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.     

Ultrasonic study of melittin effects on phospholipid model membranes.

Low dose effects of melittin on dilute suspensions of dipalmitoylphosphatidylcholine multilamellar vesicles are investigated by studying the acoustic properties of the system. The temperature dependencies of sound velocity and absorption have been measured at 7.2 MHz in the temperature range of 20-55 degrees C, for different peptide/lipid molar ratios, R. The most pronounced effects were observed at R = 5 x 10(-3), in the vicinity of the pretransition, with a simultaneous increase in sound absorption and velocity. This indicates that melittin affects the polar head group region of the bilayer resulting in a decrease in mobility of the polar head groups. A nonmonotonic dependence of the main transition temperature, with an initial decrease followed by an increase as melittin is added, is interpreted as a consequence of a destabilizing action of the interfaces between mellitin-affected clusters and the unaffected phase.     

Conformation and orientation of penetratin in phospholipid membranes.

The binding, conformation and orientation of a hydrophilic vector peptide penetratin in lipid membranes and its state of self-association in solution were examined using circular dichroism (CD), analytical ultracentrifugation and fluorescence spectroscopy. In aqueous solution, penetratin exhibited a low helicity and sedimented as a monomer in the concentration range approximately 50-500 microM. The partitioning of penetratin into phospholipid vesicles was determined using tryptophan fluorescence anisotropy titrations. The apparent penetratin affinity for 20% phosphatidylserine/80% egg phosphatidylcholine vesicles was inversely related to the total peptide concentration implying repulsive peptide-peptide interactions on the lipid surface. The circular dichroism spectra of the peptide when bound to unaligned 20% phosphatidylserine/80% egg phosphatidylcholine vesicles and aligned hydrated phospholipid multilayers were attributed to the presence of both alpha-helical and beta-turn structures. The orientation of the secondary structural elements was determined using oriented circular dichroism spectroscopy. From the known circular dichroism tensor components of the alpha-helix, it can be concluded that the orientation of the helical structures is predominantly perpendicular to the membrane surface, while that of the beta-type carbonyls is parallel to the membrane surface. On the basis of our observations, we propose a novel model for penetratin translocation.     

Hydrogen bonding in helical polypeptides from molecular dynamics simulations and amide hydrogen exchange analysis: alamethicin and melittin in methanol.

Molecular dynamics simulations of ion channel peptides alamethicin and melittin, solvated in methanol at 27 degrees C, were run with either regular alpha-helical starting structures (alamethicin, 1 ns; melittin 500 ps either with or without chloride counterions), or with the x-ray crystal coordinates of alamethicin as a starting structure (1 ns). The hydrogen bond patterns and stabilities were characterized by analysis of the dynamics trajectories with specified hydrogen bond angle and distance criteria, and were compared with hydrogen bond patterns and stabilities previously determined from high-resolution NMR structural analysis and amide hydrogen exchange measurements in methanol. The two alamethicin simulations rapidly converged to a persistent hydrogen bond pattern with a high level of 3(10) hydrogen bonding involving the amide NH's of residues 3, 4, 9, 15, and 18. The 3(10) hydrogen bonds stabilizing amide NH's of residues C-terminal to P2 and P14 were previously proposed to explain their high amide exchange stabilities. The absence, or low levels of 3(10) hydrogen bonds at the N-terminus or for A15 NH, respectively, in the melittin simulations, is also consistent with interpretations from amide exchange analysis. Perturbation of helical hydrogen bonding in the residues before P14 (Aib10-P14, alamethicin; T11-P14, melittin) was characterized in both peptides by variable hydrogen bond patterns that included pi and gamma hydrogen bonds. The general agreement in hydrogen bond patterns determined in the simulations and from spectroscopic analysis indicates that with suitable conditions (including solvent composition and counterions where required), local hydrogen-bonded secondary structure in helical peptides may be predicted from dynamics simulations from alpha-helical starting structures. Each peptide, particularly alamethicin, underwent some large amplitude structural fluctuations in which several hydrogen bonds were cooperatively broken. The recovery of the persistent hydrogen bonding patterns after these fluctuations demonstrates the stability of intramolecular hydrogen-bonded secondary structure in methanol (consistent with spectroscopic observations), and is promising for simulations on extended timescales to characterize the nature of the backbone fluctuations that underlie amide exchange from isolated helical polypeptides.     

