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 Nα and Lys-7, 21, and 23 Nε) to yield trimethylammonium groups, melittin exists as a random coil and does not show any concentration-dependent conformational transition (up to 290 μM). 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°C, which may be similar to the molten globular states that have been suggested for proteins. © 1993 John Wiley & Sons, Inc.     

Influence of the arrangement and secondary structure of melittin peptides on the formation and stability of toroidal pores

Melittin interactions with lipid bilayers and melittin formed pores are extensively studied to understand the mechanism of the toroidal pore formation. Early experimental studies suggested that melittin peptide molecules are anchored by their positively charged residues located next to the C-terminus to only one leaflet of the lipid bilayer (asymmetric arrangement). However, the recent non-linear spectroscopic experiment suggests a symmetric arrangement of the peptides with the C-terminus of the peptides anchored to both bilayers. Therefore, we present here a computational study that compares the effect of symmetric and asymmetric arrangements of melittin peptides in the toroidal pore formation. We also investigate the role of the peptide secondary structure during the pore formation. Two sets of the symmetric and asymmetric pores are prepared, one with a helical peptide from the crystal structure and the other set with a less helical peptide. We observe a stable toroidal pore being formed only in the system with a symmetric arrangement of the less helical peptides. Based on the simulation results we propose that the symmetric arrangement of the peptides might be more favorable than the asymmetric arrangement, and that the helical secondary structure is not a prerequisite for the formation of the toroidal pore.     

Dissection of antibacterial and toxic activity of melittin: a leucine zipper motif plays a crucial role in determining its hemolytic activity but not antibacterial activity

Melittin, a naturally occurring antimicrobial peptide, exhibits strong lytic activity against both eukaryotic and prokaryotic cells. Despite a tremendous amount of work done, very little is known about the amino acid sequence, which regulates its toxic activity. With the goal of understanding the basis of toxic activity and poor cell selectivity in melittin, a leucine zipper motif has been identified. To evaluate the possible structural and functional roles of this motif, melittin and its two analogs, after substituting the heptadic leucine by alanine, were synthesized and characterized. Functional studies indicated that alanine substitution in the leucine zipper motif resulted in a drastic reduction of the hemolytic activity of melittin. However, interestingly, both the designed analogs exhibited antibacterial activity comparable to melittin. Mutations caused a significant decrease in the membrane permeability of melittin in zwitterionic but not in negatively charged lipid vesicles. Although both the analogs exhibited similar secondary structures in the presence of negatively charged lipid vesicles as melittin, they failed to adopt a significant helical structure in the presence of zwitterionic lipid vesicles. Results suggest that the substitution of heptadic leucine by alanine impaired the assembly of melittin in an aqueous environment and its localization only in zwitterionic but not in negatively charged membrane. Altogether, the results suggest the identification of a structural element in melittin, which probably plays a prominent role in regulating its toxicity but not antibacterial activity. The results indicate that cell selectivity in some antimicrobial peptides can probably be introduced by modulating their assembly in an aqueous environment.     

蜂毒溶血肽的研究进展

蜂毒溶血肽 (melittin)是蜜蜂毒液的主要组分 ,由 2 6个氨基酸残基组成 ,具有两亲性和种的特异性。它的cDNA已经被克隆 ,并以融合蛋白的形式在大肠杆菌Escherichiacoli中进行表达。蜂毒溶血肽的作用机制主要包括脂酶的激活 ,产生第二信使 ,调节一些酶及离子通道 ;Ca2 +的水平调节 ,影响骨骼肌和心肌的收缩 ;作为脂类代谢的探针。由于蜂毒溶血肽结构简单 ,且抑制病毒复制 ,因此可将其用于癌症的基因治疗及爱滋病的防治。此外 ,蜂毒溶血肽还可作为对一些作用机理进行研究的模型肽。     

Structure, location, and lipid perturbations of melittin at the membrane interface

