Phospholipid Phase Transition Research Articles

Membranotropic regions of the dengue virus prM protein.

The Dengue virus (DENV) prM protein consists of two moieties, the pr and M domains. Apart from preventing the premature fusion activity of the DENV E protein, prM has several other unknown biological roles, displaying both protein-protein and membrane-protein interactions. Although the prM protein is an essential component of the DENV viral cycle, little is known about its biological functions and what regions of this protein are responsible for said functions. By performing an exhaustive study of membrane rupture induced by a prM peptide library on simple and complex model membranes as well as their ability to modulate the phospholipid phase transitions of 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine and 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-glycerol], we identified six membranotropic regions on the prM protein. Apart from the previously identified two transmembrane segments of the protein, one of these regions probably interacts with the fusion E protein and another one, the stem segment, would interact with the membrane modulating its structure. These data will help us understand the molecular mechanism of viral entry and morphogenesis, allow the identification of new targets for the treatment of Dengue virus infection, and make possible the future development of DENV entry inhibitors.

Alternative methods of determining phase transition temperatures of phospholipids that constitute liposomes on the example of DPPC and DMPC

Abstract In this work, alternatives to differential scanning calorimetry (DSC) as a method of determining the main phospholipid phase transition temperature are presented. The bilayer phase transitions from the ripple gel phase ( P β ′ ) to the liquid-crystal phase ( L α ) of 1,2-dipalmitoyl- sn -glycerol-3-phosphocholine (DPPC) and 1,2-dimyristoyl- sn -glycero-3-phosphocholine (DMPC) were studied by differential scanning calorimetry (DSC), Fourier transform infrared (FT-IR), nuclear magnetic resonance (NMR), and electron paramagnetic resonance (EPR) methods. In this work, two correlations between the DSC and FT-IR methods, and NMR and EPR methods are shown. The proposed methods allow for determining the T C temperature with a high degree of accuracy. Furthermore, a comparison of results obtained using the DSC and FT-IR methods allowed for an observation of a new DMPC phase transition. The liposomes analyzed in this work were obtained by the modified reverse-phase evaporation method (mREV).

The thermotropic behavior and major molecular species composition of the phospholipids of echinoderms

This study examines the molecular species composition and heat-induced crystalline-liquid crystalline phase transitions of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) from the muscle tissues of six species of echinoderms that were collected during the summer: the starfishes Distolasterias nipon and Asterias amurensis, the sea urchin Strongylocentrotus intermedius, and the holothurians Eupentacta fraudatrix, Cucumaria frondosa japonica, and Apostichopus japonicus. Phospholipids (PLs) were in the liquid crystalline state, which is optimal for the functioning of the cell membranes. The use of data on the molecular species composition of PLs for the interpretation of their thermotropic behavior indicated that homeoviscous adaptation is achieved by various rearrangements in the composition of the aliphatic groups of PLs. The phase transitions of PC and PE of echinoderms (except holothurians) were symbatic. The presence of a high-temperature peak on the PC thermograms of C. frondosa japonica and A. japonicus is attributable to the melting of the phospholipid domain, which is composed of molecular species with saturated aliphatic groups. Such domains are responsible for a significant shift in the temperature ranges of the phase transitions of phospholipids of holothurians and sea urchin towards temperatures above 0°C.

Hydration and Temperature-Induced Phospholipid Phase Transitions and their Influence on Desiccation Tolerance of the Nematode Caenorhabditis Elegans

The nematode Caenorhabditis elegans can bypass part of its normal larval development by forming a state with arrested metabolism. The desiccation-tolerance of this state depends on the synthesis of trehalose during a preconditioning phase, where the worm is first exposed to mildly reduced RH, rendering the dauer state attractive for studying the physiology and genetics of Anhydrobiosis. Here, we correlate physical properties of phospholipids (PLs) with the desiccation tolerance of the larvae from which PLs were extracted. The response of PL structure to transient (seconds) changes in water potential and the role of trehalose in water-mediated structural transitions were addressed. Time-resolved Rapid scan FTIR spectroscopy was used in ATR geometry to record hydration-induced difference spectra. We show by chemical analysis that a reduction in choline content in the PL headgroup composition arises during preconditioning. This leads to a stronger coupling of headgroup hydration to disorder in PL acyl chains based on the time-resolved observation of PO2-, C=O and acyl CH2 stretching modes. Trehalose enhances this effect and leads to more uniform kinetics of hydration transients and lipid transitions. In combination with spectroscopic determination of altered lipid main phase transition temperatures (Tm) in PLs upon preconditioning, the data show that chemical tuning of the kinetics and the extent of coupling of headgroup hydration to acyl chain packing changes is a key process in desiccation tolerance as it may release mechanical stress from membranes during temporal changes of water potential. Additional DSC, ITC and Langmuir-Blodget data show that trehalose interacts more favourably with PLs from preconditioned larvae to support desiccation tolerance. This work highlights that chemical tuning of lyotropic phase transitions plays a fundamental role in the trehalose-dependent desiccation tolerance of C. elegans larvae.

