Determining the membrane topology of peptides by fluorescence quenching

Determination of the topology of peptides in membranes is important for cha racterizing and understanding the interactions of peptides with membranes. We describe a method that uses fluorescence quenching arising from resonanc e energy transfer ("FRET") for determining the topology of the tryptophan r esidues of peptides partitioned into phospholipid bilayer vesicles. This is accomplished through the use of a novel lyso-phospholipid quencher (lysoMC ), N-(7-hydroxyl-4-methylcoumarin-3-acetyl) l-palmitoyl2-hydroxy-sn-glycero -3-phosphoethanolamine. The design principle was to anchor the methylcoumar in quencher in the membrane interface by attaching it to the headgroup of l yso-phosphoethanolamine. We show that lysoMC can be incorporated readily in to large unilamellar phospholipid vesicles to yield either symmetrically (b oth leaflets) or asymmetrically (outer leaflet only) labeled bilayers. Lyso MC quenches the fluorescence of membrane-bound tryptophan by the Forster me chanism with an apparent Ro that is comparable to the thickness of the hydr ocarbon core of a Lipid bilayer (similar to 25 Angstrom). Consequently, the methylcoumarin acceptor predominantly quenches tryptophans that reside in the same monolayer as the probe. The topology of a peptide's tryptophan in membranes can be determined by comparing the quenching in symmetric and asy mmetric lysoMC-labeled vesicles. Because it is essential to know that asymm etrically incorporated lysoMC remains so under all conditions, we also deve loped a second type of FRET experiment for assessing the rate of transbilay er diffusion (flip-flop) of lysoMC. Except in the presence of pore-forming peptides, there was no measurable flip-flop of lysoMC, indicating that asym metric distributions of quencher are stable. We used these methods to show that N-acetyl-tryptophan-octylamide and tryptophan-octylester rapidly equil ibrate across phosphatidylcholine (POPC) and phosphatidylglycerol (POPG) bi layers, while four amphipathic model peptides remain exclusively on the out er monolayer, The topology of the amphipathic peptide melittin bound to POP C could not be determined because it induced rapid flip-flop of lysoMC. Int erestingly, melittin did not induce lysoMC flip-flop in POPG vesicles and w as found to remain stably on the external monolayer.