Melittin is arguably the most widely studied amphipathic, membrane-lytic alpha-helical peptide. Although several lines of evidence suggest an interfacial membrane location at low concentrations, melittin's exact position and depth of penetration into the hydrocarbon core are unknown. Furthermore, the structural basis for its lytic action remains largely a matter of conjecture. Using a novel x-ray absolute-scale refinement method, we have now determined the location, orientation, and likely conformation of monomeric melittin in oriented phosphocholine lipid multilayers. Its helical axis is aligned parallel to the bilayer plane at the depth of the glycerol groups, but its average conformation differs from the crystallographic structure. As observed earlier for another amphipathic alpha-helical peptide, the lipid perturbations induced by melittin are remarkably modest. Small bilayer perturbations thus appear to be a general feature of amphipathic helices at low concentrations. In contrast, a dimeric form of melittin causes larger structural perturbations under otherwise identical conditions. These results provide direct structural evidence that self-association of amphipathic helices may be the crucial initial step toward membrane lysis.     

Attenuated total reflectance Fourier transform infrared studies of the interaction of melittin, two fragments of melittin, and delta-hemolysin with phosphatidylcholines

Attenuated total reflectance Fourier transform infrared spectroscopy (ATR FT-IR) has been used to monitor alterations in phospholipid organization in thin layers of 1,2-dipalmitoylphosphatidylcholine (DPPC) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), induced by the membrane lytic peptide melittin, its fragments 1-15 (hydrophobic fragment) and 16-26 (hydrophilic fragment), and delta-hemolysin. In addition, the secondary structures of the peptides and the orientation of helical fragments were determined with respect to the bilayer. The insertion of melittin into POPC caused large perturbations in the order and increased rates of motion of the acyl chains, as monitored by the frequency and half-width of the symmetric CH2 stretching vibration near 2850 cm-1, as well as by the ATR dichroic ratio for this mode. Changes in DPPC organization were less and were consistent with peptide-induced static disordering (gauche rotamer formation) in the acyl chains. Melittin adopted primarily an alpha-helical secondary structure, although varying small proportions of beta and/or aggregated forms were noted. The helical segments were preferentially oriented perpendicular to the bilayer plane. Several modes of melittin/lipid interaction were considered in an attempt to semiquantitatively understand the observed dichroic ratios. By considering the peptide as a bent rigid rod, a plausible model for its lytic properties has been developed. The hydrophilic fragment in DPPC showed a secondary structure with little alpha-helix present. As judged by its effect on phospholipid acyl chain organizational parameters, the fragment did not penetrate the bilayer substantially. The hydrophobic fragment in DPPC gave amide I spectral patterns consistent with a mixture of predominantly beta-antiparallel pleated sheet with a smaller fraction of alpha-helix.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluoroalcohols as structure modifiers in peptides and proteins: hexafluoroacetone hydrate stabilizes a helical conformation of melittin at low pH.

The effect of hexafluoroacetone hydrate (HFA) on the structure of the honey bee venom peptide melittin has been investigated. In aqueous solution at low pH melittin is predominantly unstructured. Addition of HFA at pH approximately 2.0 induces a structural transition from the unstructured state to a predominantly helical conformation as suggested by intense diagnostic far UV CD bands. The structural transition is highly cooperative and complete at 3.6 M (50% v/v) HFA. A similar structural transition is also observed in 2,2,2 trifluoroethanol which is complete only at a cosolvent concentration of approximately 8 M. Temperature dependent CD experiments support a 'cold denaturation' of melittin at low concentrations of HFA, suggesting that selective solvation of peptide by HFA is mediated by hydrophobic interactions. NMR studies in 3.6 M HFA establish a well-defined helical structure of melittin at low pH, as suggested by the presence of strong NH/NHi+1 NOEs throughout the sequence, along with many medium range helical NOEs. Structure calculations using NOE-driven distance constraints reveal a well-ordered helical fold with a relatively flexible segment around residues T10-G11-T12. The helical structure of melittin obtained at 3.6 M HFA at low pH is similar to those determined in methanolic solution and perdeuterated dodecylphosphocholine micelles. HFA as a cosolvent facilitates helix formation even in the highly charged C-terminal segment.     

