Effect of doxorubicin on the order of the acyl chains of anionic and zwitterionic phospholipids in liquid-crystalline mixed model membranes: absence of drug-induced segregation of lipids into extended domains.

We investigated the effect of the antineoplastic drug doxorubicin on the order of the acyl chains in liquid-crystalline mixed bilayers consisting of dioleoylphosphatidylserine (DOPS) or -phosphatidic acid (DOPA), and dioleoylphosphatidylcholine (DOPC) or -phosphatidylethanolamine (DOPE). Previous 2H-NMR studies on bilayers consisting of a single species of di[11,11-2H2]oleoyl-labeled phospholipid showed that doxorubicin does not affect the acyl chain order of pure zwitterionic phospholipid but dramatically decreases the order of anionic phospholipid [de Wolf, F. A., et al. (1991) Biochim. Biophys. Acta 1096, 67-80]. In the present work, we studied mixed bilayers in which alternatively the anionic or the zwitterionic phospholipid component was 2H-labeled so as to monitor its individual acyl chain order. Doxorubicin decreased the order parameter of the mixed anionic and zwitterionic lipids by approximately the same amount and did not induce a clear segregation of the lipid components into extended, separate domains. The drug had a comparable disordering effect on mixed bilayers of unlabeled cardiolipin and 2H-labeled zwitterionic phospholipid, indicating the absence of extensive segregation also in that case. Upon addition of doxorubicin to bilayers consisting of 67 mol% DOPE and 33 mol% anionic phospholipid, a significant part of the lipid adopted the inverted hexagonal (HII) phase at 25 degrees C. This bilayer destabilization, which occurred only in mixtures of anionic phospholipid and sufficient amounts of DOPE, might be of physiological importance. Even upon formation of extended HII-phase domains, lipid segregation was not clearly detectable, since the relative distribution of 2H-labeled anionic phospholipid and [2H]DOPE between the bilayer phase and HII phase was very similar. Our findings argue against a role of extensive anionic/zwitterionic lipid segregation in the mechanism of action and toxicity of doxorubicin.     

Lipid specificity for membrane mediated partial unfolding of cytochrome c

In this study we investigated the lipid specificity for destabilization of the native structure of horse heart cytochrome c by model membranes. From (i) the enhanced release of deuterium from deuterium-labelled cytochrome c and (ii) the increased proteolytic digestion of the protein in the presence of anionic lipids, it was concluded that these lipids are able to destabilize the native structure of cytochrome c. Changes in the absorbance at 695 nm indicated that the destabilization was accompanied by a diminished ligation of Met-80 to the heme. Beef heart cardiolipin was found to be more effective than DOPS, DOPG or DOPA, while no protein destabilization was observed in the presence of the zwitterionic lipid DOPC or, surprisingly, in the presence of E. coli cardiolipin. Experimnts with mitoplasts showed that the protein can also be destabilized in its native structure by a biological membrane.     

Saturated anionic phospholipids enhance transdermal transport by electroporation

Anionic phospholipids, but not cationic or neutral phospholipids, were found to enhance the transdermal transport of molecules by electroporation. When added as liposomes to the milieus of water-soluble molecules to be delivered through the epidermis of porcine skin by electroporation, these phospholipids enhance, by one to two orders of magnitude, the transdermal flux. Encapsulation of molecules in liposomes is not necessary. Dimyristoylphosphatidylserine (DMPS), phosphatidylserine from bovine brain (brain-PS), dioleoylphosphatidylserine (DOPS), and dioleoylphosphatidylglycerol (DOPG) were used to test factors affecting the potency of anionic lipid transport enhancers. DMPS with saturated acyl chains was found to be a much more potent transport enhancer than those with unsaturated acyl chains (DOPS and DOPG). There was no headgroup preference. Saturated DMPS was also more effective in delaying resistance recovery after pulsing, and with a greater affinity in the epidermis after pulsing. Using fluorescent carboxyl fluorescein and fluorescein isothiocyanate (FITC)-labeled Dextrans as test water-soluble molecules for transport, and rhodamine-labeled phospholipids to track anionic phospholipids, we found, by conventional and confocal fluorescence microscopy, that transport of water-soluble molecules was localized in local transport spots or regions (LTRs) created by the electroporation pulses. Anionic phospholipids, especially DMPS, were located at the center of the LTRs and spanned the entire thickness of the stratum corneum (SC). The degree of saturation of anionic phospholipids made no difference in the densities of LTRs created. We deduce that, after being driven into the epidermis by negative electric pulses, saturated anionic phospholipids mix and are retained better by the SC lipids. Anionic lipids prefer loose layers or vesicular rather than multilamellar forms, thereby prolonging the structural recovery of SC lipids to the native multilamellar form. In the presence of 1 mg/ml DMPS in the transport milieu, the flux of FITC-Dextran-4k was enhanced by 80-fold and reached 175 microg/cm(2)/min. Thus, the use of proper lipid enhancers greatly extends the upper size limit of transportable chemicals. Understanding the mechanism of lipid enhancers enables one to rationally design better enhancers for transdermal drug and vaccine delivery by electroporation.     

