Influence of poly(l-lysine) on the structure of dipalmitoylphosphatidylglycerol/water dispersions studied by X-ray scattering

The interaction between the negatively charged phospholipid DPPG and positively charged poly(l-lysine) (PLL) of different lengths was studied by X-ray scattering in the SAXS and WAXS region. As a reference pure DPPG (Na salt) was investigated over a wide temperature range (−30 to 70°C). The phase behavior of DPPG in aqueous and in buffer/salt dispersions showed a metastable subgel phase at low temperatures and a recrystallization upon heating before reaching the liquid–crystalline phase. The presence of additional salt stabilizes the bilayer structure and decreases the recrystallization temperature. Large changes in the SAXS region are not connected with changes in chain packing. In DPPG/PLL samples, the PLL is inserted between adjacent headgroup layers and liberates counterions which give rise to a freezing point depression. In the complex with DPPG PLL form an α-helical secondary structure at pH 7 and temperatures below the gel to liquid–crystalline phase transition. This prevents DPPG from recrystallization and strongly increases the stacking order. The lamellar repeat distance is decreased and fixed by the helix conformation of PLL in the gel phase. PLL with n = 14 is too short to form helices and is squeezed out reversibly from the interbilayer space upon cooling by freezing of trapped water. In dispersions with longer PLLs (n > 400) at −20°C a 1D crystallization of PLL α-helices in the aqueous layer between the headgroups takes place. A structural model is presented for the lateral periodic complex, which is similar to the known cationic lipid/DNA complex. Dedicated to Prof. K. Arnold on the occasion of his 65th birthday.     

Peptide induced demixing in PG/PE lipid mixtures: A mechanism for the specificity of antimicrobial peptides towards bacterial membranes?

Antimicrobial peptides attract a lot of interest as potential candidates to overcome bacterial resistance. So far, nearly all the proposed scenarios for their mechanism of action are associated with perforating and breaking down bacterial membranes after a binding process. In this study we obtained additional information on peptide induced demixing of bacterial membranes as a possible mechanism of specificity of antimicrobial peptides. We used DSC and FT-IR to study the influence of a linear and cyclic arginine- and tryptophan-rich antimicrobial peptide having the same sequence (RRWWRF) on the thermotropic phase transitions of lipid membranes. The cyclization of the peptide was found to enhance its antimicrobial activity and selectivity ( Dathe, M. Nikolenko, H. Klose, J. Bienert, M. Biochemistry 43 (2004) 9140-9150). A particular preference of the binding of the peptides to DPPG headgroups compared to other headgroups of negatively charged phospholipids, namely DMPA, DPPS and cardiolipin was observed. The main transition temperature of DPPG bilayers was considerably decreased by the bound peptides. The peptides caused a substantial down-shift of the transition of DPPG/DMPC. In contrast, they induced a demixing in DPPG/DPPE bilayers and led to the appearance of two peaks in the DSC curves indicating a DPPG-peptide-enriched domain and a DPPE-enriched domain. These results could be confirmed by FT-IR-spectroscopic measurements. We therefore propose that the observed peptide-induced lipid demixing in PG/PE-membranes could be a further specific effect of the antimicrobial peptides operating only on bacterial membranes, which contain appreciable amounts of PE and PG, and which could in principle also occur in liquid-crystalline membranes.     

Membrane association and selectivity of the antimicrobial peptide NK‐2: a molecular dynamics simulation study

