Bilayer conformation of fusion peptide of influenza virus hemagglutinin: a molecular dynamics simulation study

Unraveling the conformation of membrane-bound viral fusion peptides is essential for understanding how those peptides destabilize the bilayer topology of lipids that is important for virus-cell membrane fusion. Here, molecular dynamics (MD) simulations were performed to investigate the conformation of the 20 amino acids long fusion peptide of influenza hemagglutinin of strain X31 bound to a dimyristoyl phosphatidylcholine (DMPC) bilayer. The simulations revealed that the peptide adopts a kinked conformation, in agreement with the NMR structures of a related peptide in detergent micelles. The peptide is located at the amphipathic interface between the headgroups and hydrocarbon chains of the lipid by an energetically favorable arrangement: The hydrophobic side chains of the peptides are embedded into the hydrophobic region and the hydrophilic side chains are in the headgroup region. The N-terminus of the peptide is localized close to the amphipathic interface. The molecular dynamics simulations also revealed that the peptide affects the surrounding bilayer structure. The average hydrophobic thickness of the lipid phase close to the N-terminus is reduced in comparison with the average hydrophobic thickness of a pure dimyristoyl phosphatidylcholine bilayer.     

The gene expression and deficiency phenotypes of Cockayne syndrome B protein in Caenorhabditis elegans

Maculatin 1.1 is an antimicrobial peptide isolated from the Australian tree frog Litoria genimaculata that adopts an amphipathic, alpha-helical structure in solution. Its orientation and conformation when incorporated to pre-formed DMPG (1,2-dimyristoyl-sn-glycero-3-phosphoglycerol) and DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) vesicles was determined using polarised Fourier transform infrared-attenuated total reflection infrared and deuterium exchange experiments. For DMPG membranes, our results show insertion of 70% of the maculatin 1.1 molecules, with an angle of insertion of approximately 35 degrees to the membrane normal and with a predominant alpha-helical structure. These results suggest that maculatin 1.1 acts through a pore-forming mechanism to lyse bacterial membranes. A similar degree of insertion in DMPG (65%) and alpha-helical structure was observed for a biologically inactive, less amphipathic maculatin 1.1 analogue, P15A, although the helix tilt was found to be greater (46 degrees) than for maculatin 1.1. Similar experiments performed using DMPC liposomes showed poor insertion, less than 5%, for both maculatin 1.1 and its analogue. In addition, the shape of the amide I band in these samples is consistent with alpha-helix, beta-structure and disordered structures being present in similar proportion. These results clearly show that maculatin 1.1 inserts preferentially in negatively charged membranes (DMPG) which mimic the negatively charged membrane of Gram-positive bacteria. We attribute the high percentage of insertion of the biologically inactive analogue in DMPG to the fact that its concentration on the membrane surface in our experiments is likely to be much higher than that found in physiological conditions.     

The membrane-binding properties of a class A amphipathic peptide

The membrane-binding properties of a class A amphipathic peptide (18D) were investigated using two different immobilized model membrane systems. The first system involved the use of surface plasmon resonance (SPR) to study the binding of 18D to dimyristylphosphatidylcholine (DMPC) and dimyristylphosphatidylglycerol (DMPG), which allowed peptide binding to be monitored in real time. The SPR experiments indicated stronger binding of 18D to DMPG than DMPC, which kinetic analysis revealed was due to a faster on-rate. The second model membrane system involved immobilized membrane chromatography in which the binding of 18D to either DMPC or DMPG monolayers covalently linked to silica particles was analysed by elution chromatography. Stronger binding affinity of 18D was also obtained with the negatively charged phosphatidylglycerol (PG) monolayer compared to the phosphatidylcholine (PC) monolayer, which was consistent with the SPR results. Non-linear binding behaviour of 18D to the immobilized lipid monolayers was also observed, which suggests that the peptide undergoes conformational and orientational changes upon binding to the immobilized PC and PG ligands. Significant band broadening was also observed on both monolayers, with larger bandwidths obtained on the PC surface, indicating slower binding and orientation kinetics with the zwitterionic surface. The dependence of logk' on the percentage of methanol also demonstrated a bimodal interaction whereby hydrophobic forces predominated at higher temperatures and methanol concentrations, while at lower temperatures, electrostatic and other polar forces also made a contribution to the affinity of the peptides for the lipid monolayer particularly. Overall, these results demonstrate the complementary use of these two lipid biosensors which allows the role of hydrophobic and electrostatic forces in peptide–membrane interactions to be studied and insight gained into the kinetic factors associated with these interactions.     