Influence of Arenicin on Phase Transitions and Ordering of Lipids in 2D Model Membranes

Antimicrobial peptides (AMPs) are important effector molecules of the innate immune system of all species. AMPs are highly selective and can be used as lead structures for the development of new drugs complementing standard antibiotic therapies. Understanding the crucial parameters of peptide-membrane interactions is necessary for elucidation of the molecular mechanisms of action. Phospholipid monolayers, as simple 2D models of the membrane surface, can be effectively used for studies of peptide-membrane interactions. The present study is focused on the recently discovered peptide arenicin-1 (Ar-1), which possesses antibacterial and antifungal activities. A linear derivative with serine residues instead of cysteines (C/S-Ar-1) was additionally used to investigate the influence of the AMP on the phase behavior of lipid monolayers at the air/liquid interface. Using the Langmuir balance technique and IRRAS allows us to conclude that both original and modified arenicins reveal a strong influence on the phase transition of anionic phospholipids (fluidization of the lipid hydrocarbon chains), whereas the thermodynamic properties of the zwitterionic phospholipid layers are not affected. A strong effect of the modified peptide on the ordering of negatively charged phospholipids at the air-water interface compared to zwitterionic phospholipids has been observed using GIXD measurements, supported by IRRAS simulations for the spectral range corresponding to the lipid hydrocarbon chains. At lateral pressures above 30 mN/m, both peptides are squeezed out from zwitterionic lipid monolayers, but remains attached to and partly incorporated in anionic lipid monolayers. This study points at the importance of the interplay between hydrophobic and electrostatic interactions for the membrane disruption by AMPs.

Effect of the Phospholipid Chain Length and Head Group on Beta‐Phase Formation of Poly(9,9‐dioctylfluorene) Enclosed in Liposomes

We have studied the effect of head group and alkyl chain length on β-phase formation in poly(9,9-dioctylfluorene) (PFO) solubilized in phospholipid liposomes. Systems studied have three different alkyl chain lengths (1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine [DMPC], 1,2-didodecanoyl-sn-glycero-3-phosphatidylcholine [DLPC], 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine [DPPC]) and head groups (1,2-dimyristoyl-sn-glycero-3-phosphate monosodium salt [DMPA], 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine [DMPE] and 1,2-dimyristoyl-sn-glycero-3-phospho-l-serine sodium salt [DMPS]). Changes in liposome size upon addition of PFO are followed by dynamic light scattering. All the phospholipids induce the formation of PFO β-phase, which is followed by the emission intensity and deconvolution of the absorption spectra. Both the head group and alkyl chain length affect the yield of β-phase. The photophysics of PFO incorporated in liposomes is characterized by stationary and time-resolved fluorescence, whereas the polymer-phospholipid interactions have been studied by the effect of the PFO concentration on the phospholipid phase transitions (differential scanning calorimetry [DSC]).

