High cationic charge and bilayer interface-binding helices in a regulatory lipid glycosyltransferase

In the prokaryote Acholeplasma laidlawii, membrane bilayer properties are sensed and regulated by two interface glycosyltransferases (GTs), synthesizing major nonbilayer- (alMGS GT) and bilayer-prone glucolipids. These enzymes are of similar structure, as many soluble GTs, but are sensitive to lipid charge and curvature stress properties. Multivariate and bioinformatic sequence analyses show that such interface enzymes, in relation to soluble ones of similar fold, are characterized by high cationic charge, certain distances between small and cationic amino acids, and by amphipathic helices. Varying surface contents of Lys/Arg pairs and Trp indicate different membrane-binding subclasses. A predicted potential (cationic) binding helix from alMGS was structurally verified by solution NMR and CD. The helix conformation was induced by a zwitterionic as well as anionic lipid environment, and the peptide was confined to the bilayer interface. Bilayer affinity of the peptide, analyzed by surface plasmon resonance, was higher than that for soluble membrane-seeking proteins/peptides and rose with anionic lipid content. Interface intercalation was supported by phase equilibria in membrane lipid mixtures, analyzed by 31P NMR and DSC. An analogous, potentially binding helix has a similar location in the structurally determined Escherichia coli cell wall precursor GT MurG. These two helices have little sequence conservation in alMGS and MurG homologues but maintain their amphipathic character. The evolutionary modification of the alMGS binding helix and its location close to the acceptor substrate site imply a functional importance in enzyme catalysis, potentially providing a mechanism by which glycolipid synthesis will be sensitive to membrane surface charge and intrinsic curvature strain.     

Irreversible binding and activity control of the 1,2-diacylglycerol 3-glucosyltransferase from Acholeplasma laidlawii at an anionic lipid bilayer surface

1,2-Diacylglycerol 3-glucosyltransferase is associated with the membrane surface catalyzing the synthesis of the major nonbilayer-prone lipid alpha-monoglucosyl diacylglycerol (MGlcDAG) from 1,2-DAG in the cell wall-less Acholeplasma laidlawii. Phosphatidylglycerol (PG), but not neutral or zwitterionic lipids, seems to be essential for an active conformation and function of the enzyme. Surface plasmon resonance analysis was employed to study association of the enzyme with lipid bilayers. Binding kinetics could be well fitted only to a two-state model, implying also a (second) conformational step. The enzyme bound less efficiently to liposomes containing only zwitterionic lipids, whereas increasing molar fractions of the anionic PG or cardiolipin (CL) strongly promoted binding by improved association (k(a1)), and especially a decreased rate of return (k(d2)) from the second state. This yielded a very low overall dissociation constant (K(D)), corresponding to an essentially irreversible membrane association. Both liposome binding and consecutive activity of the enzyme correlated with the PG concentration. The importance of the electrostatic interactions with anionic lipids was shown by quenching of both binding and activity with increasing NaCl concentrations, and corroborated in vivo for an active enzyme-green fluorescent protein hybrid in Escherichia coli. Nonbilayer-prone lipids substantially enhanced enzyme-liposome binding by promoting a changed conformation (decreasing k(d2)), similar to the anionic lipids, indicating the importance of hydrophobic interactions and a curvature packing stress. For CL and the nonbilayer lipids, effects on enzyme binding and consecutive activity were not correlated, suggesting a separate lipid control of activity. Similar features were recorded with polylysine (cationic) and polyglutamate (anionic) peptides present, but here probably dependent on the selective charge interactions with the enzyme N- and C-domains, respectively. A lipid-dependent conformational change and PG association of the enzyme were verified by circular dichroism, intrinsic tryptophan, and pyrene-probe fluorescence analyses, respectively. It is concluded that an electrostatic association of the enzyme with the membrane surface is accompanied by hydrophobic interactions and a conformational change. However, specific lipids, the curvature packing stress, and proteins or small molecules bound to the enzyme can modulate the activity of the bound A. laidlawii MGlcDAG synthase.     