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).     

Lipid specific penetration of melittin into phospholipid model membranes

The relative depth of penetration of melittin into egg phosphatidylcholine and bovine heart cardiolipin model membranes was investigated using fluorescence spectroscopy techniques. The tryptophan intrinsic fluorescence shift suggests a more hydrophobic surrounding of this residue in cardiolipin, while the accessibility for charged and uncharged aqueous quenchers is decreased in the cardiolipin system when compared with the phosphatidylcholine-bound situation. A lipid incorporated hydrophobic, collisional quencher and a resonance energy transfer acceptor on the other hand are more effective in quenching the tryptophan fluorescence of cardiolipin bound melittin. The combination of these results is interpreted as prove of a deeper positioning of the tryptophan containing part of the peptide molecule in the cardiolipin system in comparison with the situation in phosphatidylcholine. Models that take this difference into account are presented, which try to explain the opposite effect of melittin binding to the two lipid systems with respect to supramolecular structure, as reported in the preceding article (Batenburg, A.M., Hibbeln, J.C.L., Verkleij, A.J. and De Kruijff, B. (1987) Biochim. Biophys. Acta 903, 142-154).     

Kinetics of phospholipid exchange between bilayer membranes.

In an accompanying publication by Duckwitz-Peterlein, Eilenberger and Overath ((1977) Biochim. Biophys. Acta 469,311--325) it is shown that the exchange of lipid molecules between negatively charged vesicles consisting of total phospholipid extracts from Escherichia coli occurs by the transfer of single lipid monomers or small micelles through the water. Here a kinetic interpretation is presented in terms of a rate constant, k--, for the escape of lipid molecules from the vesicle bilayer into the water. The evaluated rate constants are kP- = (0.86 +/- 0.05) - 10(-5) S-1 and ke- = (1.09 +/- 0.13) - 10(-6) s-1 for phospholipid molecules with trans-delta 9-hexadecenoate and trans-delta 9-octadecenoate, respectively, as the predominant acyl chain component. The rate constants are discussed in terms of the acyl chain and polar head group composition of the lipids.     

The structure of melittin in lipid bilayer membranes

0.15 M inorganic phosphate dramatically increased the α-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 α-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).     

Location and orientation of Triclosan in phospholipid model membranes

Triclosan is a hydrophobic antibacterial agent used in dermatological preparations and oral hygiene products. Although the molecular mechanism of action of this molecule has been attributed to inhibition of fatty acid biosynthesis, earlier work in our laboratories strongly suggested that the antibacterial action of Triclosan is mediated at least partly through its membranotropic effects. In order to assess its location in phospholipid membranes, high-resolution magic-angle spinning natural abundance ¹³C NMR of Triclosan embedded within egg yolk lecithin model membranes has been used to obtain ¹³C spin–lattice relaxation times for both Triclosan and lecithin carbon atoms in the presence of Gd³⁺ ions. The results indicate that Triclosan is localized in the upper region of the phospholipid membrane, its hydroxyl group residing in the vicinity of the C=O/C2 carbon atoms of the acyl chain of the phospholipid, and the rest of the Triclosan molecule is probably aligned in a nearly perpendicular orientation with respect to the phospholipid molecule. Intercalation of Triclosan into bacterial cell membranes likely compromises the functional integrity of those membranes, thereby accounting for at least some of this compounds antibacterial effects.Abbreviations COLOC correlation by long-range coupling - EYL egg yolk lecithin - HETCOR heteronuclear chemical-shift correlation - MAS magic-angle spinning - MLV multilamellar vesicles     

Comparison of the conformation and orientation of alamethicin and melittin in lipid membranes