Membrane interaction and perturbation mechanisms induced by two cationic cell penetrating peptides with distinct charge distribution

Independently from the cell penetrating peptide uptake mechanism (endocytic or not), the interaction of the peptide with the lipid bilayer remains a common issue that needs further investigation. The cell penetrating or antimicrobial properties of exogenous peptides require probably different preliminary interactions with the plasma membrane. Herein, we have employed (31)P NMR, differential scanning calorimetry and CD to study the membrane interaction and perturbation mechanisms of two basic peptides with similar length but distinct charge distribution, penetratin (non-amphipathic) and RL16, a secondary amphipathic peptide. The peptide effects on the thermotropic phase behavior of large multilamellar vesicles of dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG) and dipalmitoleoyl phosphatidylethanolamine (DiPoPE) were investigated. We have found that, even though both peptides are cationic, their interaction with zwitterionic versus anionic lipids is markedly distinct. Penetratin greatly affects the temperature, enthalpy and cooperativity of DMPG main phase transition but does not affect those of DMPC while RL16 presents opposite effects. Additionally, it was found that penetratin induces a negative curvature whereas RL16 induces a positive one, since a decrease in the fluid lamellar to inverted hexagonal phase transition temperature of DiPoPE (T(H)) was observed for penetratin and an increase for RL16. Contrary to penetratin, (31)P NMR of samples containing DMPC MLVs and RL16 shows an isotropic signal indicative of the formation of small vesicles, concomitant with a great decrease in sample turbidity both below and at the phase transition temperature. Opposite effects were also observed on DMPG where both peptides provoke strong aggregation and precipitation. Both CPPs adopt helical structures when contacting with anionic lipids, and possess a dual behavior by either presenting their cationic or hydrophobic domains towards the phospholipid face, depending on the lipid nature (anionic vs zwitterionic, respectively). Surprisingly, the increase of electrostatic interactions at the water membrane interface prevents the insertion of RL16 hydrophobic region in the bilayer, but is essential for the interaction of penetratin. Modulation of amphipathic profiles and charge distribution of CPPs can alter the balance of hydrophobic and electrostatic membrane interaction leading to translocation or and membrane permeabilisation. Penetratin has a relative pure CPP behavior whereas RL16 presents mixed CPP/AMP properties. A better understanding of those processes is essential to unveil their cell translocation mechanism.     

Orientation and lipid-peptide interactions of gramicidin A in lipid membranes: polarized attenuated total reflection infrared spectroscopy and spin-label electron spin resonance

Gramicidin A was incorporated at a peptide/lipid ratio of 1:10 mol/mol in aligned bilayers of dimyristoyl phosphatidylcholine (DMPC), phosphatidylserine (DMPS), phosphatidylglycerol (DMPG), and phosphatidylethanolamine (DMPE), from trifluoroethanol. Orientations of the peptide and lipid chains were determined by polarized attenuated total reflection infrared spectroscopy. Lipid-peptide interactions with gramicidin A in DMPC bilayers were studied with different spin-labeled lipid species by using electron spin resonance spectroscopy. In DMPC membranes, the orientation of the lipid chains is comparable to that in the absence of peptide, in both gel and fluid phases. In gel-phase DMPC, the effective tilt of the peptide exceeds that of the lipid chains, but in the fluid phase both are similar. For gramicidin A in DMPS, DMPG, and DMPE, the degree of orientation of the peptide and lipid chains is less than in DMPC. In the fluid phase of DMPS, DMPG, and DMPE, gramicidin A is also less well oriented than are the lipid chains. In DMPE especially, gramicidin A is largely disordered. In DMPC membranes, three to four lipids per monomer experience direct motional restriction on interaction with gramicidin A. This is approximately half the number of lipids expected to contact the intramembranous perimeter of the gramicidin A monomer. A selectivity for certain negatively charged lipids is found in the interaction with gramicidin A in DMPC. These results are discussed in terms of the integration of gramicidin A channels in lipid bilayers, and of the interactions of lipids with integral membrane proteins.     