Structural aspects of the interaction of bee venom peptide melittin with phospholipids

In aqueous solution, melittin structure, investigated by CD and ¹H-nmr, depends on pH and ionic composition, which also regulate the aggregation state of the peptide. When interacting with phospholipids, however, melittin exhibits a right-handed helical conformation without any evidence of oligomeric association. The overall bilayer structure of phospholipid aqueous dispersions is also maintained in the presence of melittin, although the permeability to aqueous solutes is considerably increased. Small-angle neutron-diffraction analysis of oriented multilayers confirms the existence of a lamellar profile, despite the presence of the peptide throughout each bilayer and exchangeable protons almost reaching the center of the hydrophobic alkyl chains region.     

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.     

The interaction of bee melittin with lipid bilayer membranes

The influence of melittin and the related 8-26 peptide on the stability and electrical properties of bilayer lipid membranes is reported. Melittin, unlike the 8-26 peptide, has a dramatic influence on lipid membranes, causing rupture at dilute concentrations. The circular dichroism of melittin demonstrated that under physiological conditions, in water, melittin is in extended conformation, which is enhanced in aqueous ethanol. However in 'membrane-like' conditions it is essentially alpha-helical. Secondary structure predictions were used to locate possible alpha-helical nucleation centres and a model of melittin was built according to these predictions. It is postulated that melittin causes a wedge effect in membranes.     

Orientation of melittin in phospholipid bilayers. A polarized attenuated total reflection infrared study.

The helical order parameter of the 26-residue amphiphilic bee venom peptide melittin was measured by polarized attenuated total reflection infrared spectroscopy (ATR-IR) in dry phospholipid multibilayers (MBLs) and when bound to single supported planar bilayers (SPBs) under D2O. Melittin adopted an alpha-helical conformation in MBLs of dipalmitoyl-phosphatidylcholine (DPPC), 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), a 4:1 mixture of POPC and 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG), and when bound to SPBs of POPC:POPG (4:1). The order parameter of the alpha-helix in the bilayers depended mainly on the type of membrane preparation, and only little on the phospholipid composition of the bilayers. On hydrated SPBs, the helical order parameter was negative, indicating that the alpha-helix long axis of melittin was preferentially oriented parallel to the plane of the supported membrane. However, in dry MBLs, the helical order parameter was positive, indicating that the alpha-helix of melittin was preferentially oriented parallel to the phospholipid fatty acyl chains. It is concluded that the orientation of melittin in membranes depends on the degree of hydration of the model membranes rather than on the technique which is used for its determination. ATR-IR spectroscopy of polypeptides in or associated with supported planar membranes in D2O may become a useful tool for the determination of their orientation in and on membranes.     

Antibacterial peptides designed as analogs or hybrids of cecropins and melittin.

Eight new analogs of cecropin A, two new analogs of melittin and 30 hybrid peptides containing sequences from cecropins and melittin have been synthesized. The lengths of the peptides have varied from 37 residues (the length of cecropin A) to 18 residues. The peptides have been assayed for lysis of sheep red blood cells and for antibacterial activity against two Gram negative and three Gram positive bacteria. The best analogs of cecropin A maintained the anti-Escherichia coli activity of the parental peptide, and were not lytic for red blood cells. Melittin and its replacement analogs were all lytic for red blood cells, but an analog with transposed segments was not. Several of the hybrid peptides were found to be both non-hemolytic and highly active against all test bacteria. The data were used to define the structural requirements for antibacterial activity.     