The nonbilayer/bilayer lipid balance in membranes. Regulatory enzyme in Acholeplasma laidlawii is stimulated by metabolic phosphates, activator phospholipids, and double-stranded DNA

In membranes of Acholeplasma laidlawii a single glucosyltransferase step between the major, nonbilayer-prone monoglucosyl-diacylglycerol (MGlcDAG) and the bilayer-forming diglucosyl-diacylglycerol (DGlcDAG) is important for maintenance of lipid phase equilibria and curvature packing stress. This DGlcDAG synthase is activated in a cooperative fashion by phosphatidylglycerol (PG), but in vivo PG amounts are not enough for efficient DGlcDAG synthesis. In vitro, phospholipids with an sn-glycero-3-phosphate backbone, and no positive head group charge, functioned as activators. Different metabolic, soluble phosphates could supplement PG for activation, depending on type, amount, and valency. Especially efficient were the glycolytic intermediates fructose 1,6-bisphosphate and ATP, active at cellular concentrations on the DGlcDAG but not on the preceding MGlcDAG synthase. Potencies of different phosphatidylinositol (foreign lipid) derivatives differed with numbers and positions of their phosphate moieties. A selective stimulation of the DGlcDAG, but not the MGlcDAG synthase, by minor amounts of double-stranded DNA was additive to the best phospholipid activators. These results support two types of activator sites on the enzyme: (i) lipid-phosphate ones close to the membrane interphase, and (ii) soluble (or particulate)-phosphate ones further out from the surface. Thereby, the nonbilayer (MGlcDAG) to bilayer (DGlcDAG) lipid balance may be integrated with the metabolic status of the cell and potentially also to membrane and cell division.     

Sequence properties of the 1,2-diacylglycerol 3-glucosyltransferase from Acholeplasma laidlawii membranes. Recognition of a large group of lipid glycosyltransferases in eubacteria and archaea

Synthesis of the nonbilayer-prone alpha-monoglucosyldiacylglycerol (MGlcDAG) is crucial for bilayer packing properties and the lipid surface charge density in the membrane of Acholeplasma laidlawii. The gene for the responsible, membrane-bound glucosyltransferase (alMGS) (EC ) was sequenced and functionally cloned in Escherichia coli, yielding MGlcDAG in the recombinants. Similar amino acid sequences were encoded in the genomes of several Gram-positive bacteria (especially pathogens), thermophiles, archaea, and a few eukaryotes. All of these contained the typical EX(7)E catalytic motif of the CAZy family 4 of alpha-glycosyltransferases. The synthesis of MGlcDAG by a close sequence analog from Streptococcus pneumoniae (spMGS) was verified by polymerase chain reaction cloning, corroborating a connection between sequence and functional similarity for these proteins. However, alMGS and spMGS varied in dependence on anionic phospholipid activators phosphatidylglycerol and cardiolipin, suggesting certain regulatory differences. Fold predictions strongly indicated a similarity for alMGS (and spMGS) with the two-domain structure of the E. coli MurG cell envelope glycosyltransferase and several amphipathic membrane-binding segments in various proteins. On the basis of this structure, the alMGS sequence charge distribution, and anionic phospholipid dependence, a model for the bilayer surface binding and activity is proposed for this regulatory enzyme.     

Lipid-engineered Escherichia coli membranes reveal critical lipid headgroup size for protein function