Kinetic and conformational properties of a novel T-cell antigen receptor transmembrane peptide in model membranes

Core peptide (CP; GLRILLLKV) is a 9-amino acid peptide derived from the transmembrane sequence of the T-cell antigen receptor (TCR) alpha-subunit. CP inhibits T-cell activation both in vitro and in vivo by disruption of the TCR at the membrane level. To elucidate CP interactions with lipids, surface plasmon resonance (SPR) and circular dichroism (CD) were used to examine CP binding and secondary structure in the presence of either the anionic dimyristoyl-L-alpha-phosphatidyl-DL-glycerol (DMPG), or the zwitterionic dimyristoyl-L-alpha-phoshatidyl choline (DMPC).Using lipid monolayers and bilayers, SPR experiments demonstrated that irreversible peptide-lipid binding required the hydrophobic interior provided by a membrane bilayer. The importance of electrostatic interactions between CP and phospholipids was highlighted on lipid monolayers as CP bound reversibly to anionic DMPG monolayers, with no detectable binding observed on neutral DMPC monolayers.CD revealed a dose-dependent conformational change of CP from a dominantly random coil structure to that of beta-structure as the concentration of lipid increased relative to CP. This occurred only in the presence of the anionic DMPG at a lipid : peptide molar ratio of 1.6:1 as no conformational change was observed when the zwitterionic DMPC was tested up to a lipid : peptide ratio of 8.4 : 1.     

Membrane interactions of synthetic peptides corresponding to amphipathic helical segments of the human immunodeficiency virus type-1 envelope glycoprotein.

The human and simian immunodeficiency virus envelope glycoproteins, which mediate virus-induced cell fusion, contain two putative amphipathic helical segments with large helical hydrophobic moments near their carboxyl-terminal ends. In an attempt to elucidate the biological role of these amphipathic helical segments, we have synthesized peptides corresponding to residues 768-788 and 826-854 of HIV-1/WMJ-22 gp160. Circular dichroism studies of the peptides showed that the alpha helicity of the peptides increased with the addition of dimyristoyl phosphatidylcholine (DMPC) indicating that the peptides form lipid-associating amphipathic helixes. The peptides solubilized turbid suspensions of DMPC vesicles, and electron microscopy of peptide-DMPC mixtures revealed the formation of discoidal complexes, suggesting that the peptides bind to and perturb lipid bilayers. The peptides were found to lyse lipid vesicles and caused carboxyfluorescein leakage from dye-entrapped egg phosphatidylcholine liposomes. The peptides also lysed human erythrocytes and were found to be toxic to cell cultures. At subtoxic concentrations, the peptides effectively inhibited the fusion of CD4+ cells infected with recombinant vaccinia virus expressing human immunodeficiency virus (HIV)-1 envelope proteins. Based on these results, and reported studies on the mutational analysis of HIV envelope proteins, we suggest that the amphipathic helical segments near the carboxyl terminus of HIV envelope proteins may play a role in lysis of HIV-infected cells and also may modulate the extent of cell fusion observed during HIV infection of CD4+ cells.     

