Modulation of tryptophan environment in membrane-bound melittin by negatively charged phospholipids: implications in membrane organization and function.

Melittin is a cationic hemolytic peptide isolated from the European honey bee, Apis mellifera. Since the association of the peptide in the membrane is linked with its physiological effects, a detailed understanding of the interaction of melittin with membranes is crucial. We have investigated the interaction of melittin with membranes of varying surface charge in the context of recent studies which show that the presence of negatively charged lipids in the membrane inhibits membrane lysis by melittin. The sole tryptophan residue in melittin has previously been shown to be critical for its hemolytic activity. The organization and dynamics of the tryptophan residue thus become important to understand the peptide activity in membranes of different charge types. Wavelength-selective fluorescence was utilized to monitor the tryptophan environment of membrane-bound melittin. Melittin exhibits a red edge excitation shift (REES) of 5 nm when bound to zwitterionic membranes while in negatively charged membranes, the magnitude of REES is reduced to 2-3 nm. Further, wavelength dependence of fluorescence polarization and near-UV circular dichroism spectra reveal characteristic differences in the tryptophan environment for melittin bound to zwitterionic and anionic membranes. These studies are supported by time-resolved fluorescence measurements of membrane-bound melittin. Tryptophan penetration depths for melittin bound to zwitterionic and anionic membranes were analyzed by the parallax method [Chattopadhyay, A., and London, E. (1987) Biochemistry 26, 39-45] utilizing differential fluorescence quenching obtained with phospholipids spin-labeled at two different depths. Our results provide further insight into molecular details of membrane lysis by melittin and the modulation of lytic activity by negatively charged lipids.