Escherichia coli membranes have a substantial bilayer curvature stress due to a large fraction of the nonbilayer-prone lipid phosphatidylethanolamine, and a mutant (AD93) lacking this lipid is severely crippled in several membrane-associated processes. Introduction of four lipid glycosyltransferases from Acholeplasma laidlawii and Arabidopsis thaliana, synthesizing large amounts of two nonbilayer-prone, and two bilayer-forming gluco- and galacto-lipids, (i) restored the curvature stress with the two nonbilayer lipids, and (ii) diluted the high negative lipid surface charge in all AD93 bilayers. Surprisingly, the bilayer-forming diglucosyl-diacylglycerol was almost as good in improving AD93 membrane processes as the two nonbilayer-prone glucosyl-diacylglycerol and galactosyl-diacylglycerol lipids, strongly suggesting that lipid surface charge dilution by these neutral lipids is very important for E. coli. Increased acyl chain length and unsaturation, plus cardiolipin (nonbilayer-prone) content, were probably also beneficial in the modified strains. However, despite a correct transmembrane topology for the transporter LacY in the diglucosyl-diacylglycerol clone, active transport failed in the absence of a nonbilayer-prone glycolipid. The corresponding digalactosyl-diacylglycerol bilayer lipid did not restore AD93 membrane processes, despite analogous acyl chain and cardiolipin contents. Chain ordering, probed by bis-pyrene lipids, was substantially lower in the digalactosyl-diacylglycerol strain lipids due to its extended headgroup. Hence, a low surface charge density of anionic lipids is important in E. coli membranes, but is inefficient if the headgroup of the diluting lipid is too large. This strongly indicates that a certain magnitude of the curvature stress is crucial for the bilayer in vivo.     

Key role of the diglucosyldiacylglycerol synthase for the nonbilayer-bilayer lipid balance of Acholeplasma laidlawii membranes

In the single membrane of Acholeplasma laidlawii, a specific glucosyltransferase (DGlcDAG synthase) synthesizes the major, bilayer-forming lipid diglucosyldiacylglycerol (DGlcDAG) from the preceding major, nonbilayer-prone monoglucosyldiacylglycerol (MGlcDAG). This is crucial for the maintenance of phase equilibria close to a potential bilayer-nonbilayer transition and a nearly constant spontaneous curvature for the membrane bilayer lipid mixture. The glucolipid pathway is also balanced against the phosphatidylglycerol (PG) pathway to maintain a certain lipid surface charge density. The DGlcDAG synthase was purified approximately 5000-fold by three chromatographic techniques and identified as a minor 40 kDa membrane protein. In CHAPS mixed micelles, a cooperative dependence on anionic lipid activators was confirmed, with PG as the best. The dependence of the enzyme on the soluble UDP-glucose substrate followed Michaelis-Menten kinetics, while the kinetics for the other (lipid) substrate MGlcDAG exhibited cooperativity, with Hill coefficients in the range of 3-5. Vmax and the Hill coefficient, but not Km, for the MGlcDAG substrate were increased by increased PG concentrations, but above 3 mol % MGlcDAG, the rate of synthesis was constant. Hence, the DGlcDAG synthase is more affected by the lipid activator than by the lipid substrate at physiological lipid concentrations. The enzyme was shown to be sensitive to curvature "stress" changes, i.e., was stimulated by various nonbilayer lipids but inhibited by certain others. Certain phosphates were also stimulatory. With the two purified MGlcDAG and DGlcDAG synthases reconstituted together in the presence of a potent nonbilayer lipid, the strong responses in the amounts of MGlcDAG and DGlcDAG synthesized mimicked the responses in vivo. This supports the important regulatory functions of these enzymes.     

Charge-dependent translocation of the Trojan peptide penetratin across lipid membranes

We studied the interaction of the cell-penetrating peptide penetratin with mixed dioleoylphosphatidylcholine/dioleoylphoshatidylglycerol (DOPC/DOPG) unilamellar vesicles as a function of the molar fraction of anionic lipid, X(PG), by means of isothermal titration calorimetry. The work was aimed at getting a better understanding of factors that affect the peptide binding to lipid membranes and its permeation through the bilayer. The binding was well described by a surface partitioning equilibrium using an effective charge of the peptide of z(P) approximately 5.1 +/- 0.5. The peptide first binds to the outer surface of the vesicles, the effective binding capacity of which increases with X(PG). At X(PG) approximately 0.5 and a molar ratio of bound peptide-to-lipid of approximately 1/20 the membranes become permeable and penetratin binds also to the inner monolayer after internalization. The results were rationalized in terms of an "electroporation-like" mechanism, according to which the asymmetrical distribution of the peptide between the outer and inner surfaces of the charged bilayer causes a transmembrane electrical field, which alters the lateral and the curvature stress acting within the membrane. At a threshold value these effects induce internalization of penetratin presumably via inversely curved transient structures.     