Effect of leucine to phenylalanine substitution on the nonpolar face of a class A amphipathic helical peptide on its interaction with lipid: high resolution solution NMR studies of 4F-dimyristoylphosphatidylcholine discoidal complex

Model class A amphipathic helical peptides mimic several properties of apolipoprotein A-I (apoA-I), the major protein component of high density lipoproteins. Previously, we reported the NMR structures of Ac-18A-NH(2) (renamed as 2F because of two phenylalanines), the base-line model class A amphipathic helical peptide in the presence of lipid ( Mishra, V. K., Anantharamaiah, G. M., Segrest, J. P., Palgunachari, M. N., Chaddha, M., Simon Sham, S. W., and Krishna, N. R. (2006) J Biol. Chem. 281, 6511-6519 ). Substitution of two Leu residues on the nonpolar face (Leu(3) and Leu(14)) with Phe residues produced the peptide 4F (so named because of four phenylalanines), which has been extensively studied for its anti-inflammatory and antiatherogenic properties. Like 2F, 4F also forms discoidal nascent high density lipoprotein-like particles with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). Since subtle structural changes in the peptide-lipid complexes have been shown to be responsible for their antiatherogenic properties, we undertook high resolution NMR studies to deduce detailed structure of 4F in 4F.DMPC discs. Like 2F, 4F adopts a well defined amphipathic alpha-helical structure in association with the lipid at a 1:1 peptide/lipid weight ratio. Nuclear Overhauser effect (NOE) spectroscopy revealed a number of intermolecular close contacts between the aromatic residues in the hydrophobic face of the helix and the lipid acyl chain protons. Similar to 2F, the pattern of observed peptide-lipid NOEs is consistent with a parallel orientation of the amphipathic alpha helix, with respect to the plane of the lipid bilayer, on the edge of the disc (the belt model). However, in contrast to 2F in 2F.DMPC, 4F in the 4F.DMPC complex is located closer to the lipid headgroup as evidenced by a number of NOEs between 4F and DMPC headgroup protons. These NOEs are absent in the 2F.DMPC complex. In addition, the conformation of the DMPC sn-3 chain in 4F.DMPC complex is different than in the 2F.DMPC complex as evidenced by the NOE between lipid 2.CH and betaCH(2) protons in 4F.DMPC, but not in 2F.DMPC, complex. Based on the results of this study, we infer that the antiatherogenic properties of 4F may result from its preferential interaction with lipid headgroups.     

In situ study by polarization modulated Fourier transform infrared spectroscopy of the structure and orientation of lipids and amphipathic peptides at the air-water interface.

Free amphipathic peptides and peptides bound to dimyristoylphosphatidylcholine (DMPC) were studied directly at the air/water interface using polarization modulation infrared reflection absorption spectroscopy (PMIRRAS). Such differential reflectivity measurements proved to be a sensitive and efficient technique to investigate in situ the respective conformations and orientations of lipid and peptide molecules in pure and mixed films. Data obtained for melittin, a natural hemolytic peptide, are compared to those of L15K7, an ideally amphipathic synthetic peptide constituted by only apolar Leu and polar Lys residues. For pure peptidic films, the intensity, shape, and position of the amide I and II bands indicate that the L15K7 peptide adopts a totally alpha-helical structure, whereas the structure of melittin is mainly alpha-helical and presents some unordered domains. The L15K7 alpha-helix axis is oriented essentially parallel to the air-water interface plane; it differs for melittin. When injected into the subphase, L15K7 and melittin insert into preformed expanded DMPC monolayers and can be detected by PMIRRAS, even at low peptide content (> 50 DMPC molecules per peptide). In such conditions, peptides have the same secondary structure and orientation as in pure peptidic films.     

Apolipoprotein E: phospholipid binding studies with synthetic peptides from the carboxyl terminus.

