The effect of the lipid bilayer state on fluorescence intensity of fluorescein-PE in a saturated lipid bilayer

Fluorescein-PE is a fluorescence probe that is used as a membrane label or a sensor of surface associated processes. Fluorescein-PE fluorescence intensity depends not only on bulk pH, but also on the local electrostatic potential, which affects the local membrane interface proton concentration. The pH sensitivity and hydrophilic character of the fluorescein moiety was used to detect conformational changes at the lipid bilayer surface. When located in the dipalmitoylphosphatidylcholine (DPPC) bilayer, probe fluorescence depends on conformational changes that occur during phase transitions. Relative fluorescence intensity changes more at pretransition than at the main phase transition temperature, indicating that interface conformation affects the condition in the vicinity of the membrane. Local electrostatic potential depends on surface charge density, the local dielectric constant, salt concentration and water organisation. Initial increase in fluorescence intensity at temperatures preceding that of pretransition can be explained by the decreased value of the dielectric constant in the lipid polar headgroups region related in turn to decreased water organisation within the membrane interface. The abrupt decrease in fluorescence intensity at temperatures between 25 degrees C and 35 degrees C (DPPC pretransition) is likely to be caused by an increased value of the electrostatic potential, induced by an elevated value of the dielectric constant within the phosphate group region. Further increase in the fluorescence intensity at temperatures above that of the gel-liquid phase transition correlates with the calculated decreased surface electrostatic potential. Above the main phase transition temperature, fluorescence intensity increase at a salt concentration of 140 mM is larger than with 14 mM. This results from a sharp decline of the electrostatic potential induced by the phosphocholine dipole as a function of distance from the membrane surface.     

Characterization of the fluorophore 4-heptadecyl-7-hydroxycoumarin: a probe for the head-group region of lipid bilayers and biological membranes

The fluorophore 4-heptadecyl-7-hydroxycoumarin was used as a probe to study the properties of phospholipid bilayers at the lipid-water interface. To this end, the steady-state fluorescence anisotropy, the differential polarized phase fluorometry, and the emission lifetime of the fluorophore were measured in isotropic viscous medium, in lipid vesicles, and in the membrane of vesicular stomatitis virus. In the isotropic medium (glycerol), the probe showed an increase in the steady-state fluorescence anisotropy with a decrease in temperature, but the emission lifetime was unaffected by the change in temperature. In glycerol, the observed and predicted values for maximum differential tangents of the probe were identical, indicating that in isotropic medium 4-heptadecyl-7-hydroxycoumarin is a free rotator. Nuclear magnetic resonance and differential scanning calorimetric studies with lipid vesicles containing 1-2 mol % of the fluorophore indicated that the packaging density of the choline head groups was affected in the presence of the probe with almost no effect on the fatty acyl chains. The fluorophore partitioned equally well in the gel and liquid-crystalline phase of the lipids in the membrane, and the phase transition of the bilayer lipids was reflected in the steady-state fluorescence anisotropy of the probe. The presence of cholesterol in the lipid vesicles had a relatively small effect on the dynamics of lipids in the liquid-crystalline state, but a significant disordering effect was noted in the gel state. One of the most favorable properties of the probe is that its emission lifetime was unaffected by the physical state of the lipids or by the temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of the fluorescent probe N-(lissamine Rhodamine B sulfonyl)dipalmitoylphosphatidylethanolamine with phosphatidylcholine bilayers

The surface density of the fluorescent probe N-(lissamine Rhodamine B sulfonyl)dipalmitoylphosphatidylcholine is the same in the two lipid leaflets of phosphatidylcholine bilayers containing the probe. In the liquid-crystalline state, the probe molecules aggregate above a threshold amount, approximately 0.2 mol/mol phospholipids. Above this threshold value, the surface density of the free probe molecules is constant, and all probe molecules added are incorporated in the aggregated form. The aggregation of the probe increases by approximately 20% when the medium pH is lowered to 4. In the gel state, the probe aggregation is higher than that in the liquid-crystalline state, and the free probe molecules distribute unevenly in the bilayer surface. Even though the results obtained in our model system cannot be directly extrapolated to all model systems, we point out that care is to be taken in the use of the probe. In fact, only in membranes in the liquid-crystalline state in which the amount of probe molecules to phospholipid molecules is lower than 1:7 the fluorescence response of the probe is independent of the pH changes and of the molecular aggregation.     

