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PubMedDate not supplied

All D-amino acid hexapeptide inhibitors of melittin's cytolytic activity derived from synthetic combinatorial libraries.

The identification of peptides that inhibit the biological functions of proteins was used as a means to explore protein/ligand interactions involved in molecular recognition processes. This approach is based on the use of synthetic combinatorial libraries (SCLs) for the rapid identification of individual peptides that block the interaction of proteins with their biological targets. Thus, each peptide mixture of an all-D-amino acid hexapeptide SCL in a positional scanning format was screened for its ability to inhibit the hemolytic activity of melittin, a model self-assembling protein. The potent inhibitory activity of the identified individual peptides suggests that protein-like complexes are able to specifically bind to peptides having an all-D configuration. These results also show that SCLs are useful for the identification of short, non-hydrolysable sequences having potential intracellular inhibitory activities.

Amino Acid Sequence↗
PubMedDate not supplied

Molecular parameters in melittin immunogenicity.

Based on immunogenicity studies, two T-cell epitopes in melittin were found to be functional in guinea pigs, one being centrally located, the other one residing in the C-terminal chain. In Balb/c mice only the central epitope was found to be active. A human T-cell clone was found by T-cell proliferation studies to employ strictly the C-terminal chain. Truncation of melittin peptides at the N-terminus did not markedly affect the capacity of guinea pigs to develop anti-IgG responses towards peptidic epitopes and towards a C-terminally attached haptenic group. Attachment of various substituents inside and outside the T-cell epitopic areas had no marked effect on antibody responses. In contrast, the substituents positioned within a T-cell epitope abolished T-cell proliferation. This difference between whole animal data and cellular in vitro responses is presently not understood.

Amino Acid Sequence↗
PubMed1999

A dual-probe fluorescence method to examine selective perturbations of membrane permeability by melittin.

A new fluorescence method has been developed to measure simultaneously and independently the release of fluorophores from two vesicle populations. Calcein and sulforhodamine B were used as a probe couple: the leakage of these probes from vesicles can be recorded independently since they can be excited simultaneously at 510 nm, and their individual fluorescence can be isolated by measuring the fluorescence signal at 525 and 590 nm, using a T-shape fluorometer. Controls show that both probes are suitable for the leakage assay based on fluorescence self-quenching, that they do not interact physically or chemically at the concentrations used in the method, and that they leak in a similar fashion from a given vesicle type. This dual-probe technique is applied to examine the specificity of the release relative to the cholesterol content of the vesicles for melittin, a toxin. This new approach shows in a straightforward manner that melittin-induced release for a given population can be modulated by the presence of vesicles with another lipid composition and this competitive release is associated with a preferential distribution of the peptide on the targeted vesicles.

Cell Membrane Permeability↗
PubMed2001

Targeted destruction of androgen-sensitive and -insensitive prostate cancer cells and xenografts through luteinizing hormone receptors.

BACKGROUND: We have prepared a conjugate of a lytic peptide (hecate) and a 15-amino acid segment of the beta-chain of LH to test the concept that this conjugate will target cancer cells expressing LH receptors. METHODS: Hecate-betaLH was added in vitro to cultures of Chinese hamster ovary (CHO) cells with and without LH receptors and to prostate cancer cells in the presence or absence of steroids, follicle-stimulating hormone (FSH), epidermal growth factor (EGF), or betaLH. PC-3 xenografts were established in male athymic nude mice and treated once a week for 3 weeks with hecate-betaLH via the lateral tail vein. RESULTS: The conjugate showed concentration-dependent toxicity for the following prostate cancer cell lines: BRF 41 T>DU145>PC-3>LNCaP, according to their LH receptor capacities. Steroid removal reduced sensitivity to the drug in a reversible manner. Hecate-betaLH reduced the tumor burden in the nude mice from 60 to 12.5 mg/g body weight. CONCLUSIONS: We conclude that the hecate-betaLH conjugate selectively kills androgen-dependent and-independent prostate cancer cells both in vivo and in vitro; its toxicity depends on the number of LH receptor sites present.

