Nanopore Tweezer Design and Operating Principles for Resolving Native Protein Conformational Dynamics

Area of Science:

• Biophysics
• Biochemistry
• Nanotechnology

Background:

• Single-molecule techniques offer real-time insights into protein dynamics, overcoming limitations of ensemble averaging.
• Nanopore tweezers utilize ionic current changes through engineered nanopores to detect protein conformational states.
• Nanopore design and experimental conditions can significantly impact measurement accuracy and protein behavior.

Purpose of the Study:

• To systematically investigate the influence of nanopore architecture and recording parameters on protein dynamics measurements.
• To determine optimal conditions for studying the conformational transitions of the West Nile virus NS2B/NS3 protease.

Main Methods:

• Systematic examination of nanopore luminal surface properties, pore size, and applied voltage.
• Monitoring ionic current signatures to analyze protein capture, residence time, and conformational-state resolution.
• Utilizing West Nile virus NS2B/NS3 protease as a model system for flaviviral enzyme dynamics.

Main Results:

• Protein dynamics measurements are sensitive to minor changes in nanopore lumen and applied voltage.
• Nanopore size critically affects the resolution of conformational states.
• An analyte-specific "Goldilocks" operating window was identified to optimize measurements.

Conclusions:

• Rational nanopore design and parameter selection are essential for accurate single-molecule biophysics.
• The identified "Goldilocks" window balances signal quality with minimal perturbation of native protein dynamics.
• This study provides a framework for optimizing nanopore tweezers for diverse protein studies.