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Tosteson, M.T., Tosteson D.C. The sting. Melittin forms channels in lipid bilayers. Biophysical Journal 36: 109-116 (1981) Abstract Melittin, a toxin of bee venom, is a cationic polypeptide composed of 26 amino acids. The six residues of the C-terminal end are polar and 19 of the 20 residues of the N-terminal end are hydrophobic. Exposure of the lecithin bilayer to melittin results in the formation of channels that are more permeable to anions that to cations. Unilateral addition of melittin produces a voltage-dependent increase in membrane conductance when the side where the polypeptide is present in made positive but not when it is made negative. At a fixed voltage, the conductance increases with the fourth power of the melittin concentration in the aqueous phase. At a fixed peptide concentration, the conductance increases approximately e-fold per 6-mV increase in the electrical potential difference across the membrane. These results suggest that four melittin monomers are needed to form a channel and, furthermore, that a minimum of four equivalent electronic charges need to be displaced by the electrical field to explain the voltage dependence of the conductance. Hristova K, et al. Structure, location, and lipid perturbations of melittin at the membrane interface. Biochim Biophys Acta. 1990 May 7;1031(2):143-61. Abstract The molecular mechanisms underlying the various effects of melittin on membranes have not been completely defined and much of the evidence described indicates that different molecular mechanisms may underlie different actions of the peptide. Ideas about the formation of transbilayer aggregates of melittin under the influence of a transbilayer potential, and for bilayer structural perturbation arising from the location of the peptide helix within the head group region of the membrane have been made based on the crystal structure of the peptide, the kinetics and concentration dependence of melittins membrane actions, together with simple ideas about the conformational properties of amphipathic helical peptides and their interactions with membranes. Physical studies of the interaction of melittin with model membranes have been useful in determining the potential of the peptide to adopt different locations, orientations and association states within membranes under different conditions, but the relationship of the results obtained to the actions of melittin in cell membranes or under the influence of a membrane potential are unclear. Experimental definition of the interaction of melittin with more complex membranes, including the erythrocyte membrane or in bilayers under the influence of a transmembrane potential, will require direct study in these membranes. Experiments employing labeled melittins for ESR, NMR or fluorescence experiments are promising both for their sensitivity (ESR and fluorescence) and the ability to focus on the peptide within the background of endogenous proteins within cell membranes. The study of melittin in model membranes has been useful for the development of methodology for determination of membrane protein structures. Despite the structural complexity of integral membrane proteins, it is interesting that in some respects their study be more straightforward, lacking as they do the elusive properties of melittin (and other structurally labile membrane peptides) which limit the possibility of defining their interaction with membranes in terms of a single conformation, location, orientation and association state within the membrane. Terwilliger, TC., Eisenberg, D. The structure of melittin. I. Structure determination and partial refinement. J. Biol. Chem., Vol. 257, Issue 11, 6010-6015, 06, 1982 Abstract Melittin is the principal protein component of bee venom and is thought to function as a lytic agent. Despite its predominantly hydrophobic character, melittin is soluble as a tetramer in aqueous salt solutions. We report here on the determination of the crystal structure of tetrameric melittin at 2.8-A resolution by the method of multiple isomorphous replacement,followed by partial atomic refinement at 2.0-A resolution. The melittin tetramer contains a noncrystallographic 2-fold axis of symmetry in addition to a crystallographic 2-fold axis, so that the four polypeptide chains have nearly identical structures. The noncrystallographic 2-fold axis was utilized twice during the determination of the structure. The multipleisomorphous replacement electron density map was averaged over this 2-fold axis before model building and strict noncrystallographic symmetry was assumed during the initial stages of atomic refinement. The 2.8-A resolution electron density map suggests that the melittin monomer contains two alpha- helical regions separated by a non-alpha-helical segment atresidues 11 and 12. Difference maps at 2.0-A resolution tend to confirm this structure and reveal that at least six solvent molecules are bound to the melittin tetramer in the crystal. The relatively high occupancies of four of these suggest that they