Phospholipid-cationic lipid interactions: influences on membrane and vesicle properties

Liposomes composed of synthetic dialkyl cationic lipids and zwitterionic phospholipids such as dioleoylphosphatidylethanolamine have been studied extensively as vehicles for gene delivery, but the broader potentials of these cationic liposomes for drug delivery have not. An understanding of phospholipid-cationic lipid interactions is essential for rational development of this potential. We evaluated the effect of the cationic lipid DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium) on liposome physical properties such as size and membrane domain structure. DSC (differential scanning calorimetry) showed progressive decrease and broadening of the phase transition temperature of dipalmitoylphosphatidylcholine (DPPC) with increasing fraction of DOTAP, in the range of 0.4-20 mol%. Laurdan (6-dodecanolyldimethylamino-naphthalene), a fluorescent probe of membrane domain structure, showed that DOTAP and DPPC remained miscible at all ratios tested. DOTAP reduced the size of spontaneously-forming PC-containing liposomes, regardless of the acyl chain length and degree of saturation. The anionic lipid DOPG (dioleoylphosphatidylglycerol) had similar effects on DPPC membrane fluidity and size. However, DOTAP/DOPC (50/50) vesicles were taken up avidly by OVCAR-3 human ovarian tumor cells, in contrast to DOPG/DOPC (50/50) liposomes. Overall, DOTAP exerts potent effects on bilayer physical properties, and may provide advantages for drug delivery.     

Effect of lipid composition on the topography of membrane-associated hydrophobic helices: stabilization of transmembrane topography by anionic lipids

To investigate the effect of lipid structure upon the membrane topography of hydrophobic helices, the behavior of hydrophobic peptides was studied in model membrane vesicles. To define topography, fluorescence and fluorescence quenching methods were used to determine the location of a Trp at the center of the hydrophobic sequence. For peptides with cationic residues flanking the hydrophobic sequence, the stability of the transmembrane (TM) configuration (relative to a membrane-bound non-TM state) increased as a function of lipid composition on the order: 1:1 (mol:mol) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC):1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine ∼ 6:4 POPC:cholesterol < POPC ∼ dioleoylphosphatidylcholine (DOPC) < 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] sodium salt (DOPG) ≤ 1,2-dioleoyl-sn-glycero-3-[phospho-l-serine] sodium salt (DOPS), indicating that the anionic lipids DOPG and DOPS most strongly stabilized the TM configuration. TM stabilization was near maximal at 20-30 mol% anionic lipid, which are physiologically relevant values. TM stabilization by anionic lipid was observed for hydrophobic sequences with a diverse set of sequences (including polyAla), diverse lengths (from 12 to 22 residues), and various cationic flanking residues (H, R, or K), but not when the flanking residues were uncharged. TM stabilization by anionic lipid was also dependent on the number of cationic residues flanking the hydrophobic sequence, but was still significant with only one cationic residue flanking each end of the peptide. These observations are consistent with TM-stabilizing effects being electrostatic in origin. However, Trp located more deeply in DOPS vesicles relative to DOPG vesicles, and peptides in DOPS vesicles showed increased helix formation relative to DOPG and all other lipid compositions. These observations fit a model in which DOPS anchors flanking residues near the membrane surface more strongly than does DOPG and/or increases the stability of the TM state to a greater degree than DOPG. We conclude that anionic lipids can have significant and headgroup structure-specific effects upon membrane protein topography.     

