STOPPED-FLOW FLUOROMETRIC STUDY OF THE INTERACTION OF MELITTIN WITH PHOSPHOLIPID-BILAYERS - IMPORTANCE OF THE PHYSICAL STATE OF THE BILAYERAND THE ACYL-CHAIN LENGTH

Stopped-flow fluorometry has been employed to study the effects of mel ittin, the major protein component of bee venom, on dimyrisloylphospha tidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) small un ilamellar vesicles (SUVs) on the millisecond time scale, before melitt in-induced vesicle fusion takes place. Use is made of trimethylammoniu mphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), which is an oriented flu orescent probe that anchors itself to the bilayer-water interface and is aligned parallel to the normal to the bilayer surface; its fluoresc ence anisotropy reports on the ''fluidity'' of the bilayer. For DMPC b ilayers, melittin is found to decrease their fluidity only at their me lting transition temperature. This perturbation appears to be exerted almost instantaneously on the millisecond time scale of the measuremen ts, as deduced from the fact that its rate is comparable to that obtai ned by following the change in the fluorescence of the single tryptoph an residue of melittin upon inserting itself into the bilayer. The per turbation is felt in the bilayer over a distance of at least 50 Angstr om, with measurements of transfer of electronic energy indicating that the protein is not sequestered in the neighborhood of TMA-DPH. The le ngth of the acyl chains is found to be an important physical parameter in the melittin-membrane interaction: unlike the case of DMPC SUVs, m elittin does not alter the fluidity of DPPC SUVs and has a considerabl y greater affinity for them. These results are discussed in terms of t he concept of elastic distortion of the lipids, which results from a m ismatch between the protein and the acyl chains that are attempting to accommodate it. Melittin is also found to cause a small (similar to 1 0%) enhancement in the total fluorescence intensity of TMA-DPH, which is interpreted as indicating a reduction in the degree of hydration of the bilayer.