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Shrinking gate fluorescence correlation spectroscopy yields equilibrium constants and separates photophysics from

Tim Schröder1, Johann Bohlen1, Sarah E Ochmann1

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Summary

This study introduces shrinking-gate fluorescence correlation spectroscopy (sg-FCS) to accurately measure biomolecular transition rates. This novel method enhances Förster resonance energy transfer (FRET) analysis for studying structural dynamics.

Keywords:
DNA origamiFörster resonance energy transferautocorrelation analysisbiophysicstime-gated fluorescence correlation spectroscopy

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Area of Science:

  • Biophysics
  • Spectroscopy
  • Molecular Dynamics

Background:

  • Fluorescence correlation spectroscopy (FCS) combined with Förster resonance energy transfer (FRET) is vital for studying biomolecular conformational changes.
  • Determining transition and equilibrium constants in FRET experiments with two intensity levels remains challenging.

Purpose of the Study:

  • To develop a method for accurately quantifying transition rate constants and equilibrium constants in FRET systems.
  • To enhance the analysis of time-correlated single-photon data for biomolecular dynamics.

Main Methods:

  • Combining intensity correlation analysis with fluorescence lifetime information using a microtime gating approach.
  • Developing the shrinking-gate fluorescence correlation spectroscopy (sg-FCS) algorithm.
  • Validating sg-FCS with simulations and experimental models, including DNA origami and graphene quenchers.

Main Results:

  • sg-FCS successfully determines forward and backward transition rate constants and equilibrium constants for two-level fluorescence intensity systems.
  • The method distinguishes photophysical effects from dynamic intensity changes, even with dark quenchers like graphene.
  • sg-FCS elucidated the mechanism of a FRET-based membrane charge sensor.

Conclusions:

  • sg-FCS provides a robust and easily implementable algorithm for analyzing FRET dynamics without prior knowledge.
  • This technique broadens the application of FCS and FRET in studying complex biomolecular systems and sensor mechanisms.