Membrane topology of a 14-mer model amphipathic peptide: a solid-state NMR spectroscopy study

We have investigated the interaction between a synthetic amphipathic 14-mer peptide and model membranes by solid-state NMR. The 14-mer peptide is composed of leucines and phenylalanines modified by the addition of crown ethers and forms a helical amphipathic structure in solution and bound to lipid membranes. To shed light on its membrane topology, 31P, 2H, 15N solid-state NMR experiments have been performed on the 14-mer peptide in interaction with mechanically oriented bilayers of dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), and dipalmitoylphosphatidylcholine (DPPC). The 31P, 2H, and 15N NMR results indicate that the 14-mer peptide remains at the surface of the DLPC, DMPC, and DPPC bilayers stacked between glass plates and perturbs the lipid orientation relative to the magnetic field direction. Its membrane topology is similar in DLPC and DMPC bilayers, whereas the peptide seems to be more deeply inserted in DPPC bilayers, as revealed by the greater orientational and motional disorder of the DPPC lipid headgroup and acyl chains. 15N{31P} rotational echo double resonance experiments have also been used to measure the intermolecular dipole-dipole interaction between the 14-mer peptide and the phospholipid headgroup of DMPC multilamellar vesicles, and the results indicate that the 14-mer peptide is in contact with the polar region of the DMPC lipids. On the basis of these studies, the mechanism of membrane perturbation of the 14-mer peptide is associated to the induction of a positive curvature strain induced by the peptide lying on the bilayer surface and seems to be independent of the bilayer hydrophobic thickness.     

Interdigitation of phosphatidylcholine and phosphatidylethanolamine mixed with complexes of acidic lipids and polymyxin B or polymyxin B nonapeptide

A fatty acid spin label, 16-doxyl-stearic acid, was used to determine the percent interdigitated lipid in mixtures of a neutral phospholipid and an acidic phospholipid. Interdigitation of the acidic lipid was induced with polymyxin B (PMB) at a mole ratio of PMB to acidic lipid of 1:5. This compound does not bind significantly to neutral lipids or induce interdigitation of the neutral lipids by themselves. The neutral lipids used were dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), or dipalmitoylphosphatidylethanolamine (DPPE), and the acidic lipids were dipalmitoylphosphatidylglycerol (DPPG) or dipalmitoylphosphatidic acid (DPPA). The percent interdigitated lipid was determined from the percent of the spin label which is motionally restricted, assuming that the spin label is homogeneously distributed in the lipid. Assuming further that 100% of the acidic lipid is interdigitated at this saturating concentration of PMB, the percentage of the neutral lipid which can become interdigitated along with it was calculated. The results indicate that about 20 mole % DPPC can be incorporated into and become interdigitated in the interdigitated bilayer of PMB/DPPG at 4 degrees C. As the temperature approaches the phase transition temperature, the lipid becomes progressively less interdigitated; this occurs to a greater degree for the mixtures than for the single acidic lipid. Thus the presence of DPPC promotes transformation of the acidic lipid to a non-interdigitated bilayer at higher temperatures. At the temperature of the lipid phase transition little or none of the lipid in the mixture is interdigitated. Thus the lipid phase transition detected by calorimetry is not that of the interdigitated bilayer. The shorter chain length DMPC can be incorporated to a greater extent than DPPC, 30-50 mol%, in the interdigitated bilayer of PMB-DPPG. This may be a result of reduced exposure of the terminal methyl groups of the shorter myristoyl chains at the polar/apolar interface of the interdigitated bilayer. Less than 29% of the total lipid was interdigitated in a DPPC/DPPA/PMB 1:1:0.2 mixture indicating that none of the DPPC in this mixture becomes interdigitated. This is attributed to the lateral interlipid hydrogen bonding interactions of DPPA which inhibits formation of an interdigitated bilayer. DPPE was found to be incorporated into the interdigitated bilayer of PMB-DPPG to a similar extent as DPPC if the amount of PMB added is sufficient to bind to only the DPPG in the mixture. Differential scanning calorimetry showed that the remaining non-interdigitated DPPE-enriched mixture phase separates into its own domain.(ABSTRACT TRUNCATED AT 400 WORDS)     

