We investigated the effects of various concentrations of staphylococcal delta-lysin on the thermotropic phase behavior of large multilamellar dimyristoylphosphatidylcholine (DMPC) vesicles by differential scanning calorimetry (DSC), P-31 nuclear magnetic resonance (NMR) and Fourier transform infrared (FTIR) spectroscopy, and X-ray diffraction. The DSC studies revealed that at all concentrations, the addition of delta-lysin progressively decreases the enthalpy of the pretransition of DMPC bilayers without significantly affecting its temperature or cooperativity. Similarly, the addition of smaller quantities of peptide has little effect on the temperature of the main phase transition of DMPC bilayers but does reduce the cooperativity and enthalpy of this transition somewhat. However, at higher peptide concentrations, a second phase transition with a slightly increased temperature and a markedly reduced cooperativity and enthalpy is also induced, and this latter phase transition resolves itself into two components at the highest peptide concentrations that are tested. Moreover, our P-31 NMR spectroscopic studies reveal that at relatively low delta-lysin concentrations, essentially all of the phospholipid molecules produce spectra characteristic of the lamellar phase, whereas at the higher peptide concentrations, an increasing proportion exhibit an isotropic signal. Also, at the highest delta-lysin concentrations that are. studied, the isotropic component of the P-31 NMR spectrum also resolves itself into two components. At the highest peptide concentration that was tested, we are also able to effect a macroscopic separation of our sample into two fractions by centrifugation, a pellet containing relatively smaller amounts of delta-lysin and a supernatant containing larger amounts of peptide relative to the amount of lipid present. We are also able to show that the more cooperative phase transition detected calorimetrically, and the lamellar phase P-31 NMR signal, arise from the pelleted material, while the less cooperative phase transition and the isotropic P-31 NMR signal arise from the supernatant. In addition, we demonstrate by X-ray diffraction that the pelleted material corresponds to delta-lysin-containing large multilamellar vesicles and the supernatant to a mixture of delta-lysin-containing small unilamellar vesicles and discoidal particles. We also show by FTIR spectroscopy that delta-lysin exists predominantly in the alpha-helical conformation in aqueous solution or when interacting with DMPC, and that a large fraction of the peptide bonds undergo H-D exchange in D2O, However, upon interaction with DMPC, the fraction of exchangeable amide protons decreases. We also demonstrate by this technique that both of the phase transitions detected by DSC correspond to phospholipid hydrocarbon chain-melting phase transitions. Finally, we show by several techniques that. the absolute concentrations of delta-lysin and the thermal history, as well as the lipid:peptide ratio, can affect the thermotropic phase behavior and morphology of peptide-lipid aggregates.