In an effort to better understand the initial mechanism of selectivity and membrane association of the synthetic antimicrobial peptide NK‐2, we have applied molecular dynamics simulation techniques to elucidate the interaction of the peptide with the membrane interfaces. A homogeneous dipalmitoylphosphatidylglycerol (DPPG) and a homogeneous dipalmitoylphosphatidylethanolamine (DPPE) bilayers were taken as model systems for the cytoplasmic bacterial and human erythrocyte membranes, respectively. The results of our simulations on DPPG and DPPE model membranes in the gel phase show that the binding of the peptide, which is considerably stronger for the negatively charged DPPG lipid bilayer than for the zwitterionic DPPE, is mostly governed by electrostatic interactions between negatively charged residues in the membrane and positively charged residues in the peptide. In addition, a characteristic distribution of positively charged residues along the helix facilitates a peptide orientation parallel to the membrane interface. Once the peptides reside close to the membrane surface of DPPG with the more hydrophobic side chains embedded into the membrane interface, the peptide initially disturbs the respective bilayer integrity by a decrease of the order parameter of lipid acyl chain close to the head group region, and by a slightly decrease in bilayer thickness. We found that the peptide retains a high content of helical structure on the zwitterionic membrane‐water interface, while the loss of α‐helicity is observed within a peptide adsorbed onto negatively charged lipid membranes. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Poly-l-lysines and poly-l-arginines induce leakage of negatively charged phospholipid vesicles and translocate through the lipid bilayer upon electrostatic binding to the membrane

Poly-l-lysines (PLL) and poly-l-arginines (PLA) of different polymer chain lengths interact strongly with negatively charged phospholipid vesicles mainly due to their different electrical charges. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) and their mixtures (1/1 mol/mol) with the respective phosphatidylcholines of equivalent chain length were chosen as model membrane systems that form at room temperature either the fluid L_α or the gel phase L_β lipid bilayer membranes, respectively. Leakage experiments revealed that the fluid POPG membranes are more perturbed compared to the gel phase DPPG membranes upon peptide binding. Furthermore, it was found that pure PG membranes are more prone to release the vesicle contents as a result of pore formation than the lipid mixtures POPG/POPC and DPPG/DPPC. For the longer polymers (≥ 44 amino acids) maximal dye-release was observed when the molar ratio of the concentrations of amino acid residues to charged lipid molecules reached a value of R_P = 0.5, i.e. when the outer membrane layer was theoretically entirely covered by the polymer. At ratios lower or higher than 0.5 leakage dropped significantly. Furthermore, PLL and PLA insertions and/or translocations through lipid membranes were analyzed by using FITC-labeled polymers by monitoring their fluorescence intensity upon membrane binding. Short PLL molecules and PLA molecules of all lengths seemed to translocate through both fluid and gel phase lipid bilayers. Comparison of the PLL and PLA fluorescence assay results showed that PLA interacts stronger with phospholipid membranes compared to PLL. Isothermal titration calorimetry (ITC) measurements were performed to give further insight into these mechanisms and to support the findings obtained by fluorescence assays. Cryo-transmission electron microscopy (cryo-TEM) was used to visualize changes in the vesicles' morphology after addition of the polypeptides.     

Membrane destabilization induced by the human immunodeficiency virus type-1 fusion peptide

Summary The human immunodeficiency virus type-1 (HIV-1) fusion peptide, corresponding to a sequence of 23 amino acid residues at the N-terminus of the spike transmembrane subunit gp41, has the capacity to destabilize negatively charged and neutral large unilamellar vesicles, representing, respectively, the acidic and the neutral fraction of the plasma membrane lipids of viral target cells. As revealed by infrared spectroscopy, the peptide associated with the vesicles may exist in different conformations. In negatively charged membranes the structure is mainly an α-helix, while in Ca²⁺-neutralized negatively charged membranes the conformation switches to a predominantly extended conformation. In membranes composed of zwitterionic phospholipids and cholesterol, the peptide also adopts a predominant extended structure. The α-helical structure permeabilizes negatively charged vesicles but does not induce membrane fusion. The peptide in β-type conformation, on the other hand, permeabilizes neutral membranes and triggers fusion. As seen by³¹P NMR, the latter structure also exhibits the capacity to alter the lamellar organization of the membrane.     

