The interaction of melittin (MLT), a 26 amino acid peptide isolated from bee venom, with diacylglycerophosphocholine has been examined by application of light scattering and fluorescence techniques. Melittin interacts most strongly with ditetradecanoylglycerophosphocholine (DMPC) and dipentadecanoylglycerophosphocholine (DPenPC) at their phase transition temperatures (Tc). Light scattering shows that the interaction with DMPC at melittin:lipid ratios of 1:400 or greater is multiphasic with respect to time. At 25°C, there is first an increase in turbidity, then an apparently cooperative decrease in light scattering, and finally a clearing of the solution. The apparent rates of all stages of the interaction increase markedly with increase in MLT:lipid ratio. The fluorescence anisotropy (rss) of diphenylhexatriene (DPH) embedded in the lipid bilayers shows a biphasic increase to an apparent equilibrium value. Attainment of the latter coincides with a minimum intensity plateau in the light scattering signal. Quasi-elastic light scattering (QLS) and electron microscopy data show that at MLT:lipid ratios of 1:400 to 1:100, homogeneous mixed (protein-lipid) micelles are formed with dimensions of 210-250 Å. The micelles apparently do not have a trapped volume and are assumed to be nonvesicular structures. Measurements of rss and differential phase fluorometry were used to show that the structure formed at equilibrium exhibits an order parameter for DPH that is in excess of that observed for DPH in the gel phase of DMPC or DPenPC vesicles only. The MLT-DMPC or MLT-DPenPC complexes monitored by changes in rss for DPH exhibit a highly cooperative thermal transition at 31 and 41 °C (compared with 24 and 33°C for DMPC and DPenPC vesicles, respectively) when melittin:lipid ratios are ca. 1:400 to 1:100. However, at higher ratios of peptide:lipid the transition becomes increasingly broadened, and at a 1:1 peptide:lipid ratio there is no detectable phase transition. The pattern of DPH fluorescence anisotropy changes is mirrored by that of a cationic analogue, trimethylammonium diphenylhexatriene (TMA-DPH), which was used to minimize the probability of adsorption of the fluorophore near the cationic peptide. QLS indicates a marked increase in the size of the MLT-lipid mixed micelles from ca. 220 to 1220 Å and delineates an apparent phase transition. Measurements of rss of DPH made on thermally equilibrated samples show no difference in profile whether the temperature is increased or decreased. However, QLS data on micellar size as the temperature is scanned showed marked hysteresis, and the micelles do not return to their original sizes. We deduce that with the saturated phospholipids, DMPC and DPenPC, MLT at low MLT:lipid ratios forms mixed micelles that (i) are of defined size, (ii) are highly ordered, and (iii) exhibit highly cooperative phase transitions at temperatures considerably higher than those of pure lipid. The last two properties are unprecedented among peptide-lipid complexes. The phase behavior may, however, depend on peptide:lipid ratios and the thermal history of the system.
|Original language||English (US)|
|Number of pages||9|
|State||Published - 1982|
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