Biological activities of C-terminal 15-residue synthetic fragment of melittin: design of an analog with improved antibacterial activity

Melittin, the 26-residue predominant toxic peptide from bee venom, exhibits potent antibacterial activity in addition to its hemolytic activity. The synthetic peptide of 15 residues corresponding to its C-terminal end (MCF), which encompasses its most amphiphilic segment, is now being shown to possess antibacterial activity about 5-7 times less compared to that of melittin. MCF, however, is 300 times less hemolytic. An analog of MCF, MCFA, in which two cationic residues have been transpositioned to the N-terminal region from the C-terminal region, exhibits antibacterial activity comparable to that of melittin, but is only marginally more hemolytic than MCF. The biophysical properties of the peptides, like folding and aggregation, correlate well with their biological properties.     

Conformational analysis of the 20-residue membrane-bound portion of melittin by conformational space annealing

The conformational space of the 20-residue membrane-bound portion of melittin has been investigated extensively with the conformational space annealing (CSA) method and the ECEPP/3 (Empirical Conformational Energy Program for Peptides) algorithm. Starting from random conformations, the CSA method finds that there are at least five different classes of conformations, within 4 kcal/mol, which have distinct backbone structures. We find that the lowest energy conformation of this peptide from previous investigations is not the global minimum-energy conformation (GMEC); but it belongs to the second lowest energy class of the five classes found here. In four independent runs, one conformation is found repeatedly as the lowest energy conformation of the peptide (two of the four lowest energy conformations are identical; the other two have essentially identical backbone conformations but slightly different side-chain conformations). We propose this conformation, whose energy is lower than that found previously by 1.9 kcal/mol, as the GMEC of the ECEPP/3 force field. The structure of the proposed GMEC is less helical and more compact than the previous one. It appears that the CSA method can find several classes of conformations of a 20-residue peptide starting from random conformations utilizing only its amino acid sequence information. The proposed GMEC has also been found with a modified electrostatically driven Monte Carlo method [D. R. Ripoll, A. Liwo, and H.A. Scheraga (1998) “New Developments of the Electrostatically Driven Monte Carlo Method: Test on the Membrane-Bound Portion of Melittin,” Biopolymers, Vol. 46, pp. 117–126]. © 1998 John Wiley & Sons, Inc. Biopoly 46: 103–115, 1998     

High pressure FTIR study of interaction of melittin with dimyristoylphosphatidyl glycerol bilayers.

Infrared spectra of hydrated dimyristoylphosphatidyl glycerol (DMPG) and of aqueous dispersions of melittin and DMPG at peptide:lipid molar ratios of 1:10 and 1:4 were recorded as a function of pressure from atmospheric to 22 kbar. Spectral features corresponding to vibrations of the amide linkages in melittin and to various functional groups in DMPG (carbonyl, methlylene, phosphate) were monitored in order to investigate the structure and dynamics of melittin:DMPG dispersions. Melittin was found to cause conformational and orientational disordering of the acyl chains in DMPG bilayers. The magnitude of these disorders was higher for higher concentration of melittin in DMPG. The higher concentration of melittin was also found to disrupt the DMPG bilayers through interactions with the lipid head groups. Such disruption may be related to some of the biological properties of melittin.     

Mechanism of the conformational transition of melittin.

It is known that, while melittin at micromolar concentrations is unfolded under conditions of low ionic strength at neutral pH, it adopts a tetrameric alpha-helical structure under conditions of high ionic strength, at alkaline pH, or at high peptide concentrations. To understand the mechanism of the conformational transition of melittin, we examined in detail the conformation of melittin under various conditions by far-UV circular dichroism at 20 degrees C. We found that the helical conformation is also stabilized by strong acids such as perchloric acid. The effects of various acids varied largely and were similar to those of the corresponding salts, indicating that the anions are responsible for the salt- or acid-induced transitions. The order of effectiveness of various monovalent anions was consistent with the electroselectivity series of anions toward anion-exchange resins, indicating that the anion binding is responsible for the salt- or acid-induced transitions. From the NaCl-, HCl-, and alkaline pH-induced conformational transitions, we constructed a phase diagram of the anion- and pH-dependent conformational transition. The phase diagram was similar in shape to that of acid-denatured apomyoglobin [Goto, Y., & Fink, A.L. (1990) J. Mol. Biol. 214, 803-805] or that of the amphiphilic Lys, Leu model polypeptide [Goto, Y., & Aimoto, S. (1991) J. Mol. Biol. 218, 387-396], suggesting a common mechanism of the conformational transition. The anion-, pH-, and peptide concentration-dependent conformational transition of melittin was explained on the basis of an equation in which the conformational transition is linked to proton and anion binding to the titratable groups.     