We have previously shown that the synthetic peptide apoE(129-169) forms lipid-peptide complexes with dimyristoylphosphatidylcholine (DMPC) with an L:P molar ratio of 125:1; the peptide in the isolated complex contains approximately 56% alpha-helicity. These results verify the presence of an amphipathic alpha-helix in this region of apoE as predicted by Chou-Fasman analysis and hydrophobicity calculations. To further define the lipid binding regions of apoE, we have synthesized four peptides, apoE(211-243), -(202-243), -(267-286), and -(263-286), from the carboxyl terminus of apoE and studied their lipid binding properties; apoE(202-243) contains two potential amphipathic helices. Although all four peptides formed alpha-helices in the helix-forming solvent 30% hexafluoropropanol, we found that only apoE(263-286) formed a stable complex with DMPC. The peptide contained approximately 80% alpha-helicity, and its Trp fluorescence spectrum was blue-shifted by 20 nm in the complex which had an L:P ratio of 163:1. We conclude that this sequence is a newly identified lipid binding region of apoE and that the amphipathic helices 203-221 and 226-243 are too hydrophilic to bind phospholipid.     

Studies of synthetic peptide analogs of the amphipathic helix. Structure of complexes with dimyristoyl phosphatidylcholine

The amphipathic helix hypothesis for the lipid-associating domains of exchangeable plasma apolipoproteins has been further studied by analysis of the structure of the complexes formed between four synthetic peptide analogs of the amphipathic helix and dimyristoyl phosphatidylcholine (DMPC). Density gradient ultracentrifugation, negative stain electron microscopy, nondenaturing gradient gel electrophoresis, 1H NMR, high sensitivity differential scanning calorimetry, and circular dichroism were the techniques used in these studies. The two analogs Asp-Trp-Leu-Lys-Ala-Phe-Tyr-Asp-Lys-Val-Ala-Glu-Lys-Leu-Lys-Glu-Ala-Phe (18A) and 18A-Pro-18A whose sequences most strongly mimic native amphipathic sequences were found also most strongly to mimic apolipoprotein A-I in DMPC complex structure. The covalently linked dimer of the prototype amphipathic analog 18A, 18A-Pro-18A, appears to have greater lipid affinity than 18A. This presumably is the result of the cooperativity provided by two covalently linked lipid-associating domains in 18A-Pro-18A. The studies further suggest that the charge-reversed analog of the prototype 18A, reverse-18A, has the lowest lipid affinity of the four analogs studied and forms only marginally stable discoidal DMPC complexes. We postulate that this low lipid affinity is due predominantly, but not necessarily exclusively, to the lack of a hydrophobic contribution of lysine residues at the polar-nonpolar interface of reverse-18A versus 18A. The intermediate lipid affinity of des-Val10-18A, the fourth analog peptide, to produce a rank order of 18A-Pro-18A greater than 18A greater than des-Val10-18A greater than reverse-18A, supports this interpretation. Des-Val10-18A which has Val deleted from 18A has an amphipathic helical structure partially disrupted by the shift of 2 lysine residues away from the polar-nonpolar interface.     

Effects of L- or D-Pro incorporation into hydrophobic or hydrophilic helix face of amphipathic alpha-helical model peptide on structure and cell selectivity

A synthetic amphipathic alpha-helical model peptide, KLW, displays non-cell selective cytotoxicity. To investigate the effects of L- or D-Pro kink incorporation into hydrophobic or hydrophilic helix face of KLW on structure, cell selectivity, and membrane-binding affinity, we designed a series of four peptides, in which Leu(9) and Lys(11) in the hydrophobic and hydrophilic helix face of KLW, respectively, are substituted with L- or D-Pro. A L- or D-Pro substitution (KLW-L9P or KLW-L9p) of Leu(9) at the hydrophobic helix face of KLW induced a more significant reduction in hemolytic activity with improved antibacterial activity than that (KLW-K11P or KLW-K11p) of Lys(11) in the hydrophilic helix face. In addition, D-Pro-containing peptides (KLW-L9p and KLW-K11p) displayed less hemolytic activity than L-Pro-containing peptides (KLW-L9P and KLW-K11P). Tryptophan fluorescence studies revealed that bacterial cell selectivity of KLW-L9P, KLW-L9p, and KLW-K11p is closely related to selective interactions with negatively charged phospholipids. CD analysis revealed that L- or D-Pro incorporation into KLW reduces the alpha-helicity of the peptide and D-Pro incorporation induces more significant disruption in alpha-helical structure than L-Pro incorporation. Our results collectively suggest that D-Pro incorporation into the hydrophobic helix face of non-cell selective amphipathic alpha-helical peptides may be useful for the design of novel antimicrobial peptides possessing high bacterial cell selectivity without hemolytic activity.     