Lateral organization in Acholeplasma laidlawii lipid bilayer models containing endogenous pyrene probes.

In membranes of the small prokaryote Acholeplasma laidlawii bilayer- and nonbilayer-prone glycolipids are major species, similar to chloroplast membranes. Enzymes of the glucolipid pathway keep certain important packing properties of the bilayer in vivo, visualized especially as a monolayer curvature stress ('spontaneous curvature'). Two key enzymes depend in a cooperative fashion on substantial amounts of the endogenous anionic lipid phosphatidylglycerol (PG) for activity. The lateral organization of five unsaturated A. laidlawii lipids was analyzed in liposome model bilayers with the use of endogenously produced pyrene-lipid probes, and extensive experimental designs. Of all lipids analyzed, PG especially promoted interactions with the precursor diacylglycerol (DAG), as revealed from pyrene excimer ratio (Ie/Im) responses. Significant interactions were also recorded within the major nonbilayer-prone monoglucosylDAG (MGlcDAG) lipids. The anionic precursor phosphatidic acid (PA) was without effects. Hence, a heterogeneous lateral lipid organization was present in these liquid-crystalline bilayers. The MGlcDAG synthase when binding at the PG bilayer interface, decreased acyl chain ordering (increase of membrane free volume) according to a bis-pyrene-lipid probe, but the enzyme did not influence the bulk lateral lipid organization as recorded from DAG or PG probes. It is concluded that the concentration of the substrate DAG by PG is beneficial for the MGlcDAG synthase, but that binding in a proper orientation/conformation seems most important for activity.     

The Effect of PS Content on the Ability of Natural Membranes to Fuse with Positively Charged Liposomes and Lipoplexes

Supramolecular aggregates containing cationic lipids have been widely used as transfection mediators due to their ability to interact with negatively charged DNA molecules and biological membranes. First steps of the process leading to transfection are partly electrostatic, partly hydrophobic interactions of liposomes/lipoplexes with cell and/or endosomal membrane. Negatively charged compounds of biological membranes, namely glycolipids, glycoproteins and phosphatidylserine (PS), are responsible for such events as adsorption, hemifusion, fusion, poration and destabilization of natural membranes upon contact with cationic liposomes/lipoplexes. The present communication describes the dependence of interaction of cationic liposomes with natural and artificial membranes on the negative charge of the target membrane, charges which in most cases were generated by charging the PS content or its exposure. The model for the target membranes were liposomes of variable content of PS or PG (phosphatidylglycerol) and erythrocyte membranes in which the PS and other anionic compound content/exposure was modified in several ways. Membranes of increased anionic phospholipid content displayed increased fusion with DOTAP (1,2-dioleoyl-3-trimethylammoniumpropane) liposomes, while erythrocyte membranes partly depleted of glycocalix, its sialic acid, in particular, showed a decreased fusion ability. The role of the anionic component is also supported by the fact that erythrocyte membrane inside-out vesicles fused easily with cationic liposomes. The data obtained on erythrocyte ghosts of normal and disrupted asymmetry, in particular, those obtained in the presence of Ca²⁺, indicate the role of lipid flip-flop movement catalyzed by scramblase. The ATP-depletion of erythrocytes also induced an increased sensitivity to hemoglobin leakage upon interactions with DOTAP liposomes. Calcein leakage from anionic liposomes incubated with DOTAP liposomes was also dependent on surface charge of the target membranes. In all experiments with the asymmetric membranes the fusion level markedly increased with an increase of temperature, which supports the role of membrane lipid mobility. The decrease in positive charge by binding of plasmid DNA and the increase in ionic strength decreased the ability of DOTAP liposomes/lipoplexes to fuse with erythrocyte ghosts. Lower pH promotes fusion between erythrocyte ghosts and DOTAP liposomes and lipoplexes. The obtained results indicate that electrostatic interactions together with increased mobility of membrane lipids and susceptibility to form structures of negative curvature play a major role in the fusion of DOTAP liposomes with natural and artificial membranes.     