Amino Acid Sequence↗
PubMed2006

A novel role of phospholipase A2 in mediating spinal cord secondary injury.

OBJECTIVE: To investigate whether phospholipase A2 (PLA2) plays a role in the pathogenesis of spinal cord injury (SCI). METHODS: Biochemical, Western blot, histological, immunohistochemical, electron microscopic, electrophysiological, and behavior assessments were performed to investigate (1) SCI-induced PLA2 activity, expression, and cellular localization after a contusive SCI; and (2) the effects of exogenous PLA2 on spinal cord neuronal death in vitro and tissue damage, inflammation, and function in vivo. RESULTS: After SCI, both PLA2 activity and cytosolic PLA2 expression increased significantly, with cytosolic PLA2 expression being localized mainly in neurons and oligodendrocytes. Both PLA2 and melittin, an activator of endogenous PLA2, induced spinal neuronal death in vitro, which was substantially reversed by mepacrine, a PLA2 inhibitor. When PLA2 or melittin was microinjected into the normal spinal cord, the former induced confined demyelination and latter diffuse tissue necrosis. Both injections induced inflammation, oxidation, and tissue damage, resulting in corresponding electrophysiological and behavioral impairments. Importantly, the PLA2-induced demyelination was significantly reversed by mepacrine. INTERPRETATION: PLA2, increased significantly after SCI, may play a key role in mediating neuronal death and oligodendrocyte demyelination following SCI. Blocking PLA2 action may represent a novel repair strategy to reduce tissue damage and increase function after SCI.

Aldehydes↗
PubMed2004

Antiarthritic effect of bee venom: inhibition of inflammation mediator generation by suppression of NF-kappaB through interaction with the p50 subunit.

OBJECTIVE: To investigate the molecular mechanisms of the antiarthritic effects of bee venom (BV) and melittin (a major component of BV) in a murine macrophage cell line (Raw 264.7) and in synoviocytes obtained from patients with rheumatoid arthritis. METHODS: We evaluated the antiarthritic effects of BV in a rat model of carrageenan-induced acute edema in the paw and in a rat model of chronic adjuvant-induced arthritis. The inhibitory effects of BV and melittin on inflammatory gene expression were measured by Western blotting, and the generation of prostaglandin E(2) (PGE(2)) and nitric oxide (NO) and the intracellular calcium level were assayed. NF-kappaB DNA binding and transcriptional activity were determined by gel mobility shift assay or by luciferase assay. Direct binding of BV and melittin to the p50 subunit of NF-kappaB was determined with a surface plasmon resonance analyzer. RESULTS: BV (0.8 and 1.6 mug/kg) reduced the effects of carrageenan- and adjuvant-induced arthritis. This reducing effect was consistent with the inhibitory effects of BV (0.5, 1, and 5 mug/ml) and melittin (5 and 10 mug/ml) on lipopolysaccharide (LPS; 1 mug/ml)-induced expression of cyclooxygenase 2, cytosolic phospholipase A(2), inducible NO synthase, generation of PGE(2) and NO, and the intracellular calcium level. BV and melittin prevented LPS-induced transcriptional and DNA binding activity of NF-kappaB via the inhibition of IkappaB release and p50 translocation. BV (affinity [K(d)] = 4.6 x 10(-6)M) and melittin (K(d) = 1.2 x 10(-8)M) bound directly to p50. CONCLUSION: Target inactivation of NF-kappaB by directly binding to the p50 subunit is an important mechanism of the antiarthritic effects of BV.

Animals↗
PubMed2003

Conformational analysis of melittin in solution phase: vibrational circular dichroism study.

The vibrational absorption and vibrational circular dichroism (VCD) spectra of melittin in D(2)O solutions at different pH values, different salt concentrations, or different 2,2,2-trifluoroethanol (TFE) concentrations are recorded in the amide I' (1850-1600 cm(-1)) region. Two models are used to simulate this peptide in different conditions, and a coupled oscillator program is used to obtain the calculated absorption and VCD spectra. This study indicates that melittin adopts a mixed structure in D(2)O solution at low pH, low salt concentration, or low TFE concentration. With an increase in pH, salt concentration, or TFE concentration, the structure changes to alpha-helix and further increases lead to aggregation. These results demonstrate the versatility of VCD in probing the conformations of peptides under different environmental perturbations.

