Dynamic gating by ϕ-clamp loop controls peptide translocation through the anthrax toxin nanopore

Area of Science:

• Biophysics
• Molecular Biology
• Nanotechnology

Background:

• The protective antigen (PA) nanopore's phi-clamp is crucial for peptide translocation but its mechanism is debated.
• A key question is how the nanopore avoids overly stable clamp-polymer interactions.

Purpose of the Study:

• To investigate the gating mechanism of the phi-clamp in peptide translocation through the PA nanopore.
• To elucidate the role of phenylalanine 427 (F427) in clamp dynamics and peptide sensing.

Main Methods:

• Single-channel electrophysiology was used on wild-type PA and F427 mutant proteins.
• Thermodynamic and kinetic analyses were performed to characterize clamp-peptide interactions.

Main Results:

• Wild-type F427 forms a stable hydrophobic trap, acting as a specific chemical sensor for peptide hydrophobicity.
• Mutating F427 abolished this sensing but intermediate states persisted, indicating larger-scale loop dynamics.
• Translocation occurs through a dynamic equilibrium between constricted (high-affinity) and dilated (low-affinity) states.

Conclusions:

• The phi-clamp facilitates transport by dynamically gating between high-affinity and low-affinity states.
• This conformational gating mechanism allows the nanopore to overcome energetic traps.
• Findings provide a framework for engineering advanced dynamical nanopore biosensors.