Comparative study on the interaction of cell-penetrating polycationic polymers with lipid membranes

Cell-penetrating peptides are arginine- and lysine-rich cationic peptides that can readily enter cells not only by themselves but also carrying other macromolecular cargos. In fact, we have reported that polycationic polymer such as poly-l-lysine (PLL) and poly-l-arginine (PLA) translocate through negatively charged phospholipid liposome membranes. In this work, we made a comparative study of the interaction of PLL or PLA with lipid membranes consisting of negatively charged phospholipids to understand the role of basic amino acid residue (i.e. arginine and lysine) in the membrane-penetrating activity of polypeptides. PLA and PLL translocated into giant unilamellar vesicle composed of soybean phospholipids. ζ-potential and turbidity measurements demonstrated the electrostatic binding of PLL and PLA to large unilamellar vesicle (LUV). Fluorescence studies using membrane probes revealed that the binding of PLA and PLL to LUV affects the hydration and packing of the membrane interface region, in which the membrane insertion of PLA appeared to be greater than PLL. Differential scanning calorimetry showed that the enthalpy of the gel to liquid-crystalline phase transition for dipalmitoyl phosphatidylglycerol vesicle was greatly reduced by binding of PLL and PLA, in which the reduction is much larger in PLA than in PLL. Circular dichroism measurements in 2,2,2-trifluoroethanol/water mixture or in the presence of LUV indicated that the propensity of PLA to form α-helical structure is greater than PLL. Consistently, attenuated total reflection-Fourier transform infrared spectroscopy revealed that there is greater α-helical structure in PLA bound to LUV compared to PLL, which has much less ordered structure. Furthermore, isothermal titration calorimetry measurements demonstrated that the contribution of enthalpy to the energetics of binding to LUV is two-fold larger in PLA than in PLL. These results suggest that the stronger interaction of arginine residue with negatively charged phospholipid membranes compared to lysine residue appears to facilitate the conformational change in cationic polypeptide and its insertion into lipid membrane interior.     

Aggregation of cateslytin beta-sheets on negatively charged lipids promotes rigid membrane domains. A new mode of action for antimicrobial peptides?

Cateslytin, a positively charged (5+) arginine-rich antimicrobial peptide (bCgA, RSMRLSFRARGYGFR), was chemically synthesized and studied against membranes that mimic bacterial or mammalian systems. Circular dichroism, polarized attenuated total reflection infrared spectroscopy, (1)H high-resolution MAS NMR, and (2)H and (31)P solid state NMR were used to follow the interaction from peptide and membrane points of view. Cateslytin, which is unstructured in solution, is converted into antiparallel beta-sheets that aggregate mainly flat at the surface of negatively charged bacterial mimetic membranes. Arginine residues are involved in the binding to negatively charged lipids. Following the interaction of the cateslytin peptide, rigid and thicker membrane domains enriched in negatively charged lipids are found. Much less interaction is detected with neutral mammalian model membranes, as reflected by only minor percentages of beta-sheets or helices in the peptide secondary structure. No membrane destruction was detected for both bacterial and mammalian model membranes. A molecular model is proposed in which zones of different rigidity and thickness bring about phase boundary defects that ultimately lead to permeability induction and peptide crossing through bacterial membranes.     

Interactions of a synthetic Leu–Lys-rich antimicrobial peptide with phospholipid bilayers

The interaction of the synthetic antimicrobial peptide P5 (KWKKLLKKPLLKKLLKKL-NH₂) with model phospholipid membranes was studied using solid-state NMR and circular dichroism (CD) spectroscopy. P5 peptide had little secondary structure in buffer, but addition of large unilamellar vesicles (LUV) composed of dimyristoylphosphatidylcholine (DMPC) increased the β-sheet content to ~20%. Addition of negatively charged LUV, DMPC–dimyristoylphosphatidylglycerol (DMPG) 2:1, led to a substantial (~40%) increase of the α-helical conformation. The peptide structure did not change significantly above and below the phospholipid phase transition temperature. P5 peptide interacted differently with DMPC bilayers with deuterated acyl chains (d₅₄-DMPC) and mixed d₅₄-DMPC–DMPG bilayers, used to mimic eukaryotic and prokaryotic membranes, respectively. In DMPC vesicles, P5 peptide had no significant interaction apart from slightly perturbing the upper region of the lipid acyl chain with minimum effect at the terminal methyl groups. By contrast, in the DMPC–DMPG vesicles the peptide increased disorder throughout the entire acyl chain of DMPC in the mixed bilayer. P5 promoted disordering of the headgroup of neutral membranes, observed by ³¹P NMR. However, no perturbations in the T ₁ relaxation nor the T ₂- values were observed at 30°C, although a slight change in the dynamics of the headgroup at 20°C was noticeable compared with peptide-free vesicles. However, the P5 peptide caused similar perturbations of the headgroup of negatively charged vesicles at both temperatures. These data correlate with the non-haemolytic activity of the P5 peptide against red blood cells (neutral membranes) while inhibiting bacterial growth (negatively charged membranes).     

