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BBA - Biomembranes (v.1768, #10)

Editorial Board (pp. ii).

Heterodimerization of y⁺LAT-1 and 4F2hc visualized by acceptor photobleaching FRET microscopy by Maaria Kleemola; Minna Toivonen; Juha Mykkänen; Olli Simell; Kirsi Huoponen; Kaisa M. Heiskanen (pp. 2345-2354).

y⁺LAT-1 and 4F2hc are the subunits of a transporter complex for cationic amino acids, located mainly in the basolateral plasma membrane of epithelial cells in the small intestine and renal tubules. Mutations in y⁺LAT-1 impair the transport function of this complex and cause a selective aminoaciduria, lysinuric protein intolerance (LPI, OMIM #222700), associated with severe, complex clinical symptoms. The subunits of an active transporter co-localize in the plasma membrane, but the exact process of dimerization is unclear since direct evidence for the assembly of this transporter in intact human cells has not been available. In this study, we used fluorescence resonance energy transfer (FRET) microscopy to investigate the interactions of y⁺LAT-1 and 4F2hc in HEK293 cells expressing y⁺LAT-1 and 4F2hc fused with ECFP or EYFP. FRET was quantified by measuring fluorescence intensity changes in the donor fluorophore (ECFP) after the photobleaching of the acceptor (EYFP). Increased donor fluorescence could be detected throughout the cell, from the endoplasmic reticulum and Golgi complex to the plasma membrane. Therefore, our data prove the interaction of y⁺LAT-1 and 4F2hc prior to the plasma membrane and thus provide evidence for 4F2hc functioning as a chaperone in assisting the transport of y⁺LAT-1 to the plasma membrane.

Keywords: y; ⁺; LAT-1; 4F2hc; Heteromeric amino acid transporters; Protein–protein interaction; FRET

Solution structures and model membrane interactions of lactoferrampin, an antimicrobial peptide derived from bovine lactoferrin by Evan F. Haney; Fanny Lau; Hans J. Vogel (pp. 2355-2364).

Bovine lactoferrampin (LFampinB) has been identified as a novel antimicrobial peptide, which is derived from the N-terminal lobe of bovine lactoferrin. In this study, the solution structure of LFampinB bound to negatively charged sodium dodecyl sulphate micelles and zwitterionic dodecyl phosphocholine micelles was determined using 2-dimensional nuclear magnetic resonance (NMR) spectroscopy. The interaction between LFampinB and multilamellar phospholipid vesicles, containing choline and glycerol head groups, was examined using differential scanning calorimetry (DSC). In addition, the interaction between the N-terminal tryptophan residue and model membranes of varying composition was analyzed by fluorescence spectroscopy. LFampinB adopts an amphipathic alpha-helical conformation across the first 11 residues of the peptide but remains relatively unstructured at the C-terminus. The hydrophobic surface of the amphipathic helix is bordered by the side chains of Trp1 and Phe11, and is seen in both micelle-bound structures. The fluorescence results suggest that Trp1 inserts into the membrane at the lipid/water interface. The phenyl side chain of Phe11 is oriented in the same direction as the indole ring of Trp1, allowing these two residues to serve as anchors for the lipid bilayer. The DSC results also indicate that LFampinB interacts with glycerol head groups in multilamellar vesicles but has little effect on acyl chain packing. Our results support a two step model of antimicrobial activity where the initial attraction of LFampinB is mediated by the cluster of positive charges on the C-terminus followed by the formation of the N-terminal helix which binds to the surface of the bacterial lipid bilayer.

Keywords: Abbreviations; LFampinB; bovine lactoferrampin; NOESY; nuclear Overhauser effect spectroscopy; TOCSY; total correlation spectroscopy; DPC; dodecylphosphocholine; SDS; sodium dodecylsulfate; DSS; 2,2-dimethyl-2-silapentane-5-sulphonic acid; ePC; egg α-phosphatidylcholine; ePG; egg α-phosphatidylglycerol; ePE; egg α-phosphatidylethanolamine; PA; phosphatidic acid; MLV; multilamellar vesicle; LUV; large unilamellar vesicle; K; _sv; Stern–Volmer constant; MIC; minimum inhibitory concentration; MBC; minimum bactericidal concentration; DPPC; 1,2-dipalmitoyl phosphatidylcholine; DPPG; 1,2-dipalmitoyl phosphatidylglycerol; DSC; differential scanning calorimetryAntimicrobial peptide; Lactoferrampin; Lactoferrin; Nuclear magnetic resonance; Micelle-bound peptide structure; Peptide–membrane interactions

Leakage-free membrane fusion induced by the hydrolytic activity of PlcHR₂, a novel phospholipase C/sphingomyelinase from Pseudomonas aeruginosa by L.-Ruth Montes; Maitane Ibarguren; Félix M. Goñi; Martin Stonehouse; Michael L. Vasil; Alicia Alonso (pp. 2365-2372).

PlcHR₂ is the paradigm member of a novel phospholipase C/phosphatase superfamily, with members in a variety of bacterial species. This paper describes the phospholipase C and sphingomyelinase activities of PlcHR₂ when the substrate is in the form of large unilamellar vesicles, and the subsequent effects of lipid hydrolysis on vesicle and bilayer stability, including vesicle fusion. PlcHR₂ cleaves phosphatidylcholine and sphingomyelin at equal rates, but is inactive on phospholipids that lack choline head groups. Calcium in the millimolar range does not modify in any significant way the hydrolytic activity of PlcHR₂ on choline-containing phospholipids. The catalytic activity of the enzyme induces vesicle fusion, as demonstrated by the concomitant observation of intervesicular total lipid mixing, inner monolayer-lipid mixing, and aqueous contents mixing. No release of vesicular contents is detected under these conditions. The presence of phosphatidylserine in the vesicle composition does not modify significantly PlcHR₂-induced liposome aggregation, as long as Ca²⁺ is present, but completely abolishes fusion, even in the presence of the cation. Each of the various enzyme-induced phenomena have their characteristic latency periods, that increase in the order lipid hydrolysis2 products than early ones, e.g. aggregation. When the above results are examined in the context of the membrane effects of other phospholipid phosphocholine hydrolases it can be concluded that aggregation is necessary, but not sufficient for membrane fusion to occur, that diacylglycerol is far more fusogenic than ceramide, and that vesicle membrane permeabilization occurs independently from vesicle fusion.

