Melittin-phospholipid interaction Evidence for melittin aggregation

The fluorescence spectra of the single tryptophan residue of melittin in 0.15 M potassium phosphate solution and when bound to egg phosphatidylcholine bilayer liposomes practically coincide and exhibit a large blue shift relative to that in aqueous solution. The rotational correlation time of the protein increases substantially in the salt solution relative to that in aqueous solution. It is inferred that the protein binds to the phospholipid in an aggregated form, most probably as a tetramer.     

Conformation and aggregation of melittin: effect of pH and concentration of sodium dodecyl sulfate

The conformation of melittin, a surface-active polypeptide, in solution was studied by CD spectra between 190 and 240 nm. The molecule was essentially unordered (possibly with a trace of helix) in water without salt at neutral pH. Upon deprotonation of four of the six cationic groups at pH 12 the polypeptide became partially helical (about 35%). The addition of NaDodSO₄ to an aqueous melittin solution first caused the solution to become turbid but it became clear again in excess surfactant solution. The conformational changes depended on the molar NaDodSO₄/melittin ratio, R. With R from 2.34 to 23.4, the melittin solution was turbid and the polypeptide conformation was probably a mixture of α-helix and β-sheets. This was supported by the ir spectrum of the turbid solution, which indicated the presence of both conformations. With R = 46.8 or 468 (1 or 10 mM NaDodSO₄) the polypeptide conformation was characteristic of an α-helix, about 70–80% of the molecule, regardless of whether the surfactant was above or below its critical micelle concentration. This compared well with the x-ray results of 92% helix in crystals. The lower helicity of melittin in NaDodSO₄ solution might be attributed to the end effects that destabilize the first and last turn of an helix at its N- and C-terminus, respectively.     

The effect of melittin on proliferation and death of thymocytes

The effect of melittin, an activator of phospholipase A₂, on proliferation and death of rat thymocytes in a broad concentration range was studied. Cell proliferation was estimated by the accumulation of colchicin metaphases, necrotic death was determined from lysis and staining of cells with trypan blue, and apoptosis was assessed from the type of DNA fragmentation, the amount of fragmented DNA, and the percentage of cells with subdiploid DNA. It was shown that low melittin concentrations (below 5 μg/ml) stimulate thymocyte proliferation. At high melittin concentrations, thymocytes die by the primary necrosis type. Throughout the concentration range studied, melittin does not produce apoptosis in thymocytes. Conversely, high melittin concentrations even inhibit thymocyte apoptosis in the control and after irradiation. An inhibitor of RNA synthesis actinomycin D does not affect thymocyte death in the presence of melittin. It is concluded that the activation of phospholipase A₂ can induce necrosis but not apoptosis and thus is not a necessary step in the signaling cascade that initiates apoptosis in thymocytes.     

The effect of anions on the hemolytic activity of melittin

Anions causing the melittin aggregation in the solution are shown to slow down the lytic process not preventing it completely. The equalization of the oncotic gradient through the erythrocytic membrane by the addition of polyethylene glycols of different molecular weight into the extracellular medium made it possible to establish the colloid-osmotic mechanism of hemolysis and to estimate the diameter of melittin pores. The diameter depends on the polypeptide concentration and makes up 20-30 A with its content of (6 divided by 12).10(-6) M.     

The aggregation state of spin-labeled melittin in solution and bound to phospholipid membranes: evidence that membrane-bound melittin is monomeric

Spin-labeled derivatives of the bee venom protein, melittin, were obtained by reacting on the average one of the four amino groups of the protein with succinimidyl-2,2,5,5-tetramethyl-3-pyrroline-1-oxyl-3-carboxylate. All 16 statistically possible reaction products with 0, 1, 2, 3 or 4 spin labels per protein were then separated in a single pass with reversed phase high performance liquid chromatography. With the help of trypsin digestion and diode array detection it was possible to assign the primary structure of all 16 eluting fractions. All fractions with only one spin label per protein were purified for electron paramagnetic resonance measurements. The labeling sites cover different regions of the protein: one is at the N-terminus, one at lysine-7, and two are near the C-terminus at lysine-21 and lysine-23, respectively. This set of specifically labeled melittins was used to study the structure and dynamics of melittin in aqueous solutions and when bound to neutral or negatively charged membranes. In aqueous solution a reduction in rotational correlation time and appearance of spin-spin interaction was observed during salt-induced transition from a random coil monomer to a mostly alpha-helical tetramer. Membrane binding to phospholipid bilayers in low or high ionic strength was reflected only in a further decrease in mobility. The absence of any spin interaction in the membrane-bound state suggests that melittin is monomeric under these conditions. All derivatives were able to detect these structural changes, but melittin labeled at the N-terminal amino group was especially valuable. Because of postulated intramolecular hydrogen bonding, this label reflects directly the motion of the entire protein or tetramer. Broadening experiments with chromium oxalate show that all labeled sites are at least partially exposed to the aqueous phase when melittin is bound to membranes. This suggests that an alpha-helical melittin monomer binds to membranes with its axis parallel to the membrane surface.     