High-level expression of a truncated 1,3-1,4-β-<Emphasis Type="SmallCaps">d</Emphasis>-glucanase from <Emphasis Type="Italic">Fibrobacter succinogenes</Emphasis> in <Emphasis Type="Italic">Pichia pastoris</Emphasis> by optimization of codons and fermentation

1,3-1,4-β-d-glucanase is an important endoglycosidase in the brewing and animal feed industries. To achieve high-level expression of recombinant glucanase in Pichia pastoris, we designed sequences encoding the α-factor signal peptide from Saccharomyces cerevisiae and the truncated 1,3-1,4-β-d-glucanase from Fibrobacter succinogenes as a whole. The codons encoding the 52 amino acids of the signal peptide and 106 residues of the glucanase protein were optimized for expression in P. pastoris; 189 nucleotides were changed. The G + C content was adjusted to 48–49%, and AT-rich stretches were eliminated to avoid premature termination. The messenger ribonucleic acid secondary structure near the AUG start codon was also adjusted to ensure efficient translation; the resulting glucanase production was twofold higher compared with that achieved with gene structure optimization alone. We also propose a new fermentation strategy for the induction phase, in which 5/95% glycerol/methanol mixed feed was used in days 1–3 and 100% methanol was used on days 4–6. By comparison with methanol feed and glycerol/methanol-mixed feed alone, the yield of recombinant glucanase increased by 38.5 and 16.5%, respectively. The expressed optimized recombinant 1,3-1,4-β-d-glucanase constituted ~90% of the total secreted protein, reaching up to 3 g l⁻¹ in the medium.     

Binding of cationic pentapeptides with modified side chain lengths to negatively charged lipid membranes: Complex interplay of electrostatic and hydrophobic interactions

Basic amino acids play a key role in the binding of membrane associated proteins to negatively charged membranes. However, side chains of basic amino acids like lysine do not only provide a positive charge, but also a flexible hydrocarbon spacer that enables hydrophobic interactions. We studied the influence of hydrophobic contributions to the binding by varying the side chain length of pentapeptides with ammonium groups starting with lysine to lysine analogs with shorter side chains, namely ornithine (Orn), α,γ-diaminobutyric acid (Dab) and α, β-diaminopropionic acid (Dap). The binding to negatively charged phosphatidylglycerol (PG) membranes was investigated by calorimetry, FT-infrared spectroscopy (FT-IR) and monolayer techniques. The binding was influenced by counteracting and sometimes compensating contributions. The influence of the bound peptides on the lipid phase behavior depends on the length of the peptide side chains. Isothermal titration calorimetry (ITC) experiments showed exothermic and endothermic effects compensating to a different extent as a function of side chain length. The increase in lipid phase transition temperature was more significant for peptides with shorter side chains. FTIR-spectroscopy revealed changes in hydration of the lipid bilayer interface after peptide binding. Using monolayer techniques, the contributions of electrostatic and hydrophobic effects could clearly be observed. Peptides with short side chains induced a pronounced decrease in surface pressure of PG monolayers whereas peptides with additional hydrophobic interactions decreased the surface pressure much less or even lead to an increase, indicating insertion of the hydrophobic part of the side chain into the lipid monolayer.     