Keywords: Abbreviations; ANTS; 8-Aminonaphthalene-1,3,6-trisulfonic acid; Cer; ceramide; Ch; cholesterol; 2D-IR; two-dimensional infrared; DAG; diacylglycerol; DPX; p; -xylenebis(pyridinium bromide); LUV; large unilamellar vesicle; PC; phosphatidylcholine; PE; phosphatidylethanolamine; PI; phosphatidylinositol; PS; phosphatidylserine; PC-PLC; phosphatidylcholine-specific phospholipase C; PLC; phospholipase C; SM; sphingomyelin; SMase; SphingomyelinasePlcHR2; Bacterial pathogenesis; Membrane fusion; Membrane leakage; Phospholipase C; Sphingomyelinase; Bacterial toxin; Pseudomonas

Natural lipid extracts and biomembrane-mimicking lipid compositions are disposed to form nonlamellar phases, and they release DNA from lipoplexes most efficiently by Rumiana Koynova; Robert C. MacDonald (pp. 2373-2382).

A viewpoint now emerging is that a critical factor in lipid-mediated transfection (lipofection) is the structural evolution of lipoplexes upon interacting and mixing with cellular lipids. Here we report our finding that lipid mixtures mimicking biomembrane lipid compositions are superior to pure anionic liposomes in their ability to release DNA from lipoplexes (cationic lipid/DNA complexes), even though they have a much lower negative charge density (and thus lower capacity to neutralize the positive charge of the lipoplex lipids). Flow fluorometry revealed that the portion of DNA released after a 30-min incubation of the cationic O-ethylphosphatidylcholine lipoplexes with the anionic phosphatidylserine or phosphatidylglycerol was 19% and 37%, respectively, whereas a mixture mimicking biomembranes (MM: phosphatidylcholine/phosphatidylethanolamine/phosphatidylserine /cholesterol 45:20:20:15 w/w) and polar lipid extract from bovine liver released 62% and 74%, respectively, of the DNA content. A possible reason for this superior power in releasing DNA by the natural lipid mixtures was suggested by structural experiments: while pure anionic lipids typically form lamellae, the natural lipid mixtures exhibited a surprising predilection to form nonlamellar phases. Thus, the MM mixture arranged into lamellar arrays at physiological temperature, but began to convert to the hexagonal phase at a slightly higher temperature, ∼40–45 °C. A propensity to form nonlamellar phases (hexagonal, cubic, micellar) at close to physiological temperatures was also found with the lipid extracts from natural tissues (from bovine liver, brain, and heart). This result reveals that electrostatic interactions are only one of the factors involved in lipid-mediated DNA delivery. The tendency of lipid bilayers to form nonlamellar phases has been described in terms of bilayer “frustration” which imposes a nonzero intrinsic curvature of the two opposing monolayers. Because the stored curvature elastic energy in a “frustrated” bilayer seems to be comparable to the binding energy between cationic lipid and DNA, the balance between these two energies could play a significant role in the lipoplex–membrane interactions and DNA release energetics.

Keywords: Lipid phase transition; Surface charge; Cationic lipid; DNA unbinding; Transfection

Role of transmembrane segment M8 in the biogenesis and function of yeast plasma-membrane H⁺-ATPase by Guadalupe Guerra; Valery V. Petrov; Kenneth E. Allen; Manuel Miranda; Juan Pablo Pardo; Carolyn W. Slayman (pp. 2383-2392).

Of the four transmembrane helices (M4, M5, M6, and M8) that pack together to form the ion-binding sites of P₂-type ATPases, M8 has until now received the least attention. The present study has used alanine-scanning mutagenesis to map structure–function relationships throughout M8 of the yeast plasma-membrane H⁺-ATPase. Mutant forms of the ATPase were expressed in secretory vesicles and at the plasma membrane for measurements of ATP hydrolysis and ATP-dependent H⁺ pumping. In secretory vesicles, Ala substitutions at a cluster of four positions near the extracytoplasmic end of M8 led to partial uncoupling of H⁺ transport from ATP hydrolysis, while substitution of Ser-800 (close to the middle of M8) by Ala increased the apparent stoichiometry of H⁺ transport. A similar increase has previously been reported following the substitution of Glu-803 by Gln (Petrov, V. et al., J. Biol. Chem. 275:15709–15718, 2000) at a position known to contribute directly to Ca²⁺ binding in the Ca²⁺-ATPase of sarcoplasmic reticulum (Toyoshima, C., et al., Nature 405: 647–655, 2000). Four other mutations in M8 interfered with H⁺-ATPase folding and trafficking to the plasma membrane; based on homology modeling, they occupy positions that appear important for the proper bundling of M8 with M5, M6, M7, and M10. Taken together, these results point to a key role for M8 in the biogenesis, stability, and physiological functioning of the H⁺-ATPase.

Lipid tail chain asymmetry and the strength of membrane-induced interactions between membrane proteins by Nily Dan (pp. 2393-2399).

Many lipids are composed of asymmetric tail chains that differ by their molecular weight (MW) and/or degree of saturation. Previous studies found that membrane moduli vary with the degree of lipid tail asymmetry. However, to date little is known regarding the effect (if any) of tail asymmetry on the membrane-induced interactions between embedded proteins. In this paper we use a self-consistent field model to examine the effect of lipid tail asymmetry on membrane proteins. We first examine the case where the overall tail length (sum of both chains) is held constant, which implies that the membrane thickness remains constant as well, independent of tail asymmetry. We find that, in these systems, the membrane area stretch and bending moduli decrease with increasing chain asymmetry, thereby reducing the magnitude of the membrane-induced barrier to protein aggregation. Since in symmetric lipid bilayers the energy barrier is typically of order ∼1–2 times the thermal energy kT, the asymmetry-induced reduction in barrier height may increase the probability of protein aggregation significantly. In systems where one tail chain is held constant, increasing asymmetry involves changes in the bilayer thickness which are found to dominate any effect arising from the asymmetry.