Raman spectra of planar supported lipid bilayers

Raman scattering has been used to obtain high quality vibrational spectra of planar supported lipid bilayers (pslb's) at the silica/water interface without the use of resonance or surface enhancement. A total internal reflection geometry was used both to increase the bilayer signal and to suppress the water background. Polarization control permits the determination of four components of the Raman tensor, of which three are independent for a uniaxial film. Spectra are reported of the phospholipids DMPC, DPPC, and POPC, in the C-H stretching region and the fingerprint region. The temperature-dependent polarized spectra of POPC show only small changes over the range 14-41 degrees C. The corresponding spectra of DMPC and DPPC bilayers show large thermal changes consistent with a decreasing tilt angle from the surface normal and increasing chain ordering at lower temperatures. The thermal behavior of DMPC pslb's is similar to that of vesicles of the same lipid in bulk suspension. In contrast to calorimetry, which shows a sharp phase transition (L alpha-L beta') with decreasing temperature, the changes in the Raman spectra occur over a temperature range of ca. 10 degrees C commencing at the calorimetric phase transition temperature.     

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.     

Effects of lindane on membrane fluidity: intramolecular excimerization of a pyrene derivative and polarization of diphenylhexatriene.

Fluorescence polarization studies of 1,6-diphenyl-1,3,5-hexatriene (DPH) have been compared with the excimer/monomer fluorescence intensity ratio (I'/I) of 1,3-di(2-pyrenyl)propane, (2Py(3)2Py). This ratio permits evaluation of changes in fluidity of the outer regions of the bilayer, where 2Py(3)2Py preferentially distributes. On the other hand, fluorescence polarization of DPH reports the structural order of the bilayer core. In the fluid phase of DMPC bilayers, for lindane concentrations higher than 25 microM, the excimer/monomer fluorescence intensity ratio (I'/I) decreases, thus reflecting an order increase of the probe environment. However, in the same conditions, the fluorescence polarization of DPH is almost insensitive to any perturbation. Identical results have been obtained in other pure lipid bilayers, namely DPPC and DSPC. However, both probes detect disordering effects of lindane in the gel phase of these lipids. The pyrene probe, unlike DPH, is very sensitive to the pretransitions of DPPC and DSPC, removed in the presence of lindane. Both probes fail to detect any apparent effect of lindane in DMPC bilayers enriched with high cholesterol content (greater than 30 mol%). However, in DMPC bilayers with low cholesterol content (less than 30 mol%), for temperatures below the phase transition of DMPC, both probes detect fluidizing effects induced by lindane. Nevertheless, above the phase transition of DMPC, 2Py(3)2Py detects ordering effects of lindane, whereas DPH detects hardly any effect. These results in DMPC bilayers with low cholesterol content are qualitatively similar to those described for DMPC without cholesterol.     

Homooligopeptides composed of hydrophobic amino acid residues interact in a specific manner by taking α-helix or β-structure toward lipid bilayers

In order to investigate the role of each amino acid residue in determining the secondary structure of the transmembrane segment of membrane proteins in a lipid bilayer, we made a conformational analysis by CD for lipid-soluble homooligopeptides, benzyloxycarbonyl-(Z-) Aaa_n-OEt (n = 5-7), composed of Ala, Leu, Val, and Phe, in three media of trifluoroethanol, sodium dodecyl sulfaie micelle, and phospholipid liposomes. The lipid-peptide interaction was also studied through the observation of bilayer phase transition by differential scanning cahrimetry (DSC). The CD studies showed that peptides except for Phe oligomers are present as a mainly random structure in trifluoroethanol, as a mixture of α-helix, β-sheet, β-turn, and /or random in micelles above the critical micellization concentration and preferably as an extended structure of α-helical or β-structure in dipalmitoyl-D,L -α-phosphatidylcholine (DPPC) liposomes of gel state. That the β-structure content of Val oligomers in lipid bilayers is much higher than that in micelles and the oligopeptides of Leu (n = 7) and Ala (n = 6) can take an α-helical structure with one to two turns in lipid bilayers despite their short chain lengths indicates that lipid bilayers can stabilize the extended structure of both α-helical and β-structures of the peptides. The DSC study for bilayer phase transition of DPPC / peptide mixtures showed that the Leu oligomer virtually affects neither the temperature nor the enthalpy of the transition, while Val and Ala oligomers slightly reduce the transition enthalpy without altering the transition temperature. In contrast, the Phe oligomer affects the phase transition in much more complicated manner. The decreasing tendency of the transition enthalpy was more pronounced for the Ala oligomer as compared with the Leu and Val oligomers, which means that the isopropyl group of the side chain has a less perturbing effect on the lipid acyl chain than the methyl group of Ala. © 1995 John Wiley & Sons, Inc.     