The secondary structure of alamethicin in lipid membranes below and above the lipid phase transition temperature Tt is determined by Raman spectroscopy and circular dichroism (CD) measurements. In both cases structural data are obtained by fitting the experimental spectra by a superposition of the spectra of 15 reference proteins of known three-dimensional structure. According to the Raman experiments, in a lipid bilayer above Tt alamethicin is helical from residue 1 to 12, whereas below Tt the helix extends from residue 1 to 16. The remaining C-terminal part is nonhelical up to the end residue 20 both above and below Tt. A considerable lower helix content is derived from CD, namely, 38% and 46% above and below Tt, respectively, in agreement with several reported values for CD in the literature. It is shown that the commonly used set of CD spectra of water-soluble reference proteins is unsuitable to describe the CD spectra of alamethicin correctly. Therefore the secondary structure of alamethicin as derived from CD measurements is at the present state of analysis unreliable. In contrast to the case of alamethicin, the CD spectra of melittin in lipid membranes are correctly described by the reference protein spectra. The helix content of melittin is determined thereby to be 72% in lipid membranes above Tt and 75% below Tt. The data are in accord with a structure where the hydrophobic part of melittin adopts a bent helix as determined recently by Raman spectroscopy [Vogel, H., & J?hnig, F. (1986) Biophys. J. 50, 573]. The orientational order parameters of the helical parts of alamethicin and of melittin in a lipid membrane are deduced from the difference between a corresponding CD spectrum of a polypeptide in planar multibilayers and that in lipid vesicles. The presented method for determining helix order parameters is new and may be generally applicable to other membrane proteins. The orientation of the helical part of both polypeptides depends on the physical state of the lipid bilayer at maximal membrane hydration and in the ordered lipid state furthermore on the degree of membrane hydration. Under conditions where alamethicin and melittin are incorporated in an aggregated form in a fluid lipid membrane at maximal water content the helical segments are oriented preferentially parallel to the membrane normal. Cooling such lipid membranes to a temperature below Tt changes the orientation of the helical part of alamethicin as well as melittin toward the membrane plane.(ABSTRACT TRUNCATED AT 400 WORDS)     

Kinetics of phospholipid exchange between bilayer membranes

In an accompanying publication by Duckwitz-Peterlein, Eilenberger and Overath ((1977) Biochim. Biophys. Acta 469, 311–325) it is shown that the exchange of lipid molecules between negatively charged vesicles consisting of total phospholipid extracts from Escherichia coli occurs by the transfer of single lipid monomers or small micelles through the water. Here a kinetic interpretation is presented in terms of a rate constant, $\text{k}{}_{\mathrm{?}}$, for the escape of lipid molecules from the vesicle bilayer into the water. The evaluated rate constants are $\text{k}{}_{\mathrm{?}}{}^{\mathrm{<mtext>P</mtext>}}\text{= (0.86 ± 0.05) · 10}{}^{\mathrm{?5}}\text{s}{}^{\mathrm{?1}}$ and $\text{k}{}_{\mathrm{?}}{}^{\mathrm{<mtext>E</mtext>}}\text{= (1.09 ± 0.13) · 10}{}^{\mathrm{?6}}\text{s}{}^{\mathrm{?1}}$ for phospholipid molecules with $\text{trans-}\text{Δ}{}^9\text{-hexadecenoate}$ and $\text{trans-}\text{Δ}{}^9\text{-octadecenoate}$, respectively, as the predominant acyl chain component. The rate constants are discussed in terms of the acyl chain and polar head group composition of the lipids.     

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.     

The interaction of amino-deuteromethylated melittin with phospholipid membranes studied by deuterium NMR

Melittin, deuteromethylated on each of the four amino groups (Gly-1 N alpha and Lys-7, 21, and 23 N epsilon), was prepared by reductive methylation using deuteroformaldehyde and NaBD3CN. Deuterium NMR spectra were obtained for the modified peptide (D-melittin) bound to phospholipid bilayers and erythrocyte ghosts. D-Melittin at 4 mol% (peptide:lipid) induced reversible transitions between extended bilayers and micelles at the phase-transition temperature in dimyristoylphosphatidylcholine (DMPC) bilayers. These changes in lipid morphology did not occur at 1 mol% D-melittin: DMPC and the peptide was highly motionally restricted in gel in gel-phase lipid.     

