Deciphering Kinetic Information from Single-Molecule FRET Data That Show Slow Transitions

Area of Science:

• Biophysics
• Biochemistry
• Molecular Biology

Background:

• Single-molecule Förster Resonance Energy Transfer (smFRET) is a powerful biophysical technique.
• smFRET measurements are often limited by weak signals and short measurement times.
• Ultraslow molecular conformational transitions (transition times comparable to or longer than measurement times) pose a challenge for kinetic analysis.

Purpose of the Study:

• To develop a method for accurately extracting mean transition times from single-molecule data with ultraslow kinetics.
• To address the limitations of short measurement times in smFRET experiments.

Main Methods:

• Introduced the sHaRPer (serialized Handshaking Repeated Permutation with end removal) scheme.
• Concatenated multiple short time traces to create longer effective measurement times.
• Provided mathematical criteria for data acquisition frequency (f), measurement time (δt), and mean transition time (⟨τ⟩) to ensure accurate estimation of ⟨τ⟩orig.

Main Results:

• The sHaRPer method enables reliable estimation of mean transition times for ultraslow kinetics.
• Established criteria for selecting appropriate f, δt, and ⟨τ⟩ to minimize estimation errors.
• Provided guidelines to avoid distortion of kinetic phase time constants when using kinetic partitioning.

Conclusions:

• The sHaRPer method offers a practical solution for analyzing single-molecule data with slow transition kinetics.
• This approach enhances the utility of smFRET for studying complex biological dynamics.
• The study provides a practical guide for implementing the sHaRPer method.