Investigation of the interaction between melittin and dipalmitoylphosphatidylglycerol bilayers by vibrational spectroscopy.

Melittin is shown to affect the structure of the charged phospholipid dipalmitoylphosphatidylglycerol (DPPG). In the gel phase, the presence of melittin leads to (i) an increased lipid interchain vibrational coupling, (ii) a shift of the rectangular to hexagonal lipid packing transition toward low temperatures, (iii) a very small conformational disordering effect, (iv) a decrease of the polarity or hydrogen bonding capability of the lipid ester group surrounding, (v) an important decrease of the water content in the complexes where the remaining water has a more disordered structure than bulk water, and (vi) an interlamellar repeat distance of 79 A. All these observations are rationalized by the following model: adjacent bilayers of DPPG are bridged by tetramers of melittin through electrostatic interactions inducing surface charge neutralization and partial dehydration of the complexes. Melittin also affects the thermotropic behavior of DPPG. When a small amount of the toxin is present, its affinity for charged lipids is such that a phase separation occurs, the domains being stable enough to have their own gel to liquid-crystalline phase transition. In the fluid state, a deeper penetration into the lipid matrix is proposed based on the downshift of the phase transition and the low vibrational interchain coupling. This study brings out general features of cationic species/anionic lipid complexes. The charge neutralization leads to stronger interchain coupling, and electrostatic bridging of adjacent bilayers seems to be common. The hydrophobicity of the peptide is a key factor in the modulation of the gel to liquid-crystalline phase transition and in its insertion in the fluid lipid matrix.     

蜂毒溶血肽对鸡红细胞及膜的生化作用

本文采用荧光分光光度、薄层层析、原子吸收、荧光显微图像等多种生化技术,系统研究了蜂毒肽作用于鸡红细胞及膜的生化机理。结果表明：蜂毒肽影响红细胞膜上及胞内两种酶的功能。它抑制膜Na⁺-K⁺-ATPase活性,导致胞内外离子转运异常,K⁺浓度失衡;它也抑制细胞内葡萄糖-6-磷酸脱氢酶活性,其正电区域干扰胞内带负电小分子的作用,影响红细胞正常代谢。蜂毒肽干扰膜中阴离子通道的转运功能,使细胞渗透压改变,引起膨胀而溶血。蜂毒肽对有核红细胞核内DNA没有作用,与其他抗微生物多肽作用的靶向不同。据此认为,抗菌蛋白类抗生素对细菌作用的生化机理与传统抗生素不同,这是细菌对其不易产生耐药性的重要原因。     

Murine IgE and IgG responses to melittin and its analogs.

Melittin, a bee-venom peptide of 26 amino acids, induces IgE and IgG responses in man and animals. The antibody response was shown previously to be specific primarily for the C-terminal 6 residues and its T cell epitope in H-2d restricted mice was shown to be in residue 11-19 of melittin. To study the relationship of peptide structure and immunogenicity in mice, we have prepared a series of melittin analogs varied in length and composition at the C-terminus. Immunogenicity of the analogs for IgG and IgE responses was found to correlate with two factors: a peptide length of more than 24 residues and the presence of a hydrophilic C-terminal region preferably with two to four cationic groups. These factors result in the ability of peptide to bind to cell membranes. Analogs that possess these features are good immunogens whereas those lacking any of these features are weak immunogens.