Onset of DNA aggregation in presence of monovalent and multivalent counterions

We address theoretically aggregation of DNA segments by multivalent polyamines such as spermine and spermidine. In experiments, the aggregation occurs above a certain threshold concentration of multivalent ions. We demonstrate that the dependence of this threshold on the concentration of DNA has a simple form. When the DNA concentration c(DNA) is smaller than the monovalent salt concentration, the threshold multivalent ion concentration depends linearly on c(DNA), having the form alphac(DNA) + beta. The coefficients alpha and beta are related to the density profile of multivalent counterions around isolated DNA chains, at the onset of their aggregation. This analysis agrees extremely well with recent detailed measurements on DNA aggregation in the presence of spermine. From the fit to the experimental data, the number of condensed multivalent counterions per DNA chain can be deduced. A few other conclusions can then be reached: 1), the number of condensed spermine ions at the onset of aggregation decreases with the addition of monovalent salt; 2), the Poisson-Boltzmann theory overestimates the number of condensed multivalent ions at high monovalent salt concentrations; and 3), our analysis of the data indicates that the DNA charge is not overcompensated by spermine at the onset of aggregation.     

The effect of divalent cations on aggregation of Dictyostelium discoideum.

Following the initiation of development, amoebae of Dictyostelium discoideum aggregate chemotactically toward cyclic AMP (cAMP). Adenyl cyclase, cAMP phosphodiesterase, and cAMP binding sites all increase 20--40 fold during the first few hours of development. It has been shown that addition of 1 mM EDTA and 5 mM MgCl2 accelerates the aggregation process. Likewise, the calcium ionophore, A23187, leads to precocious aggregation while 4 X 10(-5) M progesterone considerably delays it. These treatments have now been shown to result in increased accumulation of adenyl cyclase in the case of EDTA and Mg2+ or the ionophore and greatly decreased accumulation in the case of the steroid. Treatment with EDTA and Mg2+ or the ionophore has been shown not only to accelerate aggregation in wild-type amoebae but to overcome complete blocks to aggregation in certain mutant strains. We have found that addition of Mn2+ will also permit aggregation of mutant cells otherwise unable to aggregate. This divalent ion, unlike EDTA and Mg2+ or the ionophore, was shown to directly stimulate adenyl cyclase. Calcium ions were also found to affect the enzyme such that at Ca2+ concentrations found within the cells the great majority of the activity is inhibited. Manganese ions can overcome the inhibition by Ca2+. These findings show that conditions which stimulate aggregation result in increased activity of adenyl cyclase either by increased accumulation of the enzyme or by increased activity of the available enzyme, and support the proposed central role of adenyl cyclase in aggregation.     

The structure of melittin in membranes.

The conformation of the polypeptide melittin in lipid membranes as determined by Raman spectroscopy is a bent alpha-helix formed by the mainly hydrophobic residues 1-21, and a nonhelical COOH-terminal segment of the hydrophilic residues 22-26. Fluorescence quenching experiments on residue Trp19 reveal that all COOH-termini are located on that side of a vesicular membrane to which melittin was added. By means of fluorescence energy transfer between unmodified and modified Trp19 residues, melittin is shown to aggregate in membranes predominantly in the form of tetramers. These and previous results on the location and orientation of melittin permit the development of a model for the structure of melittin tetramers in membranes. The hydrophilic sides of four bilayer-spanning helices face each other to form a hydrophilic pore through the membrane.     

The actions of melittin on membranes

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.     

Comparison of p25 presequence peptide and melittin. Red blood cell haemolysis and band 3 aggregation.

The 25 residue presequence (p25) for subunit IV of yeast cytochrome oxidase had previously been shown to possess structural and behavioural characteristics in common with the bee venom polypeptide, melittin. The present study extends the results of leakage experiments on model-membrane systems to the haemolysis of human erythrocytes, which both peptides are shown to accomplish in a manner sensitive to membrane potential. In addition, the laser flash-induced transient dichroism technique for measuring protein rotational diffusion has been used to show that both peptides aggregate band 3, the major integral membrane protein of the erythrocyte. Aggregation cannot be reversed by high ionic strength; this serves to differentiate these peptides from other positively charged species such as polylysine that aggregate band 3 at low ionic strength. These results suggest that aggregation of membrane proteins may possibly prove to be a feature of the interaction of p25 signal peptide with mitochondrial membranes.     