Keywords: Transmembrane protein; Bending modulus; Area modulus

Membrane interactions and the effect of metal ions of the amyloidogenic fragment Aβ(25–35) in comparison to Aβ(1–42) by Tong-Lay Lau; John D. Gehman; John D. Wade; Keyla Perez; Colin L. Masters; Kevin J. Barnham; Frances Separovic (pp. 2400-2408).

Aβ(1−42) peptide, found as aggregated species in Alzheimer's disease brain, is linked to the onset of Alzheimer's disease. Many reports have linked metals to inducing Aβ aggregation and amyloid plaque formation. Aβ(25–35), a fragment from the C-terminal end of Aβ(1−42), lacks the metal coordinating sites found in the full-length peptide and is neurotoxic to cortical cortex cell cultures. We report solid-state NMR studies of Aβ(25–35) in model lipid membrane systems of anionic phospholipids and cholesterol, and compare structural changes to those of Aβ(1–42). When added after vesicle formation, Aβ(25–35) was found to interact with the lipid headgroups and slightly perturb the lipid acyl-chain region; when Aβ(25–35) was included during vesicle formation, it inserted deeper into the bilayer. While Aβ(25–35) retained the same β-sheet structure irrespective of the mode of addition, the longer Aβ(1–42) appeared to have an increase in β-sheet structure at the C-terminus when added to phospholipid liposomes after vesicle formation. Since the Aβ(25–35) fragment is also neurotoxic, the full-length peptide may have more than one pathway for toxicity.

Keywords: Amyloid Aβ; Peptide–lipid interactions; Phospholipid membranes; Solid-state NMR; Structure; Metal interactions

Two-dimensional infrared correlation spectroscopy study of the interaction of oxidized and reduced cytochrome c with phospholipid model membranes by Angela Bernabeu; Lellys M. Contreras; José Villalaín (pp. 2409-2420).

We have used two-dimensional infrared correlation spectroscopy (2D-IR) to study the interaction and conformation of cytochrome c in the presence of a binary phospholipid mixture composed of a zwitterionic perdeuterated phospholipid and a negatively-charged one. The influence of the main temperature phase transition of the phospholipid model membranes on the conformation of cytochrome c has been evaluated by monitoring both the Amide I′ band of the protein and the CH₂ and CD₂ stretching bands of the phospholipids. Synchronous 2D-IR analysis has been used to determine the different secondary structure components of cytochrome c which are involved in the specific interaction with the phospholipids, revealing the existence of a specific interaction between the protein with cardiolipin-containing vesicles but not with phosphatidic acid-containing ones. Interestingly, 2D-IR is capable of showing the existence of significant changes in the protein conformation at the same time that the phospholipid transition occurs. In summary, 2D-IR revealed an important effect of the phospholipid phase transition of cardiolipin on the secondary structure of oxidized cytochrome c but not to either reduced cytochrome c or in the presence of phosphatidic acid, demonstrating the existence of specific intermolecular interactions between cardiolipin and cytochrome c.

Keywords: Abbreviations; 2D-IR; Two-dimensional infrared spectroscopy; DMPA; 1,2-Dimyristoyl-; sn; -Glycero-3-Phosphate; DMPC; _d; 1,2-Dimyristoyl-d; ₅₄; -; sn; -Glycero-3-Phosphocoline; MLV; Multilamellar vesicles; TMCL; 1,1′,2,2′-Tetramyristoyl-Cardiolipin; T; _m; Temperature of the gel-to–liquid crystalline phase transition; IR; Infrared spectroscopyCardiolipin; Cytochrome; c; Phosphatidic acid; Two-dimensional infrared spectroscopy; Protein–lipid interaction

Biophysical analysis of the interaction of granulysin-derived peptides with enterobacterial endotoxins by Xi Chen; Jörg Howe; Jörg Andrä; Manfred Rössle; Walter Richter; Ana Paula Galvão da Silva; Alan M. Krensky; Carol Clayberger; Klaus Brandenburg (pp. 2421-2431).

To combat infections by Gram-negative bacteria, it is not only necessary to kill the bacteria but also to neutralize pathogenicity factors such as endotoxin (lipopolysaccharide, LPS). The development of antimicrobial peptides based on mammalian endotoxin-binding proteins is a promising tool in the fight against bacterial infections, and septic shock syndrome. Here, synthetic peptides derived from granulysin (Gra-pep) were investigated in microbiological and biophysical assays to understand their interaction with LPS. We analyzed the influence of the binding of Gra-pep on (1) the acyl chain melting of the hydrophobic moiety of LPS, lipid A, by Fourier-transform spectroscopy, (2) the aggregate structure of LPS by small-angle X-ray scattering and cryo-transmission electron microscopy, and 3) the enthalpy change by isothermal titration calorimetry. In addition, the influence of Gra-pep on the incorporation of LPS and LPS-LBP (lipopolysaccharide-binding protein) complexes into negatively charged liposomes was monitored. Our findings demonstrate a characteristic change in the aggregate structure of LPS into multilamellar stacks in the presence of Gra-pep, but little or no change of acyl chain fluidity. Neutralization of LPS by Gra-pep is not due to a scavenging effect in solution, but rather proceeds after incorporation into target membranes, suggesting a requisite membrane-bound step.

Keywords: Endotoxin; Lipopolysaccharide; Antimicrobial peptide; Sepsis; Granulysin

NMR studies of membranes composed of glycolipids and phospholipids by Vasco Castro; Sergey V. Dvinskikh; Göran Widmalm; Dick Sandström; Arnold Maliniak (pp. 2432-2437).