Phase transitions of phospholipids as a criterion for assessing the capacity for thermal adaptation in fish

This study examines the crystal-liquid crystal phase transitions of the major phospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), from muscle tissue of marine fish living at temperatures of 0–4.1°C (the Pacific cod Gadus macrocephalus, banded Irish lord Hemilepidotus gilberti, Pacific halibut Hippoglossus hippoglossus, black edged sculpin Gymnocanthus herzensteini, dark colored flounder Pleuronectes obscurus, and plain sculpin Myoxocephalus jaok), as well as of fish living at 14 and 18°C (Pacific redfin Tribolodon brandti). The PC and PE phase-transition thermograms of all the investigated species displayed specific profiles. The largest share of the thermogram area at temperatures higher than those of the habitat was found for the PC (28–40%) and PE (47–82%) of the black-edged sculpin, dark-colored flounder, and the plain sculpin, which have reduced physiological activity at low temperatures. In the Pacific cod, banded Irish lord, and the Pacific redfin, this parameter was much lower, 0–18% (PC) and 0–39% (PE). The thermotropic behavior PC and PE was symbate in all fish, except for the cod and the plain sculpin. The transition enthalpy of PC in all the investigated species was 2.8 times higher than that of PE. To interpret the varied PC and PE thermogram profiles of fish with similar fatty-acid compositions, the data on the composition of the molecular species of these phospholipids appeared to be the most informative. This study suggests that each fish species has its own strategy for thermal adaptation, which is realized through a certain set of phospholipid molecular species.

Reorientation of hydration water during the thermotropic main phase transition of 1-palmitoyl-2-oleolyl-sn-glycero-3-phosphocholine (POPC) bilayers at low degrees of hydration

Calorimetric scans of a number of low-hydrated phospholipids provide quite complex heat capacity profiles during the thermotropic main phase transition. This short study proposes some explanations that are based on experimental data obtained from four different methods (DSC, 31P-NMR, 2H-NMR, X-ray diffraction).The preliminary results show for POPC in which way DSC data are related to the melting of the hydrocarbon chains. Furthermore, the quadrupolar splitting data obtained from the 2H-NMR spectra of hydration water reveal how the water reorientation might contribute to the main phase transition of phospholipids.

Thermotropic and barotropic phase behavior of phosphatidylcholine bilayers.

Bilayers formed by phospholipids are frequently used as model biological membranes in various life science studies. A characteristic feature of phospholipid bilayers is to undergo a structural change called a phase transition in response to environmental changes of their surroundings. In this review, we focus our attention on phase transitions of some major phospholipids contained in biological membranes, phosphatidylcholines (PCs), depending on temperature and pressure. Bilayers of dipalmitoylphosphatidylcholine (DPPC), which is the most representative lipid in model membrane studies, will first be explained. Then, the bilayer phase behavior of various kinds of PCs with different molecular structures is revealed from the temperature–pressure phase diagrams, and the difference in phase stability among these PC bilayers is discussed in connection with the molecular structure of the PC molecules. Furthermore, the solvent effect on the phase behavior is also described briefly.

NS4A and NS4B proteins from dengue virus: Membranotropic regions

Proteins NS4A and NS4B from Dengue Virus (DENV) are highly hydrophobic transmembrane proteins which are responsible, at least in part, for the membrane arrangements leading to the formation of the viral replication complex, essential for the viral life cycle. In this work we have identified the membranotropic regions of DENV NS4A and NS4B proteins by performing an exhaustive study of membrane rupture induced by NS4A and NS4B peptide libraries on simple and complex model membranes as well as their ability to modulate the phospholipid phase transitions Pβ′–Lα of DMPC and Lβ–Lα/Lα–HII of DEPE. Protein NS4A presents three membrane active regions coincident with putative transmembrane segments, whereas NS4B presented up to nine membrane active regions, four of them presumably putative transmembrane segments. These data recognize the existence of different membrane-active segments on these proteins and support their role in the formation of the replication complex and therefore directly implicated in the DENV life cycle.

A modified squeeze-out mechanism for generating high surface pressures with pulmonary surfactant

The exact mechanism by which pulmonary surfactant films reach the very low surface tensions required to stabilize the alveoli at end expiration remains uncertain. We utilized the nanoscale sensitivity of atomic force microscopy (AFM) to examine phospholipid (PL) phase transition and multilayer formation for two Langmuir–Blodgett (LB) systems: a simple 3 PL surfactant-like mixture and the more complex bovine lipid extract surfactant (BLES). AFM height images demonstrated that both systems develop two types of liquid condensed (LC) domains (micro- and nano-sized) within a liquid expanded phase (LE). The 3 PL mixture failed to form significant multilayers at high surface pressure (π while BLES forms an extensive network of multilayer structures containing up to three bilayers. A close examination of the progression of multilayer formation reveals that multilayers start to form at the edge of the solid-like LC domains and also in the fluid-like LE phase. We used the elemental analysis capability of time-of-flight secondary ion mass spectrometry (ToF-SIMS) to show that multilayer structures are enriched in unsaturated PLs while the saturated PLs are concentrated in the remaining interfacial monolayer. This supports a modified squeeze-out model where film compression results in the hydrophobic surfactant protein-dependent formation of unsaturated PL-rich multilayers which remain functionally associated with a monolayer enriched in disaturated PL species. This allows the surface film to attain low surface tensions during compression and maintain values near equilibrium during expansion.