X-ray diffraction measurement of the monolayer spontaneous curvature of dioleoylphosphatidylglycerol

Phosphatidylglycerol (PG) is an anionic lipid commonly found in large proportions in the cell membranes of bacteria and plants and, to a lesser extent, in animal cells. PG plays an important role in the regulation and determination of the elastic properties of the membrane. Using small angle X-ray scattering experiments, we obtain that the monolayer spontaneous curvature of dioleoylphosphatidylglycerol (DOPG) is -1/150+/-0.021 nm(-1) when measured in 150 mM NaCl. When the experiments are carried out in 150 mM NaCl and 20mM MgCl(2), the value obtained for the monolayer spontaneous curvature is -1/8.7+/-0.037 nm(-1). These values are of importance in modelling the effects of curvature elastic stress in membrane lipid homeostasis in the bacterium Acholeplasma laidlawii [Alley, S.H., Barahona, M., Ces, O., Templer, R.H., in press. Biophysical regulation of lipid biosynthesis in the plasma membrane. Biophys. J.] and indicate that divalent cations can play a significant role in altering curvature elastic stress.     

Tunable biomimetic bacterial membranes from binary and ternary lipid mixtures and their application in antimicrobial testing

The reconstruction of accurate yet simplified mimetic models of cell membranes is a very challenging goal of synthetic biology. To date, most of the research focuses on the development of eukaryotic cell membranes, while reconstitution of their prokaryotic counterparts has not been fully addressed, and the proposed models do not reflect well the complexity of bacterial cell envelopes. Here, we describe the reconstitution of biomimetic bacterial membranes with an increasing level of complexity, developed from binary and ternary lipid mixtures. Giant unilamellar vesicles composed of phosphatidylcholine (PC) and phosphatidylethanolamine (PE); PC and phosphatidylglycerol (PG); PE and PG; PE, PG and cardiolipin (CA) at varying molar ratios were successfully prepared by the electroformation method. Each of the proposed mimetic models focuses on reproducing specific membrane features such as membrane charge, curvature, leaflets asymmetry, or the presence of phase separation. GUVs were characterized in terms of size distribution, surface charge, and lateral organization. Finally, the developed models were tested against the lipopeptide antibiotic daptomycin. The obtained results showed a clear dependency of daptomycin binding efficiency on the amount of negatively charged lipid species present in the membrane. We anticipate that the models proposed here can be applied not only in antimicrobial testing but also serve as platforms for studying fundamental biological processes in bacteria as well as their interaction with physiologically relevant biomolecules.     

Negatively charged phospholipids restore prePhoE translocation across phosphatidylglycerol-depleted Escherichia coli inner membranes

Translocation of outer membrane precursor proteins across the Escherichia coli inner membrane is severely hampered in lipid biosynthetic mutants with strongly reduced phosphatidylglycerol (PG) levels (De Vrije, T., De Swart, R. L., Dowhan, W., Tommassen, J., and De Kruijff, B. (1988) Nature 334, 173-175; Lill, R., Dowhan, W., and Wickner, W. (1990) Cell 60, 271-280). Two independent methods were used to demonstrate that anionic lipids by virtue of their negative head-group charge are involved in membrane translocation of the precursor of the pore protein PhoE. Using a lipid transfer protein-based method we show that introduction from lipid vesicles of PG and other acidic phospholipids but not of phosphatidylcholine restores efficient translocation across the membrane of PG-depleted inner membrane vesicles. Moreover, translocation was found to be proportional to the PG content in vesicles isolated from strain HDL11 in which the PG content was altered by varying the synthesis of the PG-phosphate synthase.     