Fourier transform infrared spectroscopic studies of the interaction of the antimicrobial peptide gramicidin S with lipid micelles and with lipid monolayer and bilayer membranes

We have utilized Fourier transform infrared spectroscopy to study the interaction of the antimicrobial peptide gramicidin S (GS) with lipid micelles and with lipid monolayer and bilayer membranes as a function of temperature and of the phase state of the lipid. Since the conformation of GS does not change under the experimental conditions employed in this study, we could utilize the dependence of the frequency of the amide I band of the central beta-sheet region of this peptide on the polarity and hydrogen-bonding potential of its environment to probe GS interaction with and location in these lipid model membrane systems. We find that the GS is completely or partially excluded from the gel states of all of the lipid bilayers examined in this study but strongly partitions into lipid micelles, monolayers, or bilayers in the liquid-crystalline state. Moreover, in general, the penetration of GS into zwitterionic and uncharged lipid bilayer coincides closely with the gel to liquid-crystalline phase transition of the lipid. However, GS begins to penetrate into the gel-state bilayers of anionic phospholipids prior to the actual chain-melting phase transition, while in cationic lipid bilayers, GS does not partition strongly into the liquid-crystalline bilayer until temperatures well above the chain-melting phase transition are reached. In the liquid-crystalline state, the polarity of the environment of GS indicates that this peptide is located primarily at the polar/apolar interfacial region of the bilayer near the glycerol backbone region of the lipid molecule. However, the depth of GS penetration into this interfacial region can vary somewhat depending on the structure and charge of the lipid molecule. In general, GS associates most strongly with and penetrates most deeply into more disordered bilayers with a negative surface charge, although the detailed chemical structure of the lipid molecule and physical organization of the lipid aggregate (micelle versus monolayer versus bilayer) also have minor effects on these processes.     

The phenyltetraene lysophospholipid analog PTE-ET-18-OMe as a fluorescent anisotropy probe of liquid ordered membrane domains (lipid rafts) and ceramide-rich membrane domains

The conjugated phenyltetraene PTE-ET-18-OMe (all-(E)-1-O-(15'-phenylpentadeca-8',10',12',14'-tetraenyl)-2-O-methyl-rac-glycero-3-phosphocholine) is a recently developed fluorescent lysophospholipid analog of edelfosine, (Quesada et al. (2004) J. Med. Chem. 47, 5333-5335). We investigated the use of this analog as a probe of membrane structure. PTE-ET-18-OMe was found to have several properties that are favorable for fluorescence anisotropy (polarization) experiments in membranes, including low fluorescence in water and moderately strong association with lipid bilayers. PTE-ET-18-OMe has absorbance and fluorescence properties similar to those of diphenylhexatriene (DPH) probes, with about as large a difference between its fluorescence anisotropy in liquid disordered (Ld) and ordered states (gel and Lo) as observed for DPH. Also like DPH, PTE-ET-18-OMe has a moderate affinity for both gel state ordered domains and Lo state ordered domains (rafts). However, unlike fluorescent sterols or DPH (Megha and London (2004) J. Biol. Chem. 279, 9997-10004), PTE-ET-18-OMe is not displaced from ordered domains by ceramide. Also unlike DPH, PTE-ET-18-OMe shows only slow exchange between the inner and outer leaflets of membrane bilayers, and can thus be used to examine anisotropy of an individual leaflet of a lipid bilayer. Since PTE-ET-18-OMe is a zwitterionic molecule, it should not be as influenced by electrostatic interactions as are other probes that do not cross the lipid bilayer but have a net charge. We conclude that PTE-ET-18-OMe has some unique properties that should make it a useful fluorescence probe of membrane structure.     