Liposomes for cryopreservation of bovine sperm

In this study, the effect of various unilamellar liposomes on cryopreservation of bovine spermatozoa has been investigated. Liposomes were composed of saturated lipids with various acyl chain lengths: DSPC (18:0), DPPC (16:0), DMPC (14:0), or DLPC (12:0). Alternatively, liposomes were prepared using unsaturated egg phosphatidylcholine (EPC) or DOPC (18:1, neutral), alone or in combination with lipids with various head groups: DOPS (negatively charged), DOPG (negatively charged), and DOPE (neutral). Fourier transform infrared spectroscopy studies showed that bovine sperm membranes display a gradual phase transition from 10 to 24 ^oC. Phase transition temperatures of the liposomes varied from −20 to +53 ^oC. Sperm was incubated in the presence of liposomes for either 6 or 24 h at 4 °C prior to freezing. Postfreeze survival rates were determined based on the percentage of progressively motile cells as well as the percentage of acrosome- and plasma membrane-intact cells. With DOPC liposomes a postthaw progressive motility of 43% was obtained compared with 59% using standard egg yolk freezing extender. Postthaw progressive motility increased up to 52% using DOPC:DOPG (9:1) liposomes, whereas DOPC:DOPS or DOPC:DOPE liposomes did not increase survival compared with DOPC liposomes. Among the saturated lipids, only DMPC was found to increase cryosurvival, up to 44% based on progressive motility. DLPC liposomes caused a complete loss in cell viability, already prior to freezing, whereas DPPC and DSPC liposomes neither positively nor negatively affected cryosurvival. Taken together, the higher postthaw survival obtained with DOPC:DOPG liposomes as compared with DOPC liposomes can likely be attributed to increased liposome-sperm interactions between the charged phosphatidylglycerol groups and charged regions in the sperm membranes. Interestingly, the lipid phase state of the liposomes during preincubation is not the decisive factor for their cryoprotective action.     

Anionic amphiphile and phospholipid-induced conformational changes in human neutrophil flavocytochrome b observed by fluorescence resonance energy transfer

The integral membrane protein flavocytochrome b (Cyt b) comprises the catalytic core of the human phagocyte NADPH oxidase complex and serves to initiate a cascade of reactive oxygen species that participate in the elimination of infectious agents. Superoxide production by the NADPH oxidase complex has been shown to be specifically regulated by the enzymatic generation of lipid second messengers following phagocyte activation. In the present study, a Cyt b-specific monoclonal antibody (mAb 44.1) was labeled with Cascade Blue (CCB) and used in resonance energy transfer (RET) studies probing the effects of a panel of lipid species on the structure of Cyt b. The binding of CCB-mAb 44.1 to immunoaffinity-purified Cyt b was both highly specific and resulted in significant quenching of the steady state donor fluorescence. Titration of the CCB-mAb 44.1:Cyt b complex with the anionic amphiphile lithium dodecyl sulfate (LDS) resulted in a saturable relaxation of fluorescence quenching due to conformational changes in Cyt b at concentrations of the amphiphile required for maximum rates of superoxide production by Cyt b in cell-free assays. Similar results were observed for the anionic amphiphile arachidonic acid (AA), although no relaxation of fluorescence quenching was observed for arachidonate methyl ester (AA-ME). Saturable relaxation of fluorescence quenching was also observed with the anionic, 18:1 phospholipids phosphatidic acid (DOPA) and phosphatidylserine (DOPS), while no relaxation was observed upon addition of the neutral 18:1 lipids phosphatidylcholine (DOPC), phosphatidylethanolamine (DOPE) or diacylglycerol (DAG) at similar levels. Further examination of a variety of phosphatidic acid (PA) species demonstrated DOPA to both potently induce conformational changes in Cyt b and to cause more dramatic conformational changes than PA species with shorter, saturated acyl chains. The data presented in this study support the hypothesis that second messenger lipids, such as AA and PA, directly bind to flavocytochrome b and modulate conformational states relevant to the activation of superoxide production.     

