Measuring Protein Binding to Lipid Vesicles by Fluorescence Cross-Correlation Spectroscopy
![Measuring Protein Binding to Lipid Vesicles by Fluorescence Cross-Correlation Spectroscopy](/image/eyJ1cmkiOiJodHRwOi8vc3RhY2thZGVtaWMuaGVyb2t1YXBwLmNvbS9pbWFnZT9pbWFnZV9pZD0xNzQxMiIsImZvcm1hdCI6IndlYnAiLCJxdWFsaXR5IjoxMDAsIm5vQ2FjaGUiOnRydWV9.webp)
Abstract
Fluorescence correlation spectroscopy has been previously used to investigate peptide and protein binding to lipid membranes, as it allows for very low amounts of sample, short measurement times and equilibrium binding conditions. Labeling only one of the binding partners, however, comes with certain drawbacks, as it relies on identifying binding events by a change in diffusion coefficient. Since peptide and protein aggregation can obscure specific binding, and since non-stoichiometric binding necessitates the explicit choice of a statistical distribution for the number of bound ligands, we additionally label the liposomes and perform dual-color fluorescence cross-correlation spectroscopy (dcFCCS). We develop a theoretical framework showing that dcFCCS amplitudes allow calculation of the degree of ligand binding and the concentration of unbound ligand, leading to a model-independent binding curve. As the degree of labeling of the ligands does not factor into the measured quantities, it is permissible to mix labeled and unlabeled ligand, thereby extending the range of usable protein concentrations and accessible dissociation constants,
Publisher URL: http://www.cell.com/biophysj/fulltext/S0006-3495(17)30673-2
DOI: 10.1016/j.bpj.2017.06.023
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