Using micropatterned lipid bilayer arrays to measure the effect of membrane composition on merocyanine 540 binding

The lipophilic dye merocyanine 540 (MC540) was used to model small molecule-membrane interactions using micropatterned lipid bilayer arrays (MLBAs) prepared using a 3D Continuous Flow Microspotter (CFM). Fluorescence microscopy was used to monitor MC540 binding to fifteen different bilayer compositions simultaneously. MC540 fluorescence was two times greater for bilayers composed of liquid-crystalline (l.c.) phase lipids (1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)) compared to bilayers in the gel phase (1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)). The effect cholesterol (CHO) had on MC540 binding to the membrane was found to be dependent on the lipid component; cholesterol decreased MC540 binding in DMPC, DPPC and DSPC bilayers while having little to no effect on the remaining l.c. phase lipids. MC540 fluorescence was also lowered when 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DOPS) was incorporated into DOPC bilayers. The increase in the surface charge density appears to decrease the occurrence of highly fluorescent monomers and increase the formation of weakly fluorescent dimers via electrostatic repulsion. This paper demonstrates that MLBAs are a useful tool for preparing high density reproducible bilayer arrays to study small molecule-membrane interactions in a high-throughput manner.     

Depolarization of dehydroergosterol in phospholipid bilayers

The behavior in phospholipid bilayers of low concentrations of dehydroergosterol, a fluorescent cholesterol mimic, has been examined by fluorometry and calorimetry. In contrast to many fluorescent membrane probes, dehydroergosterol shows a decrease in fluorescence anisotropy when the matrix phospholipid goes from the liquid-crystalline to the gel state. This was observed in three systems in which the matrix lipid was either dipalmitoyl- or dimyristoylphosphatidylcholine or dilauroylphosphatidylethanolamine. The decrease in anisotropy is the result of a large increase in the fluorescence life time of dehydroergosterol in these bilayer systems which is probably the result of thermal quenching of dehydroergosterol by neighboring molecules. The rotation of dehydroergosterol in these bilayers can be described in terms of the thermal coefficient of frictional resistance offered by the environment (Weber et al. (1984) Biochemistry 23, 6785-6788). The thermal coefficients are observed to change abruptly at the onset and completion temperatures of the gel to liquid-crystalline phase transition temperatures of the three matrix phospholipids. These changes are, however, much smaller than are the corresponding changes in the thermal coefficient observed for the fluorescent probe diphenylhexatriene in dilauroylphosphatidylethanolamine bilayers. The difference in behavior of the two fluorescent probes may be the result of lateral phase separation of dehydroergosterol similar to that reported for cholesterol in similar systems.     

Labeling of the cytoplasmic domain of the influenza virus hemagglutinin with fluorescein reveals sites of interaction with membrane lipid bilayers

The hemagglutinin (HA) glycoprotein of influenza virus was labeled in its cytoplasmic domain with fluorescein. Reactive amino groups in the external domain were blocked by modification of the intact virus with the membrane-impermeable reagent isethionyl acetimidate. The HA was then solubilized with the detergent octyl glucoside, and the single lysine in the cytoplasmic domain was reacted with fluorescein isothiocyanate. This protocol resulted in the incorporation of 1.3 mol of fluorescein/mol of HA. Using a virus strain lacking lysine in the cytoplasmic domain of HA, it was determined that 0.47 mol of fluorescein/mol of HA was located at an additional site(s). The fluorescein groups at both sites exist in an environment of reduced polarity as shown by a shift in excitation and emission maxima and a shift in the pKa of the fluorescein groups. The fluorescence polarization and the pKa of the fluorescein groups were greater when the HA was incorporated into liposomes than when in detergent solution. These data indicate that the fluorescein groups interact directly with the lipid bilayer, probably in the phospholipid head-group region. The fluorescence properties of the labeled HA were not responsive to the gel to liquid-crystal phase transition in the lipid bilayer. These results indicate that the boundary between the cytoplasmic domain and the hydrophobic sequence that anchors the protein to the lipid bilayer is located in the head-group region of the bilayer.     

The phenyltetraene lysophospholipid analog PTE-ET-18-OMe as a fluorescent anisotropy probe of liquid ordered membrane domains (lipid rafts) and ceramide-rich membrane domains

