Dual-color fluorescence-burst analysis to study pore formation and protein–protein interactions

Dual-color fluorescence-burst analysis (DCBFA) enables to study leakage of fluorescently labeled (macro) molecules from liposomes that are labeled with a second, spectrally non-overlapping fluorophore. The fluorescent bursts that reside from the liposomes diffusing through the focal volume of a confocal microscope will coincide with those from the encapsulated size-marker molecules. The internal concentration of size-marker molecules can be quantitatively calculated from the fluorescence bursts at a single liposome level. DCFBA has been successfully used to study the effective pore-size of the mechanosensitive channel of large-conductance MscL and the pore-forming mechanism of the antimicrobial peptide melittin from bee venom. In addition, DCFBA can be used to quantitatively measure the binding of proteins to liposomes and to membrane proteins. In this paper, we provide an overview of the method and discuss the experimental details of DCFBA.