Lipid membranes composed of monogalactosyldiacylglycerol (MGDG) and dimyristoylphosphatidylcholine (DMPC) were studied by means of NMR spectroscopy. The macroscopic phase behaviour was investigated by³¹P NMR under stationary conditions, whereas microscopic properties such as segmental ordering were probed by two-dimensional¹H–¹³C separated local field experiments under magic-angle spinning conditions. Our results clearly show that ordering/disordering effects occur for the headgroups as well as for the acyl chains when the sample composition is varied. In particular, the¹H–¹³C dipolar couplings within the galactose headgroup of MGDG exhibited significant concentration dependence.

Keywords: Solid-state NMR; Carbon-13; Phosphorus-31; Dipolar couplings; Separated local field; Lyotropic liquid crystals; Biomembranes

CFTR inhibition by glibenclamide requires a positive charge in cytoplasmic loop three by Patricia Melin; Eric Hosy; Michel Vivaudou; Frédéric Becq (pp. 2438-2446).

The sulfonylurea glibenclamide is widely used as an open-channel blocker of the CFTR chloride channel. Here, we used site-directed mutagenesis to identify glibenclamide site of interaction: a positively charged residue K978, located in the cytoplasmic loop 3. Charge-neutralizing mutations K978A, K978Q, K978S abolished the inhibition of forskolin-activated CFTR chloride current by glibenclamide but not by CFTR_inh-172. The charge-conservative mutation K978R did not alter glibenclamide sensitivity of CFTR current. Mutations of the neighbouring R975 (R975A, R975S, R975Q) did not affect electrophysiological and pharmacological properties of CFTR. No alteration of halide selectivity was observed with any of these CFTR mutant channels. This study identifies a novel potential inhibitor site within the CFTR molecule, and suggests a novel role of cytoplasmic loop three, within the second transmembrane domain of CFTR protein. This work is the first to report on the role of a residue in a cytoplasmic loop in the mechanism of action of the channel blocker glibenclamide.

Keywords: Abbreviations; ABC; ATP binding cassette; CFTR; cystic fibrosis transmembrane conductance regulator; MRP; multidrug resistance-associated protein; SUR; sulfonylurea receptor; CL; cytoplasmic loop; TMD; transmembrane domain; Fsk; forskolin; Glib; glibenclamide; HEK; human embryonic kidneyCystic Fibrosis; CFTR chloride channel; Site of action; Glibenclamide; cytoplasmic loop three

Interactions of tryptophan-rich cathelicidin antimicrobial peptides with model membranes studied by differential scanning calorimetry by Valery V. Andrushchenko; Hans J. Vogel; Elmar J. Prenner (pp. 2447-2458).

The 13-residue cathelicidins indolicidin and tritrpticin are part of a group of relatively short tryptophan-rich antimicrobial peptides that hold potential as future substitutes for antibiotics. Differential scanning calorimetry (DSC) has been applied here to study the effect of indolicidin and tritrpticin as well as five tritrpticin analogs on the phase transition behaviour of model membranes made up of zwitterionic dimyristoylphosphatidylcholine (DMPC, DMPC/cholesterol) and anionic dimyristoylphosphatidyl glycerol (DMPG) phospholipids. Most of the peptides studied significantly modified the phase transition profile, suggesting the importance of hydrophobic forces for the peptide interactions with the lipid bilayers and their insertion into the bilayer. Indolicidin and tritrpticin are both known to be flexible in aqueous solution, but they adopt turn–turn structures when they bind to and insert in a membrane surface. Pro-to-Ala substitutions in tritrpticin, which result in the formation of a stable α-helix in this peptide, lead to a substantial increase in the peptide interactions with both zwitterionic and anionic phospholipid vesicles. In contrast, the substitution of the three Trp residues by Tyr or Phe resulted in a significant decrease of the peptide's interaction with anionic vesicles and virtually eliminated binding of these peptides to the zwitterionic vesicles. An increase of the cationic charge of the peptide induced much smaller changes to the peptide interaction with all lipid systems than substitution of particular amino acids or modification of the peptide conformation. The presence of multiple lipid domains with a non-uniform peptide distribution was noticed. Slow equilibration of the lipid–peptide systems due to peptide redistribution was observed in some cases. Generally good agreement between the present DSC data and peptide antimicrobial activity data was obtained.

Keywords: Abbreviations; DMPC; dimyristoylphosphatidylcholine; DMPG; dimyristoylphosphatidyl glycerol; DPC; dodecylphosphocholine; DSC; differential scanning calorimetry; Indo; indolicidin; ITC; isothermal titration calorimetry; PC; phosphatidylcholine; PG; phosphatidylglycerol; Tritrp; tritrpticinAntimicrobial peptides; DSC; Indolicidin; Lipid phase transition; Tritrpticin

Photodynamic activity and binding of sulfonated metallophthalocyanines to phospholipid membranes: Contribution of metal-phosphate coordination by Alina A. Pashkovskaya; Elena A. Sokolenko; Valeri S. Sokolov; Elena A. Kotova; Yuri N. Antonenko (pp. 2459-2465).

Photosensitized efficacy of tetrasulfonated phthalocyanines of zinc, aluminum and nickel (ZnPcS₄, AlPcS₄ and NiPcS₄, respectively) as studied by gramicidin channel (gA) photoinactivation was compared with adsorption of the dyes on the surface of a bilayer lipid membrane as measured by the inner field compensation method. The adsorption of the negatively charged phthalocyanines on diphytanoylphosphatidylcholine (DPhPC) membranes led to formation of a negative boundary potential difference between the membrane/water interfaces. Good correlation was shown between the photodynamic activity and the membrane binding of the three metallophthalocyanines. ZnPcS₄ appeared to be the most potent of these photosensitizers, while NiPcS₄ was completely ineffective. All of these phthalocyanines displayed no binding and negligible gA photoinactivation with membranes formed of glycerol monooleate (GMO), whereas Rose Bengal exhibited significant binding and photodynamic efficacy with GMO membranes. Gramicidin photoinactivation in the presence of AlPcS₄, being insensitive to the ionic strength of the bathing solution, was inhibited by fluoride and attenuated by phosphate ions. A blue shift of the fluorescence peak position of ZnPcS₄ dissolved in ethanol was elicited by phosphate, similarly to fluoride, which was indicative of the coordination interaction of these ions with the central metal atom of the phthalocyanine macrocycle. This interaction was enhanced in the medium modeling the water–membrane interface. The results obtained imply that binding of tetrasulfonated metallophthalocyanines to phospholipid membranes is determined primarily by metal–phosphate coordination.