The conjugated phenyltetraene PTE-ET-18-OMe (all-(E)-1-O-(15′-phenylpentadeca-8′,10′,12′,14′-tetraenyl)-2-O-methyl-rac-glycero-3-phosphocholine) is a recently developed fluorescent lysophospholipid analog of edelfosine, (Quesada et al. (2004) J. Med. Chem. 47, 5333-5335). We investigated the use of this analog as a probe of membrane structure. PTE-ET-18-OMe was found to have several properties that are favorable for fluorescence anisotropy (polarization) experiments in membranes, including low fluorescence in water and moderately strong association with lipid bilayers. PTE-ET-18-OMe has absorbance and fluorescence properties similar to those of diphenylhexatriene (DPH) probes, with about as large a difference between its fluorescence anisotropy in liquid disordered (Ld) and ordered states (gel and Lo) as observed for DPH. Also like DPH, PTE-ET-18-OMe has a moderate affinity for both gel state ordered domains and Lo state ordered domains (rafts). However, unlike fluorescent sterols or DPH (Megha and London (2004) J. Biol. Chem. 279, 9997-10004), PTE-ET-18-OMe is not displaced from ordered domains by ceramide. Also unlike DPH, PTE-ET-18-OMe shows only slow exchange between the inner and outer leaflets of membrane bilayers, and can thus be used to examine anisotropy of an individual leaflet of a lipid bilayer. Since PTE-ET-18-OMe is a zwitterionic molecule, it should not be as influenced by electrostatic interactions as are other probes that do not cross the lipid bilayer but have a net charge. We conclude that PTE-ET-18-OMe has some unique properties that should make it a useful fluorescence probe of membrane structure.     

Regulation of calcium channel activity by lipid domain formation in planar lipid bilayers

The sarcoplasmic reticulum channel (ryanodine receptor) from cardiac myocytes was reconstituted into planar lipid bilayers consisting of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) and 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) in varying ratios. The channel activity parameters, i.e., open probability and average open time and its resolved short and long components, were determined as a function of POPE mole fraction (X(PE)) at 22.4 degrees C. Interestingly, all of these parameters exhibited a narrow and pronounced peak at X(PE) approximately 0.80. Differential scanning calorimetric measurements on POPE/POPC liposomes with increasing X(PE) indicated that the lipid bilayer enters a composition-driven transition from the liquid-crystalline state to the gel state at 22.4 degrees C when X(PE) approaches 0.80. Thus, the peaking of the reconstituted channel activity at X(PE) approximately 0.80 in the planar bilayer could result from the appearance of gel/liquid-crystalline domain boundaries at this POPE content. Lipid packing at domain boundaries is known to be looser as compared to the homogenous gel or liquid-crystalline state. We propose that the attractive potential of packing defects at lipid domain boundaries and entropic excluded-volume effects could result in the direct interactions of the transmembrane region of the channel protein with the lipid-packing defects at the lipid/protein interface, which could thus provide a favorable environment for the open state of the protein. The present findings indicate that the activity of the sarcoplasmic reticulum calcium channel could be modulated by lipid domain formation upon slight changes in membrane lipid composition in vivo.     

Modulation of (Na+,K+)-ATPase activity by the lipid bilayer examined with dansylated phosphatidylserine

The fluorescent probe 8-(dimethylamino)naphthalene-1-sulfonylphosphatidylserine (Dns-PS) was incorporated into purified lamb kidney Na+- and K+-stimulated adenosinetriphosphatase (EC 3.6.1.3) [(Na+,K+)-ATPase] by using a purified phospholipid exchange protein. Phospholipase C was used to reduce phospholipid content. Up to 40% of the phospholipid could be hydrolyzed with only 10% inhibition of the (Na+,K+)-ATPase, but when 67% of the phospholipid was hydrolyzed, the enzyme was inhibited 53%. To examine the effect of protein on the phospholipid bilayer, the fluorescent parameters of the probe incorporated into the enzyme preparation were contrasted with the same parameters for the probe incorporated into the total lipid extract of the preparation. The polarization of fluorescence of the probe in the lipid extract was 0.118 while in the enzyme preparation it was 0.218. This reflected a decrease in fluidity of the glycerol region of the phospholipid bilayer which was mediated by the protein. This effect increased as the phospholipid content of the (Na+,K+)-ATPase preparation was reduced so that with maximal phospholipid reduction the polarization of fluorescence was 0.262. The protein caused a decrease in the transition temperature from gel to fluid states of the bilayer detected by polarization of the probe. The midpoint temperature transition of the enzyme preparation decreased from 33 degrees C when all phospholipids were present to 20 degrees C when 67% of the phospholipids were hydrolyzed. This decrease was not observed for the lipid extract of these samples. A direct correlation between the (Na+,K+)-ATPase specific activity and the polarization of fluorescence of Dns-PS was found. The reduction in phospholipid content did not affect the steady-state level of phosphorylation of the enzyme by ATP but did affect the rate of dephosphorylation which would require conformational changes of the enzymes. The data showed that the fluidity of the phospholipid bilayer can modulate the activity of the (Na+,K+)-ATPase.     