Circular Dichroism↗
PubMed2004

Structure and solvation of melittin in hexafluoroacetone/water.

Intermolecular (1)H[(19)F] and (1)H[(1)H] nuclear Overhauser effects have been used to explore interaction of solvent components with melittin dissolved in 50% hexafluoroacetone trihydrate (HFA)/water. Standard nuclear Overhauser effect experiments and an analysis of C(alpha)H proton chemical shifts confirm that the conformation of the peptide in this solvent is alpha-helical from residues Ala4 to Thr11 and from Leu13 to Arg24. The two helical regions are not collinear; the interhelix angle (144 +/- 20 degrees ) found in this work is near that observed in the solid state and previous NMR studies. Intermolecular NOEs arising from interactions between spins of the solvent and the solute indicate that both fluoroalcohol and water molecules are strongly enough bound to the peptide that solvent-solute complexes persist for > or =2 ns. Preferential interactions of HFA with many hydrophobic side chains of the peptide are apparent while water molecules appear to be localized near hydrophilic side chains. These results indicate that interactions of both HFA and water are qualitatively different from those present when the peptide is dissolved in 35% hexafluoro-2-propanol/water, a chemically similar helix-supporting solvent system.

Acetone↗
PubMed2006

Effect of ionic strength on folding and aggregation of the hemolytic peptide melittin in solution.

Melittin is a cationic, amphipathic, hemolytic peptide composed of 26 amino acid residues. It is intrinsically fluorescent due to the presence of a single tryptophan residue, which has been shown to be crucial for its hemolytic activity. It undergoes a structural transition from a random coil monomer to an alpha-helical tetramer at high ionic strength. Although the aggregation behavior of melittin in solution is well characterized, dynamic information associated with the aggregation of melittin is lacking. In this paper, we have monitored the effect of ionic strength on the dynamics and aggregation behavior of melittin in aqueous solution by utilizing sensitive fluorescence approaches, which include the red edge excitation shift (REES) approach. Importantly, we demonstrate that REES is sensitive to the self-association of melittin induced by ionic strength. The change in environment experienced by melittin tryptophan(s) is supported by changes in fluorescence emission maximum, polarization, and lifetime. In addition, the accessibility of the tryptophan residue was probed by fluorescence quenching experiments using acrylamide and trichloroethanol as soluble and hydrophobic quenchers, respectively. Circular dichroism studies confirm the ionic strength-induced change in the secondary structure of melittin. Taken together, these results constitute the first report showing that REES could be used as a sensitive tool to monitor the aggregation behavior of melittin in particular and other proteins and peptides in general.

Melitten↗
PubMed1990

On the multiple-minima problem in the conformational analysis of polypeptides. IV. Application of the electrostatically driven Monte Carlo method to the 20-residue membrane-bound portion of melittin.

The conformational space of the membrane-bound portion of melittin has been searched using the electrostatically driven Monte Carlo (EDMC) method with the ECEPP/2 (empirical conformational energy program for peptides) algorithm. The former methodology assumes that a polypeptide or protein molecule is driven toward the native structure by the combined action of electrostatic interactions and stochastic conformational changes associated with thermal movements. The algorithm produces a Monte Carlo search in the conformational hyperspace of the polypeptide using electrostatic predictions and a random sampling technique, combined with local minimization of the energy function, to locate low-energy conformations. As a result of 8 test calculations on the 20-residue membrane-bound portion of melittin, starting from six arbitrary and two completely random conformations, the method was able to locate a very low-energy region of the potential with a well-defined structure for the backbone. In all of the cases under study, the method found a cluster of similar low-energy conformations that agree well with the structure deduced from x-ray diffraction experiments and with one computed earlier by the build-up procedure.

Amino Acid Sequence↗
PubMed1992

Conformational studies of anionic melittin analogues: effect of peptide concentration, pH, ionic strength, and temperature--models for protein folding and halophilic proteins.

