Probing Position-Dependent Diffusion in Folding Reactions Using Single-Molecule Force Spectroscopy.

Area of Science:

• Biophysics
• Computational Biology
• Molecular Dynamics

Background:

• Protein and nucleic acid folding involves navigating complex energy landscapes.
• The diffusion coefficient (D) is crucial for understanding folding dynamics but its position-dependence is difficult to measure experimentally.
• Force spectroscopy is a common technique for studying molecular folding.

Purpose of the Study:

• To investigate the position-dependence of the diffusion coefficient (D) during DNA hairpin folding.
• To reconcile conflicting results from different experimental methods used to measure D.
• To identify and account for experimental artifacts in force spectroscopy measurements.

Main Methods:

• Analysis of round-trip times in constant-force extension trajectories from force spectroscopy.
• Analysis of fall times in force jump measurements.
• Comparison of experimental data with computational simulations.
• Application of Kramers's theory to hairpin kinetics with varying energy barrier positions.

Main Results:

• Round-trip time analysis suggested D varied significantly, while fall time analysis indicated D was relatively constant.
• Computational simulations revealed that experimental artifacts in force spectroscopy masked the intrinsic position-dependence of D.
• Kramers's theory application suggested D did not vary substantially with barrier position along the DNA hairpin stem.

Conclusions:

• Experimental artifacts in force spectroscopy present significant challenges in accurately measuring position-dependent diffusion coefficients.
• The diffusion coefficient in DNA hairpin folding may be less position-dependent than previously suggested by some experimental interpretations.
• This study highlights the critical need for careful artifact analysis when interpreting folding dynamics from force spectroscopy data.