Cholesterol esterase accelerates intestinal cholesterol absorption

Lipid bilayers of dimyristoyl phosphatidylcholine (DMPC) containing opioid peptide dynorphin A(1-17) are found to be spontaneously aligned to the applied magnetic field near at the phase transition temperature between the gel and liquid crystalline states (T(m)=24 degrees C), as examined by 31P NMR spectroscopy. The specific interaction between the peptide and lipid bilayer leading to this property was also examined by optical microscopy, light scattering, and potassium ion-selective electrode, together with a comparative study on dynorphin A(1-13). A substantial change in the light scattering intensity was noted for DMPC containing dynorphin A(1-17) near at T(m) but not for the system containing A(1-13). Besides, reversible change in morphology of bilayer, from small lipid particles to large vesicles, was observed by optical microscope at T(m). These results indicate that lysis and fusion of the lipid bilayers are induced by the presence of dynorphin A(1-17). It turned out that the bilayers are spontaneously aligned to the magnetic field above T(m) in parallel with the bilayer surface, because a single 31P NMR signal appeared at the perpendicular position of the 31P chemical shift tensor. In contrast, no such magnetic ordering was noted for DMPC bilayers containing dynorphin A(1-13). It was proved that DMPC bilayer in the presence of dynorphin A(1-17) forms vesicles above T(m), because leakage of potassium ion from the lipid bilayers was observed by potassium ion-selective electrode after adding Triton X-100. It is concluded that DMPC bilayer consists of elongated vesicles with the long axis parallel to the magnetic field, together with the data of microscopic observation of cylindrical shape of the vesicles. Further, the long axis is found to be at least five times longer than the short axis of the elongated vesicles in view of simulated 31P NMR lineshape.     

Phosphatidylinositol induces fluid phase formation and packing defects in phosphatidylcholine model membranes

Liposomes consisted of phosphatidylinositol (PI) and phosphatidylcholine (PC) have been utilized as delivery vehicle for drugs and proteins. In the present work, we studied the effect of soy PI on physical properties of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes such as phase state of lipid bilayer, lipid packing and phase properties using multiple orthogonal biophysical techniques. The 6-dodecanoyl-2-dimethylamino naphthalene (Laurdan) fluorescence studies showed that presence of PI induces the formation of fluid phases in DMPC. Differential scanning calorimetry (DSC), temperature dependent fluorescence anisotropy measurements, and generalized polarization values for Laurdan showed that the presence of as low as 10mol% of PI induces substantial broadening and shift to lower temperature of phase transition of DMPC. The fluorescence emission intensity of DPH labeled, PI containing DMPC lipid bilayer decreased possibly due to deeper penetration of water molecules in lipid bilayer. In order to further delineate the effect of PI on the physico chemical properties of DMPC is due to either significant hydrophobic mismatch between the acyl chains of the DMPC and that of soy PI or due to the inositol head group, we systematically replaced soy PI with PC species of similar acyl chain composition (DPPC and 18:2 (Cis) PC) or with diacylglycerol (DAG), respectively. The anisotropy of PC membrane containing soy PI showed largest fluidity change compared to other compositions. The data suggests that addition of PI alters structure and dynamics of DMPC bilayer in that it promotes deeper water penetration in the bilayer, induces fluid phase characteristics and causes lipid packing defects that involve its inositol head group.     