The effect of temperature on histone GRK aggregation

The aggregation of histone GRK, induced by the addition of salt, is found to be highly dependent on temperature. The rate of this aggregation decreases with decreasing temperature and can be essentially stopped at low temperatures.     

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.     

The structure of melittin in lipid bilayer membranes.

0.15 M inorganic phosphate dramatically increased the alpha-helix content of melittin in aqueous solution. When melittin interacted with egg yolk phosphatidylcholine liposomes in the absence of inorganic phosphate, it was converted to an alpha-helix rich form, as postulated by Dawson et al. (Dawson, C.R., Drake, A.F. Helliwell, J. and Hider, R.C. (1978) Biochim. Biophys. Acta 510, 75--86).     

Electron microscopic observation of the aggregation of membrane proteins in human erythrocyte by melittin

Human erythrocytes and erythrocyte ghost membranes were treated with native and modified melittins, up to 250 nmol/mg membrane protein. Native melittin induced aggregation of intramembranous particles (IMPs, observed by freeze-fracture electron microscopy), and created large, smooth bilayer areas devoid of IMP. The degree of IMP aggregation increased with increasing concentration of melittin, corresponding to hemolysis results. Membrane ghosts were slightly more susceptible to IMP aggregation than membranes on intact cells. The potency of inducing IMP aggregation was ranked in the order of: native melittin greater than acetylated melittin greater than succinylated melittin = 0. The concentration range of melittin which caused IMP aggregation corresponded to that which caused the immobilization of band 3 proteins as detected by measurement of rotational mobility by transient dichroism (Dufton et al. (1984) Eur. J. Biophys. 11, 17-24). Because both IMP aggregation and band 3 protein immobilization decreased with decreasing positive charge of the melittins used, the nature of melittin-protein interaction is likely to be at least in part electrostatic in the case of human erythrocyte membranes. Possible roles of IMP aggregation and the consequent creation of 'exposed' bilayer areas in the cytotoxic reaction of melittins are discussed.     

Kerr effect of charged dipolar macromolecules without condensed counterions in conducting solution.

The theoretical treatment of the Kerr constant of rigid, dipolar, conducting ellipsoidal macromolecules of O'Konski and Krause (1970. J. Phys. Chem. 74:3243) has been extended to very low ionic strength solutions for charged macromolecules. The O'Konski and Krause theoretical treatment postulated a surface conductivity directly on the surface of each macromolecule. For charged macromolecules, this surface conductivity was generally assumed to be caused by movement of condensed counterions on the macromolecules. In the present work, it has been assumed that, at very low ionic strength, the average counterion is at the Debye characteristic distance from the surface of each charged macromolecule and contributes to surface conductivity at that distance, with no additional surface conductivity on the true surface of the macromolecule. Essentially, these considerations change the calculated interaction energy of the macromolecule with an externally applied electric field via a change in both the internal field components and in the reaction field of the macromolecular dipole. The new interaction energy is used to calculate the orientation distribution function of the macromolecules in solution and this distribution function can, in principle, be used to calculate the steady state electric linear or circular dichroism, electric light scattering, anisotropy of conductivity, etc., using the appropriate theoretical treatment for each of these quantities.     

Protein aggregation. The effect of deuterium oxide on large protein aggregates of C-phycocyanin

The amount of 11s aggregate in phycocyanin, normally stimulated by hydrophobic forces, is dramatically increased by the presence of deuterium oxide. Proteins in which hydrophobic forces are not proposed as a mechanism for aggregation are unaffected by deuterium oxide. These observations are consistent with the lower critical micelle concentration reported for ionic detergents in deuterium oxide. Phycocyanin samples containing a majority of material sedimenting faster than 11s were also investigated in the presence of deuterium oxide with the following findings: the most rapidly sedimenting species in water buffer is 24s; in deuterium oxide more than 10% of the protein sediments at 67s and substantial amounts of other species with sedimentation coefficients larger than 24s are present. These large quantities of species sedimenting faster than 24s are found in deuterium oxide buffers from pD5.5 to 7.0. Sucrose-density-gradient studies in deuterium oxide at pD6.0 confirm the presence of large amounts of more rapidly sedimenting species. Spectrophotometric studies on fractions from the sucrose-density-gradient experiments indicate with the presence of higher aggregates a red shift of the visible-absorption maximum and an enhancement of the E(620)/E(280) ratio. Fluorescence-emission studies show a greater relative fluorescence efficiency for these higher aggregates and are consistent with the suggested enhancement of higher aggregates in deuterium oxide. The existence of phycocyanin aggregates of such a large size is suggested to be of importance in vivo, with phycocyanin playing a role as a structural protein.