are ions of crystallization rather than water molecules. DeGrado, W.F. et al. Kinetics and mechanism of hemolysis induced by melittin and by a synthetic melittin analogue. Biophys J. 1982 January; 37(1): 329–338. Abstract The cytotoxic peptide from honeybee venom, melittin, and a synthetic peptide analogue of it lyse human erythrocytes in a biphasic process. The kinetics of the lysis in 0.30 M sucrose, 0.01 M sodium phosphate, pH 7.30 at 4 degrees C were investigated. Our results show that melittin rapidly binds to the outer surface of the erythrocyte membrane, and the surface-bound monomers produce transient openings through which approximately 40 hemoglobin molecules can escape. Concomitantly, the melittin loses its ability to effect the process, presumably by translocation through the bilayer. The half-life for this process is 1.2 min. In a much slower process, dimers of this internalized melittin again produce transient membrane openings in a steady state. On a molar basis, the synthetic peptide analogue produces a fast process comparable to that caused by melittin, but is more efficient in the slow phase. Escape of hemoglobin and of carbonic anhydrase through the openings is diffusion controlled. These results suggest that the functional units necessary for the activity of melittin-like cytotoxic peptides are a 20 amino acid amphiphilic alpha-helix with a hydrophobic:hydrophilic ratio greater than 1 and a short segment with a high concentration of positive charges. Boman, H.G. et al. Antibacterial and antimalarial properties of peptides that are cecropin-melittin hybrids. Dept of Microbiology, University of Stockholm, Sweden. FEBS Lett. 1989 Dec 18;259(1):103-6. Abstract Solid phase synthesis was used to produce 5 hybrid peptides containing sequences from the antibacterial peptide, cecropin A, and from the bee venom toxin, melittin. Four of these chimeric peptides showed good antibacterial activity against representative Gram-negative and Gram-positive bacterial species. The best hybrid, cecropin A(1-13)-melittin(1-13) was 100-fold more active than cecropin A against Staphylococcus aureus. It was also a 10-fold better antimalarial agent than cecropin B or magainin 2. Sheep red cells were lysed by melittin at low concentrations, but not by the hybrid molecules, even at 50 times higher concentrations. Lauterwein, J. et al. Physicochemical studies of the protein-lipid interactions in melittin-containing micelles. Biochim Biophys Acta. 1979 Sep 21;556(2):244-64. Abstract Complexes of melittin with detergents and phospholipids have been characterized by fluorescence, circular dichroism, ultracentrifugation, quasi-elastic light scattering and 1H nuclear magnetic resonance (NMR) experiments. By ultracentrifugation and quasi-elastic light-scattering measurements it is shown that melittin forms stoichiometrically well-defined complexes with dodecylphosphocholine micelles consisting of one melittin molecule and approximately forty detergent molecules. Evidence from fluorescence, circular dichroism and 1H nuclear magnetic resonance experiments indicates that the conformation of melittin bound to micelles of various detergents or of diheptanoyl phosphatidylcholine is largely independent of the type of lipid and furthermore appears to be quite closely related to the conformation of melittin bound to phosphatidylcholine bilayers. 1H NMR is used to investigate the conformation of micelle-bound melittin in more detail and to compare certain aspects of the melittin conformation in the micelles with the spatial structures of monomeric and self-aggregated tetrameric melittin in aqueous solution. The experience gained with this system demonstrates that high resolution NMR of complexes of membrane proteins with micelles provides a viable method for conformational studies of membrane proteins. Ghosh, A. K. et al. Modulation of Tryptophan Environment in Membrane-Bound Melittin by Negatively Charged Phospholipids: Implications in Membrane Organization and Function. Biochemistry, 36 (47), 14291 -14305, 1997. bi971933j S0006-2960(97)01933-8 Abstract 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. Comte, M. et al. Ca2+-dependent high-affinity complex formation between calmodulin and melittin.Biochem J. 1983 January 1; 209(1): 269–272. Abstract The amphiphatic polypeptide melittin migrates as an equimolar complex with bovine brain calmodulin when monitored by gel disc electrophoresis or gel filtration in the presence of Ca2+, even in 4M-urea. The complex disassociates in the presence of EDTA and urea. The affinity is of the same order as that of calmodulin for its target enzymes, and more than 1000-fold higher than that of calmodulin for basic peptide hormones or hydrophobic drugs. The activation of brain phosphodiesterase by calmodulin is inhibited by melittin. The kinetics of inhibition suggest competition between the enzyme and melittin for calmodulin. The calmodulin-melittin interaction may constitute a model for that existing between calmodulin and its target enzymes.
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