Dependence of M13 major coat protein oligomerization and lateral segregation on bilayer composition

M13 major coat protein was derivatized with BODIPY (n-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-yl)methyl iodoacetamide), and its aggregation was studied in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and DOPC/1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DOPG) or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)/DOPG (model systems of membranes with hydrophobic thickness matching that of the protein) using photophysical methodologies (time-resolved and steady-state self-quenching, absorption, and emission spectra). It was concluded that the protein is essentially monomeric, even in the absence of anionic phospholipids. The protein was also incorporated in pure bilayers of lipids with a strong mismatch with the protein transmembrane length, 1,2-dierucoyl-sn-glycero-3-phosphocholine (DEuPC, longer lipid) and 1,2-dimyristoleoyl-sn-glycero-3-phosphocholine (DMoPC, shorter lipid), and in lipidic mixtures containing DOPC and one of these lipids. The protein was aggregated in the pure vesicles of mismatching lipid but remained essentially monomeric in the mixtures as detected from BODIPY fluorescence emission self-quenching. From fluorescence resonance energy transfer (FRET) measurements (donor-n-(iodoacetyl)aminoethyl-1-sulfonaphthylamine (IAEDANS)-labeled protein; acceptor-BODIPY labeled protein), it was concluded that in the DEuPC/DOPC and DMoPC/DOPC lipid mixtures, domains enriched in the protein and the matching lipid (DOPC) are formed.     

Structural changes in a binary mixed phospholipid bilayer of DOPG and DOPS upon saposin C interaction at acidic pH utilizing 31P and 2H solid-state NMR spectroscopy

Saposin C (Sap C) is known to stimulate the catalytic activity of the lysosomal enzyme glucosylceramidase (GCase) that facilitates the hydrolysis of glucosylceramide to ceramide and glucose. Both Sap C and acidic phospholipids are required for full activity of GCase. In order to better understand this interaction, mixed bilayer samples prepared from dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylserine (DOPS) (5:3 ratio) and Sap C were investigated using (2)H and (31)P solid-state NMR spectroscopy at temperatures ranging from 25 to 50 degrees C at pH 4.7. The Sap C concentrations used to carry out these experiments were 0 mol%, 1 mol% and 3 mol% with respect to the phospholipids. The molecular order parameters (S(CD)) were calculated from the dePaked (2)H solid-state NMR spectra of Distearoyl-d70-phosphatidylglycerol (DSPG-d70) incorporated with DOPG and DOPS binary mixed bilayers. The S(CD) profiles indicate that the addition of Sap C to the negatively charged phospholipids is concentration dependent. S(CD) profiles of 1 mol% of the Sap C protein show only a very slight decrease in the acyl chain order. However, the S(CD) profiles of the 3 mol% of Sap C protein indicate that the interaction is predominantly increasing the disorder in the first half of the acyl chain near the head group (C1-C8) indicating that the amino and the carboxyl termini of Sap C are not inserting deep into the DOPG and DOPS mixed bilayers. The (31)P solid-state NMR spectra show that the chemical shift anisotropy (CSA) for both phospholipids decrease and the spectral broadening increases upon addition of Sap C to the mixed bilayers. The data indicate that Sap C interacts similarly with the head groups of both acidic phospholipids and that Sap C has no preference to DOPS over DOPG. Moreover, our solid-state NMR spectroscopic data agree with the structural model previously proposed in the literature [X. Qi, G.A. Grabowski, Differential membrane interactions of saposins A and C. Implication for the functional specificity, J. Biol. Chem. 276 (2001) 27010-27017] [1].     