The membrane-active regions of the dengue virus proteins C and E

We have identified the membranotropic regions of proteins C and E of DENV virus by performing an exhaustive study of membrane rupture induced by two C and E-derived peptide libraries on model membranes having different phospholipid compositions as well as its ability to modulate the DEPE Lβ–Lα and Lα–HII phospholipid phase transitions. Protein C presents one hydrophobic leakage-prone region coincidental with a proposed membrane interacting domain, whereas protein E presents five membrane-rupture zones coincidental with different significant zones of the protein, i.e., the fusion peptide, a proline-rich sequence, a sequence containing a hydrophobic pocket as well as the stem and transmembrane domains of the protein. The identification of these membrane-active segments supports their role in viral membrane fusion, formation of the replication complex and morphogenesis and therefore attractive targets for development of new anti-viral compounds.

β-Phase Formation of Poly(9,9-dioctylfluorene) Induced by Liposome Phospholipid Bilayers

The well-structured β-phase emission of the neutral poly(9,9-dioctylfluorene) (PFO) is observed in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers, either as polydisperse aqueous liposomes or as the lamellar phase in thin films, and has been characterized by absorption, fluorescence (steady-state and time-resolved), and fluorescence anisotropy spectroscopy. Inclusion of PFO in DMPC liposomes provides a way of obtaining the ordered structure of this neutral polymer in aqueous suspensions. Quantification of the increase of the PFO β-phase in DMPC liposomes with the increase in polymer concentration is followed by deconvolution of the absorption spectra. In solid films, the presence of the phospholipids enhances the β-phase formation. In addition, the effect of the PFO concentration on the phospholipid phase transitions has been studied by differential scanning calorimetry (in liposome) and polarized light thermal microscopy (in solid film), confirming PFO/DMPC interactions in both liposome and films. The liposome size and structure in the presence and absence of polymer were characterized by dynamic light scattering and transmission electron microscopy, which showed relatively modest changes in liposome shape but a decrease in size upon incorporation of PFO.

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

The interaction of the synthetic antimicrobial peptide P5 (KWKKLLKKPLLKKLLKKL-NH(2)) 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 (d(54)-DMPC) and mixed d(54)-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 (31)P 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).

Interaction of plant alkaloid, berberine, with zwitterionic and negatively charged phospholipid bilayers

Interaction of plant alkaloid, berberine, with zwitterionic and negatively charged phospholipid bilayers Berberine exhibits many pharmacological activities e.g. antibacterial, anti-inflammatory, antiproliferative and apoptosis-inducing. Interaction of berberine with model membranes was studied for the first time using differential scanning calorimetry, fluorescence spectroscopy and turbidity measurements. Influence of berberine on thermotropic properties of bilayers formed from zwitterionic DMPC was insignificant, whereas in bilayers formed from negatively charged DMPG berberine reduced the temperature and cooperativity of main phospholipid phase transition. In higher concentrations berberine induced complex double-peak transition, with the new peak appearing in temperature higher than the original one. It suggested the interaction of the alkaloid with lipid headgroup region of the bilayer. Additionally, berberine quenched fluorescence of Prodan to a higher extent than Laurdan that pointed to stronger interaction with membrane segments close to its surface. Berberine-induced fluorescence quenching of both probes was more pronounced in DPPG than in DPPC bilayers. It was concluded that electrostatic interactions governed berberine association with model membrane.