Melittin (MLT), a 26-residue cationic (net charge +5 at pH 7.2) peptide from bee venom, is well known to be a monomeric, approximately random coil; but when its charges are reduced by titration, by acetylation (net charge +2) or succinylation (net charge -2), or by screening by salt, it goes over to tetrameric alpha-helix. The conversion is promoted by raising the peptide concentration. The tetramer is held together by hydrophobic forces. We have changed the net charge to -6 by acylation with acetylcitric anhydride (a new acylating agent); this anionic derivative forms tetrameric helix at neutral pH, without salt, and at relatively low concentration, conditions under which the cationic MLT does not become helical. Thus, a high net charge is not sufficient to prevent association and helix formation. We have synthesized an anionic melittin analogue of MLT (E-MLT; net charge -4) in which all five lysine and arginine residues are replaced with glutamate, and acetyl and succinyl derivatives of E-MLT (net charges -5 and -6). All three of these are resistant to helix formation. They require much higher NaCl or NaClO4 concentration for helix formation than does MLT. Even CaCl2, MgCl2, and spermine.4HCl are less effective in helicizing E-MLT than MLT. MLT, at pH 7.2, shows increasing helix as the peptide concentration increases (8-120 microM), but E-MLT and its acyl derivatives do not. MLT and acylated MLTs in the helical tetramer show both cold- and heat-induced unfolding, with maximum stability near room temperature. At high temperature, a significant amount of residual structure remains. Heating (to 100 degrees C) monomeric MLT (i.e., MLT at low concentration) or E-MLT results in a monotonic increase in negative ellipticity. In 1.0 M NaCl, E-MLT (at sufficiently high concentration) also shows cold and hot unfolding. The results are discussed in respect to charge-charge and charge-dipole interactions, and hydrophobic effects. E-MLT is also discussed in relation to proteins of halophilic bacteria, which have higher proportions of anionic residues than do corresponding proteins of nonhalophiles.

Amino Acid Sequence↗
PubMed1993

Necessary conditions for avoiding incorrect polypeptide folds in conformational search by energy minimization.

Low energy conformations have been generated for melittin, pancreatic polypeptide, and ribonuclease S-peptide, both in the vicinity of x-ray structures by energy refinement and by an unconstrained search over the entire conformational space. Since the structural polymorphism of these medium-sized peptides in crystal and solution is moderate, comparing the calculated conformation to x-ray and nmr data provides information on local and global behavior of potential functions. Local analysis includes standardization calculations, which show that models with standard geometry can approximate good resolution x-ray data with less than 0.5 A rms deviation (RMSD). However, the atomic coordinates are shifted up to 2 A RMSD by local energy minimization, and thus 2 A is generally the smallest RMSD value one can target in a conformational search using the same energy evaluation models. The unconstrained search was performed by a buildup-type method based on dynamic programming. To accelerate the generation of structures in the conformational search, we used the ECEPP potential, defined in terms of standard polypeptide geometry. A number of low energy conformations were further refined by relaxing the assumption of standard bond lengths and bond angles through the use of the CHARMM potential, and the hydrophobic folding energies of Eisenberg and McLachlan were calculated. Each conformation is described in terms of the RMSD from the native, hydrogen-bonding structure, solvent-accessible surface area, and the ratio of surfaces corresponding to nonpolar and polar residues. The unconstrained search finds conformations that are different from the native, sometimes substantially, and in addition, have lower conformational energies than the native. The origin of deviations is different for each of the three peptides, but in all examples the refined x-ray structures have lower energies than the calculated incorrect folds when (1) the assumption of standard bond lengths and bond angles is relaxed; (2) a small and constant effective dielectric permittivity (epsilon < 10) is used; and (3) the hydrophobic folding energy is incorporated into the potential.

Amino Acid Sequence↗
PubMed1993

Permethylation alters the conformational transitions and the complexing ability of melittin: a model for methylated proteins.