Concentration-dependent behavior of nisin interaction with supported bilayer lipid membrane

Nisin is a positively charged antibacterial peptide that binds to the negatively charged membranes of gram-positive bacteria. The initial interaction of the peptide with the model membrane of negatively charged DPPG (dipalmitoylphosphatidylglycerol) was studied by cyclic voltammetry and a.c. impedance spectroscopy. Nisin could induce pores in the supported bilayer lipid membrane, thus, it led to the marker ions Fe(CN)(6)(3-/4-) crossing the lipid membrane and giving the redox reaction on the glassy carbon electrode (GCE). Experimental results suggested that the pore formation on supported bilayer lipid membrane was dependent on the concentration of nisin and it included three main concentration stages: low, middling, high concentration.     

Grafting of polylysine with polyethylenoxide prevents demixing of O-pyromellitylgramicidin in lipid membranes

Both natural and synthetic polycations can induce demixing of negatively charged components in artificial and possibly in natural membranes. This process can result in formation of clusters (binding of several components to a polycation chain) and/or domains (aggregation of clusters and formation of a separate phase enriched in some particular component). In order to distinguish between these two phenomena, a model lipid membrane system containing ion channels, formed by a negatively charged peptide, O-pyromellitylgramicidin, and polycations of different structures was used. Microelectrophoresis of liposomes, changes in boundary potential of planar bilayers, the shape of compression curves and potentials of lipid and lipid/peptide monolayers were used to monitor the electrostatic factors in polymer adsorption to the membrane and peptide-polymer interactions. The synthesized PEO-grafted polylysine, PLL-PEO20000, did not induce peptide demixing monitored by stabilization of the gramicidin channels, in contrast to parent polylysine (PLL). Both polymers were shown to bind effectively to negatively charged liposomes and lipid monolayers, suggesting that the ineffectiveness of PLL-PEO20000 was not due to reduction of its binding. It was hypothesized that PLL-PEO20000 could not induce domain formation due to steric hindrance of long PEO chains preventing lateral fusion of clusters. Another copolymer, PLL-PEO4000, having four PEO chains of 4000 Da, exhibited intermediate effect between PLL and PLL-PEO20000, which shows the importance of the copolymer architecture for the effect on the lateral distribution of OPg channels. The model system can be relevant to regulation of lateral organization of ion channels and other components in natural membrane systems.     

Interaction of the cholesterol reducing agent simvastatin with zwitterionic DPPC and charged DPPG phospholipid membranes

Simvastatin is a lipid-lowering drug in the pharmaceutical group statins. Interaction of a drug with lipids may define its role in the system and be critical for its pharmacological activity. We examined the interactions of simvastatin with zwitterionic dipalmitoyl phosphatidylcholine (DPPC) and anionic dipalmitoyl phosphatidylglycerol (DPPG) multilamellar vesicles (MLVs) as a function of temperature at different simvastatin concentrations using Fourier transform infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC). The FTIR results indicate that the effect of simvastatin on membrane structure and dynamics depends on the type of membrane lipids. In anionic DPPG MLVs, high simvastatin concentrations (12, 18, 24 mol%) change the position of the CH₂ antisymmetric stretching mode to lower wavenumber values, implying an ordering effect. However, in zwitterionic DPPC MLVs, high concentrations of simvastatin disorder systems both in the gel and liquid crystalline phases. Moreover, in DPPG and DPPC MLVs, simvastatin has opposite dual effects on membrane dynamics. The bandwidth of the CH₂ antisymmetric stretching modes increases in DPPG MLVs, implying an increase in the dynamics, whereas it decreases in DPPC MLVs. Simvastatin caused broadening of the phase transition peaks and formation of shoulders on the phase transition peaks in DSC curves, indicating multi-domain formations in the phospholipid membranes. Because physical features of membranes such as lipid order and fluidity may be changed with the bioactivity of drugs, opposing effects of simvastatin on the order and dynamics of neutral and charged phospholipids may be critical to deduce the action mechanism of the drug and estimate drug-membrane interactions.     