Cholesterol modulation of lipid intermixing in phospholipid and glycosphingolipid mixtures. Evaluation using fluorescent lipid probes and brominated lipid quenchers.

Carbazole- and indole-labeled phospholipids have been used to monitor the homo- or heterogeneity of lipid mixing in several types of lipid bilayers combining a brominated and a nonbrominated lipid with varying amounts of cholesterol. Experimental quenching curves (relating the normalized probe fluorescence intensity to the mole fraction of brominated lipid) show a characteristic smooth, monophasic form for homogeneous liquid-crystalline lipid mixtures. However, for mixtures exhibiting lipid lateral segregation, such curves show marked perturbations in form over the region of composition where segregation occurs. Using this approach, it is found that high mole fractions of cholesterol (40-50 mol %) promote the formation of apparently homogeneous solutions in mixtures of disaturated and monounsaturated phosphatidylcholines (PCs) that exhibit extensive thermotropic phase separations in the absence of sterol. At only slightly lower levels of cholesterol, however, these systems exhibit inhomogeneous lipid mixing over a wide range of relative proportions of the two PC components. Mixtures of cerebroside and monounsaturated PCs, even at high bilayer cholesterol contents, exhibit significant inhomogeneity in lipid mixing over a wide range of cerebroside/PC ratios. Phase-separating PC/PC and PC/cerebroside mixtures can readily form long-lived metastable solutions when the level of the higher-melting component in the liquid-crystalline phase exceeds its equilibrium solubility by as much as 20-30 mol %; this tendency is significantly increased by cholesterol. Cholesterol shows no significant ability to enhance lipid intermixing in a third type of phase-separating lipid system, combining a monounsaturated PC with a monounsaturated phosphatidic acid--calcium complex. Experiments using cleavable phospholipid conjugates, linking a fluorescent lipid to a brominated lipid, suggest that each fluorescent molecule probes a local lipid domain comprising approximately less than 40-50 nearby acyl chains.     

Effect of membrane tension on the electric field and dipole potential of lipid bilayer membrane

The dipole potential of lipid bilayer membrane controls the difference in permeability of the membrane to oppositely charged ions. We have combined molecular dynamics (MD) simulations and experimental studies to determine changes in electric field and electrostatic potential of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid bilayer in response to applied membrane tension. MD simulations based on CHARMM36 force field showed that electrostatic potential of DOPC bilayer decreases by ~45mV in the physiologically relevant range of membrane tension values (0 to 15dyn/cm). The electrostatic field exhibits a peak (~0.8×10(9)V/m) near the water/lipid interface which shifts by 0.9? towards the bilayer center at 15dyn/cm. Maximum membrane tension of 15dyn/cm caused 6.4% increase in area per lipid, 4.7% decrease in bilayer thickness and 1.4% increase in the volume of the bilayer. Dipole-potential sensitive fluorescent probes were used to detect membrane tension induced changes in DOPC vesicles exposed to osmotic stress. Experiments confirmed that dipole potential of DOPC bilayer decreases at higher membrane tensions. These results are suggestive of a potentially new mechanosensing mechanism by which mechanically induced structural changes in the lipid bilayer membrane could modulate the function of membrane proteins by altering electrostatic interactions and energetics of protein conformational states.     

