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.     

Effect of calcium on phospholipid interaction with pulmonary surfactant protein C.

Porcine pulmonary surfactant-associated protein SP-C was incorporated into bilayers of chain-perdeuterated dipalmitoylphosphatidylglycerol (DPPG-d62) and chain-perdeuterated dipalmitoyl-phosphatidylcholine (DPPC-d62) and into bilayers containing 70 mol% dipalmitoyl-phosphatidylcholine (DPPC) and 30 mol% DPPG-d62 or 70 mol% DPPC-d62 and 30 mol% dipalmitoylphosphatidylglycerol (DPPG). The effect of SP-C on the phase behavior, lipid chain order, and dynamics in these bilayers was examined by using deuterium nuclear magnetic resonance. SP-C was found to have a similar effect on the chain order and phase behavior of DPPC-d62 and DPPG-d62 in bilayers with a single lipid component. In gel phase DPPC/DPPG (7:3) bilayers with one or the other lipid component chain-perdeuterated, SP-C was found to affect first spectral moment more strongly for DPPG-d62 than for DPPC-d62. This may indicate that SP-C induced a nonrandom lateral distribution in the mixed lipid bilayer. SP-C was also found to influence motions responsible for deuteron transverse relaxation in both the gel and liquid crystalline phases. The presence of 5 mM Ca2+ in the aqueous phase substantially altered the effect of SP-C on transverse relaxation in the bilayer.     

Effects of diacylglycerols on the structure of phosphatidylcholine bilayers: a 2H and 31P NMR study

The interaction of four diacylglycerols (DAGs) with multilamellar phospholipid bilayers consisting either of dipalmitoylphosphatidylcholine (DPPC) or of a mixture of DPPC and bovine liver phosphatidylcholine (BL-PC) extracts was investigated by a combination of 31P and 2H NMR spectrometry. We found that saturated and unsaturated long-chain DAGs induce different types of perturbations into the bilayer structure. The saturated DAGs dipalmitin and distearin induce lateral phase separation of the lipids into (i) DAG-enriched gellike domains and (ii) relatively DAG-free regions in the liquid-crystalline phase. In the latter regions, the order parameters along the fatty acyl chains of DPPC are practically identical with the control. This phase separation effect was observed in both model systems studied, and its extent is dependent upon DAG concentration and temperature. Only bilayer phases were present upon addition of dipalmitin or distearin at all concentrations and temperatures studied. The unsaturated DAGs diolein and DAG derived from egg PC (egg-DAG) affect PC bilayers in the following two ways: (i) by increasing the order parameters of the side chains, as observed for both DPPC and BL-PC model systems; (ii) by inducing nonbilayer lipid phases, as observed for BL-PC, but not DPPC. At a concentration of 25 mol % of an unsaturated DAG in mixed PC bilayers, a peak corresponding to isotropic lipid conformation appeared and increased in intensity with increase in temperature, while at 32 mol % hexagonal and bilayer phases coexisted.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermotropic and barotropic phase transitions of N-methylated dipalmitoylphosphatidylethanolamine bilayers