Kinetics of cholesterol and phospholipid exchange from membranes containing cross-linked proteins or cross-linked phosphatidylethanolamines

Mono- and dipalmitoylphosphatidylethanolamine derivatives have been synthesized and used to evaluate the role of cross-links between the amino groups of two phospholipid molecules in the rate of cholesterol movement between membranes. Incorporation of the cross-linked phospholipids into small unilamellar vesicles (the donor species) decreased the rate of spontaneous cholesterol exchange with acceptor membranes (small unilamellar vesicles or Mycoplasma gallisepticum cells). These results suggest that the cross-linking of aminophospholipids by reactive intermediates, which may be one of the degenerative transformations associated with peroxidation of unsaturated lipids and cellular aging, can inhibit cholesterol exchangeability in biological membranes. The rates of spontaneous [14C]cholesterol and protein-mediated 14C-labeled phospholipid exchange from diamide-treated mycoplasma and erythrocyte membranes have also been measured. The formation of extensive disulfide bonds in the membrane proteins of M. gallisepticum enhanced the 14C-labeled phospholipid exchange rate but did not affect the rate of [14C]cholesterol exchange. The rates of radiolabeled cholesterol and phospholipid exchange between erythrocyte ghosts and vesicles were both enhanced (but to different extents) when ghosts were treated with diamide. These observations suggest that diamide-induced oxidative cross-linking of sulfhydryl groups in membrane proteins does not lead to random defects in the lipid domain.     

Different states of self-association of melittin in phospholipid bilayers

Melittin is known to self-associate as tetramers in solutions of high ionic strength. Here, an N-bromosuccinimide oxidized-Trp₁₉ melittin is prepared. This derivative can act as an acceptor of the fluorescence of native melittin and is used in order to observe a possible self-association of melittin in phospholipid bilayers.Resonance energy transfer was shown to occur in solutions of high ionic strength, showing that oxidized melittin can associate with native melittin.In phospholipid bilayers, no association is detected in the absence of NaCl. In its presence, an equilibrium between monomeric melittin and oligomeric species is observed. These species are not dimers, but any other degree of association may account for our experimental results. Significant differences in characteristic transfer efficiency reveal differences in the structure of these oligomers according to the length or state of phospholipids (fluid or at the transition temperature). These bound complexes are also different from the soluble hetero-oligomer.Some models of bound complexes are proposed which may explain the leakage and the further disruption of vesicles or cells induced by melittin.Abbreviations NBS N-bromosuccinimide - NATA N-acetyl tryptophanamide - DMPC dimyristoyl phosphatidylcholine - DPPC dipalmitoyl phosphatidylcholine - PG phosphatidylglycerol - EPC egg phosphatidylcholine - O-melittin oxindole-melittin - RET resonance energy transfer - EDTA ethylene diamine tetracetic acid - Mel melittin     

Neutron diffraction from cannabinoids in phospholipid membranes

Neutron diffraction measurements have been utilized to study the effects of delta 9-tetrahydrocannabinol (delta 9-THC) and delta 8-tetrahydrocannabinol (delta 8-THC) incorporated in phospholipid membranes of dipalmitoylphosphatidylcholine (DPPC). Low-angle diffraction indicated that these cannabinoids induce increases in interlamellar spacing similar to those produced by cholesterol. Wide-angle diffraction indicated significant differences in how the intralamellar structure is affected by the inclusion of either cannabinoids or cholesterol. Similar weight percentages of cholesterol and cannabinoids in membranes yielded different thermal analysis profiles but the profiles for membranes with either delta 8 or delta 9-THC were similar. Since the neutron diffraction results for inclusions of delta 8 and delta 9-THC were also similar, this suggests that the difference in psychoactivity of delta 8 and delta 9-THC is probably due to interactions with membrane proteins rather than with phospholipids.     