Mechanism of liposome destabilization by polycationic amino acids

Polycationic amino acids induce the leakage and fusion of liposomes containing anionic lipids. We have investigated the nature and extent of the changes in membrane physical properties caused by these polypeptides which could result in the observed membrane destabilization. We found that in the range of pH 5 to pH 7 both poly-l-histidine and poly-l-lysine were ineffective in shifting the bilayer to hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine, either in the presence of absence of 1-palmitoyl-2-oleoylphosphatidylserine. We also studied the gel to liquid crystalline phase transition properties of 11 mixtures of phosphatidylserine and phosphatidylethanolamine, both in dimyristoyl forms as well as the 1-palmitoyl-2-oleoyl forms, as a function of pH and in the presence and absence of polycationic amino acids. We observed that these two lipids were largely miscible at all pH values and in the presence and absence of the polypeptides. However, there was some increased tendency for phase separation at higher pH and in the absence of polypeptide. Thus neither changes in curvature strain nor lateral phase separation induced by the polycationic amino acids could account for their marked ability to induce leakage and fusion.Phosphatidylethanolamine labelled with pyrene on one of the acyl chains gives rise to fluorescent emission from both monomer and excimer forms. The ratio of emission intensity from these two forms is indicative of lateral phase separation and the degree of lateral mobility of this probe. In equimolar mixtures of the 1-palmitoyl-2-oleoyl forms of phosphatidylserine and phosphatidylethanolamine in the liquid crystalline phase at 30 °C we find little effect of pH on the ratio of excimer to monomer emission intensity. However poly-l-lysine markedly lowers the fraction of excimer emission from these liposomes through the pH range from 5 to 7. Poly-l-histidine lowers the excimer to monomer emission ratio at pH 5 but not at pH 7. This is opposite to what one would expect for lateral phase separation and is interpreted at being the consequence of the polypeptide lowering the rate of lateral diffusion of the lipids. This effect of poly-l-histidine is observed over a range of temperatures from 0 to 40°C in both gel and liquid crystalline phases. There is no evidence from the behaviour of the pyrene fluorescent probe for lipid interdigitation. We conclude that the promotion of leakage and fusion in anionic liposomes by polycationic amino acids is not a result of large changes in the physical properties or arrangements of the lipids but rather to a surface binding of the polyamino acids.Abbreviations DSC differential scanning calorimetry - DEPE dielaidoylphosphatidylethanolamine - POPS 1-palmitoyl-2-oleoylphosphatidylethanolamine - DMPS dimyristoylphosphatidylserine - DMPE dimyristoylphosphatidylethanolamine - POPE 1-palmitoyl-2-oleoylphosphatidylethanolamine - T_H bilayer to hexagonal phase transition temperature - pyr-PE 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphoethanolaline - E/M ratio of intensities of excimer to monomer emission     

The effects of radioprotectant and potential antioxidant agent amifostine on the structure and dynamics of DPPC and DPPG liposomes

Agents capable of scavenging ROS have attracted attention recently because of their potential use as antioxidative agents. Amifostine, a ROS scavenger, has the potential to be used as an antioxidant in therapeutic applications. In this study, the effect of amifostine on neutral zwitterionic dipalmitoylphosphatidylcholine (DPPC) and anionic dipalmitoylphosphatidylglycerol (DPPG) model membranes' structure and dynamics is aimed to be examined by Fourier transform infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC). Our results revealed that amifostine at concentrations used (1–24 mol%) does not induce any important alteration in the shape of phase transition curve and phase transition temperature in the DPPC and DPPG membranes. High concentrations of amifostine slightly increased the acyl chain flexibility of DPPC membranes in the liquid crystalline phase and DPPG membranes in the gel phase. A lessening in the dynamics of DPPC liposomes was observed for all concentrations of amifostine in both phases but slight dual effect was observed only in the gel phase as a decrease in dynamics at low concentrations and an increase at higher concentrations of amifostine in DPPG liposomes. Additionally, strong hydrogen bonding was observed for both CO and PO₂⁻ groups in case of DPPC and for PO₂⁻ groups in case of DPPG. Dehydration around the CO regions occurred in case of DPPG. Accordingly, amifostine is proposed to be interacting strongly with zwitterionic and negatively charged membrane head groups and glycerol backbone in all concentrations and because of this interaction it causes some changes in lipid order and dynamics especially at high concentrations.     