Phase equilibria of model milk membrane lipid systems

The phase behaviour of mixtures of recombined milk membrane lipids dioleoylphosphatidylcholine (DOPC), sphingomyelin (SM), dioleoylphosphatidylethanolamine (DOPE), phosphatidylinositol (PI) and dioleoylphosphatidylserine (DOPS) in 60% water was examined as a function of temperature between 5 and 90 degrees C. The aim was to examine under which lipid composition the average properties turn from balanced over to hydrophobic. The phase boundaries were determined by small angle X-ray diffraction (SAXD) and differential scanning calorimetry (DSC). The lamellar phase was dominating in the DOPC/SM/DOPE system. The phase boundary for the reversed hexagonal phase was only observed at high DOPE content within the examined temperature interval. The anionic phospholipids PI and DOPS induced a swollen lamellar phase, but no significant change of the transition between the lamellar phase and the reversed hexagonal phase was observed.     

Lipid bilayers cushioned with polyelectrolyte-based films on doped silicon surfaces

Silicon semiconductors with a thin surface layer of silica were first modified with polyelectrolytes (polyethyleneimine, polystyrene sulfonate and poly(allylamine)) via a facile layer-by-layer deposition approach. Subsequently, lipid vesicles were added to the preformed polymeric cushion, resulting in the adsorption of intact vesicles or fusion and lipid bilayer formation. To study involved interactions we employed optical reflectometry, electrochemical impedance spectroscopy and fluorescent recovery after photobleaching. Three phospholipids with different charge of polar head groups, i.e. 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) were used to prepare vesicles with varying surface charge. We observed that only lipid vesicles composed from 1:1 (mole:mole) mixture of DOPC/DOPS have the ability to fuse onto an oppositely charged terminal layer of polyelectrolyte giving a lipid bilayer with a resistance of >100 kΩ. With optical reflectometry we found that the vesicle surface charge is directly related to the amount of mass adsorbed onto the surface. An interesting observation was that zwitterionic polar head groups of DOPC allow the adsorption on both positively and negatively charged surfaces. As found with fluorescent recovery after photobleaching, positively charged surface governed by the presence of poly(allylamine) as the terminal layer resulted in intact DOPC lipid vesicles adsorption whereas in the case of a negatively charged silica surface formation of lipid bilayers was observed, as expected from literature.     

Effects of phospholipid composition on MinD-membrane interactions in vitro and in vivo

The peripheral membrane ATPase MinD is a component of the Min system responsible for correct placement of the division site in Escherichia coli cells. By rapidly migrating from one cell pole to the other, MinD helps to block unwanted septation events at the poles. MinD is an amphitropic protein that is localized to the membrane in its ATP-bound form. A C-terminal domain essential for membrane localization is predicted to be an amphipathic alpha-helix with hydrophobic residues interacting with lipid acyl chains and cationic residues on the opposite face of the helix interacting with the head groups of anionic phospholipids (Szeto, T. H., Rowland, S. L., Rothfield, L. I., and King, G. F. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 15693-15698). To investigate whether E. coli MinD displays a preference for anionic phospholipids, we first examined the localization dynamics of a green fluorescent protein-tagged derivative of MinD expressed in a mutant of E. coli that lacks phosphatidylethanolamine. In these cells, which contain only anionic phospholipids (phosphatidylglycerol and cardiolipin), green fluorescent protein-MinD assembled into dynamic focal clusters instead of the broad zones typical of cells with normal phospholipid content. In experiments with liposomes composed of only zwitterionic, only anionic, or a mixture of anionic and zwitterionic phospholipids, purified MinD bound to these liposomes in the presence of ATP with positive cooperativity with respect to the protein concentration and exhibited Hill coefficients of about 2. Oligomerization of MinD on the liposome surface also was detected by fluorescence resonance energy transfer between MinD molecules labeled with different fluorescent probes. The affinity of MinD-ATP for anionic liposomes as well as liposomes composed of both anionic and zwitterionic phospholipids increased 9- and 2-fold, respectively, relative to zwitterionic liposomes. The degree of acyl chain unsaturation contributed positively to binding strength. These results suggest that MinD has a preference for anionic phospholipids and that MinD oscillation behavior, and therefore cell division site selection, may be regulated by membrane phospholipid composition.     