In order to understand the effect of polar head group modification on the thermotropic and barotropic phase behavior of phospholipid bilayer membranes, the phase transitions of dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidyl-N-methylethanolamine (DPMePE), dipalmitoylphosphatidyl-N,N-dimethylethanolamine (DPMe2PE) and dipalmitoylphosphatidylcholine (DPPC) bilayer membranes were observed by differential scanning calorimetry and high-pressure optical methods. The temperatures of the so-called main transition from the gel (L(beta)) or ripple gel (P(beta)') phase to the liquid crystalline (L(alpha)) phase were almost linearly elevated by applying pressure. The slope of the temperature-pressure boundary, dT/dp, was in the range of 0.220-0.264 K MPa(-1) depending on the number of methyl groups in the head group of lipids. The main-transition temperatures of N-methylated DPPEs decreased with increasing size of head group by stepwise N-methylation. On the other hand, there was no significant difference in thermodynamic quantities of the main transition between the phospholipids. With respect to the transition from the subgel (L(c)) phase to the lamellar gel (L(beta) or L(beta)') phase, the transition temperatures were also elevated by applying pressure. In the case of DPPE bilayer the L(c)/L(beta) transition appeared at a pressure higher than 21.8 MPa. At a pressure below 21.8 MPa the L(c)/L(alpha) transition was observed at a temperature higher than the main-transition temperature. The main (L(beta)/L(alpha)) transition can be recognized as the transformation between metastable phases in the range from ambient pressure to 21.8 MPa. Polymorphism in the gel phase is characteristic of DPPC bilayer membrane unlike other lipid bilayers used in this study: the L(beta)', P(beta)' and pressure-induced interdigitated gel (L(beta)I) phases were observed only in the DPPC bilayer. Regarding the bilayers of DPPE, DPMePE and DPMe2PE, the interdigitation of acyl chain did not appear even at pressures as high as 200 MPa.     

Atomic force microscopy studies of ganglioside GM1 domains in phosphatidylcholine and phosphatidylcholine/cholesterol bilayers.

The distribution of ganglioside in supported lipid bilayers has been studied by atomic force microscopy. Hybrid dipalmitoylphosphatidylcholine (DPPC)/dipalmitoylphosphatidylethanolamine (DPPE) and (2:1 DPPC/cholesterol)/DPPE bilayers were prepared using the Langmuir Blodgett technique. Egg PC and DPPC bilayers were prepared by vesicle fusion. Addition of ganglioside GM1 to each of the lipid bilayers resulted in the formation of heterogeneous surfaces that had numerous small raised domains (30--200 nm in diameter). Incubation of these bilayers with cholera toxin B subunit resulted in the detection of small protein aggregates, indicating specific binding of the protein to the GM1-rich microdomains. Similar results were obtained for DPPC, DPPC/cholesterol, and egg PC, demonstrating that the overall bilayer morphology was not dependent on the method of bilayer preparation or the fluidity of the lipid mixture. However, bilayers produced by vesicle fusion provided evidence for asymmetrically distributed GM1 domains that probably reflect the presence of ganglioside in both inner and outer monolayers of the initial vesicle. The results are discussed in relation to recent inconsistencies in the estimation of sizes of lipid rafts in model and natural membranes. It is hypothesized that small ganglioside-rich microdomains may exist within larger ordered domains in both natural and model membranes.     

Development and modeling of arsenic-trioxide–loaded thermosensitive liposomes for anticancer drug delivery

In this article, a novel delivery system for the anticancer drug, arsenic trioxide (ATO), is characterized. The release of ATO from DPPC liposomes with MPPC lysolipid incorporated into the bilayer was measured. Upon heating the liposomes to 37°C, there was a 15–25% release over 24 hours. The ATO release from the DPPC and DPPC:MPPC (5%) systems leveled off after 10 hours at 37°C, whereas the DPPC:MPPC (10%) liposomes continue to release ATO over the 24-hour time span. Upon heating the liposomes rapidly to 42°C, the release rate was substantially increased. The systems containing lysolipids exhibited a very rapid release of a significant amount of arsenic in the first hour. In the first hour, the DPPC:MPPC (5%) liposomes released 40% of the arsenic and the DPPC:MPPC (10%) liposomes released 55% of the arsenic. Arsenic release from pure DPPC liposomes was comparable at 37 and 42°C, indicating that the presence of a lysolipid is necessary for a significant enhancement of the release rate. A coarse-grained molecular dynamics (CGMD) model was used to investigate the enhanced permeability of lysolipid-incorporated liposomes and lipid bilayers. The CG liposomes did not form a gel phase when cooled due to the high curvature; however, permeability was still significantly lower below the liquid-to-gel phase-transition temperature. Simulations of flat DPPC:MPPC bilayers revealed that a peak in the permeability did coincide with the phase transition from the gel to LC state when the lysolipid, MPPC, was present. No pores were observed in the simulations, so it is unlikely this was the permeability-enhancing mechanism.     

