Probing ligand protein binding equilibria with fluorescence fluctuation spectroscopy

Area of Science:

• Biophysics
• Analytical Chemistry
• Biochemistry

Background:

• Fluorescence fluctuation experiments measure molecular brightness and concentration.
• Normalized variance g(0) alone is insufficient to resolve individual species in complex samples.
• Ligand-protein titration is key to linking molecular brightness and concentration.

Purpose of the Study:

• To develop and validate a method for separating and characterizing molecular species using fluorescence fluctuation analysis.
• To investigate the influence of binding parameters on fluctuation amplitude.
• To demonstrate the capability of g(0) analysis in identifying molecular heterogeneity in biomolecular systems.

Main Methods:

• Analysis of fluctuation amplitude g(0) in fluorescence fluctuation experiments.
• Titration of fluorescent ligands with proteins to establish binding models.
• Global analysis of fluctuation amplitude and fluorescence intensity.
• Validation using fluorescence lifetime experiments.

Main Results:

• g(0) analysis successfully separated species in binary dye mixtures.
• Binding parameters like dissociation coefficient and stoichiometry significantly influence fluctuation amplitude.
• Molecular heterogeneity of a hapten-antibody complex was identified and characterized.
• Results from g(0) analysis were confirmed by independent fluorescence lifetime measurements.

Conclusions:

• Fluorescence fluctuation analysis, particularly g(0) analysis combined with titration, is a powerful tool for resolving molecular species and characterizing binding.
• The method accurately determines dissociation coefficients, binding stoichiometry, and reveals molecular heterogeneity.
• This technique has significant implications for understanding complex biomolecular systems and identifying molecular variations.