title: Dual-color fluorescence-burst analysis to study pore formation and protein-protein interactions.
abstract: 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.
authors.affiliation: Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors.forename: Geert
authors.initials: G
authors.lastname: van den Bogaart
authors.forename: Ilja
authors.initials: I
authors.lastname: Kusters
authors.forename: Jeanette
authors.initials: J
authors.lastname: Velásquez
authors.forename: Jacek T
authors.initials: JT
authors.lastname: Mika
authors.forename: Victor
authors.initials: V
authors.lastname: Krasnikov
authors.forename: Arnold J M
authors.initials: AJ
authors.lastname: Driessen
authors.forename: Bert
authors.initials: B
authors.lastname: Poolman
mesh_terms: D029968:Escherichia coli Proteins / Q000737:chemistry / Q000502:physiology; D005456:Fluorescent Dyes; D007473:Ion Channels / Q000737:chemistry / Q000502:physiology; D008081:Liposomes / Q000737:chemistry*; D008555:Melitten / Q000737:chemistry / Q000502:physiology; D008565:Membrane Proteins / Q000737:chemistry*; D018613:Microscopy, Confocal; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D025941:Protein Interaction Mapping
chemical_list: D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D007473:Ion Channels; D008081:Liposomes; D008565:Membrane Proteins; C086596:MscL protein, E coli; D008555:Melitten