Fluorescence-quenching-resolved spectra of melittin in lipid bilayers.
The interaction of bee venom melittin with dimyristoylphosphatidylcholine (DMPC) unilamellar vesicles has been studied by means of fluorescence quenching of the single tryptophan residue of the protein, at lipid-to-peptide ratio, Ri = 50 and at high ionic strength (2 M NaCl). The method of fluorescence-quenching-resolved spectra (FQRS), applied in this study with potassium iodide as a quencher, enabled us to decompose the tryptophan emission spectrum of liposome-bound melittin into components, at temperatures above as well as below the main phase transition temperature (Tt) of DMPC. One of the two resolved spectra exhibits maximum at 342 and 338 nm for experiments above and below Tt, respectively, and is similar to the maximum of tryptophan emission found for tetrameric melittin in solution (340 nm). This spectrum is characterized by the Stern-Volmer quenching constant, Ksv, of about 4 M-1 and it represents the fraction of melittin molecules whose tryptophan residues are exposed to the solvent to a degree comparable with tetrameric species in solution. The other spectrum component, corresponding to the quencher-inaccessible fraction of tryptophan molecules (Ksv = 0 M-1) has its maximum blue-shifted up to 15 nm, indicating a decrease in polarity of the environment. For experiments above Tt, the blue spectrum component revealed the excitation-wavelength dependence, originating probably from the relaxation processes between the excited tryptophan molecules and lipid polar head groups. We conclude that melittin bound to DMPC liposomes exists in two lipid-associated forms; one, with tryptophan residues exposed to the solvent and the other, penetrating the membrane interior, with tryptophan residues located in close proximity to the phospholipid polar head groups of the outer vesicle lipid layer. We also discuss our data with current models of melittin-bilayer interactions.
Kaszycki
P
P
Department of Biochemistry, Jagiellonian University, Kraków, Poland.
Wasylewski
Z
Z
D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004134:Dimyristoylphosphatidylcholine; D066298:In Vitro Techniques; D007454:Iodides / Q000737:chemistry; D008051:Lipid Bilayers; D008555:Melitten / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry
D007454:Iodides; D008051:Lipid Bilayers; D008555:Melitten; D014364:Tryptophan; D004134:Dimyristoylphosphatidylcholine