Simultaneous probing of hydration and polarity of lipid bilayers with 3-hydroxyflavone fluorescent dyes

The penetration of water into the hydrophobic interior leads to polarity and hydration profiles across lipid membranes which are fundamental in the maintenance of membrane architecture as well as in transport and insertion processes into the membrane. The present paper is an original attempt to evaluate simultaneously polarity and hydration properties of lipid bilayers by a fluorescence approach. We applied two 3-hydroxyflavone probes anchored in lipid bilayers at a relatively precise depth through their attached ammonium groups. They are present in two forms: either in H-bond-free form displaying a two-band emission due to an excited state intramolecular proton transfer reaction (ESIPT), or in H-bonded form displaying a single-band emission with no ESIPT. The individual emission profiles of these forms were obtained by deconvolution of the probes' fluorescence spectra. The polarity of the probe surrounding the bilayer was estimated from the two-band spectra of the H-bond-free form, while the local hydration was estimated from the relative contribution of the two forms. Our results confirm that by increasing the lipid order (phase transition from fluid to gel phase, addition of cholesterol or decrease in the lipid unsaturation), the polarity and to a lesser extent, the hydration of the bilayers decrease simultaneously. In contrast, when fluidity (i.e. lipid order) is kept invariant, increase of temperature and of bilayer curvature leads to a higher bilayer hydration with no effect on the polarity. Furthermore, no correlation was found between dipole potential and the hydration of the bilayers.     

The binding of a neutral aromatic molecule to a negatively-charged lipid membrane. I. Thermodynamics and mode of binding

This paper presents a detailed study of the binding of the fluorescent dye N-phenyl naphthylamine (NPN) to bilayers composed of the negatively-charged phospholipid methylphosphatidic acid. Binding to the liquid-crystalline membrane is enthalpy-driven. It is shown by determination of the binding constant and confirmed by n.m.r. that most of the dye ("guest") molecules reside between the lipid hydrocarbon chains at a fixed distance from the head-group, and are not distributed uniformly throughout the hydrocarbon phase. Each guest molecule is surrounded by about four lipid molecules. Transition of the membrane from the liquid-crystalline to the crystalline state results in almost total expulsion of the bound NPN into the water phase. Electrostatic theory is developed to find the effect of electrostatics upon the binding of a neutral molecule to charged membranes. Although the charge product is zero, electrostatic interactions play a part in determining the strength of binding, if each guest molecule incorporated increases the area of the membrane. For NPN this increase was found to be ca. 41 A(2).     

Assembly of the major light-harvesting complex II in lipid nanodiscs

Self-aggregation of isolated plant light-harvesting complexes (LHCs) upon detergent extraction is associated with fluorescence quenching and is used as an in vitro model to study the photophysical processes of nonphotochemical quenching (NPQ). In the NPQ state, in vivo induced under excess solar light conditions, harmful excitation energy is safely dissipated as heat. To prevent self-aggregation and probe the conformations of LHCs in a lipid environment devoid from detergent interactions, we assembled LHCII trimer complexes into lipid nanodiscs consisting of a bilayer lipid matrix surrounded by a membrane scaffold protein (MSP). The LHCII nanodiscs were characterized by fluorescence spectroscopy and found to be in an unquenched, fluorescent state. Remarkably, the absorbance spectra of LHCII in lipid nanodiscs show fine structure in the carotenoid and Q_y region that is different from unquenched, detergent-solubilized LHCII but similar to that of self-aggregated, quenched LHCII in low-detergent buffer without magnesium ions. The nanodisc data presented here suggest that 1), LHCII pigment-protein complexes undergo conformational changes upon assembly in nanodiscs that are not correlated with downregulation of its light-harvesting function; and 2), these effects can be separated from quenching and aggregation-related phenomena. This will expand our present view of the conformational flexibility of LHCII in different microenvironments.     

Prodan as a membrane surface fluorescence probe: partitioning between water and phospholipid phases.

Fluorescence spectral features of 6-propionyl-2-dimethylaminonaphthalene (Prodan) in phospholipid vesicles of different phase states are investigated. Like the spectra of 6-lauroyl-2-dimethylaminonaphthalene (Laurdan), the steady-state excitation and emission spectra of Prodan are sensitive to the polarity of the environment, showing a relevant shift due to the dipolar relaxation phenomenon. Because of the different lengths of their acyl residues, the partitioning of the two probes between water and the membrane bilayer differs profoundly. To account for the contribution of Prodan fluorescence arising from water, we introduce a three-wavelength generalized polarization method that makes it possible to separate the spectral properties of Prodan in the lipid phase and in water, and to determine the probe partitioning between phospholipid and water and between the gel and the liquid-crystalline phases of phospholipids. In contrast to Laurdan, Prodan preferentially partitions in the liquid-crystalline phase with respect to the gel and is sensitive to the polar head pretransition, and its partition coefficient between the membrane and water depends on the phase state, i.e., on the packing of the bilayer. Prodan is sensitive to polarity variations occurring closer to the bilayer surface than those detected by Laurdan.     