Mixing of perfluorinated carboxylic acids with dipalmitoylphosphatidylcholine

Perfluorinated acids are emerging as an important class of persistent environmental pollutant, thus raising human health concerns. To understand the behavior of these compounds in biological systems, the mixing behavior of two perfluorinated acids, perfluorododecanoic and perfluorotetradecanoic acid, with dipalmitoylphosphatidylcholine (DPPC) was studied in monolayers at the air-water interface and in fully hydrated DPPC bilayers. The mixing behavior of both acids was indicative of an attractive interaction and partial miscibility with DPPC at the air-water interface. In the bilayer studies, the fluorinated acids cause peak broadening and elimination of the pretransition of DPPC. The onset temperature of the main phase transition remains constant in the presence of the fluorinated acids suggesting immiscibilities in the gel phase. Below X(DPPC) = 0.97 significant peak broadening of the main phase transition can be observed. These results suggest strong interaction between the respective acid and DPPC, and that both acids are able to partition into the lipid bilayer. However, their mixing behavior is far from ideal, thus suggesting the presence of domains or lipid aggregates with high acid concentrations which may (adversely) impact the function of biological mono- and bilayers.     

Mixing of perfluorinated carboxylic acids with dipalmitoylphosphatidylcholine

Perfluorinated acids are emerging as an important class of persistent environmental pollutant, thus raising human health concerns. To understand the behavior of these compounds in biological systems, the mixing behavior of two perfluorinated acids, perfluorododecanoic and perfluorotetradecanoic acid, with dipalmitoylphosphatidylcholine (DPPC) was studied in monolayers at the air-water interface and in fully hydrated DPPC bilayers. The mixing behavior of both acids was indicative of an attractive interaction and partial miscibility with DPPC at the air-water interface. In the bilayer studies, the fluorinated acids cause peak broadening and elimination of the pretransition of DPPC. The onset temperature of the main phase transition remains constant in the presence of the fluorinated acids suggesting immiscibilities in the gel phase. Below X(DPPC)=0.97 significant peak broadening of the main phase transition can be observed. These results suggest strong interaction between the respective acid and DPPC, and that both acids are able to partition into the lipid bilayer. However, their mixing behavior is far from ideal, thus suggesting the presence of domains or lipid aggregates with high acid concentrations which may (adversely) impact the function of biological mono- and bilayers.     

Thermotropic and barotropic phase transitions in bilayer membranes of ether-linked phospholipids with varying alkyl chain lengths

The bilayer phase transitions of a series of ether-linked phospholipids, 1,2-dialkylphosphatidylcholines containing linear saturated alkyl chain (C(n)=12, 14, 16 and 18), were observed by differential scanning calorimetry (DSC) under ambient pressure and light-transmittance measurements under high pressure. The thermodynamic quantities of the pre- and main-transitions for the ether-linked PC bilayer membranes were calculated and compared with those of a series of ester-linked PCs, 1,2-diacylphosphatidylcholines. The thermodynamic quantities of the main transition for the ether-linked PC bilayers showed distinct dependence on alkyl-chain length and were slightly different from those of the ester-linked PC bilayers. From the comparison of thermodynamic quantities for the main transition between both PC bilayers, we revealed that the attractive interaction in the gel phase for the ether-linked PC bilayers is weaker than that for the ester-linked PC bilayers. Regarding the pretransition, although changes in enthalpy and entropy for both PC bilayers were comparable to each other, the volume changes of the ether-linked PC bilayers roughly doubled those of the ester-linked PC bilayers. The larger volume change results from the smallest partial molar volume of the ether-linked PC molecule in the interdigitated gel phase. Further, we constructed the temperature-pressure phase diagrams for the ether-linked PC bilayers by using the phase-transition data. The region of the interdigitated gel phase in the phase diagrams was extended by applying pressure and by increasing the alkyl-chain length of the molecule. Comparing the phase diagrams with those for the ester-linked PC bilayers, it was proved that the phase behavior of the ester-linked PC bilayers under high temperature and pressure is almost equivalent to that of the ether-linked PC bilayers in the vicinity of ambient pressure.     

