Phase Behavior Of Model Membranes Research Articles

Accumulation of styrene oligomers alters lipid membrane phase order and miscibility

Growth of plastic waste in the natural environment, and in particular in the oceans, has raised the accumulation of polystyrene and other polymeric species in eukyarotic cells to the level of a credible and systemic threat. Oligomers, the smallest products of polymer degradation or incomplete polymerization reactions, are the first species to leach out of macroscopic or nanoscopic plastic materials. However, the fundamental mechanisms of interaction between oligomers and polymers with the different cell components are yet to be elucidated. Simulations performed on lipid bilayers showed changes in membrane mechanical properties induced by polystyrene, but experimental results performed on cell membranes or on cell membrane models are still missing. We focus here on understanding how embedded styrene oligomers affect the phase behavior of model membranes using a combination of scattering, fluorescence, and calorimetric techniques. Our results show that styrene oligomers disrupt the phase behavior of lipid membranes, modifying the thermodynamics of the transition through a spatial modulation of lipid composition.

The influence of NBD fluorescent probe on model membranes containing POPC and DPPC.

To investigate the effect of fluorescent probe on the properties of membranes, we studied model membranes composed of 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1-palmitoyl 2-oleoyl-sn-glycero-3-phosphocholine (POPC) in the presence and absence of fluorescent probe. The morphology of giant unilamellar vesicles (GUVs) has been observed as a function of temperature and composition by fluorescence microscopy using NBD-DOPE or C6-NBD-PC as the probe. The phase behavior of model membranes containing no fluorescent probe was investigated by 2H-NMR spectroscopy. We found that the bright phase observed on GUVs was the fluid phase enriched in POPC and the dark phase was the gel phase enriched in DPPC. NBD-DOPE and C6-NBD-PC preferentially participated in the fluid-phase domains when GUVs were in the gel + fluid phase coexistence. Inclusion of both fluorescent probes (1 mol%) lowered the transition temperature of POPC/DPPC membranes. In addition, C6-NBD-PC exhibited a stronger effect than NBD-DOPE, which was considered to be associated with the structures of fluorescent molecules.

Modulation of Gel Phase Model Membranes by Vitamin D-Related Proteins

Severe vitamin D deficiency has long been linked to dysfunctions of the musculoskeletal system, and more recent studies have also associated vitamin D deficiency with other conditions, ranging from certain cancers to cardiovascular disease and diabetes. Vitamin D is a steroidal hormone that can be biosynthesized from 7-dehydrocholesterol by the action of UV light on skin (vitamin D3 only), or obtained from dietary sources (vitamins D3 and D2). Vitamins D3 and D2 are inactive precursor molecules that must be successively hydroxylated in the kidney and liver in order to become active. The vitamin D binding protein (DBP, GC-globulin) is responsible for transporting the lipophilic vitamin D throughout the body, and the vitamin D receptor (VDR) is the transcription factor that exerts the most well-established activities of vitamin D in the cell nucleus. Here, we present data based on synchrotron X-ray diffraction, 31P NMR and 2H NMR describing the effects of DBP and VDR in modulating the phase behavior of model membranes composed of DPPC, brain-sphingomyelin and cholesterol. Initial results suggest that DBP particularly interacts with the bilayers to reduce the gel-fluid lamellar phase transition and increase lipid mixing in ternary membranes, with the effect of VDR being somewhat less significant. These data have consequences for our understanding of how the action of soluble proteins that bind lipophilic, membrane-based molecules may alter bilayer structure and dynamics as they interact with the membrane surface to identify and extract their target.

Thermotropic Behavior of Cardiolipin and Dimyristoylphosphatidylcholine Bilayers in the Presence and Absence of Calcium

Cardiolipin is an unusual lipid in that it is comprised of two diacylglycerols linked by a glycerol bridge. It is in found primarily in membranes in which electron transport is coupled to phosphorylation. There it plays a critical role in effective functioning of the electron transport chain. The mechanism by which cardiolipin plays this role may be through effects on bilayer properties as well as through direct interactions with protein complexes. In this study we investigate the influence of increasing the composition of tetramyristoyl cardiolipin (TMCL) on the thermotropic behavior of dimyristoyl phosphatidylcholine (DMPC) bilayers using differential scanning calorimetry (DSC). DSC experiments were performed using a MicroCal VP-DSC microcalorimeter. Lipid dispersions of DMPC/TMCL were prepared in which TMCL constituted 0, 5,10, 20, 50 and 100 mole percent of the total lipid. In these studies the Tm increased from 25 oC to 40 oC as the TMCL composition increased. Ca++ has been shown to have a plethora of in vivo effects on biological membranes and to induce alterations in lipid phase behavior of model membranes. Therefore we further investigated the thermotropic behavior of DMPC/TMCL bilayers in the presence of Ca++. Above 20 mole percent TMCL a distinct phase separation was induced. The effect of Ca++ on cardiolipin/PC membranes was also investigated using 6-dodecanoyl-2-dimethylaminonaphthalene (LAURDAN). These data are consistent with an alteration of the hydration of the bilayer in the headgroup region in the presence of Ca++ and suggest that Ca++ induces tighter packing.