Chlorpromazine interaction with glycerophospholipid liposomes studied by magic angle spinning solid state (13)C-NMR and differential scanning calorimetry

Phosphatidylserine (PS) extracted from pig brain and synthetic dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) were used to make DPPC/DMPC and DPPC/PS large unilamellar liposomes with a diameter of approximately 1 microm. Chlorpromazine-HCl (CPZ), an amphipathic cationic psychotropic drug of the phenothiazine group, is known to partition into lipid bilayer membranes of liposomes with partition coefficients depending on the acyl chain length and to alter the bilayer structure in a manner depending on the phospholipid headgroups. The effects of adding CPZ to these membranes were studied by differential scanning calorimetry and proton cross polarization solid state magic angle spinning (13)C-nuclear magnetic resonance spectroscopy (CP-MAS-(13)C-NMR). CP-MAS-(13)C-NMR spectra of the DPPC (60%)/DMPC (40%) and the DPPC (54%)/DMPC (36%)/CPZ (10%) liposomes, show that CPZ has low or no interaction with the phospholipids of this neutral and densely packed bilayer. Conversely, the DPPC (54%)/PS (36%)/CPZ (10%) bilayer at 25 degrees C demonstrates interaction of CPZ with the phospholipid headgroups (PS). This CPZ interaction causes about 30% of the acyl chains to enter the gauche conformation with low or no CPZ interdigitation among the acyl chains at this temperature (25 degrees C). The DPPC (54%)/PS (36%)/CPZ (10%) bilayer at a sample temperature of 37 degrees C (T(C)=31.2 degrees C), shows CPZ interdigitation among the phospholipids as deduced from the finding that approximately 30% of the phospholipid acyl chains carbon resonances shift low-field by 5-15 ppm.     

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)     

Modelling the phase equilibria in two-component membranes of phospholipids with different acyl-chain lengths

A phenomenological model is proposed to describe the membrane phase equilibria in binary mixtures of saturated phospholipids with different acyl-chain lengths. The model is formulated in terms of thermodynamic and thermomechanic properties of the pure lipid bilayers, specifically the chain-melting transition temperature and enthalpy, the hydrophobic bilayer thickness, and the lateral area compressibility modulus. The model is studied using a regular solution theory made up of a set of interaction parameters which directly identify that part of the lipid-lipid interaction which is due to hydrophobic mismatch of saturated chains of different lengths. It is then found that there is effectively a single universal interaction parameter which, in the full composition range, describes the phase equilibria in mixtures of DMPC/DPPC, DPPC/DSPC, DMPC/DSPC, and DLPC/DSPC, in excellent agreement with experimental measurements. The model is used to predict the variation with temperature and composition of the specific heat, as well as of the average membrane thickness and area in each of the phases. Given the value of the universal interaction parameter, the model is then used to predict the phase diagrams of binary mixtures of phospholipids with different polar head groups, e.g., DPPC/DPPE, DMPC/DPPE and DMPE/DSPC. By comparison with experimental results for these mixtures, it is shown that difference in acyl-chain lengths gives the major contribution to deviation from ideal mixing. Application of the model to mixtures with non-saturated lipids is also discussed.     

Experimental evidence for hydrophobic matching and membrane-mediated interactions in lipid bilayers containing gramicidin.

Hydrophobic matching, in which transmembrane proteins cause the surrounding lipid bilayer to adjust its hydrocarbon thickness to match the length of the hydrophobic surface of the protein, is a commonly accepted idea in membrane biophysics. To test this idea, gramicidin (gD) was embedded in 1, 2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) and 1, 2-myristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers at the peptide/lipid molar ratio of 1:10. Circular dichroism (CD) was measured to ensure that the gramicidin was in the beta6.3 helix form. The bilayer thickness (the phosphate-to-phosphate distance, or PtP) was measured by x-ray lamellar diffraction. In the Lalpha phase near full hydration, PtP is 30.8 A for pure DLPC, 32.1 A for the DLPC/gD mixture, 35.3 A for pure DMPC, and 32.7 A for the DMPC/gD mixture. Gramicidin apparently stretches DLPC and thins DMPC toward a common thickness as expected by hydrophobic matching. Concurrently, gramicidin-gramicidin correlations were measured by x-ray in-plane scattering. In the fluid phase, the gramicidin-gramicidin nearest-neighbor separation is 26.8 A in DLPC, but shortens to 23.3 A in DMPC. These experiments confirm the conjecture that when proteins are embedded in a membrane, hydrophobic matching creates a strain field in the lipid bilayer that in turn gives rise to a membrane-mediated attractive potential between proteins.     