Effects of alterations of the amino-terminal glycine of influenza hemagglutinin fusion peptide on its structure,organization and membrane interactions

Mutations of the glycine residue at the amino terminus of HA2 have been shown to have a large effect on the fusion activity of HA2, the extent of which apparently correlates with the side chain bulkiness of the substituting amino acids. To investigate into the cause of abrogation in fusogenicity and virus-promoted fusion mechanism, we synthesized several peptides in which this glycine was substituted by serine, glutamic acid, or lysine. 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl sn-glycero-3-phosphoglycerol (DMPG) were used as model membranes in the fluorescence, circular dichroism (CD), and FTIR measurements while sodium dodecyl sulfate was used in NMR studies. We found that, for the less active variants, affinity to membrane, degree of solvent dehydration, lipid perturbation, depth of insertion, and helicity were less. Comparison of affinity to membrane bilayer among these analogs revealed that binding of the fusion peptide is determined largely by the hydrophobic effect. Additionally, the orientation is closer to the membrane normal for the wild-type fusion peptide in the helix form while the inactive analogs inserted more parallel to the membrane surface.     

Association of beta-Arrestin 1 with the type 1A angiotensin II receptor involves phosphorylation of the receptor carboxyl terminus and correlates with receptor internalization

Arrestins bind to phosphorylated G protein-coupled receptors and participate in receptor desensitization and endocytosis. Although arrestins traffic with activated type 1 (AT(1A)) angiotensin II (AngII) receptors, the contribution of arrestins to AT(1A) receptor internalization is controversial, and the physical association of arrestins with the AT(1A) receptor has not been established. In this study, by coimmunoprecipitating AT(1A) receptors and beta-arrestin 1, we provide direct evidence for an association between arrestins and the AT(1A) receptor that was agonist- and time-dependent and contingent upon the level of beta-arrestin 1 expression. Serial truncation of the receptor carboxyl terminus resulted in a graded loss of beta-arrestin 1 association, which correlated with decreases in receptor phosphorylation. Truncation of the AT(1A) receptor to lysine(325) prevented AngII-induced phosphorylation and beta-arrestin 1 association as well as markedly inhibiting receptor internalization, indicating a close correlation between these receptor parameters. AngII-induced association was also dramatically reduced in a phosphorylation- and internalization-impaired receptor mutant in which four serine and threonine residues in the central portion of the AT(1A) receptor carboxyl terminus (Thr(332), Ser(335), Thr(336), Ser(338)) were substituted with alanine. In contrast, substitutions in another serine/threonine-rich region (Ser(346), Ser(347), Ser(348)) and at three PKC phosphorylation sites (Ser(331), Ser(338), Ser(348)) had no effect on AngII-induced beta-arrestin 1 association or receptor internalization. While AT(1A) receptor internalization could be inhibited by a dominant-negative beta-arrestin 1 mutant (beta arr1(319-418)), treatment with hyperosmotic sucrose to inhibit internalization did not abrogate the differences in arrestin association observed between the wild-type and mutant receptors, indicating that arrestin binding precedes, and is not dependent upon, receptor internalization. Interestingly, a substituted analog of AngII, [Sar(1)Ile(4)Ile(8)]-AngII, which promotes robust phosphorylation of the receptor but does not activate receptor signaling, stimulated strong beta-arrestin 1 association with the full-length AT(1A) receptor. These results identify the central portion of the AT(1A) receptor carboxyl terminus as the important determinant for beta-arrestin 1 binding and internalization and indicate that AT(1A) receptor phosphorylation is crucial for beta-arrestin docking.     