Massive Formation of Intracellular Membrane Vesicles in Escherichia coli by a Monotopic Membrane-bound Lipid Glycosyltransferase

The morphology and curvature of biological bilayers are determined by the packing shapes and interactions of their participant molecules. Bacteria, except photosynthetic groups, usually lack intracellular membrane organelles. Strong overexpression in Escherichia coli of a foreign monotopic glycosyltransferase (named monoglycosyldiacylglycerol synthase), synthesizing a nonbilayer-prone glucolipid, induced massive formation of membrane vesicles in the cytoplasm. Vesicle assemblies were visualized in cytoplasmic zones by fluorescence microscopy. These have a very low buoyant density, substantially different from inner membranes, with a lipid content of ≥60% (w/w). Cryo-transmission electron microscopy revealed cells to be filled with membrane vesicles of various sizes and shapes, which when released were mostly spherical (diameter ≈100 nm). The protein repertoire was similar in vesicle and inner membranes and dominated by the glycosyltransferase. Membrane polar lipid composition was similar too, including the foreign glucolipid. A related glycosyltransferase and an inactive monoglycosyldiacylglycerol synthase mutant also yielded membrane vesicles, but without glucolipid synthesis, strongly indicating that vesiculation is induced by the protein itself. The high capacity for membrane vesicle formation seems inherent in the glycosyltransferase structure, and it depends on the following: (i) lateral expansion of the inner monolayer by interface binding of many molecules; (ii) membrane expansion through stimulation of phospholipid synthesis, by electrostatic binding and sequestration of anionic lipids; (iii) bilayer bending by the packing shape of excess nonbilayer-prone phospholipid or glucolipid; and (iv) potentially also the shape or penetration profile of the glycosyltransferase binding surface. These features seem to apply to several other proteins able to achieve an analogous membrane expansion.     

Effect of electrostatic interactions on phase stability of cubic phases of membranes of monoolein/dioleoylphosphatidic acid mixtures.

To elucidate effects of electrostatic interactions resulting from surface charges on structures and phase stability of cubic phases of lipid membranes, membranes of 1-monoolein (MO) and dioleoylphosphatidic acid (DOPA) (DOPA/MO membrane) mixtures have been investigated by small-angle x-ray scattering method. As increasing DOPA concentration in the DOPA/MO membrane at 30 wt% lipid concentration, a phase transition from Q(224) to Q(229) phase occurred at 0.6 mol% DOPA, and at and above 25 mol% DOPA, DOPA/MO membranes were in the L(alpha) phase. As NaCl concentration in the bulk phase increased, for 10% DOPA/90% MO membrane in excess water, a Q(229) to Q(224) phase transition occurred at 60 mM NaCl, and then a Q(224) to H(II) phase transition occurred at 1.2 M NaCl. Similarly, for 30% DOPA/70% MO membrane in excess water, at low NaCl concentrations it was in the L(alpha) phase, but at and above 0.50 M NaCl it was in the Q(224) phase, and then at 0.65 M NaCl a Q(224) to H(II) phase transition occurred. These results indicate that the electrostatic interactions in the membrane interface make the Q(229) phase more stable than the Q(224) phase, and that, at larger electrostatic interactions, the L(alpha) phase is more stable than the cubic phases (Q(224) and Q(229)). We have found that the addition of tetradecane to the MO membrane induced a Q(224)-to-H(II) phase transition and also that to the 30% DOPA/70% MO membrane induced an L(alpha)-to-H(II) phase transition. By using these membranes, the effect of the electrostatic interactions resulting from the membrane surface charge (DOPA) on the spontaneous curvature of the monolayer membrane has been investigated. The increase in DOPA concentration in the DOPA/MO membrane reduced the absolute value of spontaneous curvature of the membrane. In the 30% DOPA/70% MO membrane, the absolute value of spontaneous curvature of the membrane increased with an increase in NaCl concentration. On the basis of these new results, the phase stability of DOPA/MO membranes can be reasonably explained by the spontaneous curvature of the monolayer membrane and a curvature elastic energy of the membrane.     

Reconstituted phosphatidylserine synthase from Escherichia coli is activated by anionic phospholipids and micelle-forming amphiphiles.