Structure and Phase Behavior of Cholesterol Containing Membranes in the Presence of Ethanol

Molecular dynamics (MD) of cholesterol containing membranes is used to examine the structural changes and phase behavior of lipids in the presence of varying ethanol concentrations over a range of temperatures. Alcohols are known to cause changes in the phase transitions of phospholipids as well as inducing the formation of an interdigitated phase of reduced thickness, where the hydrophobic tails of the top and bottom lipids intercalate causing an increase in the area per lipid as well as the solvent exposed surface of the headgroups. Atomistic MD simulations using the Gromacs 4.0 software allows analysis of structural changes in lipid volume, area per lipid, and hydrogen bonding among others at the molecular level. Pure 1,2-Dioleoyl-sn-Glycero-3-Phosphocholine (DOPC) and DOPC/cholesterol membranes were constructed to have 128 lipid molecules under full hydration. DOPC/cholesterol systems contain 10, 20, and 30 mole % cholesterol, and those containing ethanol had additional 5, 10, 15 and 20 % V/V ethanol molecules in the solvent. All systems were simulated at 6 different temperatures which span relevant biological processes for biofuel production; also experimentally observed phase changes occur at some of these temperatures. The effects of ethanol on biological membranes are of considerable importance to the studies of biofuels, enology, and medicine.

Anhydrobiosis in yeast: Stabilization by exogenous lactose

We have found that incubation in lactose solutions (0.75 M) of yeast culture Saccharomyces cerevisiae sensitive to dehydration damage increased the stability of the cells during dehydration. Simultaneously with this increase in viability, a decrease in plasma membrane permeability during rehydration was seen. Using Fourier transform infrared spectroscopy to measure lipid phase transitions, we observed that the lactose treatment depressed the membrane phospholipid phase transition temperature in a sensitive culture of dry yeast. As a result, it leads to the decrease in the damages of molecular organization of membranes during rehydration of dry yeast cells, thus reducing leakage from the cells.

Thermodynamics of Bilayer Phase Transitions of Phospholipids

Thermodynamics of Bilayer Phase Transitions of Phospholipids

Screening a Peptide Library by DSC and SAXD: Comparison with the Biological Function of the Parent Proteins

We have recently identified the membranotropic regions of the hepatitis C virus proteins E1, E2, core and p7 proteins by observing the effect of protein-derived peptide libraries on model membrane integrity. We have studied in this work the ability of selected sequences of these proteins to modulate the Lβ-Lα and Lα-HII phospholipid phase transitions as well as check the viability of using both DSC and SAXD to screen a protein-derived peptide library. We demonstrate that it is feasible to screen a library of peptides corresponding to one or several proteins by both SAXD and DSC. This methodological combination should allow the identification of essential regions of membrane-interacting proteins which might be implicated in the molecular mechanism of membrane fusion and/or budding.

Comparative Studies On Bovine And Rat Pulmonary Surfactants Using AFM

Recent studies employing a variety of microscopic techniques have revealed that phospholipid (PL) phase separations and transitions may play important roles in determining the biophysical properties of pulmonary surfactant. Most of these microscopic studies used model systems which were composed of simple mixtures of PL with or without hydrophobic surfactant proteins and cholesterol. The present work compared modified natural (lipid extract) bovine and rat surfactants using atomic force microscopy (AFM). AFM revealed PL phase separation upon compression of both surfactant monolayers, and a monolayer-to-multilayer transition at surface pressure 40-50 mN/m. Similar to bovine surfactant, the tilted-condensed (TC) phase in rat surfactant consisted of domains both on micrometer and nanometer scales. Upon film compression, the microdomains were dissociated into nanodomains, thus forming a more homogeneous two-phase mixture. Differences between rat and bovine surfactants were: (1) more TC domains were formed at lower surface pressures in rat than in bovine surfactant; and (2) an interesting domain-in-domain structure was exclusively observed in rat surfactant. These structural differences were attributed to the higher cholesterol content of rat surfactant (∼ 10 vs ∼2.5 wt%). To further investigate the effects of cholesterol on the structure of surfactant films, we have studied cholesterol-depleted bovine surfactant (∼ 0%) prepared by repetitive acetone extraction. Removal of cholesterol from bovine surfactant induced significant variations in film structure. More importantly, the film structure can be effectively restored by recombining cholesterol with the cholesterol-depleted bovine surfactant. Recombinant bovine surfactant with 10% cholesterol showed domain-in-domain structures similar to those found with rat surfactant. These interspecies studies of the micro- and nano- structures of natural pulmonary surfactants add insight into the biophysical interpretation of phospholipid phase transition and separation, in particular the role of cholesterol.