Disentangling Timescales of Molecular Kinetics with spFRET using ALEX-FCS

Area of Science:

• Biophysics
• Chemical Physics
• Molecular Dynamics

Background:

• Single-pair Förster resonance energy transfer (spFRET) offers sub-nanometer accuracy for molecular dynamics.
• Combining spFRET with fluorescence correlation spectroscopy (FCS) and alternating excitation (ALEX) enhances data analysis for freely diffusing molecules.
• Extracting subtle conformational dynamics and diffusion differences in complex systems remains challenging.

Purpose of the Study:

• To rigorously analyze the range of molecular lifetimes measurable with spFRET-ALEX-FCS.
• To simulate experiments on systems with distinct conformational states and diffusion coefficients, like nucleosomes.
• To establish a framework for analyzing complex molecular dynamics using these combined techniques.

Main Methods:

• Simulated spFRET-ALEX-FCS experiments.
• Modeled molecules with two conformations differing in FRET efficiency and diffusion coefficients.
• Utilized burst analysis for background reduction and lifetime extraction.

Main Results:

• Successfully distinguished small changes in diffusion coefficients between conformations.
• Demonstrated accurate determination of conformational lifetimes between 100 µs and 100 ms via burst selection.
• Validated the utility of spFRET-ALEX-FCS for probing molecular dynamics.

Conclusions:

• spFRET-ALEX-FCS is a powerful combination for characterizing molecular dynamics and conformational changes.
• The simulation framework can be extended to more complex biological systems.
• This approach enables precise measurement of lifetimes crucial for understanding molecular mechanisms.