Effect of the vesicular stomatitis virus matrix protein on the lateral organization of lipid bilayers containing phosphatidylglycerol: use of fluorescent phospholipid analogues

In order to investigate the mode of interaction of peripheral membrane proteins with the lipid bilayer, the basic (pI approximately 9.1) matrix (M) protein of vesicular stomatitis virus was reconstituted with small unilamellar vesicles (SUV) containing phospholipids with acidic head groups. The lateral organization of lipids in such reconstituted membranes was probed by fluorescent phospholipid analogues labeled with pyrene fatty acids. The excimer/monomer (E/M) fluorescence intensity ratios of the intrinsic pyrene phospholipid probes were measured at various temperatures in M protein reconstituted SUV composed of 50 mol % each of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG). The M protein showed relatively small effects on the E/M ratio either in the gel or in the liquid-crystalline phase. However, during the gel to liquid-crystalline phase transition, the M protein induced a large increase in the E/M ratio due to phase separation of lipids into a neutral DPPC-rich phase and DPPG domains presumably bound to M protein. Similar phase separation of bilayer lipids was also observed in the M protein reconstituted with mixed lipid vesicles containing one low-melting lipid component (1-palmitoyl-2-oleoylphosphatidylcholine or 1-palmitoyl-2-oleoylphosphatidylglycerol) or a low mole percent of cholesterol. The self-quenching of 4-nitro-2,1,3-benzoxadiazole (NBD) fluorescence, as a measure of lipid clustering in the bilayer, was also studied in M protein reconstituted DPPC-DPPG vesicles containing 5 mol % NBD-phosphatidylethanolamine (NBD-PE). The quenching of NBD-PE was enhanced at least 2-fold in M protein reconstituted vesicles at temperatures within or below the phase transition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of a bacterial trehalose lipid on phosphatidylglycerol membranes

Bacterial trehalose lipids are biosurfactants with potential application in the biomedical/healthcare industry due to their interesting biological properties. Given the amphiphilic nature of trehalose lipids, the understanding of the molecular mechanism of their biological action requires that the interaction between biosurfactant and membranes is known. In this study we examine the interactions between a trehalose lipid from Rhodococcus sp. and dimyristoylphosphatidylglycerol membranes by means of differential scanning calorimetry, X-ray diffraction, infrared spectroscopy and fluorescence polarization. We report that there are extensive interactions between trehalose lipid and dimyristoylphosphatidylglycerol involving the perturbation of the thermotropic gel to liquid-crystalline phase transition of the phospholipid, the increase of fluidity of the phosphatidylglycerol acyl chains and dehydration of the interfacial region of the bilayer, and the modulation of the order of the phospholipid bilayer. The observations are interpreted in terms of structural perturbations affecting the function of the membrane that might underline the biological actions of the trehalose lipid.     

Fluorescence studies of dipalmitoylphosphatidylcholine vesicles reconstituted with the glycoprotein of vesicular stomatitis virus

The vesicular stomatitis virus glycoprotein (G) was reconstituted into dipalmitoylphosphatidylcholine (DPPC) vesicles by detergent dialysis. The DPPC gel to liquid-crystalline phase transition of the DPPC-G protein vesicles was monitored by the fluorescence anisotrophy of trans-paranaric acid, 16-(9-anthroyloxy)palmitoylglucocerebroside, 1,6-diphenyl-1,3,5-hexatriene, and 4-heptadecyl-7-hydroxycoumarin. The DPPC transition temperature measured by all four fluorescent probes was lowered in the presence of the G protein and the DPPC gel state was disordered by the G protein as evidenced by a decreased fluorescence anisotropy for all four probes below the phase-transition temperature. A possible ordering of the DPPC liquid-crystalline state by the G protein was indicated by an increased anisotropy of trans-paranaric acid and 16-(9-anthroyloxy)palmitoylglucocerebroside in the liquid-crystalline state of DPPC-G protein vesicles. The G protein in addition affected the ionization of the 4-heptadecyl-7-hydroxycoumarin in lipid vesicles, increasing the apparent pK of the probe from 9.05 to 9.45.