Structural changes in a binary mixed phospholipid bilayer of DOPG and DOPS upon saposin C interaction at acidic pH utilizing P and H solid-state NMR spectroscopy

Saposin C (Sap C) is known to stimulate the catalytic activity of the lysosomal enzyme glucosylceramidase (GCase) that facilitates the hydrolysis of glucosylceramide to ceramide and glucose. Both Sap C and acidic phospholipids are required for full activity of GCase. In order to better understand this interaction, mixed bilayer samples prepared from dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylserine (DOPS) (5:3 ratio) and Sap C were investigated using ²H and ³¹P solid-state NMR spectroscopy at temperatures ranging from 25 to 50 °C at pH 4.7. The Sap C concentrations used to carry out these experiments were 0 mol%, 1 mol% and 3 mol% with respect to the phospholipids. The molecular order parameters (S_CD) were calculated from the dePaked ²H solid-state NMR spectra of Distearoyl-d70-phosphatidylglycerol (DSPG-d70) incorporated with DOPG and DOPS binary mixed bilayers. The S_CD profiles indicate that the addition of Sap C to the negatively charged phospholipids is concentration dependent. S_CD profiles of 1 mol% of the Sap C protein show only a very slight decrease in the acyl chain order. However, the S_CD profiles of the 3 mol% of Sap C protein indicate that the interaction is predominantly increasing the disorder in the first half of the acyl chain near the head group (C1-C8) indicating that the amino and the carboxyl termini of Sap C are not inserting deep into the DOPG and DOPS mixed bilayers. The ³¹P solid-state NMR spectra show that the chemical shift anisotropy (CSA) for both phospholipids decrease and the spectral broadening increases upon addition of Sap C to the mixed bilayers. The data indicate that Sap C interacts similarly with the head groups of both acidic phospholipids and that Sap C has no preference to DOPS over DOPG. Moreover, our solid-state NMR spectroscopic data agree with the structural model previously proposed in the literature [X. Qi, G.A. Grabowski, Differential membrane interactions of saposins A and C. Implication for the functional specificity, J. Biol. Chem. 276 (2001) 27010-27017] [1].     

Binding of peripheral proteins to mixed lipid membranes: effect of lipid demixing upon binding

Binding isotherms have been determined for the association of horse heart cytochrome c with dioleoyl phosphatidylglycerol (DOPG)/dioleoyl phosphatidylcholine (DOPC) bilayer membranes over a range of lipid compositions and ionic strengths. In the absence of protein, the DOPG and DOPC lipids mix nearly ideally. The binding isotherms have been analyzed using double layer theory to account for the electrostatics, either the Van der Waals or scaled particle theory equation of state to describe the protein surface distribution, and a statistical thermodynamic formulation consistent with the mass-action law to describe the lipid distribution. Basic parameters governing the electrostatics and intrinsic binding are established from the binding to membranes composed of anionic lipid (DOPG) alone. Both the Van der Waals and scaled particle equations of state can describe the effects of protein distribution on the DOPG binding isotherms equally well, but with different values of the maximum binding stoichiometry (13 lipids/protein for Van der Waals and 8 lipids/protein for scaled particle theory). With these parameters set, it is then possible to derive the association constant, Kr, of DOPG relative to DOPC for surface association with bound cytochrome c by using the binding isotherms obtained with the mixed lipid membranes. A value of Kr (DOPG:DOPC) = 3.3-4.8, depending on the lipid stoichiometry, is determined that consistently describes the binding at different lipid compositions and different ionic strengths. Using the value of Kr obtained it is possible to derive the average in-plane lipid distribution and the enhancement in protein binding induced by lipid redistribution using the statistical thermodynamic theory.     

