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Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions
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Characterizing multiple molecular States in single-molecule multiparameter fluorescence detection by probability

Stanislav Kalinin1, Suren Felekyan, Alessandro Valeri

  • 1Institut für Physikalische Chemie, Lehrstuhl für Molekulare Physikalische Chemie, Heinrich-Heine-Universität, Universitätsstrasse 1, Geb 26.32, 40225 Düsseldorf, Germany. stanislav.kalinin@uni-duesseldorf.de

The Journal of Physical Chemistry. B
|June 24, 2008
PubMed
Summary

Probability distribution analysis (PDA) is enhanced to quantitatively analyze complex single-molecule FRET and polarization data. This extended theory accurately models multiple fluorescent states and high concentrations, achieving Angstrom-level resolution for molecular distance measurements.

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Area of Science:

  • Biophysics
  • Physical Chemistry
  • Spectroscopy

Background:

  • Single-molecule (SM) experiments, such as Förster resonance energy transfer (FRET) and fluorescence polarization, generate complex data.
  • Existing Probability Distribution Analysis (PDA) methods accurately predict histograms for simple systems but struggle with multiple states and experimental noise.
  • Analyzing complex SM data requires accounting for factors like background noise, shot noise, brightness variations, and multiple-molecule events.

Purpose of the Study:

  • To extend Probability Distribution Analysis (PDA) theory for quantitative analysis of complex single-molecule FRET and polarization data.
  • To incorporate effects of brightness variations and multiple-molecule events into the PDA framework.
  • To achieve high-resolution molecular distance measurements by distinguishing between fixed and distributed distances.

Main Methods:

  • Developed extensions to PDA theory to account for multiple non-interconverting fluorescent states.
  • Incorporated analysis of brightness variations and multiple-molecule events using experimental signal intensity distribution.
  • Applied model-free deconvolution using the maximum entropy method (MEM) and combined mean donor fluorescence lifetime analysis.

Main Results:

  • The extended PDA accurately predicts complex SM-FRET and polarization data histograms, even at high molecule concentrations.
  • The theory successfully accounts for background, shot noise, brightness variations, and multiple-molecule events.
  • Achieved ultimate resolution in FRET experiments in the range of a few Angstroms, enabling discrimination between fixed and distributed molecular distances.

Conclusions:

  • The extended PDA theory provides a robust framework for analyzing complex single-molecule biophysical data.
  • Accurate analysis of FRET and polarization experiments is possible even with multiple fluorescent states and high molecule concentrations.
  • The developed methods allow for precise molecular distance determination, advancing the field of molecular nanometer-scale measurements.