Catalytic properties of phospholipid exchange protein from bovine heart.

A protein which catalyzes the exchange of phosphatidylcholine between membranes has been purified from heart tissue homogenates up to 300-fold by acidic pH precipitation, (NH4)2SO4 precipitation, gel filtration, and ion-exchange chromatography. Binding of the protein to phosphatidylcholine liposomes as measured by Sepharose chromatography was nondetectable. However, isoelectric focusing experiments showed that individual molecules of phosphatidylcholine were transferred from liposomes to the soluble, partially purified protein. Exchange of phospholipid between liposomes and mitochondria was not affected by the presence of moderate amounts of cholesterol in liposomes. A search for competitive inhibitors among moieties similar to phosphatidylcholine failed to show strong binding sites in the hydrophilic part of the substrate. High concentrations of Na+, Ca2+ and Mg2+ impaired the exchange activity.     

31P NMR first spectral moment study of the partial magnetic orientation of phospholipid membranes.

Structural data can be obtained on proteins inserted in magnetically oriented phospholipid membranes such as bicelles, which are most often made of a mixture of long and short chain phosphatidylcholine. Possible shapes for these magnetically oriented membranes have been postulated in the literature, such as discoidal structures with a thickness of one bilayer and with the short acyl chain phosphatidylcholine on the edges. In the present paper, a geometrical study of these oriented structures is done to determine the validity of this model. The method used is based on the determination of the first spectral moment of solid-state (31)P nuclear magnetic resonance spectra. From this first moment, an order parameter is defined that allows a quantitative analysis of partially oriented spectra. The validity of this method is demonstrated in the present study for oriented samples made of DMPC, DMPC:DHPC, DMPC:DHPC:gramicidin A and adriamycin:cardiolipin.     

Effects of melittin on lipid-protein interactions in sarcoplasmic reticulum membranes.

To investigate the physical mechanism by which melittin inhibits Ca-adenosine triphosphatase (ATPase) activity in sarcoplasmic reticulum (SR) membranes, we have used electron paramagnetic resonance spectroscopy to probe the effect of melittin on lipid-protein interactions in SR. Previous studies have shown that melittin substantially restricts the rotational mobility of the Ca-ATPase but only slightly decreases the average lipid hydrocarbon chain fluidity in SR. Therefore, in the present study, we ask whether melittin has a preferential effect on Ca-ATPase boundary lipids, i.e., the annular shell of motionally restricted lipid that surrounds the protein. Paramagnetic derivatives of stearic acid and phosphatidylcholine, spin-labeled at C-14, were incorporated into SR membranes. The electronic paramagnetic resonance spectra of these probes contained two components, corresponding to motionally restricted and motionally fluid lipids, that were analyzed by spectral subtraction. The addition of increasing amounts of melittin, to the level of 10 mol melittin/mol Ca-ATPase, progressively increased the fraction of restricted lipids and increased the hyperfine splitting of both components in the composite spectra, indicating that melittin decreases the hydrocarbon chain rotational mobility for both the fluid and restricted populations of lipids. No further effects were observed above a level of 10 mol melittin/mol Ca-ATPase. In the spectra from control and melittin-containing samples, the fraction of restricted lipids decreased significantly with increasing temperature. The effect of melittin was similar to that of decreased temperature, i.e., each spectrum obtained in the presence of melittin (10:1) was nearly identical to the spectrum obtained without melittin at a temperature approximately 5 degrees C lower. The results suggest that the principal effect of melittin on SR membranes is to induce protein aggregation and this in turn, augmented by direct binding of melittin to the lipid, is responsible for the observed decreases in lipid mobility. Protein aggregation is concluded to be the main cause of inactivation of the Ca-ATPase by melittin, with possible modulation also by the decrease in mobility of the boundary layer lipids.