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Updated: Jun 20, 2026

Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions
Published on: January 30, 2018
Characterization of coupled ground state and excited state equilibria by fluorescence spectral deconvolution
Wouter Caarls1, M Soledad Celej, Alexander P Demchenko
1Laboratory of Cellular Dynamics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, D37077, Göttingen, Germany.
Abstract:
Fluorescence probes with multiparametric response based on the relative variation in the intensities of several emission bands are of great general utility. An accurate interpretation of the system requires the determination of the number, positions and intensities of the spectral components. We have developed a new algorithm for spectral deconvolution that is applicable to fluorescence probes exhibiting a two-state ground-state equilibrium and a two-state excited-state reaction. Three distinct fluorescence emission bands are resolved, with a distribution of intensities that is excitation-wavelength-dependent. The deconvolution of the spectrum into individual components is based on their representation as asymmetric Siano-Metzler log-normal functions. The application of the algorithm to the solvation response of a 3-hydroxychromone (3HC) derivative that exhibits an H-bonding-dependent excited-state intramolecular proton transfer (ESIPT) reaction allowed the separation of the spectral signatures characteristic of polarity and hydrogen bonding. This example demonstrates the ability of the method to characterize two potentially uncorrelated parameters characterizing dye environment and interactions.
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