Determining the Membrane Topology of Peptides by Fluorescence Quenching

摘要: Determination of the topology peptides in membranes is important for characterizing and understanding interactions with membranes. We describe a method that uses fluorescence quenching arising from resonance energy transfer ("FRET") determining tryptophan residues partitioned into phospholipid bilayer vesicles. This accomplished through use novel lyso-phospholipid quencher (lysoMC), N-(7-hydroxyl-4-methylcoumarin-3-acetyl)-1-palmitoyl-2-hydroxy-sn-gly cero-3-phosphoethanolamine. The design principle was to anchor methylcoumarin membrane interface by attaching it headgroup lyso-phosphoethanolamine. show lysoMC can be incorporated readily large unilamellar vesicles yield either symmetrically (both leaflets) or asymmetrically (outer leaflet only) labeled bilayers. LysoMC quenches membrane-bound Forster mechanism an apparent R(0) comparable thickness hydrocarbon core lipid (approximately 25 A). Consequently, acceptor predominantly tryptophans reside same monolayer as probe. peptide's determined comparing symmetric asymmetric lysoMC-labeled Because essential know remains so under all conditions, we also developed second type FRET experiment assessing rate transbilayer diffusion (flip-flop) lysoMC. Except presence pore-forming peptides, there no measurable flip-flop lysoMC, indicating distributions are stable. used these methods N-acetyl-tryptophan-octylamide tryptophan-octylester rapidly equilibrate across phosphatidylcholine (POPC) phosphatidylglycerol (POPG) bilayers, while four amphipathic model remain exclusively on outer monolayer. peptide melittin bound POPC could not because induced rapid Interestingly, did induce POPG found stably external