Decreased lipid order induced by microsomal cytochrome P-450 and NADPH-cytochrome P-450 reductase in model membranes: fluorescence and electron spin resonance studies

Cytochrome P-450 and NADPH-cytochrome P-450 reductase were reconstituted in unilamellar lipid vesicles prepared by the cholate dialysis technique from pure dimyristoylphosphatidylcholine (DMPC), pure dipalmitoylphosphatidylcholine (DPPC), pure dioleoylphosphatidylcholine (DOPC), and phosphatidylcholine/phosphatidylethanolamine/phosphatidylserine (PC/PE/PS) (10:5:1). As probes for the vesicles' hydrocarbon region, 1,6-diphenyl-1,3,5-hexatriene (DPH) and spin-labeled PC were used. The steady-state and time-resolved fluorescence parameters of DPH were determined as a function of temperature and composition of liposomes. Incorporation of either protein alone or together increased the steady-state fluorescence anisotropy (rs) of DPH in DOPC and PC/PE/PS (10:5:1) liposomes. In DMPC and DPPC vesicles, the proteins decreased rs significantly below the transition temperature (Tc) of the gel to liquid-crystalline phase transition. Time-resolved fluorescence measurements of DPH performed in reconstituted PC/PE/PS and DMPC proteoliposomes showed that the proteins disorder the bilayer both in the gel and in the liquid-crystalline phase. Little disordering by the proteins was observed by a spin-label located near the mid-zone of the bilayer 1-palmitoyl-2-(5-doxylstearoyl)-3-sn-phosphatidylcholine (8-doxyl-PC), whereas pronounced disordering was detected by 1-palmitoyl-2-(8-doxylpalmitoyl)-3-sn-phosphatidylcholine (5-doxyl-PC), which probes the lipid zone closer to the polar part of the membrane. Fluorescence lifetime measurements of DPH indicate an average distance of greater than or equal to 60 A between the heme of cytochrome P-450 and DPH.     

Miscibility of Sphingomyelins and Phosphatidylcholines in Unsaturated Phosphatidylcholine Bilayers

Polyunsaturated phospholipids are common in biological membranes and affect the lateral structure of bilayers. We have examined how saturated sphingomyelin (SM; palmitoyl and stearoyl SM (PSM and SSM, respectively)) and phosphatidylcholine (PC; dipalmitoyl PC and 1-palmitoyl-2-stearoyl PC (DPPC and PSPC, respectively)) segregate laterally to form ordered gel phases in increasingly unsaturated PC bilayers (sn-1: 16:0 and sn-2: 18:1...22:6; or sn-1 and sn-2: 18:1…22:6). The formation of gel phases was determined from the lifetime analysis of trans-parinaric acid. Using calorimetry, we also determined gel phase formation by PSM and DPPC in unsaturated PC mixed bilayers. Comparing PSM with DPPC, we observed that PSM formed a gel phase with less order than DPPC at comparable bilayer concentrations. The same was true when SSM was compared with PSPC. Furthermore, we observed that at equal saturated phospholipid concentration, the gel phases formed were less ordered in unsaturated PCs having 16:0 in sn-1, as compared to PCs having unsaturated acyl chains in both sn-1 and sn-2. The gel phases formed by the saturated phospholipids in unsaturated PC bilayers did not appear to achieve properties similar to pure saturated phospholipid bilayers, suggesting that complete lateral phase separation did not occur. Based on scanning calorimetry analysis, the melting of the gel phases formed by PSM and DPPC in unsaturated PC mixed bilayers (at 45 mol % saturated phospholipid) had low cooperativity and hence most likely were of mixed composition, in good agreement with trans-parinaric acid lifetime data. We conclude that both interfacial properties of the saturated phospholipids and their chain length, as well as the presence of 16:0 in sn-1 of the unsaturated PCs and the total number of cis unsaturations and acyl chain length (18 to 22) of the unsaturated PCs, all affected the formation of gel phases enriched in saturated phospholipids, under the conditions used.     