Lateral Diffusion of Membrane Proteins: Consequences of Hydrophobic Mismatch and Lipid Composition

Biological membranes are composed of a large number lipid species differing in hydrophobic length, degree of saturation, and charge and size of the headgroup. We now present data on the effect of hydrocarbon chain length of the lipids and headgroup composition on the lateral mobility of the proteins in model membranes. The trimeric glutamate transporter (GltT) and the monomeric lactose transporter (LacY) were reconstituted in giant unilamellar vesicles composed of unsaturated phosphocholine lipids of varying acyl chain length (14-22 carbon atoms) and various ratios of DOPE/DOPG/DOPC lipids. The lateral mobility of the proteins and of a fluorescent lipid analog was determined as a function of the hydrophobic thickness of the bilayer (h) and lipid composition, using fluorescence correlation spectroscopy. The diffusion coefficient of LacY decreased with increasing thickness of the bilayer, in accordance with the continuum hydrodynamic model of Saffman-Delbrück. For GltT, the mobility had its maximum at diC18:1 PC, which is close to the hydrophobic thickness of the bilayer in vivo. The lateral mobility decreased linearly with the concentration of DOPE but was not affected by the fraction of anionic lipids from DOPG. The addition of DOPG and DOPE did not affect the activity of GltT. We conclude that the hydrophobic thickness of the bilayer is a major determinant of molecule diffusion in membranes, but protein-specific properties may lead to deviations from the Saffman-Delbrück model.     

Lipolysis in model membranes in the presence of positively charged soluble proteins]

Phospholipase A2 hydrolysis of neutral and negatively charged lipid membranes modified by positively charged proteins has been studied using liposomes composed of either dioleoylphosphatidylcholine (DOPC) or dioleoylphosphatidylglycerol (DOPG) alone or their equimolar mixture in the presence of cytochrome c, histone H1, cytochrome b5, and polylysine. Twenty minutes after the reaction had been initiated, DOPC hydrolysis was 58%, while that in the equimolar mixture with DOPG was 35%. DOPG hydrolysis was more complete in binary mixtures of liposomes. The same was observed for liposomes in the presence of cytochrome c. Hydrolysis of phospholipids in binary liposomes in the presence of histone H1 was 3 times faster than that in protein-free liposomes. In the presence of polylysine the rate of DOPG hydrolysis was decreased. The results obtained are suggestive of electrostatic interactions between hydrophilic proteins and negatively charged phospholipids, the phospholipase A2 catalytic activity being affected by these interactions.     

Binding of doxorubicin to cardiolipin as compared to other anionic phospholipids—An evaluation of electrostatic effects

The binding of doxorubicin to large unilamellar vesicles consisting of cardiolipin or other anionic phospholipids was analyzed in terms of the local drug concentration at the membrane surface, according to the Gouy-Chapman theory. The analysis suggests strong positive binding cooperativity. Part of the drug binds in the uncharged form. The affinity for cardiolipin and other anionic phospholipids is comparable. A binding level of 0.5 doxorubicin per lipid-phosphorus is reached when the local concentration of free doxorubicin monomer-equivalents at the membrane surface is about 0.2–0.7 mM. This contrasts with earlier findings indicating a 300–1000 fold higher affinity for cardiolipin. The present analysis provides an explanation for this apparent discrepancy.     

tBid forms a pore in the liposome membrane

We investigated the ability of tBid (truncated form of Bid) to bind and permeabilize the liposomes (large unilamellar vesicles, LUVs) and release fluorescent marker molecules (fluorescein-isothiocyanate-conjugated dextrans, FITC-dextrans) of various molecular diameters (FD-20, FD-70, FD-250S) from LUVs. Obtained data showed that tBid was more efficient in promoting leakage of FITC-dextrans from LUVs composed of cardiolipin and dioleoylphosphatidylcholine (DOPC) than LUVs made of dioleoylphosphatidic acid or dioleoylphosphatidylglycerol and DOPC. The leakage efficiency was reduced with increasing amount of dioleoylphosphatidylethanolamine or dielaidoylphosphatidylethanolamine. Phospholipid monolayer assay and fluorescence quenching measurements revealed that tBid inserted deeply into the hydrophobic acyl chain of acidic phospholipids. Taking into account the tBid three-dimensional structure, we propose that tBid could penetrate into the hydrophobic core of membrane, resulting in the leakage of entrapped content from LUVs via a pore-forming mechanism.     

