Fluorometric studies of the interaction of bee venom melittin with model biological membranes

Stopped-flow fluorometric measurements were carried out in order to (i) measure the kinetics of melittin-induced fusion of dipalmitoylphosphatidylcholine small unilamellar vesicles at 51°C under conditions where melittin was either monomeric or tetrameric in solution; (ii) study the effects of melittin binding on the fluidity and permeability of dimyristoylphosphatidylcholine and dipalmitoylphosphatidylcholine small unilamellar vesicles for below, at and above their respective transition temperatures prior to the occurence of lipid ordering effects due to melittin-induced fusion; and (iii) study the kinetics of melittin binding to dipalmitoylphosphatidylcholine small unilamellar vesicle over a range of temperatures. In the case of (i), it was found that the rate of melittin-induced fusion is strongly dependent on the state of aggregation of melittin in solution and, to a lesser extent ions in solution. This suggests that the fusion mechanisms are different for the different states of protein aggregation. In the case of (ii) it was found that for dimyristoylphosphatidylcholine small unilamellar vesicles, the effects of melittin binding on fluidity and permeability are maximal at the lipid transition temperature, in agreement with theory. The melittin-induced lipid perturbation was also found to be long range, rather than confined to a boundary annulus of lipids, and propagated rapidly through the bilayer. Interestingly, melittin binding to dipalmitoylphosphatidylcholine small unilamellar vesicles had little to no effect on membrane fluidity or permeability, which demonstrates the importance of acyl chain length in lipid stability, (iii) Kinetic data for the binding of melittin to dimyristoylphosphatidylcholine small unilamellar vesicles was analyzed in the context of a more sophisticated model than hitherto used. There, it was found that the rates of melittin binding and disassociation decreased with decreasing temperature, as might be expected. In the case of binding, an Arrhenius analysis yielded an activation energy close to that for the melting of lipid acyl chains. The number of lipids required to bind a melittin molecule was found to decrease with decreasing temperature. Fluorescence polarization measurements analyzed using a model which considers statistical effects from the existence of multiple binding site may clarify future analyses.