Perturbations induced by alpha- and beta-endosulfan in lipid membranes: a DSC and fluorescence polarization study.

The interaction of alpha- and beta-endosulfan isomers with lipid bilayers was searched by differential scanning calorimetry (DSC) and fluorescence polarization of 2-, 6- and 12-(9-anthroyloxy) stearic acids (2-AS, 6-AS and 12-AS) and 16-(9-anthroyloxy) palmitic acid (16-AP). Both endosulfan isomers, at insecticide/lipid molar ratios ranging from 1/40 to 1/1, shift the phase transition midpoint to lower temperature values and broaden the transition profile of dipalmitoylphosphatidylcholine (DPPC) bilayers. At insecticide/lipid molar ratios of 1/40, the isomers fully abolish the bilayer pretransition. Conversely to beta-endosulfan, alpha-endosulfan promotes a new phase transition, centered at 35.4 degrees C, in addition to the main phase transition of DPPC. Therefore, the alpha-isomer may undergo a heterogeneous distribution in separate domains in the plane of the membrane, whereas the beta-isomer may undergo a homogeneous distribution. Fluorescence polarization data indicate that alpha-endosulfan increases the lipid structural order in the regions probed by 2-AS and decreases it in the regions probed by 6-AS, 12-AS and 16-AP. On the other hand, the beta-isomer produces disordering effects in the upper regions of the bilayers, probed by 2-AS, and ordering in deeper regions, probed by 6-AS, 12-AS and 16-AP, mainly in the gel phase. The incorporation of cholesterol into DPPC bilayers progressively decreases the effects of beta-isomer which are vanished at 20 mol% cholesterol. However, this and higher cholesterol concentrations did not prevent alpha-endosulfan membrane interaction, as revealed by DSC and fluorescence polarization. The distinct effects promoted by alpha- and beta-endosulfan are discussed in terms of molecular orientation and positioning within the bilayer. Apparently, the alpha-isomer preferentially locates closer to the phospholipid headgroups whereas the beta-isomer distributes in deeper domains of the bilayer.     

The alterations of lipid bilayer fluidity induced by newly synthesized phenothiazine derivative

Using fluorescence spectroscopy, calorimetry and ESR the interactions of the phenothiazine derivative 2-trifluoromethyl-10-(4-[methylsulfonylamid]buthyl)-phenothiazine (FPhMS) with lipids were studied. Calorimetry showed biphasic effect of FPhMS on main phase transition of DPPC. At molar ratios up to 0.06 drug induced decrease of transition temperature and enthalpy, while at higher concentrations it caused subsequent increase of these parameters. For all concentrations studied we observed gradual broadening of transition peaks. Fluorescence polarization revealed that in FPhMS/lipid mixtures, order in bilayers is decreased in the gel state and increased in the liquid crystalline state. ESR experiment showed that at molar ratio of 0.06, FPhMS reduces the mobility of spin probes located in both polar and hydrophobic regions. Comparing observed effects with those reported for cholesterol/lipid mixtures, we conclude that at higher concentrations FPhMS presumably induces a new mode of bilayer packing. This structure is less co-operative than an unperturbed bilayer, but locally the mobility of lipid molecules is decreased.     