l-Fucose residues on cellulose-based dialysis membranes: Quantification of membrane-associatedl-fucose and analysis of specific lectin binding

Contact of mononuclear human leukocytes with cellulose dialysis membranes may result in complement-independent cell activation, i.e. enhanced synthesis of cytokines, prostaglandins and an increase in 2-microglobulin synthesis. Cellular contact activation is specifically inhibited by the monosaccharidel-fucose suggesting that dialysis membrane associatedl-fucose residues are involved in leukocyte activation. In this study we have detected and quantitatedl-fucose on commercially-available cellulose dialysis membranes using two approaches. A sensitive enzymatic fluorescence assay detectedl-fucose after acid hydrolysis of flat sheet membranes. Values ranged from 79.3±3.6 to 90.2±5.0 pmol cm^–2 for Hemophan® or Cuprophan® respectively. Enzymatic cleavage of terminal -l-fucopyranoses with -l-fucosidase yielded 7.7±3.3 pmoll-fucose per cm² for Cuprophan. Enzymatic hydrolysis of the synthetic polymer membranes AN-69 and PC-PE did not yield detectable amounts ofl-fucose. In a second approach, binding of the fucose specific lectins ofLotus tetragonolobus andUlex europaeus (UEAI) demonstrated the presence of biologically accessiblel-fucose on the surface of cellulose membranes. Specific binding was observed with Cuprophan®, and up to 2.6±0.3 pmoll-fucose per cm² was calculated to be present from Langmuir-type adsorption isotherms. The data presented are in line with the hypothesis that surface-associatedl-fucose residues on cellulose dialysis membranes participate in leukocyte contact activation.     

Nontoxic and toxic oligopeptides with D-amino acids and unusual residues in Microcystis aeruginosa PCC 7806

Toxic and nontoxic peptides were isolated from the cyanobacterium Microcystis aeruginosa PCC 7806 by a procedure including extraction of cells with water-saturated 1-butanol, chromatography of the extract on silica gel plates and high performance liquid chromatography (HPLC) on Partisil-5. The toxin was shown to be only a minor constituent, being negatively charged and thus separable by electrophoresis, within the HPLC-purified fraction. It contained erythro-β-methyl-D-Asp, D-Glu, D-Ala, L-Leu, and L-Arg known to be part of the Microcystis peptide-toxin with M_r 994. The major part of the HPLC-purified fraction was assigned, however, to a nontoxic peptide with a M_r of 956. Partial hydrolysis studies of the nontoxic peptide(s) revealed amino acid sequences composed of D-Glu, N-methyl-Phe, and 3,4-dehydro-Pro, aside from the common L-amino acids. Cyclic linkage in the nontoxic peptide(s) appears likely.     

Deletion of all cysteines in tachyplesin I abolishes hemolytic activity and retains antimicrobial activity and lipopolysaccharide selective binding