The pH dependence of headgroup and acyl chain structure and dynamics of phosphatidylserine, studied by 2H-NMR

By varying the pH, the influence of the ionization degree on the structure and dynamics of aqueous dispersions of 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) was studied, using 2H-NMR methods. For this purpose DOPS was synthesized with deuterium labels incorporated either stereospecifically at the beta-position of the serine headgroup ([2-2H]DOPS) or at the 11-position of both acyl chains ([11,11-2H2]DOPS), allowing the effects of pH on headgroup and acyl chains to be measured in parallel. A large scale synthesis procedure of stereospecific 1,2-dioleoyl-sn-glycero-3-phospho-[2-2H]-L- serine is described. The quadrupolar splitting (delta nu q) of [2-2H]DOPS is shown to be a sensitive sensor for the degree of protonation of the molecule. Whereas the delta nu q of [2-2H]DOPS decreases upon lowering the pH, that of [11,11-2H2]DOPS gradually increases, indicating an increase in acyl chain ordering. In the pH range below the pKa value, DOPS exhibits a temperature-dependent bilayer to hexagonal HII phase transition, apparent from the 31P-NMR spectra and the occurrence of a second component in the [11,11-2H2]DOPS 2H-NMR spectrum, with a much smaller delta nu q. The HII phase component in spectra from [2-2H]DOPS coincides with the isotropic position and has no defined delta nu q. In the bilayer organization delta nu q and spin-lattice relaxation time (T1) values for the acyl chain deuterated DOPS are similar to those obtained for other lipid systems. In contrast the PS headgroup region displays a relatively rigid structure as evidenced by a large delta nu q and very small T1 values. Upon adopting the HII phase the T1 values of the acyl chain deuterons are hardly affected. The uniqueness of the PS headgroup with respect to structure and motional properties is reinforced by the occurrence of a T1 minimum at 45 degrees C in the measurement of the temperature dependence of T1 for [2-2H]DOPS in the hexagonal HII configuration. Quantitative analysis yields a correlation time (tau c) for the motions determining T1 under these conditions, of 3.45 ns.     

Real-time Observation of Lipoplex Formation and Interaction with Anionic Bilayer Vesicles

A novel development has allowed for the direct observation of single, pairwise interactions of linear DNA with cationic vesicles and of DNA-cationic lipid complexes with anionic vesicles. A new cationic phospholipid derivative, l,2-dioleoyl-sn-glycero-3-ethylphosphocholine, was used to prepare giant bilayer vesicles and to form DNA-cationic lipid complexes (lipoplexes). The cationic vesicles were electrophoretically maneuvered into contact with DNA, and similarly, complexes were brought into contact with anionic phospholipid vesicles composed of dioleoylphosphatidylglycerol (DOPG; 100%), DOPG/dioleoylphosphatidylethanolamine (DOPE; 1:1) or DOPG/dioleoylphosphatidylcholine (DOPC; 1:1). Video fluorescence microscopy revealed that upon contact with phospholipid anionic vesicles, lipoplexes exhibited four different types of behavior: adhesion, vesicle rupture, membrane perforation (manifested as vesicle shrinkage and/or content loss), and expansion of DNA (which was always concomitant with membrane perforation.) In one instance, the lipoplex was injected into the target vesicle just prior to DNA expansion. In all other instances, the DNA expanded over the outer surface of the vesicle, and expansion was faster, the larger the area of vesicle over which it expanded. Given the likelihood of incorporation of cellular anionic lipids into lipoplexes, the expansion of the DNA could be important in DNA release during cell transfection. Upon contact with naked DNA, giant cationic vesicles usually ruptured and condensed the DNA into a small particle. Contact of cationic vesicles that were partially coated with DNA usually caused the DNA to wrap around the vesicle, leading to vesicle rupture, vesicle fusion (with other attached vesicles or lipid aggregates), or simply cessation of movement. These behaviors clearly indicated that both DNA and vesicles could be partly or fully covered by the other, thus modifying surface charges, which, among others, allowed adhesion of DNA-coated vesicles with uncoated vesicles and of lipid-coated DNA with uncoated DNA.