Measuring Protein Binding to Lipid Vesicles by Fluorescence Cross-Correlation Spectroscopy

Area of Science:

• Biophysics
• Membrane Biology
• Spectroscopy

Background:

• Fluorescence correlation spectroscopy (FCS) studies peptide/protein binding to lipid membranes.
• Single-labeling in FCS has drawbacks: reliance on diffusion changes and issues with aggregation or non-stoichiometric binding.

Purpose of the Study:

• To develop and validate a dual-color fluorescence cross-correlation spectroscopy (dcFCCS) framework for accurate biophysical binding analysis.
• To enable model-independent determination of binding curves and ligand concentrations.

Main Methods:

• Implemented dcFCCS by labeling both liposomes and binding partners.
• Developed a theoretical framework to calculate binding degree and unbound ligand concentration from dcFCCS amplitudes.
• Applied the dcFCCS analysis to Sar1p protein binding to liposomes.

Main Results:

• dcFCCS allows model-independent binding curve generation.
• The method is robust to ligand labeling degree, enabling use of mixed labeled/unlabeled ligands.
• Analysis of Sar1p binding to liposomes yielded a dissociation constant (K_D) of (2.1±1.1)μM.

Conclusions:

• The presented dcFCCS framework offers a versatile and accurate method for studying ligand-membrane interactions.
• This technique overcomes limitations of traditional FCS, broadening applications in biophysical analysis.
• Applicable to various fluorescently labeled ligands and particles, including membrane protein receptors.