Effect of variations in the structure of a polyleucine-based alpha-helical transmembrane peptide on its interaction with phosphatidylethanolamine Bilayers

High-sensitivity differential scanning calorimetry and Fourier transform infrared spectroscopy were used to study the interaction of a cationic alpha-helical transmembrane peptide, acetyl-Lys2-Leu24-Lys2-amide (L24), and members of the homologous series of zwitterionic n-saturated diacyl phosphatidylethanolamines (PEs). Analogs of L24, in which the lysine residues were replaced by 2,3-diaminopropionic acid (acetyl-DAP2-Leu24-DAP2-amide (L24DAP)) or in which a leucine residue at each end of the polyleucine sequence was replaced by a tryptophan (Ac-K2-W-L22-W-K2-amide (WL22W)), were also studied to investigate the roles of lysine side-chain snorkeling and aromatic side-chain interactions with the interfacial region of phospholipid bilayers. The gel/liquid-crystalline phase transition temperature of the PE bilayers is altered by these peptides in a hydrophobic mismatch-independent manner, in contrast to the hydrophobic mismatch-dependent manner observed previously with zwitterionic phosphatidylcholine (PC) and anionic phosphatidylglycerol (PG) bilayers. Moreover, all three peptides reduce the phase transition temperature to a greater extent in PE bilayers than in PC and PG bilayers, indicating a greater disruption of PE gel-phase bilayer organization. Moreover, the lysine-anchored L24 reduces the phase transition temperature, enthalpy, and the cooperativity of PE bilayers to a much greater extent than DAP-anchored L24DAP, whereas replacement of the terminal leucines by tryptophan residues (Ac-K2-W-L22-W-K2-amide) only slightly attenuates the effects of this peptide on the chain-melting phase transition of the host PE bilayers. All three peptides form very stable alpha-helices in PE bilayers, but small conformational changes occur in response to mismatch between peptide hydrophobic length and gel-state lipid bilayer hydrophobic thickness. These results suggest that the lysine snorkeling plays a significant role in the peptide-PE interactions and that cation-pi-interactions between lysine and tryptophan residues may modulate these interactions. Altogether, these results suggest that the lipid-peptide interactions are affected not only by the hydrophobic mismatch between these peptides and the host lipid bilayer but also by the electrostatic and hydrogen-bonding interactions between the positively charged lysine residues at the termini of these peptides and the polar headgroups of PE bilayers.     

Kinetic parameters of the interactions of retinol with lipid bilayers

The process of transfer of vitamin A alcohol (retinol) between unilamellar vesicles of phosphatidylcholine was studied. The transfer was found to proceed spontaneously by hydration from the bilayer and diffusion through the aqueous phase. The rate-limiting step for transfer was the dissociation from the bilayer, a step that was characterized in bilayers of egg phosphatidylcholine (PC) by a rate constant koff = 0.64 s-1. The rate constant for association of retinol with bilayers of egg PC was also determined: kon = 2.9 x 10(6) s-1. The relative avidities for retinol of vesicles comprised of PC lipids with the various fatty acyl chains were measured. It was found that the binding affinity was determined by the composition of the lipids, such that PC with symmetric acyl chains had a lower affinity for retinol vs those with mixed chains. To clarify the mechanism underlying this observation, the rates of dissociation and association of retinol bound to vesicles of dioleoyl-PC were determined. The rate of association of retinol with bilayers strongly depended on the composition of the fatty acyl chains of the lipids. The rate of dissociation of retinol from the bilayers of PC was found to be independent of that composition. The implications of the observations for the interactions of hydrophobic ligands with lipid bilayers are discussed.     

Increased adhesion between neutral lipid bilayers: interbilayer bridges formed by tannic acid.

