Analyzing Förster resonance energy transfer with fluctuation algorithms.

Area of Science:

• Biophysics
• Biochemistry
• Physical Chemistry

Background:

• Fluorescence correlation spectroscopy (FCS) and Förster resonance energy transfer (FRET) are powerful tools for analyzing biomolecular dynamics.
• FRET-FCS is particularly effective for studying structural fluctuations, conformational flexibility, and complex formation in biomolecules.
• Structural dynamics lead to anticorrelated donor and acceptor signals, which FRET-FCS analyzes to characterize these dynamics.

Purpose of the Study:

• To review experimental implementations of FRET-FCS and present theoretical frameworks for analyzing biomolecular dynamics.
• To address challenges in extracting meaningful information from correlation amplitudes and propose strategies for robust parameter determination.
• To introduce filtered FCS (fFCS) as an advanced method for analyzing complex FRET dynamics in samples with multiple fluorescent species.

Main Methods:

• Utilizing FRET-FCS to analyze fluctuations in donor and acceptor signals over timescales from nanoseconds to seconds.
• Developing theoretical models for two-state interconverting systems and systems with donor-only labeled species.
• Implementing pulsed polarized excitation with multiparameter fluorescence detection for filtered FCS (fFCS) to enable species-specific correlation analysis.

Main Results:

• Demonstrated that mean relaxation times for structural dynamics are generally obtainable, though correlation amplitudes can be challenging to interpret.
• Presented a strategy to constrain donor and acceptor brightnesses, enabling determination of FRET efficiencies and rate constants.
• Showcased fFCS's ability to filter species based on fluorescence decays, yielding interpretable correlation functions and improving resolution of multistate kinetics.

Conclusions:

• FRET-FCS is a versatile technique for characterizing biomolecular structural dynamics, conformational flexibility, and interactions.
• Filtered FCS (fFCS) significantly enhances the capability to resolve complex and multistate kinetics by enabling species-specific analysis.
• The presented strategies improve the accuracy and reliability of FRET-FCS data analysis, particularly for complex biological systems.