Melittin, a 26-residue cytolytic peptide, is the major component (∼50﹪)of the venom of the European honey bee Apis mellifera. An array of studies have been undertaken to examine the nature of the interaction between melittin and membrane. A survey of the literature on oligomerization state and orientation of melittin in phospholipid bilayers, yielded a conflicting conclusion. Considering the high net charge of +5 to +6 for melittin, it is possible that the charged of lipid may play a role in the interaction with melittin. Therefore we use zwitterionic (DMPC) and anionic (DMPG) phospholipid vesicles to examine oligmerization state and orientation of melittin in different charge lipids.
The highly distance-sensitive fluorescent probe, rhodamine, is selected to label on the N-terminus of melittin. The aggregation numbers of melittin in phospholipid vesicles can be deduced from rhodamine self-quenching effect. In addition cobalt ion was used to quench NBD, which is labeled on melittin N-terminus, and acryamide was used to quench tryptophan of melittin. The membrane insertion depth of the N-terminal melittin in zwitterionic (DMPC) and anionic (DMPG) lipid are studied by quenching N-terminal labeled NBD fluorescence with the quencher, cobalt ions. The relative depth of tryptophan 19 in DMPC and DMPG lipid are compared with acryamide quenching experiment. Additionally, each individual residue of melittin in the position of membrane-mimic SDS micelles is acquired from 2D-NMR experiments through Mn2+ titration.
We concluded that melittin is monomeric in anionic lipid with helix axis nearly parallel to the membrane plane. In marked contrast, in zwitterionic lipid, the number of melittin is dimmer to tetramer with helix axis perpendicular to the membrane plane.
Gp41 is a part of envelope glycorprotein of the human immunodeficiency virus type 1 (HIV-1). Following binding of gp120 to CD4 and a chemokine receptor, conformational changes occur in the gp120/gp41 complex that leads to trimeric hairpin structure core. The pre-hairpin intermediate of gp41 from HIV is the target for two classes of fusion inhibitors that bind to the C-terminal region or the trimeric coiled-coil of N-terminal helices, thereby preventing formation of the fusogenic trimer of hairpins. The gp120 sequence is highly variable, rendering an ineffective target for fusion inhibitors.
A number of peptides and peptide analogs derived from the membrane proximal region of gp41 ectodomain are found to be effective inhibitors of human immunodeficiency virus type 1 (HIV-1)-mediated fusion events. Two of them, T20 (aa 638-673)、T649(aa 628-663), were found disordered and sparingly soluble in water, but became soluble upon mixing with selected, structured peptides from the amino terminal heptad repeat (NHR) region of gp41 using a simple and sensitive method of reduction in the scattering of T20 suspension. From the results on mapping the locus of interaction with T20 by employing partially overlapping peptides derived from NHR, it was concluded that the LLSGIV segment was a critical docking site for the C-terminal peptide of gp41 in its putative inhibitory action. It was also found that peptides capable of solubilizing T20 dispersion have a high content of α-helix, as well as β-sheet, conformation in aqueous solution.
Comparison the turbidity clearance assay between T20-L43L and T649-L43L interaction, it was found the sequence of aa 628-638 likely played an important role of gp41 N-terminal binding. The secondary structure analysis from CD data of T20 and T649 revealed that transformation of random-coil toα-helix on binding of NHR. And Isotheraml Tirtation Calormetry and Real Time Biomolecular interaction data demonstrated that the binding affinity of T649 to gp41 N-terminal domain is stronger than T20.||en_US|