Tannic acid (TA) is a naturally occurring polyphenolic compound that aggregates membranes and neutral phosolipid vesicles and precipitates many proteins. This study analyzes TA binding to lipid membranes and the ensuing aggregation. The optical density of dispersions of phosphatidylcholine (PC) vesicles increased upon the addition of TA and electron micrographs showed that TA caused the vesicles to aggregate and form stacks of tightly packed disks. Solution calorimetry showed that TA bound to PC bilayers with a molar binding enthalpy of -8.3 kcal/mol and zeta potential measurements revealed that TA imparted a small negative charge to PC vesicles. Monolayer studies showed that TA bound to PC with a dissociation constant of 1.5 microM and reduced the dipole potential by up to 250 mV. Both the increase in optical density and decrease in dipole potential produced by TA could be reversed by the addition of polyvinylpyrrolidone, a compound that chelates TA by providing H-bond acceptor groups. NMR, micropipette aspiration, and x-ray diffraction experiments showed that TA incorporated into liquid crystalline PC membranes, increasing the area per lipid molecule and decreasing the bilayer thickness by 2 to 4%. 2H-NMR quadrupole splitting measurements also showed that TA associated with a PC molecule for times much less than 10(-4) s. In gel phase bilayers, TA caused the hydrocarbon chains from apposing monolayers to fully interdigitate. X-ray diffraction measurements of both gel and liquid crystalline dispersions showed that TA, at a critical concentration of about 1 mM, reduced the fluid spacing between adjacent bilayers by 8-10 A. These data place severe constraints on how TA can pack between adjacent bilayers and cause vesicles to adhere. We conclude that TA promotes vesicle aggregation by reducing the fluid spacing between bilayers by the formation of transient interbilayer bridges by inserting its digallic acid residues into the interfacial regions of adjacent bilayers and spanning the interbilayer space.     

Interaction of ceramides with phosphatidylcholine, sphingomyelin and sphingomyelin/cholesterol bilayers

Ceramides (Cers) may exert their biological activity through changes in membrane structure and organization. To understand this mechanism, the effect of Cer on the biophysical properties of phosphatidylcholine, sphingomyelin (SM) and SM/cholesterol bilayers was determined using fluorescence probe techniques. The Cers were bovine brain Cer and synthetic Cers that contained a single acyl chain species. The phospholipids were 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glyero-3-phosphocholine (DPPC) and bovine brain, egg yolk and bovine erythrocyte SM. The addition of Cer to POPC and DPPC bilayers that were in the liquid-crystalline phase resulted in a linear increase in acyl chain order and decrease in membrane polarity. The addition of Cer to DPPC and SM bilayers also resulted in a linear increase in the gel to liquid-crystalline phase transition temperature (T(M)). The magnitude of the change was dependent upon Cer lipid composition and was much higher in SM bilayers than DPPC bilayers. The addition of 33 mol% cholesterol essentially eliminated the thermal transition of SM and SM/Cer bilayers. However, there is still a linear increase in acyl chain order induced by the addition of Cer. The results are interpreted as the formation of DPPC/Cer and SM/Cer lipid complexes. SM/Cer lipid complexes have higher T(M)s than the corresponding SM because the addition of Cer reduces the repulsion between the bulky headgroup and allows closer packing of the acyl chains. The biophysical properties of a SM/Cer-rich bilayer are dependent upon the amount of cholesterol present. In a cholesterol-poor membrane, a sphingomyelinase could catalyze the isothermal conversion of a liquid-crystalline SM bilayer to a gel phase SM/Cer complex at physiological temperature.     

Galactocerebroside-phospholipid interactions in bilayer membranes.

Differential scanning calorimetry (DSC) and x-ray diffraction have been used to study the interaction of hydrated N-palmitoylgalactosylsphingosine (NPGS) and dipalmitoylphosphatidylcholine (DPPC). For mixtures containing less than 23 mol% NPGS, complete miscibility of NPGS into hydrated DPPC bilayers is observed in both the bilayer gel and liquid-crystal phases. X-ray diffraction data demonstrate insignificant differences in the DPPC-bilayer gel-phase parameters on incorporation of up to 23 mol% NPGS. At greater than 23 mol% NPGS, additional high-temperature transitions occur, indicating phase separation of cerebroside. For these cerebroside concentrations, at 20 degrees C, x-ray diffraction shows two lamellar phases, hydrated DPPC-NPGS gel bilayers (d = 64 A) containing 23 mol% NPGS, and NPGS "crystal" bilayers (d = 55 A). On heating to temperatures greater than 45 degrees C, the mixed DPPC-NPGS bilayer phase undergoes chain melting, and on further increasing the temperature progressively more NPGS is incorporated into the liquid-crystal DPPC-NPGS bilayer phase. At temperatures greater than 82 degrees C (the transition temperature of hydrated NPGS), complete lipid miscibility is observed at all DPPC/NPGS molar ratios.     

