specificity and potential for high-throughput protease screening.