Keywords: Abbreviations; BLM; bilayer lipid membrane; gA; gramicidin A; DPhPC; diphytanoylphosphatidylcholine; MPcS; ₄; tetrasulfonated metallophthalocyanine; AlPcS; ₄; Aluminium tetrasulfophthalocyanine; ZnPcS; ₄; Zinc tetrasulfophthalocyanine; NiPcS; ₄; Nickel tetrasulfophthalocyanine; RB; Rose Bengal; GMO; glycerol monooleate; IFC; intramembrane field compensation method; Δ; ϕ; _b; difference of boundary potentialssensitized photoinactivation; gramicidin A; sulfonated phthalocyanine; bilayer lipid membrane

A CLC chloride channel plays an essential role in copper homeostasis in Aspergillus nidulans at increased extracellular copper concentrations by Delphine M. Oddon; Eugene Diatloff; Stephen K. Roberts (pp. 2466-2477).

A putative CLC voltage-gated anion channel gene from Aspergillus nidulans ( AnCLCA) is characterised. The expression of the AnCLCA cDNA restored the iron-limited growth of the Saccharomyces cerevisiae CLC null mutant strain ( gef1) suggesting that AnCLCA functions as a chloride channel. An AnCLCA conditional mutant was created and exhibited a strong and specific growth inhibition in the presence of extracellular copper concentrations >18 μM. This sensitivity was shown to be the result of a hyper-accumulation of copper by the conditional mutant, which generates superoxide to toxic levels inhibiting the growth. Further analysis revealed that copper dependent enzymes were disrupted in the AnCLCA conditional null mutant, specifically, a reduced activity of the copper–zinc superoxide dismutase (CuZn–SOD) and enhanced activity of the cytochrome oxidase (COX). These results suggest that AnCLCA plays a key role in copper homeostasis in A. nidulans and that a malfunction of this chloride channel results in disrupted intracellular copper trafficking.

Keywords: CLC; Chloride channel; Aspergillus nidulans; Copper homeostasis; Filamentous fungi

Role of 57–72 loop in the allosteric action of bile salts on pancreatic IB phospholipase A₂: Regulation of fat and cholesterol homeostasis by Bao-Zhu Yu; Rafael J. Apitz-Castro; Mahendra K. Jain; Otto G. Berg (pp. 2478-2490).

Mono- and biphasic kinetic effects of bile salts on the pancreatic IB phospholipase A2 (PLA2) catalyzed interfacial hydrolysis are characterized. This novel phenomenon is modeled as allosteric action of bile salts with PLA2 at the interface. The results and controls also show that these kinetic effects are not due to surface dilution or solubilization or disruption of the bilayer interface where in the mixed-micelles substrate replenishment becomes the rate-limiting step. The PLA2-catalyzed rate of hydrolysis of zwitterionic dimyristoylphosphatidylcholine (DMPC) vesicles depends on the concentration and structure of the bile salt. The sigmoidal rate increase with cholate saturates at 0.06 mole fraction and changes little at the higher mole fractions. Also, with the rate-lowering bile salts (B), such as taurochenodeoxycholate (TCDOC), the initial sigmoidal rate increase at lower mole fraction is followed by nearly complete reversal to the rate at the pre-activation level at higher mole fractions. The rate-lowering effect of TCDOC is not observed with the (62–66)-loop deleted ΔPLA2, or with the Naja venom PLA2 that is evolutionarily devoid of the loop. The rate increase is modeled with the assumption that the binding of PLA2 to DMPC interface is cooperatively promoted by bile salt followed by allosteric k_cat^⁎-activation of the bound enzyme by the anionic interface. The rate-lowering effect of bile salts is attributed to the formation of a specific catalytically inert E^⁎B complex in the interface, which is noticeably different than the 1:1 EB complex in the aqueous phase. The cholate-activated rate of hydrolysis is lowered by hypolidemic ezetimibe and guggul extract which are not interfacial competitive inhibitors of PLA2. We propose that the biphasic modulation of the pancreatic PLA2 activity by bile salts regulates gastrointestinal fat metabolism and cholesterol homeostasis.

Keywords: Abbreviations; Analytical parameters are defined in Scheme A1 in Appendix A. Structures of the bile salts and conjugates are given in Figure 1; DMPC; 1,2-dimyristoyl-; sn; -3-glycerophospho-choline; DMPM; 1,2-dimyristoyl-; sn; -3-glycerophospho-methanol; DOC; deoxycholate; DTPC; 1,2-ditetradecyl-; rac; -3-glycerophosphocholine; i-face; the interface binding surface of PLA2; LPC; 1-myristoyl-; sn; -3-glycerophospho-choline; MA; myristic acid; MJ33; 1-hexadecyl-3-(trifluoroethyl)-; rac; -glycero-2-phosphomethanol; PGU; 2-dodecanylamino-hexanol-2-phosphoglycol; PLA2; group IB phospholipase A; ₂; from pig pancreas; POPC; 1-palmitoyl-2-oleoyl-; sn; -3-glycerophospho-choline; products; LPC+MA (1:1); TCDOC; taurochenodeoxycholate; TDOC; taurodeoxycholate; TMA-DPH; trimethylammonium-diphenylhexatriene; WT; wild type PLA2

In vitro stability and content release properties of phosphatidylglyceroglycerol containing thermosensitive liposomes by Martin Hossann; Michael Wiggenhorn; Alenka Schwerdt; Kirsten Wachholz; Nicole Teichert; Hansjörg Eibl; Rolf D. Issels; Lars H. Lindner (pp. 2491-2499).