Phospholipid miscibility in ternary mixtures

The gel to liquid-crystalline phase transition of aqueous dispersions of phospholipid mixtures was investigated by means of the repartition of the spin label 2,2,6,6-tetramethylpiperidine-I-oxyl between aqueous space and lipid hydrocarbon region. The dimyristoylphosphatidylcholine (DMPC)/dibehenoylphosphatidylcholine (DBPC) and dipalmitoylphosphatidylcholine (DPPC)/DBPC phase diagrams indicate gel phase immiscibility, whereas the distearoylphosphatidylcholine (DSPC)/DBPC phase diagram indicates non-ideal gel phase miscibility at low DBPC molar fractions. Aqueous dispersions of DMPC/DPPC/DBPC ternary mixtures show two distinct phase transitions, the first associated with the melting of a DMPC/DPPC phase and the second with the melting of a DBPC phase. Aqueous dispersions of DMPC/DSPC/DBPC ternary mixtures show to phase transitions at low DSPC molar fractions; the first is probably associated with the melting of a DMPC/DSPC phase, and the second with the melting of a DBPC/DSPC phase. At high DSPC molar fractions, only one phase transition is observed; this suggests that all the lipids are mixed in gel state membranes.     

The membrane insertion of helical antimicrobial peptides from the N-terminus of Helicobacter pylori ribosomal protein L1

The interaction of two helical antimicrobial peptides, HPA3 and HPA3P with planar supported lipid membranes was quantitatively analysed using two complementary optical biosensors. The peptides are analogues of Hp(2-20) derived from the N-terminus of Helicobacter pylori ribosomal protein L1 (RpL1). The binding of these two peptide analogues to zwitterionic dimyristoyl-phosphatidylcholine (DMPC) and negatively charged membranes composed of DMPC/dimyristoylphosphatidylglycerol (DMPG) (4:1) was determined using surface plasmon resonance (SPR) and dual polarisation interferometry (DPI). Using SPR analysis, it was shown that the proline substitution in HPA3P resulted in much lower binding for both zwitterionic and anionic membranes than HPA3. Structural changes in the planar DMPC and DMPC/DMPG (4:1) bilayers induced by the binding of both Hp(2-20) analogues were then resolved in real-time with DPI. The overall process of peptide-induced changes in membrane structure was analysed by the real-time changes in bound peptide mass as a function of bilayer birefringence. The insertion of both HPA3 and HPA3P into the supported lipid bilayers resulted in a decrease in birefringence with increasing amounts of bound peptide which reflects a decrease in the order of the bilayer. The binding of HPA3 to each membrane was associated with a higher level of bound peptide and greater membrane lipid disordering and a faster and higher degree of insertion into the membrane than HPA3P. Furthermore, the binding of both HPA3 and HPA3P to negatively charged DMPC/DMPG bilayers also leads to a greater disruption of the lipid ordering. These results demonstrate the geometrical changes in the membrane upon peptide insertion and the extent of membrane structural changes can be obtained quantitatively. Moreover, monitoring the effect of peptides on a structurally characterised bilayer has provided further insight into the role of membrane structure changes in the molecular basis of peptide selectivity and activity and may assist in defining the mode of antimicrobial action.     