Deuterium NMR Study of the Effect of Phytosterol on DPPC Membranes

We investigate the phase behavior of model membranes composed of 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine(DPPC) and phytosterol using deuterium nuclear magnetic resonance (NMR). The sn-1 chain of DPPC is deuterium labeled. The deuterium NMR spectra were taken as a function of temperature and phytosterol concentration. Our data shows that phytosterol promotes the formation of the liquid-ordered phase. Moreover, this phytosterol has opposite ordering effect on the DPPC membranes below and above Tm: It decreases the order of DPPC membranes below Tm, whereas increases the chain orders of DPPC above Tm. In addition, the partial phase diagram is determined. There are two two-phase coexistence regions found below and above Tm at intermediate sterolconcentration.

The structure of the antimicrobial peptide Ac-RRWWRF-NH2 bound to micelles and its interactions with phospholipid bilayers.

The hexapeptide Ac-RRWWRF-NH2 has earlier been identified as a potent antimicrobial peptide by screening synthetic combinatorial hexapeptide libraries. In this study, it was found that this peptide had a large influence on the thermotropic phase behavior of model membranes containing the negatively charged headgroup phosphatidylglycerol, a major component of bacterial membranes. In contrast, differential scanning calorimetry showed that it had little effect on model membranes containing the zwitterionic phosphatidylcholine headgroup, the main component of erythrocyte membranes. This behavior is consistent with its biological activity and with its affinity to these membranes as determined by titration calorimetry, implying that peptide-lipid interactions play an important role in this process. The structure of this peptide bound to membrane-mimetic sodium dodecyl sulfate (SDS) and dodecylphosphocholine micelles has been determined using conventional two-dimensional nuclear magnetic resonance methods. It forms a marked amphipathic structure in SDS with its hydrophobic residues on one side of the structure and with the positively charged residues on the other side. This amphipathic structure may allow this peptide to penetrate deeper into the interfacial region of negatively charged membranes, leading to local membrane destabilization. Knowledge about the importance of electrostatic interactions of Arg and the role of Trp residues as a membrane interface anchor will provide insight into the future design of potent antimicrobial peptidomimetics.

The Effect of Peptide/Lipid Hydrophobic Mismatch on the Phase Behavior of Model Membranes Mimicking the Lipid Composition in Escherichia coli Membranes

The effect of hydrophobic peptides on the lipid phase behavior of an aqueous dispersion of dioleoylphosphatidylethanolamine and dioleoylphosphatidylglycerol (7:3 molar ratio) was studied by 31P NMR spectroscopy. The peptides (WALP n peptides, where n is the total number of amino acid residues) are designed as models for transmembrane parts of integral membrane proteins and consist of a hydrophobic sequence of alternating leucines and alanines, of variable length, that is flanked on both ends by tryptophans. The pure lipid dispersion was shown to undergo a lamellar-to-isotropic phase transition at ∼60°C. Small-angle x-ray scattering showed that at a lower water content a cubic phase belonging to the space group Pn3m is formed, suggesting also that the isotropic phase in the lipid dispersion represents a cubic liquid crystalline phase. It was found that the WALP peptides very efficiently promote formation of nonlamellar phases in this lipid system. At a peptide-to-lipid (P/L) molar ratio of 1:1000, the shortest peptide used, WALP16, lowered the lamellar-to-isotropic phase transition by ∼15°C. This effect was less for longer peptides. For all of the WALP peptides used, an increase in peptide concentration led to a further lowering of the phase transition temperature. At the highest P/L ratio (1:25) studied, WALP16 induced a reversed hexagonal liquid crystalline (H II) phase, while the longer peptides still promoted the formation of an isotropic phase. Peptides with a hydrophobic length larger than the bilayer thickness were found to be unable to inhibit formation of the isotropic phase. The results are discussed in terms of mismatch between the hydrophobic length of the peptide and the hydrophobic thickness of the lipid bilayer and its consequences for lipid-protein interactions in membranes.