The interaction of polyphenols with bilayers: conditions for increasing bilayer adhesion.

Because proteins and other molecules with a high polyphenol content are commonly involved in adhesion processes, we are investigating the interactions between polyphenols and biological materials. A naturally occurring polyphenol that binds a variety of proteins and lipids is tannic acid (TA), which contains five digallic acid residues covalently linked to a central D-glucose. A previous study has shown that TA increases the adhesion between apposing phosphatidylcholine (PC) bilayers and over a very narrow concentration range collapses the interbilayer fluid space from about 15 A to 5 A. To determine the chemical requirements a polyphenolic molecule must possess to increase bilayer adhesion, we have synthesized several simpler TA analogs that vary in their size, shape, and number of gallic acid and hydroxyl groups. X-ray diffraction, absorbance, binding, and differential scanning calorimetry measurements were used to investigate the interaction of these polyphenolic molecules with egg PC (EPC) and dipalmitoyl PC (DPPC) bilayers. Of these synthetic polyphenols, only penta-O-galloyl-alpha-D-glucose (PGG) was able to completely mimic the effects of TA by collapsing the interbilayer fluid space from 15 A to 5 A, decreasing the dipole potential by about 300 mV, increasing the transition enthalpy of DPPC liposomes, and inducing an interdigitated phase in DPPC. Binding studies indicated that the fluid space was reduced to 5 A at an EPC:PGG mole ratio of 5:1. We conclude that these polyphenols collapse the fluid space of PC bilayers because they 1) are amphipathic and partition into the bilayers interfacial region, 2) are long enough to span the interbilayer space, 3) contain several gallic acids distributed so that they can partition simultaneously into apposing bilayers, and 4) have sufficient gallic acid residues to interact with all lipid headgroups and cover the bilayer surface. Under these conditions we conclude that the polyphenols from interbilayer bridges. We argue that these bridges are stabilized by increased adhesion arising from an increased van der Waals interaction between apposing bilayers, electrostatic interactions between the pi electrons in the phenol ring and the -(N+CH3)3 groups on the PC headgroups, decreased hydration repulsion between bilayers, and hydrogen bonds between the H-bond-donating moieties on the polyphenols and H-bond-accepting groups in the bilayer.     

Perturbation of DPPC/POPG bilayers by the N-terminal helix of lung surfactant protein SP-B: a <Superscript>2</Superscript>H NMR study

SP-B_8–25 is a synthetic peptide comprising the N-terminal helix of the essential lung surfactant protein SP-B. Rat lung oxygenation studies have shown that SP-B_8–25 retains some of the function of full-length SP-B. We have used deuterium nuclear magnetic resonance (²H-NMR) to examine the influence of SP-B_8–25 on the mixing properties of saturated PC and unsaturated PG lipids in model mixed lipid bilayers containing dipalmitoylphosphatidylcholine (DPPC) and palmitoyl-oleoyl-phosphatidylglycerol (POPG), in a molar ratio of 7:3. In the absence of the peptide, ²H-NMR spectra of DPPC/POPG mixtures, with one or the other lipid component deuterated, indicate coexistence of large liquid crystal and gel domains over a range of about 10°C through the liquid crystal to gel transition of the bilayer. Addition of SP-B_8–25 has little effect on the width of the transition but the spectra through the transition range cannot be resolved into distinct liquid crystal and gel spectral components suggesting that the peptide interferes with the tendency of the DPPC and POPG lipid components in this mixture to phase separate near the bilayer transition temperature. Quadrupole echo decay observations suggest that the peptide may also reduce differences in the correlation times for local reorientation of the two lipids. These observations suggest that SP-B_8–25 promotes a more thorough mixing of saturated PC and unsaturated PG components and may be relevant to understanding the behaviour of lung surfactant material under conditions of lateral compression which might be expected to enhance the propensity for saturated and unsaturated surfactant lipid components to segregate.