Spectroscopic studies of the effects of bee venom melittin on model phospholipid membranes

The enhancement of the 0-0 vibronic transition in the fluorescence spectrum of pyrene, which is a measure of the polarity of the microenvironment of the probe, and the formation of intramolecular excimers by 1, 3-bis (1-pyrene) propane, (PC3P), have been employed for investigating the effects of bee venom melittin on distearoylphosphatidylcholine, (DSPC), bilayer liposomes. For melittin-to-lipid molar ratios > 1:165 melittin has been found to induce a pronounced increase in the enhancement in the vicinity of the phase transition temperature, T_t, of the phospholipid; this implies that the protein causes structural rearrangements in the bilayer that result in an increase in its packing density with a concomitant dislocation of the probe toward the direction of the polar head groups. Alternatively, on the basis of the concept of lipid cluster formation, the protein reduces the probability of formation of such clusters; the resulting reduction in the number of mismatches between gel and liquid domains impedes the penetration of the probe into the bilayer. Apparent activation energies of the order of RT have been obtained from the enhancement profiles. The observed increase in the T_t in the presence of the protein is in line with these interpretations.

Nanosecond time-resolved measurements have shown that the long wavelength emission by PC₃P, that peaks at ~ 480 nm, is an excited-state process with very little contribution from ground-state complex formation.

Plots of log (q_e/q_m) against 1/T were q_e and q_m are fluorescence quantum yields of excimer and monomer, respectively, have been found to be linear In the two phases of the phospholipid but to exhibit a break In the adjoining region. Such plots allowed the determination of the energies of activation, of the differences in the free energies of activation and of the entropies of activation for excimer formation in the two phases. The PC₃P molecules are in a less favorable conformation for excimer formation in the crystalline phase than in the llquld-crystalllne phase. The protein Is found not to affect significantly the excimer-forming ability of the probe below T_t but to impede it above it. In the fluid state melittin is found to increase the entropy of activation relative to that in the crystalline phase. This implies an induced disorganization of the conformations of the two pyrene moieties of PC₃ and, by extension, of the lipid structure. Furthermore, the protein causes a decrease in the free energy of activation in that phase relative to that in the crystalline phase, which implies enhancement of the fluidity of the bilayer.

The results of both studies show that melittin exerts its most pronounced effect when liquid domains of the lipid are present by altering both the fluidity and the organization of the lipid.