Insect immune activation by apolipophorin III is correlated with the lipid-binding properties of this protein

Apolipophorin III (apoLp-III) is an exchangeable insect apolipoprotein consisting of five amphipathic alpha-helices. The protein is able to open reversibly on associating with hydrophobic surfaces and plays a role both in lipid transport and induction of immune responses. Point mutations were introduced at positions 66 (N-->D) and/or 68 (K-->E) between helices 2 and 3, a region possibly serving as a hinge for the opening of the molecule when associating with lipids. The lipid-binding properties of the mutant proteins were analyzed and compared with their immune inducing activities. Structural properties of the proteins were studied by far UV circular dichroism spectroscopy and their abilities to form discoidal complexes of dimyristoyl phosphatidylcholine (DMPC) vesicles were investigated. In comparison to wild-type apoLp-III, apoLp-III(N66D/K68E), and apoLp-III(K68E) displayed significantly decreased lipid-binding abilities and immune stimulating activities, while these effects were less noticeable with apoLp-III(N66D). The secondary structure of the double mutant apoLp-III(N66D/K68E) was similar to that of wild-type apoLp-III. A noticeable reduction of alpha-helical content could be observed for the single mutants apoLp-III(N66D) and apoLp-III(K68E), which was accompanied by an increase in percentage amount of beta-turns. The stability of the secondary structure determined by heat denaturation was not affected by mutagenesis. Furthermore, the ability of all proteins to form discoidal complexes of equal size and shape in the presence of dimyristoyl phosphatidylcholine indicated that the mutagenesis did not affect the molecular architecture in the lipid-associated conformation. The relationship between reduced lipid association and reduced immune stimulating activity supports the hypothesis that apoLp-III-induced immune activation is triggered by the conformational change of the protein.     

The peculiar thermo-structural behavior of the anionic lipid DMPG

Aqueous dispersions of the anionic phospholipid dimyristoyl phosphatidylglycerol (DMPG), around 100 mM ionic strength, are known to exhibit a thermal behavior similar to that of the largely studied lipid dimyristoyl phosphatidylcholine (DMPC), which undergoes a gel to liquid crystalline phase transition at 23 degrees C, well characterized by differential scanning calorimetry (DSC), and other methods. However, at low ionic strength, DMPG has been shown to present a large gel-fluid transition region, ranging from 18 to 35 degrees C. This intermediate phase is optically transparent and characterized by a continuous change in membrane packing. Structural properties of the DMPG gel-fluid transition region will be discussed, based on results obtained by several techniques: electron spin resonance (ESR) of spin labels at the membrane surface and intercalated at different depths in the bilayer; light scattering; DSC; small angle X-ray scattering (SAXS); and fluorescence spectroscopy of probes in the bilayer.     

Derivative spectrophotometry as a tool for the determination of drug partition coefficients in water/dimyristoyl-L-alpha-phosphatidylglycerol (DMPG) liposomes.

The partition coefficients (K(p)) between lipid bilayers of dimyristoyl-L-alpha-phosphatidylglycerol (DMPG) unilamellar liposomes and water were determined using derivative spectrophotometry for chlordiazepoxide (benzodiazepine), isoniazid and rifampicin (tuberculostatic drugs) and dibucaine (local anaesthetic). A comparison of the K(p) values in water/DMPG with those in water/DMPC (dimyristoyl-L-alpha-phosphatidylcholine) revealed that for chlordiazepoxide and isoniazid, neutral drugs at physiological pH, the partition coefficients are similar in anionic (DMPG) and zwitterionic (DMPC) liposomes. However, for ionised drugs at physiological pH, the electrostatic interactions are different with DMPG and DMPC, with the cationic dibucaine having a stronger interaction with DMPG, and the anionic rifampicin having a much larger K(p) in zwitterionic DMPC. These results show that liposomes are a better model membrane than an isotropic two-phase solvent system, such as water-octanol, to predict drug-membrane partition coefficients, as they mimic better the hydrophobic part and the outer polar charged surface of the phospholipids of natural membranes.     

