title: A One Donor-Two Acceptor Lipid Bilayer FRET Assay Based on Asymmetrically Labeled Liposomes.
abstract: The fusion of two opposing membranes is essential in biological functions such as fertilization, viral entry, membrane trafficking and synaptic transmission. Before the membrane bilayers are fully connected, at some stage a hemifusion intermediate-when the outer leaflets are merged but not the inner leaflets-is formed. However, the position of hemifusion in the energy landscape and the duration of it vary and have not been fully mapped out. To date, there has not been a way to differentiate lipid mixing of the two leaflets directly in a single experiment. Herein we demonstrate labeling of the outer and inner leaflets with different fluorophores, which can be distinguished by their fluorescence lifetimes. As a proof of concept, the asymmetrically labeled liposomes were used as acceptor liposomes in a novel one donor-two acceptor Förster resonance energy transfer (FRET) assay to monitor membrane fusion reactions mediated by the synaptic proteins soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) in microfluidic devices. Initial hemifusion was clearly indicated by the acceptor fluorescence lifetime originating solely from FRET acceptors on the outer leaflet (Oregon Green 488; τFl∼ 4.8 ns). Progression to full fusion was then indicated by the significantly increasing lifetime contribution from acceptors on the inner leaflet (nitrobenzoxadiazole; τFl∼ 6.7 ns). The new labeling strategy creates many possibilities in the design of bulk and single-molecule experiments.
authors.affiliation: Research Group Biomolecular Spectroscopy and Single-Molecule Detection, Max Planck Institute for Biophysical Chemistry , Am Faßberg 11, 37077 Göttingen, Germany.
authors.forename: Chao-Chen
authors.initials: CC
authors.lastname: Lin
authors.affiliation: Laboratory for Fluid Dynamics, Pattern Formation and Biocomplexity, Max Planck Institute for Dynamics and Self-Organization , Am Faßberg 17, 37077 Göttingen, Germany.
authors.forename: Hsin-Fang
authors.initials: HF
authors.lastname: Hsu
authors.affiliation: Research Group Biomolecular Spectroscopy and Single-Molecule Detection, Max Planck Institute for Biophysical Chemistry , Am Faßberg 11, 37077 Göttingen, Germany.|Department of Biophysical Chemistry, Institute for Physical and Theoretical Chemistry, Technical University of Braunschweig , Hans-Sommer-Straße 10, 38106 Braunschweig, Germany.
authors.forename: Peter Jomo
authors.initials: PJ
authors.lastname: Walla
mesh_terms: D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer; D008051:Lipid Bilayers / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry; D010069:Oxadiazoles / Q000737:chemistry*
chemical_list: D008051:Lipid Bilayers; D008081:Liposomes; D010069:Oxadiazoles