Differential scanning microcalorimetry indicates that human defensin, HNP-2, interacts specifically with biomembrane mimetic systems.

alpha-Defensins are antimicrobial peptides with 29-35 amino acid residues and cysteine-stabilized amphiphilic, triple-stranded beta-sheet structures. We used high-precision differential scanning microcalorimetry to investigate the effects of a human neutrophil alpha-defensin, HNP-2, on the phase behavior of model membranes mimicking bacterial and erythrocyte cell membranes. In the presence of this positively charged peptide, the phase behavior of liposomes containing negatively charged phosphatidylglycerol was markedly altered even at a high lipid-to-peptide molar ratio of 500:1. Addition of HNP-2 to liposomes mimicking bacterial membranes (mixtures of dipalmitoylphosphatidylglycerol and -ethanolamine) resulted in phase separation owing to some domains being peptide-poor and others peptide-rich. The latter are characterized by an increase of the main transition temperature, most likely arising from electric shielding of the phospholipid headgroups by the peptide. On the other hand, HNP-2 did not affect the phase behavior of membranes mimicking erythrocyte membranes (equimolar mixtures of dipalmitoylphosphatidylcholine and sphingomyelin) as well as the pure single components. This is in contrast to melittin, which significantly affected the phase behavior of choline phospholipids in accordance with its unspecific lytic activity. These results support the hypothesis of preferential interaction of defensins with negatively charged membrane cell surfaces, a common feature of bacterial cell membranes, and demonstrate that HNP-2 discriminates between model membrane systems mimicking prokaryotic and eukaryotic cell membranes.

How trehalose affects the lipid phase behavior of model membranes

How trehalose affects the lipid phase behavior of model membranes

Thermotropic phase behavior of model membranes composed of phosphatidylcholines containing dl-methyl anteisobranched fatty acids. 1. Differential scanning calorimetric and 31P NMR spectroscopic studies.

The thermotropic phase behavior of aqueous dispersions of nine dl-methyl branched anteisoacylphosphatidylcholines was studied by differential scanning calorimetry and 31P nuclear magnetic resonance spectroscopy. The calorimetric studies demonstrate that these compounds all exhibit a complex phase behavior, consisting of at least two minor, low-enthalpy, gel-state transitions which occur at temperatures just prior to the onset of the gel/liquid-crystalline phase transition. In addition, at still lower temperatures, anteisobranched phosphatidylcholines containing fatty acyl chains with an odd number of carbon atoms show a major, higher enthalpy, gel-state transition, which was assigned to a conversion from a condensed to a more loosely packed gel phase. No such transition was observed for the even-numbered compounds in aqueous dispersion, but when dispersed in aqueous ethylene glycol, a major gel-state transition is clearly discernible for two of the even-numbered phospholipids. The major gel-state transition exhibits heating and cooling hysteresis and is fairly sensitive to the composition of the bulk aqueous phase. 31P NMR spectroscopic studies indicate that the major gel-state transition is accompanied by a considerable change in the mobility of the phosphate head group and that, at temperatures just prior to the onset of the gel/liquid-crystalline phase transition, the mobility of the phosphate head group is comparable to that normally exhibited by the liquid-crystalline state of most other phospholipids. The temperatures at which the gel/liquid-crystalline phase transition occurs and the enthalpy change associated with this process are considerably lower than those of the saturated n-acyl-PC's of comparable acyl chain length.(ABSTRACT TRUNCATED AT 250 WORDS)

Thermotropic phase behavior of model membranes composed of phosphatidylcholines containing dl-methyl anteisobranched fatty acids. 2. An infrared spectroscopy study.

The thermotropic phase behavior of four members of the homologous series of dl-methyl anteisobranched phosphatidylcholines was investigated by Fourier transform infrared spectroscopy. The odd-numbered phosphatidylcholines exhibit spectral changes in two distinct temperature ranges, while their even-numbered counterparts exhibit spectral changes within only a single temperature range. The high-temperature transition observed in the odd-numbered phosphatidylcholines and the single thermotropic event characteristic of the phase behavior of their even-numbered counterparts are both identified as gel/liquid-crystalline phase transitions. The low-temperature event exhibited only by the odd-numbered phospholipids is identified as a gel/gel phase transition that involves changes in the packing mode of the acyl chain methylene groups, as well as changes in the conformation of the glycerol ester interface. These infrared spectroscopic data thus suggest that at low temperatures the odd-numbered methyl anteisobranched phosphatidylcholines form a highly ordered condensed phase similar to the Lc phases of the linear saturated n-acyl-phosphatidylcholines. A comparable condensed phase was not formed by the even-numbered anteisobranched phosphatidylcholines under similar conditions. The properties of the gel states of the even-numbered anteisoacylphosphatidylcholines were generally similar to those of the high-temperature gel states of their odd-numbered counterparts. Those gel states exhibit spectral characteristics indicative of hexagonally packed but relatively mobile acyl chains. The temperature-dependent changes in the spectral characteristics of these gel states were continuous and were not resolved into the discrete but overlapping transitions observed by differential scanning calorimetry.