The activity of phosphatidylserine (PS) synthase (CDP-1, 2-diacyl-sn-glycerol: l-serine O-phosphatidyltransferase, EC 2.7.8. 8) from Escherichia coli was studied after reconstitution with lipid vesicles of various compositions. PS synthase exhibited practically no activity in the absence of a detergent and with the substrate CDP-diacylglycerol (CDP-DAG) present only in the lipid vesicles. Inclusion of octylglucoside (OG) in the assay mixture increased the activity 20- to 1000-fold, the degree of activation depending on the lipid composition of the vesicles. Inclusion of additional CDP-DAG in the assay mixture increased the activity 5- to 25-fold. When the fraction of phosphatidylglycerol (PG) was increased from 15 to 100 mol% in the vesicles the activity increased 10-fold using the assay mixture containing OG. The highest activities were exhibited with the anionic lipids synthesized by E. coli, namely PG, diphosphatidylglycerol (DPG), and phosphatidic acid, while phosphatidylinositol gave a lower activity. Cryotransmission electron microscopy showed that transformation of the vesicles to micelles brings about an activation of the enzyme that is proportional to the degree of micellization. Thus, the activity of PS synthase is modulated by the lipid aggregate structure and by the fraction and type of anionic phospholipid in the aggregates. The increase in the activity caused by PG and DPG is physiologically relevant; it may be part of a regulatory mechanism that keeps the balance between phosphatidylethanolamine, and the sum of PG and DPG, nearly constant in wild-type E. coli cells.     

Binding of a fluorescent dansylcadaverine-substance P analogue to negatively charged phospholipid membranes

We have investigated the binding of a new dansylcadaverine derivative of substance P (DNC-SP) with negatively charged small unilamellar vesicles composed of a mixture of phosphatidylcholine (PC) and either phosphatidylglycerol (PG) or phosphatidylserine (PS) using fluorescence spectroscopic techniques. The changes in fluorescence properties were used to obtain association isotherms at variable membrane negative charges and at different ionic strengths. The experimental association isotherms were analyzed using two binding approaches: (i) the Langmuir adsorption isotherm and the partition equilibrium model, that neglect the activity coefficients; and (ii) the partition equilibrium model combined with the Gouy-Chapman formalism that considers electrostatic effects. A consistent quantitative analysis of each DNC-SP binding curve at different lipid composition was achieved by means of the Gouy-Chapman approach using a peptide effective interfacial charge (v) value of (0.95 +/- 0.02), which is lower than the physical charge of the peptide. For PC/PG membranes, the partition equilibrium constant were 7.8 x 10(3) M(-1) (9/1, mol/mol) and 6.9 x 10(3) M(-1) (7/3, mol/mol), whereas for PC/PS membranes an average value of 6.8 x 10(3) M(-1) was estimated. These partition equilibrium constants were similar to those obtained for the interaction of DNC-SP with neutral PC membranes (4.9 x 10(3) M(-1)), as theoretically expected. We demonstrate that the v parameter is a determinant factor to obtain a unique value of the binding constant independently of the surface charge density of the vesicles. Also, the potential of fluorescent dansylated SP analogue in studies involving interactions with cell membranes is discussed.     

EDTA-induced Membrane Fluidization and Destabilization： Biophysical Studies on Artificial Lipid Membranes

The molecular mechanism of ethylenediaminetetraacetic acid （EDTA）-induced membrane destabilization has been studied using a combination of four biophysical techniques on artificial lipid membranes. Data from Langmuir film balance and epifluorescence microscopy revealed the fluidization and expansion effect of EDTA on phase behavior of monolayers of either 1,2-dipalmitoyl-sn-glycero-3-phosphocholine （DPPC） or mixtures of DPPC and metal-chelating lipids, such as N^a,N^a-Bis[carboxymethyl]-N^ε [（dioctadecylamino）succinyl]-L-lysine or 1,2-dioleoyl-sn-glycero-3-[N-（5-amino- 1 -carboxypentyl iminodiacetic acid） succinyl]. A plausible explanation could be drawn from the electrostatic interaction between negatively charged groups of EDTA and the positively charged choline head group of DPPC. Intercalation of EDTA into the lipid membrane induced membrane curvature as elucidated by atomic force microscopy. Growth in size and shape of the membrane protrusion was found to be time-dependent upon exposure to EDTA. Further loss of material from the lipid membrane surface was monitored in real time using a quartz crystal microbalance. This indicates membrane restabilization by exclusion of the protrusions from the surface. Loss of lipid components facilitates membrane instability, leading to membrane permeabilization and lysis.     

