Force-Dependent Folding Kinetics of Single Molecules with Multiple Intermediates and Pathways

Area of Science:

• Biophysics
• Chemical Kinetics
• Molecular Dynamics

Background:

• Single-molecule experiments commonly use equilibrium hopping to study protein folding kinetics.
• Characterizing intermediate states and their kinetic rates remains challenging with equilibrium methods.

Purpose of the Study:

• To develop and apply a nonequilibrium single-molecule approach for deriving force-dependent kinetic rates of intermediate states.
• To extract kinetic barriers and folding energies from nonequilibrium pulling experiments.
• To characterize properties of native, unfolded, and intermediate states not obtainable from equilibrium studies.

Main Methods:

• Application of the Kramers kinetic diffusive model to nonequilibrium pulling experiments.
• Analysis of single-molecule force-extension data for DNA hairpins.
• Derivation of force-dependent kinetic rates, barriers, and equilibrium folding energies.

Main Results:

• Successfully derived force-dependent kinetic rates for intermediate states using nonequilibrium pulling.
• Extracted force-dependent kinetic barriers and equilibrium folding energies.
• Demonstrated agreement between experimental results and theoretical predictions for DNA hairpins.

Conclusions:

• The proposed nonequilibrium single-molecule method provides a powerful tool for characterizing kinetic and thermodynamic properties of biomolecular states.
• This approach expands the scope of single-molecule studies to include properties previously inaccessible through equilibrium methods.
• The method is applicable to complex systems like DNA hairpins with multiple folding pathways.