Melittin exhibits a transition from random-coil monomer to helical tetramer as a function of peptide concentration [J. Bello, H. R. Bello, and E. Granados (1982) Biochemistry, Vol. 21, pp. 461-465]. When permethylated on each of the four amino groups (Gly-1 N alpha and Lys-7, 21, and 23 N epsilon) to yield trimethylammonium groups, melittin exists as a random coil and does not show any concentration-dependent conformational transition (up to 290 microM). Acylation of the amino groups of melittin with glycine or 5-aminopentanoic acid followed by permethylation increases helix formation, but to a lesser extent than for the unmethylated aminoacylmelittin derivatives. The results are discussed in relation to hydrophobicity, charge repulsions, and ion binding. Melittin, and more weakly, permethylated melittin (MLT-Me) form helical hybrids with an anionic random-coil melittin analogue (E-MLT), in which all the lysine and arginine residues of melittin were replaced by glutamate residues. The hybrid between MLT-Me and E-MLT shows a concentration-dependent increase in helicity. E-MLT, when succinylated at the N-terminal glycine (E-MLT-suc), forms a stronger hybrid with MLT-Me, possibly as a result of increased electrostatic interaction between equal but opposite charges in E-MLT-suc (net charge -6) and MLT-Me (net charge +6). The hybrids exhibit both cold- and heat-induced denaturation, similar to the phenomenon exhibited by proteins. The hybrids also exhibit significant residual structures in the temperature range of 80-100 degrees C, which may be similar to the molten globular states that have been suggested for proteins.

Amino Acid Sequence↗
PubMed1993

Helix promotion in polypeptides by polyols.

The helix content of [L-Lys(Me3)]n.ClO4 and [L-Lys(Me3)50,L-Ala50]n.ClO4 in water is markedly increased by the presence of sucrose and glycerol. For [L-Lys(Me3)]n.ClO4 the ellipticity at 222 nm changes from +2 x 10(3) deg cm2 dmole-1 in water to -44 x 10(3) in 50% glycerol. Sucrose does not promote helix formation in melittin at pH 7.2, but glycerol does. At pH 5.5 sucrose and, more so, glycerol, induce helix in melittin. Glycerol induces some helix in methylated melittin, but less than in melittin. The results are discussed in relation to excluded volume effects, delta G of transfer of peptide and hydrophobic groups from water to mixed solvents, electrostatic effects, and preferential hydration.

Amino Acid Sequence↗
PubMed1994

Alpha-helical assembly of biologically active peptides and designed helix bundle protein.

The formation of alpha-helical assembly by complexing biologically active peptides with de novo designed protein is described. The de novo designed protein described here is a cystine-linked 4-helix bundle protein constructed with 80 amino acid residues and forms a hydrophobic core region surrounded by 4 helices in an aqueous solution. The biologically active peptides, such as melittin and human growth hormone releasing factor, contain the sequences that are able to form amphiphilic helices. These peptides alone do not form the alpha-helix structure in a diluted solution with low ion strength. But on mixing with the designed helix bundle protein, the peptides are strongly bound to the protein with the induction of alpha-helical structure in the biologically active peptides. The content of induced alpha-helix is in accord with that estimated from the amphiphilic sequence. The results mean that a novel architecture composed of alpha-helices is formed. Fluorescent and temperature-scanning measurement revealed that the alpha-helical assembly is constructed with hydrophobic interaction. Also, it is shown by means of fluorescence depolarization that the assembly has a compact globular form corresponding to 1:1 complex.

Amino Acid Sequence↗
PubMed1994

Effect of succinylation on the membrane activity and conformation of a short cecropin A-melittin hybrid peptide.

A 15-residue hybrid peptide (KWKLFKKIGAVLKVL-amide) incorporating partial sequences of cecropin A and melittin causes the release of carboxyfluoresceine encapsulated in phosphatidylcholine liposomes. Succinylation of the amino groups in the N-terminus and lysine side chains inhibits the effect of this peptide on liposome permeability. Conformational analysis of the parent peptide and its succinyl derivative by CD and nmr indicates that both peptides form amphipathic alpha-helices in the presence of hexafluoro-2-propanol, but only the unmodified peptide acquires a relevant level of alpha-helical conformation in the presence of liposomes.

Amino Acid Sequence↗