Recently, we reported that 1,2-dipalmitoyl- sn-glycero-3-phosphoglyceroglycerol (DPPGOG) prolongs the circulation time of thermosensitive liposomes (TSL). Since the only TSL formulation in clinical trials applies DSPE-PEG2000 and lysophosphatidylcholine (P-lyso-PC), the objective of this study was to compare the influence of these lipids with DPPGOG on in vitro stability and heat-induced drug release properties of TSL. The content release rate was significantly increased by incorporating DPPGOG or P-lyso-PC in TSL formulations. DPPC/DSPC/DPPGOG 50:20:30 (m/m) and DPPC/P-lyso-PC/DSPE-PEG2000 90:10:4 (m/m) did not differ significantly in their release rate of carboxyfluorescein with >70% being released within the first 10s at their phase transition temperature. Furthermore, DPPC/DSPC/DPPGOG showed an improved stability at 37 °C in serum compared to the PEGylated TSL. The in vitro properties of DPPGOG-containing TSL remained unchanged when encapsulating doxorubicin instead of carboxyfluorescein. The TSL retained 89.1±4.0% of doxorubicin over 3 h at 37 °C in the presence of serum. The drug was almost completely released within 120s at 42 °C. In conclusion, DPPGOG improves the in vitro properties in TSL formulations compared to DSPE-PEG2000, since it not only increases the in vivo half-life, it even increases the content release rate without negative effect on TSL stability at 37 °C which has been seen for DSPE-PEG2000/P-lyso-PC containing TSL.

Keywords: Thermosensitive liposomes; Content release rate; Hyperthermia; Poly(ethylene glycol); Phosphatidylglyceroglycerol; Doxorubicin

Bacterial lipid composition and the antimicrobial efficacy of cationic steroid compounds (Ceragenins) by Raquel F. Epand; Paul B. Savage; Richard M. Epand (pp. 2500-2509).

Ceragenins are cationic bile salt derivatives having antimicrobial activity. The interactions of several ceragenins with phospholipid bilayers were tested in different systems. The ceragenins are capable of forming specific associations with several phospholipid species that may be involved with their antimicrobial action. Their antimicrobial activity is lower in bacteria that have a high content of phosphatidylethanolamine. Gram negative bacteria with a high content of phosphatidylethanolamine exhibit sensitivity to different ceragenins that corresponds to the extent of interaction of these compounds with phospholipids, including the ability of different ceragenins to induce leakage of aqueous contents from phosphatidylethanolamine-rich liposomes. A second class of bacteria having cell membranes composed largely of anionic lipids and having a low content of phosphatidylethanolamine are very sensitive to the action of the ceragenins but they exhibit similar minimal inhibitory concentrations with most of the ceragenins and for different strains of bacteria. Although Gram negative bacteria generally have a high content of phosphatidylethanolamine, there are a few exceptions. In addition, a mutant strain of Escherichia coli has been made that is essentially devoid of phophatidylethanolamine, although 80% of the lipid of the wild-type strain is phosphatidylethanolamine. Furthermore, certain Gram positive bacteria are also exceptions in that they can have a high content of phosphatidylethanolamine. We find that the antimicrobial action of the ceragenins correlates better with the content of phosphatidylethanolamine in the bacterial membrane than whether or not the bacteria has an outer membrane. Thus, the bacterial lipid composition can be an important factor in determining the sensitivity of bacteria to antimicrobial agents.

Keywords: Abbreviations; DEPE; dielaidoylphosphatidylethanolamine; DPPC; dipalmitoylphosphatidylcholine; DPPG; dipalmitoylphosphatidylglycerol; DOPC; dioleoylphosphatidylcholine; DOPG; dioleoylphosphatidylglycerol; ANTS; 8-aminonaphthalene-1,3,6-trisulfonic acid; DPX; p; -xylene-bis-pyridinium bromide; CSA; Cationic Antimicrobial Steroid; PE; phosphatidylethanolamine; PG; phosphatidylglycerol; CL; cardiolipin; PC; phosphatidylcholineBacterial outer membrane; Cell wall; Antimicrobial agent; Bacterial membrane; Cationic sterol derivative; Gram negative bacteria; Phosphatidylethanolamine

VDAC closure increases calcium ion flux by Wenzhi Tan; Marco Colombini (pp. 2510-2515).

VDAC is the major permeability pathway in the mitochondrial outer membrane and can control the flow of metabolites and ions. Therefore Ca²⁺ flux across the outer membrane occurs mainly through VDAC. Since both Ca²⁺ fluxes and VDAC are involved in apoptosis, we examined whether Ca²⁺ is required for channel formation by VDAC isolated from rat liver. The voltage gating of VDAC does not require Ca²⁺ and it functions normally with or without Ca²⁺. Additionally, VDAC generally shows a higher permeability to Ca²⁺ in the closed states (states with lower permeability to metabolites) than that in the open state. Thus VDAC closure, which induces apoptosis, also favors Ca²⁺ flux into mitochondria, which can also lead to permeability transition and cell death. These results are consistent with the view that VDAC closure is a pro-apoptotic signal.

Keywords: PTP; Mitochondria; Apoptosis; Voltage gating; Swelling; Planar membrane

Probing the dynamics of intact cells and nuclear envelope precursor membrane vesicles by deuterium solid state NMR spectroscopy by Marie Garnier-Lhomme; Axelle Grélard; Richard D. Byrne; Cécile Loudet; Erick J. Dufourc; Banafshé Larijani (pp. 2516-2527).