Tachyplesin I is a cyclic beta-sheet antimicrobial peptide isolated from the hemocytes of Tachypleus tridentatus. The four cysteine residues in tachyplesin I play a structural role in imparting amphipathicity to the peptide which has been shown to be essential for its activity. We investigated the role of amphipathicity using an analogue of tachyplesin I (TP-I), CDT (KWFRVYRGIYRRR-NH(2)), in which all four cysteines were deleted. Like TP-I, CDT shows antimicrobial activity and disrupts Escherichia coli outer membrane and model membranes mimicking bacterial inner membranes at micromolar concentrations. The CDT peptide does not cause hemolysis up to 200 microg/mL while TP-I showed about 10% hemolysis at 100 microg/mL and about 25% hemolysis at 150 microg/mL. Peptide-into-lipid titrations under isothermal conditions reveal that the interaction of CDT with lipid membranes is an enthalpy-driven process. Binding assays performed using fluorometry demonstrate that the peptide CDT binds and inserts into only negatively charged membranes. The peptide-induced thermotropic phase transition of MLVs formed of DMPC and the DMPC/DMPG (7:3) mixture suggests specific lipid-peptide interactions. The circular dichroism study shows that the peptide exists as an unordered structure in an aqueous buffer and adopts a more ordered beta-structure upon binding to negatively charged membrane. The NMR data suggest that CDT binding to negatively charged bilayers induces a change in the lipid headgroup conformation with the lipid headgroup moving out of the bilayer surface toward the water phase, and therefore, a barrel stave mechanism of membrane disruption is unlikely as the peptide is located near the headgroup region of lipids. The lamellar phase (31)P chemical shift spectra observed at various concentrations of the peptide in bilayers suggest that the peptide may function neither via fragmentation of bilayers nor by promoting nonlamellar structures. NMR and fluorescence data suggest that the presence of cholesterol inhibits the peptide binding to the bilayers. These properties help to explain that cysteine residues may not contribute to antimicrobial activity and that the loss of hemolytic activity is due to lack of hydrophobicity and amphipathicity.     

Coupled changes between lipid order and polypeptide conformation at the membrane surface. A 2H NMR and Raman study of polylysine-phosphatidic acid systems

Thermotropism and segmental chain order parameters of sn-2-perdeuteriated dimyristoyl-phosphatidic acid (DMPA)-water dispersions, with and without poly(L-lysine) (PLL) of different molecular weights, have been investigated by solid-state deuterium NMR spectroscopy. The segmental chain order parameter profile of this negatively charged lipid is similar to that already found for other lipids. Addition of long PLL (MW = 200,000) increases the temperature, Tc, of the lipid gel-to-fluid phase transition, whereas short PLL (MW = 4000) has practically no effect on Tc. In the fluid phase both varieties of PLL increase the "plateau" character of segmental order parameters up to carbon position 10. At the same reduced temperature, long PLL more significantly increases the segmental ordering, especially at the methyl terminal position. This leads to the conclusion that polar head-group capping and charge neutralization by PLL induce severe changes in lipid chain ordering, even down to the bilayer core. The structure of PLL bound to the lipid bilayer surface was monitored by Raman spectroscopy, following the amide I bands. Results show that the lipid gel-to-fluid phase transition triggers a conformational transition from ordered beta-sheet to random structure of short PLL, while it does not affect the strongly stabilized beta-sheet structure of long PLL. It is concluded that both short and long PLL can efficiently cap and neutralize lipid head groups, whatever their structure, and that peptide length is a key parameter in whether lipids or peptides are the driving force in conformationally coupled changes of both partners in the membrane.     

Mitochondrial creatine kinase interaction with heterogeneous monolayers: Effect on lipid lateral organization

Our study highlights the tight relationship between protein binding to monolayers and the phase-state of the phospholipids. Interaction of mitochondrial creatine kinase with phospholipidic membranes was analysed using a two-phase monolayer system containing anionic phospholipids under chain mismatch conditions. Monolayers were made up of mixtures of DMPC/DPPG or DPPC/DMPG containing 40% negatively charged phospholipids which is approximately the negative charge content of the mitochondrial inner membrane. Langmuir isotherms of these monolayers showed that they underwent a phase transition from a liquid expanded state to a liquid-condensed phase at about 2 mN/m and 5 mN/m respectively. Interface morphology modifications caused by injection of mtCK under these monolayers at low or high surface pressure were monitored by Brewster angle microscopy. This work provides evidence that the presence at the air/water interface of discrete domains with increased charge density, may lead to difference in partition of soluble proteins such as mtCK, interacting with the lipid monolayer. Conversely these proteins may help to organize charged phospholipid domains in a membrane.