Alcohol interactions with lipids: a carbon-13 nuclear magnetic resonance study using butanol labeled at C-1

The interactions of carbon-13 enriched butanol with dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) were studied using C-13 nuclear magnetic resonance. It was found that above the gel to liquid crystal phase transition the resonance from the butanol could be resolved into two signals with similar chemical shifts but different T1 values and line widths. In contrast, only one narrow resonance was observed for ethanol, which has considerably less solubility in the lipids than butanol. Thermodynamic analyses of the effects of butanol on the phase transition temperature predict much greater solubility or butanol when the lipid is above the phase transition temperature than when it is below. It was concluded that the two butanol resonances represent two slowly exchanging populations, the free butanol in the aqueous phase and butanol dissolved in the liquid crystalline region of the lipid. No bound butanol was detected below the gel to liquid crystal phase transition. Relaxation studies were performed on the resonance of the bound butanol in DPPC and DMPC, including measurements of T1, line width, and nuclear Overhauser enhancement. Theoretical analysis of the relaxation parameters indicates that the lipid-bound alcohol has very high mobility within the fluid lipid bilayer. The data are consistent with butanol being present at the aqueous boundary or head group region of the lipid.     

Physical biochemistry of a liposomal amphotericin B mixture used for patient treatment

There seems little doubt now that intravenous liposomal amphotericin B can be a useful treatment modality for the management of immunocompromised patients with suspected or proven disseminated fungal infections. Interestingly, the very significant reduction in toxicity reported when amphotericin B is part of a bilayer membrane is closely tied to the physical characteristics of the liposomes involved, although these are poorly understood at the molecular level. We record here an examination by spectroscopy and freeze-etch electron microscopy of unsonicated amphotericin B multilamellar vesicles prepared along the lines that we and others have followed for samples used in clinical trials and preclinical in vivo or in vitro studies. Our study has focussed on liposomes of 7:3 dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DMPC/DMPG) bearing 0-25 mol% amphotericin B, since this lipid mixture has been the choice for the first clinical trials. Phase transition behaviour of these liposomes was examined by electron paramagnetic resonance (EPR) spectroscopy of a nitroxide spin label partitioning into the bilayers. The same experiments were then performed on similarly prepared liposomes of the disaturated species, dipalmitoylphosphatidylcholine (DPPC), and the diunsaturated species, dielaidoylphosphatidylcholine (DEPC). Partial phase diagrams were constructed for each of the lipid/drug mixtures. Melting curves and derived phase diagrams showed evidence that amphotericin B is relatively immiscible with the solid phase of bilayer membranes. The phase diagram for DEPC/amphotericin B was very similar to that of DPPC/amphotericin B, and both exhibited less extensive temperature ranges of phase separation than did the 7:3 DMPC/DMPG mixture with amphotericin B. Between 25 and 37 degrees C the measured fluidity of the 7:3 DMPC/DMPG liposomes was similar to that of the (unsaturated fatty acid) DEPC liposomes, and considerably higher than that seen for (saturated fatty acid) DPPC liposomes. Preparations of 7:3 DMPC/DMPG, DPPC, and DEPC containing 0-25 mol% amphotericin B were examined by freeze-etch electron microscopy at 35 and 22 degrees C (to cover the temperature range of the mammalian body core and periphery). The same liposome features were present in all three liposome types studied. The appearance of individual liposomes at x 100,000 magnification reflected their molecular characteristics, which were found to be significantly heterogeneous within each batch. The lipid/drug structures were bilayer in nature, although liposomes showing considerable disruption were common, particularly at the highest drug concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Dye permeability at phase transitions in single and binary component phospholipid bilayers

By encapsulating a pH-sensitive dye, phenol red, in multilamellar liposomes of DMPC, DPPC and DMPC/DPPC mixtures, the permeability of these phospholipid bilayers to dye as a function of temperature has been studied. For both DMPC and DPPC liposomes, dye release begins well below the main gel-to-liquid-crystalline phase transition (24°C and 42°C, respectively) at temperatures corresponding to the onset of the pretransition (about 14°C and 36°C, respectively) with DPPC liposomes exhibiting a permeability anomaly at the main phase transition (42°C). The perturbation occurring in the bilayer structure that allows the release of encapsulated phenol red (approx. 5 Å diameter) is not sufficient to permit the release of encapsulated haemoglobin (approx. 20 Å diameter, negatively charged). In liposomes composed of a range of DMPC/DPPC mixtures, dye release commences at the onset of the pretransition range (determined by optical absorbance measurements) and increases with increasing temperature until the first appearance of liquid crystalline phase after which no further dye release occurs. Interestingly, the dye retaining properties of DMPC and DPPC liposomes well below their respective pretransition temperature regions are very different: DMPC liposomes release much encapsulated dye at incubation temperatures of 5°C whilst DPPC liposomes do not.     