Membrane dynamics is an essential part of many cellular mechanisms such as intracellular trafficking, membrane fusion/fission and mitotic organelle reconstitution. The dynamics of membranes is dependent primarily on their phospholipid and cholesterol composition and how these molecules are ordered in relation to one another. To determine the physical status of membranes in whole cells or purified membranes of subcellular compartments we have developed a novel application exploiting solid-state²H-NMR spectroscopy. We utilise this method to probe the dynamics of intact sperm and nuclear envelope precursor membranes. We show, using mass spectrometry, that either multilamellar or small unilamellar vesicles of deuterium-labelled palmitoyl-oleoylphosphatidylcholine can be used to probe the dynamics of sperm cells or nuclear envelope precursor membrane vesicles, respectively. Using²H-NMR we determine the order parameters of sperm cells and nuclear envelope precursor membrane vesicles. We demonstrate that whole sperm membranes are more dynamic than nuclear envelope precursor membranes due to the higher cholesterol levels of the latter. Our new application can be exploited as a generic method for monitoring membrane dynamics in whole cells, various subcellular membrane compartments and membrane domains in subcellular compartments.

Keywords: Abbreviations; DPPC; dipalmitoylphosphatidylcholine; DSC; differential scanning calorimetry; Δ; ν; _Q; quadrupolar splitting; ESI-MS/MS; electrospray ionisation tandem mass spectrometry; HPLC; high pressure liquid chromatography; MLV; multilamellar vesicle; MV0; precursor egg membrane vesicles; PtdAc; phosphatidic acid; PtdCho; phosphatidylcholine; PtdEth; phosphatidylethanolamine; PtdIns; phosphatidylinositol; PtdSer; phosphatidylserine; POPC; palmitoyl-oleoylphosphatidylcholine; S10; activated egg cytoplasm; S; _CD; order parameter; ssNMR; solid-state NMR; SUV; small unilamellar vesicles; TEM; transmission electron microscopyNuclear envelope membrane; Cholesterol; Solid-state NMR spectroscopy; Membrane dynamics

Critical amino acid residues of maurocalcine involved in pharmacology, lipid interaction and cell penetration by Kamel Mabrouk; Narendra Ram; Sylvie Boisseau; Flavie Strappazzon; Amel Rehaim; Rémy Sadoul; Hervé Darbon; Michel Ronjat; Michel De Waard (pp. 2528-2540).

Maurocalcine (MCa) is a 33-amino acid residue peptide that was initially identified in the Tunisian scorpion Scorpio maurus palmatus. This peptide triggers interest for three main reasons. First, it helps unravelling the mechanistic basis of Ca²⁺ mobilization from the sarcoplasmic reticulum because of its sequence homology with a calcium channel domain involved in excitation–contraction coupling. Second, it shows potent pharmacological properties because of its ability to activate the ryanodine receptor. Finally, it is of technological value because of its ability to carry cell-impermeable compounds across the plasma membrane. Herein, we characterized the molecular determinants that underlie the pharmacological and cell-penetrating properties of maurocalcine. We identify several key amino acid residues of the peptide that will help the design of cell-penetrating analogues devoid of pharmacological activity and cell toxicity. Close examination of the determinants underlying cell penetration of maurocalcine reveals that basic amino acid residues are required for an interaction with negatively charged lipids of the plasma membrane. Maurocalcine analogues that penetrate better have also stronger interaction with negatively charged lipids. Conversely, less effective analogues present a diminished ability to interact with these lipids. These findings will also help the design of still more potent cell penetrating analogues of maurocalcine.

Keywords: Abbreviations; BSA; bovine serum albumin; CGN; cerebellar granule neurons; CHO; chinese hamster ovary; CPP; cell penetrating peptide; DHE; dihydroethidium; DHP; dihydropyridine; DHPR; dihydropyridine receptor; DMEM; dulbecco’s modified eagle’s medium; DMSO; dimethyl sulfoxide; EDTA; ethylenediaminetetraacetic acid; FACS; fluorescence activated cell sorter; Fmoc; N; -α-fluorenylmethyloxycarbonyl; HEK293; human embryonic kidney 293 cells; HEPES; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HMP; 4-hydroxymethylphenyloxy; ¹; H-NMR; proton nuclear magnetic resonance; HPLC; high pressure liquid chromatography; MCa; maurocalcine; MCa; _b; biotinylated maurocalcine; MTT; 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide; PC; ₅₀; half-maximal penetration concentration; PBS; phosphate buffered saline; PtdIns; phosphatidylinositol; RyR; ryanodine receptor; SR; sarcoplasmic reticulum; strep-Cy5 (Cy3); streptavidine-cyanine 5 (cyanine 3); TFA; trifluoroacetic acidMaurocalcine; Ryanodine receptor; Cell penetration; Cell penetrating peptide; Lipid interaction; Cell toxicity

Superficially active water in lipid membranes and its influence on the interaction of an aqueous soluble protease by M.F. Martini; E.A. Disalvo (pp. 2541-2548).

The purpose of this paper is to demonstrate that the interaction of an aqueous soluble enzyme with lipid membranes is influenced by the lipid composition of the interphase. The results show that the interaction of an aqueous soluble protease, Rennet from Mucor miehei, depends on the exposure of the carbonyl and phosphate groups at the membrane interphase. The changes produced by the protease on the surface pressure of monolayers of dimyristoylphosphatidylcholine (DMPC); dioleoylphosphatidylcholine (DOPC); diphytanoylphosphatidylcholine (DPhPC); dipalmitoylphosphatidylcholine (DPPC); di- O-tetradecylphosphatidyl-choline [D(ether)PC]; dimyristoylphosphatidylethanolamine (DMPE); di- O-tetradecyl-phosphatidylethanolamine [D(ether)PE] were measured at different initial surface pressures. The meaning of the Δ Π vs. Π curves was interpreted in the light of the concept of interphase given by Defay and Prigogine [R. Defay, I. Prigogine, Surface Tension and Adsorption, John Wiley & Sons, New York, 1966, pp. 273–277] considering the interphase as a bidimensional solution of polar head groups. With this approach, and based on reported evidences that carbonyls and phosphates are the main hydration sites of the lipid membranes, it is suggested that the mechanism of interaction of aqueous soluble protein involves water beyond the hydration shell. At high surface pressure, only water strongly bound to carbonyl and phosphate groups is present and the interaction is not occurring. In contrast, at low surface pressures, the protease-membrane interaction is a function of acyl chain for different polar groups. This is interpreted, as a consequence of the changes in the interfacial tension produced by the displacement of water confined between the hydrated head groups.

