Single-Molecule Protein Interactions and Unfolding Revealed by Plasmon-Enhanced Fluorescence.

Area of Science:

• Biophysics
• Biochemistry
• Molecular Biology

Background:

• Ensemble-average methods obscure crucial single-molecule protein interaction kinetics.
• Current single-molecule fluorescence techniques face limitations in signal brightness and time resolution.

Purpose of the Study:

• To develop and validate a novel platform for single-molecule protein-protein interaction quantification.
• To investigate protein interaction kinetics and folding dynamics with enhanced precision.

Main Methods:

• Utilized plasmon-enhanced fluorescence microscopy for single-molecule analysis.
• Employed DNA-mediated hybridization for controlled immobilization of PDZ protein conjugated to plasmonic particles.
• Quantified protein-protein interactions and urea-mediated unfolding/refolding kinetics.

Main Results:

• Discovered previously unobserved heterogeneities in protein-protein interactions, including a distinct bound-state lifetime.
• Demonstrated the method's capability to study urea-mediated protein unfolding and refolding.
• Found bound-state lifetime to be independent of urea concentration, suggesting a two-state unfolding model.
• Observed entirely reversible folding for immobilized PDZ, unlike aggregation in solution-phase unfolding.

Conclusions:

• Single-molecule plasmon-enhanced fluorescence microscopy is a powerful new tool for monitoring transient protein-protein interactions.
• This technique offers high time resolution for studying protein folding dynamics at the single-molecule level.
• The method provides insights into protein conformational changes and binding kinetics previously inaccessible.