Effects of alterations of the amino-terminal glycine of influenza hemagglutinin fusion peptide on its structure, organization and membrane interactions

Mutations of the glycine residue at the amino terminus of HA2 have been shown to have a large effect on the fusion activity of HA2, the extent of which apparently correlates with the side chain bulkiness of the substituting amino acids. To investigate into the cause of abrogation in fusogenicity and virus-promoted fusion mechanism, we synthesized several peptides in which this glycine was substituted by serine, glutamic acid, or lysine. 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl sn-glycero-3-phosphoglycerol (DMPG) were used as model membranes in the fluorescence, circular dichroism (CD), and FTIR measurements while sodium dodecyl sulfate was used in NMR studies. We found that, for the less active variants, affinity to membrane, degree of solvent dehydration, lipid perturbation, depth of insertion, and helicity were less. Comparison of affinity to membrane bilayer among these analogs revealed that binding of the fusion peptide is determined largely by the hydrophobic effect. Additionally, the orientation is closer to the membrane normal for the wild-type fusion peptide in the helix form while the inactive analogs inserted more parallel to the membrane surface.     

Minimal peptide length for interaction of amphipathic alpha-helical peptides with phosphatidylcholine liposomes

The interactions of a series of amphipathic alpha-helical peptides containing from 6 to 18 amino acid residues with dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) were studied by optical and calorimetric methods. Several peptides rapidly decreased the turbidity of DMPC and DPPC liposomes when mixed at the phase transition temperatures of the lipids. The extent of the clearing depended upon the chain length of the peptides, with the most effective clearing attained with peptides 10-12 residues in length. An eight-residue peptide was somewhat less effective and a six-residue peptide had no effect on liposome structure. The peptides formed small micellar structures, as judged by gel filtration chromatography. The effects of the peptides on the phase transitions of the lipids were examined by differential scanning calorimetry. The peptides that were most effective in disrupting the liposomes and forming clear micelles were also most effective in reducing the enthalpy of the gel to liquid-crystalline phase transition of the lipid. The addition of DMPC or DPPC liposomes to the peptides increased the magnitude of the negative bonds at 208 and 222 nm in circular dichroism measurements, consistent with the expected formation of alpha-helical structure on binding to lipid. The extent of burial of the single tryptophan residue in the peptides was determined by fluorescence spectroscopy. In peptides that bound to lipid, the tryptophan was in a less solvent-exposed environment in the presence of lipid, as evidenced by a blue shift in the fluorescence emission maximum of the peptide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Aqueous dispersions of DMPG in low salt contain leaky vesicles

Aqueous dispersions of dimyristoyl phosphatidylglycerol (DMPG), at low ionic strength, display uncommon thermal behavior. Models for such behavior need to assign a form to the lipid aggregate. Although most studies accept the presence of lipid vesicles in the lipid gel and fluid phases, this is still controversial. With electron spin resonance (ESR) spectra of spin labels incorporated into DMPG aggregates, quantification of [(14)C]sucrose entrapped by the aggregates, and viscosity measurements, we demonstrate the existence of leaky vesicles in dispersions of DMPG at low ionic strength, in both gel and fluid phases of the lipid. As a control system, the ubiquitous lipid dimyristoyl phosphatidylcholine (DMPC) was used. For DMPG in the gel phase, spin labeling only indicated the presence of lipid bilayers, strongly suggesting that DMPG molecules are organized as vesicles and not micelles or bilayer fragments (bicelles), as the latter has a non-bilayer structure at the edges. Quantification of [(14)C]sucrose entrapping by DMPG aggregates revealed the presence of highly leaky vesicles. Due to the short hydrocarbon chains ((14)C atoms), DMPC vesicles were also found to be partially permeable to sucrose, but not as much as DMPG vesicles. Viscosity measurements, with the calculation of the intrinsic viscosity of the lipid aggregate, showed that DMPG vesicles are rather similar in the gel and fluid phases, and quite different from aggregates observed along the gel-fluid transition. Taken together, our data strongly supports that DMPG forms leaky vesicles at both gel and fluid phases.