Benchmarking dynamic analysis methods in diffusion-based single-molecule FRET.

Area of Science:

• Biophysics
• Biochemistry
• Physical Chemistry

Background:

• Single-molecule Förster resonance energy transfer (smFRET) is crucial for observing biomolecular conformational dynamics at the nanoscale.
• Extracting reliable dynamic information from diffusion-based smFRET data necessitates careful selection of analysis techniques.

Purpose of the Study:

• To benchmark widely used analysis methods for diffusion-based smFRET.
• To provide practical guidelines for selecting appropriate analysis strategies and accurately interpreting conformational dynamics.

Main Methods:

• Benchmarking of qualitative and quantitative analysis approaches, including hidden Markov modeling.
• Utilizing simulated datasets representing common experimental conditions for a two-state system.

Main Results:

• Clarification of the timescales reported by different methods.
• Determination of the number of data bursts required for reliable kinetic rate inference.
• Evaluation of FRET efficiency requirements and accuracy of kinetic rate recovery.

Conclusions:

• The study offers practical guidelines for optimizing experimental design and analysis in diffusion-based smFRET.
• Accurate interpretation of biomolecular conformational dynamics is facilitated by informed method selection.