Dimer ribbons of ATP synthase shape the inner mitochondrial membrane

ATP synthase converts the electrochemical potential at the inner mitochondrial membrane into chemical energy, producing the ATP that powers the cell. Using electron cryo-tomography we show that the ATP synthase of mammalian mitochondria is arranged in long approximately 1-microm rows of dimeric supercomplexes, located at the apex of cristae membranes. The dimer ribbons enforce a strong local curvature on the membrane with a 17-nm outer radius. Calculations of the electrostatic field strength indicate a significant increase in charge density, and thus in the local pH gradient of approximately 0.5 units in regions of high membrane curvature. We conclude that the mitochondrial cristae act as proton traps, and that the proton sink of the ATP synthase at the apex of the compartment favours effective ATP synthesis under proton-limited conditions. We propose that the mitochondrial ATP synthase organises itself into dimer ribbons to optimise its own performance.     

A phosphatidylserine decarboxylase activity in root cells of oat (Avena sativa) is involved in altering membrane phospholipid composition during drought stress acclimation.

During acclimation to drought stress, the lipid composition of oat root cell membranes is altered. The level of phosphatidylethanolamine (PE), a non-bilayer forming lipid, is increased relative to the bilayer-forming lipid phosphatidylcholine (PC). These changes are believed to increase stress tolerance by increasing the flexibility of the membranes. To elucidate if de novo lipid synthesis is involved in altering membrane lipid composition, oat plants, acclimated or non-acclimated, were incubated in vivo with radioactively labelled lipid precursors. The labelling pattern indicated that de novo synthesis, at least partly, is causing the alterations. In plants, phospholipids can be synthesized by the Kennedy pathway, with addition of activated head groups to diacylglycerol (DAG) or, alternatively, via the CDP-DAG pathway, where phospahtidylserine (PS) is decarboxylated to form PE. To reveal the importance of the respective pathways during acclimation, we studied the effect of a decarboxylase inhibitor and the relative incorporation of [(3)H]-serine and [(14)C]-ethanolamine in vivo. Activities of CTP:ethanolaminephosphate cytidyltransferase (EC 2.7.7.14), phosphatidylserine decarboxylase (EC 4.1.1.65) and phosphatidylserine synthase; CDP-DAG:L-serine o-phosphatidyltransferase (EC 2.7.8.8) were measured and additionally, the presence of a PS decarboxylase (PSD1) in oat was confirmed by immunoblotting. The results suggest that PE synthesis via the Kennedy pathway is downregulated during acclimation and that synthesis by PS decarboxylation, via the CDP-DAG pathway, is increased, mainly through an increased activity of PS synthase.     

Examining the contributions of lipid shape and headgroup charge on bilayer behavior

To better understand bilayer property dependency on lipid electrostatics and headgroup size, we use atomistic molecular dynamics simulations to study negatively charged and neutral lipid membranes. We compare the negatively charged phosphatidic acid (PA), which at physiological pH and salt concentration has a negative spontaneous curvature, with the negatively charged phosphatidylglycerol (PG) and neutrally charged phosphatidylcholine (PC), both of which have zero spontaneous curvature. The PA lipids are simulated using two different sets of partial charges for the headgroup and the varied charge distribution between the two PA systems results in significantly different locations for the Na⁺ ions relative to the water/membrane interface. For one PA system, the Na⁺ ions are localized around the phosphate group. In the second PA system, the Na⁺ ions are located near the ester carbonyl atoms, which coincides with the preferred location site for the PG Na⁺ ions. We find that the Na⁺ ion location has a larger effect on bilayer fluidity properties than lipid headgroup size, where the A_lipid and acyl chain order parameter values are more similar between the PA and PG bilayers that have Na⁺ ions located near the ester groups than between the two PA bilayers.