The journal of physical chemistry. B

A One Donor-Two Acceptor Lipid Bilayer FRET Assay Based on Asymmetrically Labeled Liposomes.

PMID 27762543


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; τ