Phase behavior and nanoscale structure of phospholipid membranes incorporated with acylated C14-peptides

The thermotropic phase behavior and lateral structure of dipalmitoylphosphatidylcholine (DPPC) lipid bilayers containing an acylated peptide has been characterized by differential scanning calorimetry (DSC) on vesicles and atomic force microscopy (AFM) on mica-supported bilayers. The acylated peptide, which is a synthetic decapeptide N-terminally linked to a C14 acyl chain (C14-peptide), is incorporated into DPPC bilayers in amounts ranging from 0-20 mol %. The calorimetric scans of the two-component system demonstrate a distinct influence of the C14-peptide on the lipid bilayer thermodynamics. This is manifested as a concentration-dependent downshift of both the main phase transition and the pretransition. In addition, the main phase transition peak is significantly broadened, indicating phase coexistence. In the AFM imaging scans we found that the C14-peptide, when added to supported gel phase DPPC bilayers, inserts preferentially into preexisting defect regions and has a noticeable influence on the organization of the surrounding lipids. The presence of the C14-peptide gives rise to a laterally heterogeneous bilayer structure with coexisting lipid domains characterized by a 10 A height difference. The AFM images also show that the appearance of the ripple phase of the DPPC lipid bilayers is unaffected by the C14-peptide. The experimental results are supported by molecular dynamics simulations, which show that the C14-peptide has a disordering effect on the lipid acyl chains and causes a lateral expansion of the lipid bilayer. These effects are most pronounced for gel-like bilayer structures and support the observed downshift in the phase-transition temperature. Moreover, the molecular dynamics data indicate a tendency of a tryptophan residue in the peptide sequence to position itself in the bilayer headgroup region.     

Phospholipid structure determines the effects of peptides on membranes. Differential scanning calorimetry studies with pentagastrin-related peptides

The effect of phospholipid structure on the interaction between small peptides and phospholipid membranes has been studied by high-sensitivity differential scanning calorimetry. The peptides used, N-Boc-beta-Ala-Trp-Met-Arg-Phe-NH2 and N-Boc-beta-Ala-Trp-Met-Lys-Phe-NH2, are basic analogs of the hormone pentagastrin. These peptides split the gel-to-liquid crystalline phase transition of synthetic phosphatidylcholines into two components. For dimyristoyl (DMPC), dipalmitoyl (DPPC) and 1-stearoyl-2-oleoyl (SOPC) phosphatidylcholines, one component remains at the temperature corresponding to that of pure lipid and the other one is shifted towards higher temperatures. With increasing peptide concentration there is a gradual increase in the enthalpy of the high-temperature component at the expense of the low-temperature one, and there is also an increase in the total enthalpy of the transition. A mixture of the peptide with distearoylphosphatidylcholine (DSPC) behaves differently, with the transition occurring at a temperature below that of the pure lipid increasing with peptide concentration. The susceptibility of various phosphatidylcholines to perturbation by the peptides increases in the order DMPC greater than SOPC greater than DPPC greater than DSPC. The effect of these peptides on the phase transitions of acidic phosphatidylglycerols is generally greater than with the corresponding phosphatidylcholines, but the dependence on the length of lipid hydrocarbon chains is similar. Perturbation of the thermotropic phase transition is strongest for dimyristoylphosphatidylglycerol, followed by the dipalmitoyl and the distearoyl analogs. The effect of the peptides on the phase transition of dimyristoylphosphatidylserine is significantly smaller compared to that observed with dimyristoylphosphatidylglycerol and it is further reduced for dimyristoylphosphatidic acid. The phase transition of this latter lipid remains virtually unchanged, even in the presence of high concentrations of the peptide. Similar resistance to the perturbation of the phase transitions by the peptides is observed for synthetic phosphatidylethanolamine. The different susceptibility of various phospholipids to perturbation by the peptides is suggested to be related to different degrees of intermolecular interaction between phospholipid molecules, and particularly to different abilities of phospholipids to form intermolecular hydrogen bonding.