Keywords: Abbreviations; PC; phosphatidylcholine; DPPC; dipalmitoylphosphatidylcholine; DMPC; dimyristoylphosphatidylcholine; DOPC; dioleoylphosphatidylcholine; DPhPC; diphytanoylphosphatidylcholine; D(eter)PC; di-; O; -tetradecylphosphatidylcholine; PE; phosphatidylethanolamine; DMPE; dimyristoylphosphatidylethanolamine; D(eter)PE; di-; O; -tetradecylphosphatidylethanolamine; Phlo; phloretin; Π; surface pressure; Π; i; initial surface pressure; Δ; Π; change of surface pressureLipid monolayer; Surface pressure; Superficially active water; Interphase; Lipid–protein interaction

Membrane interactions of the hydrophobic segment of diacylglycerol kinase epsilon by Evgenia Glukhov; Yulia V. Shulga; Raquel F. Epand; Armela O. Dicu; Matthew K. Topham; Charles M. Deber; Richard M. Epand (pp. 2549-2558).

Diacylglycerol kinase epsilon (DGKε) is unique among mammalian DGK isoforms in having a segment of hydrophobic amino acids as a putative membrane anchor. To model the conformation, and stoichiometry of this segment in membrane-mimetic environments, we have prepared a peptide corresponding to this hydrophobic segment of DGKε of sequence KKKKLILWTLCSVLLPVFITFWKKKKK-NH₂. Flanking Lys residues mimic the natural setting of this peptide in DGKε, while facilitating peptide synthesis and characterization. Circular dichroism and fluorescence spectroscopic analysis demonstrated that the peptide has increased helical content and significant blue shifts in the presence of anionic – but not zwitterionic – bilayer membranes. When labeled with fluorophores that can undergo fluorescence resonance energy transfer, the peptide was found to dimerize – a result also observed from migration rates on SDS-PAGE gels under both reducing and non-reducing disulfide bridge conditions. The peptide was shown to preferentially interact with cholesterol in lipid films comprised of homogeneous mixtures of cholesterol and phosphatidylcholine, yet the presence of cholesterol in hydrated vesicle bilayers decreases its helical content. The peptide was also able to inhibit the activity of DGKε protein in vitro. Our overall findings suggest that the peptide ultimately cannot leave the bulk water for attachment/insertion into the outer leaflet of an erythrocyte-like bilayer, yet its core sequence is sufficiently hydrophobic to insert into membrane core regions when membrane attachment is promoted by electrostatic attraction to anionic lipid head groups of the inner leaflet of an erythrocyte-like bilayer.

Keywords: Abbreviations; CD; circular dichroism; DAG; diacylglycerol; DGK; diacylglycerol kinase; DGKε; epsilon isoform of DGK; DGKζ; zeta isoform of DGK; DMPS; 1,2-dimyristoylphosphatidylserine; DSC; differential scanning calorimetry; SAG; 1-stearoyl-2-arachidonoyl glycerol; DOG; 1,2-dioleoyl glycerol; DOPC; 1,2-dioleoylphosphatidylcholine; FRET; fluorescence resonance energy transfer; L22W39-K; ₉; KKKKLILWTLCSVLLPVFITFWKKKKK-NH; ₂; LPC; lysophosphatidylcholine; OG; octyl glucoside; MRE; mean residue ellipticity; PA; phosphatidic acid; POPC; 1-palmitoyl-2-oleoylphosphatidylcholine; SOPC; 1-stearoyl-2-oleoylphosphatidylcholine; SUV; small unilamellar vesicle; TDW; triply distilled water; TM; transmembraneDiacylglycerol kinase; Hydrophobic segment; Transmembrane helix; Membrane protein oligomerization

The Enterobacter aerogenes outer membrane efflux proteins TolC and EefC have different channel properties by Muriel Masi; Nathalie Saint; Gérard Molle; Jean-Marie Pagès (pp. 2559-2567).

The outer membrane proteins TolC and EefC from Enterobacter aerogenes are involved in multidrug resistance as part of two resistance-nodulation-division efflux systems. To gain more understanding in the molecular mechanism underlying drug efflux, we have undertaken an electrophysiological characterization of the channel properties of these two proteins. TolC and EefC were purified in their native trimeric form and then reconstituted in proteoliposomes for patch-clamp experiments and in planar lipid bilayers. Both proteins generated a small single channel conductance of about 80 pS in 0.5 M KCl, indicating a common gated structure. The resultant pores were stable, and no voltage-dependent openings or closures were observed. EefC has a low ionic selectivity ( P_K/ P_Cl=∼3), whereas TolC is more selective to cations ( P_K/ P_Cl=∼30). This may provide a possible explanation for the difference in drug selectivity between the AcrAB-TolC and EefABC efflux systems observed in vivo. The pore-forming activity of both TolC and EefC was severely inhibited by divalent cations entering from the extracellular side. Another characteristic of the TolC and EefC channels was the systematic closure induced by acidic pH. These results are discussed in respect to the physiological functions and structural models of TolC and EefC.

Keywords: E. aerogenes; Outer membrane; Multidrug resistance; Efflux pumps; Channel-forming proteins

Transbilayer distribution of phospholipids in bacteriophage membranes by Simonas Laurinavičius; Dennis H. Bamford; Pentti Somerharju (pp. 2568-2577).

We have previously demonstrated that the membranes of several bacteriophages contain more phosphatidylglycerol (PG) and less phosphatidylethanolamine (PE) than the host membrane from where they are derived. Here, we determined the transbilayer distribution of PG and PE in the membranes of bacteriophages PM2, PRD1, Bam35 and phi6 using selective modification of PG and PE in the outer membrane leaflet with sodium periodate or trinitrobenzene sulfonic acid, respectively. In phi6, the transbilayer distributions of PG, PE and cardiolipin could also be analyzed by selective hydrolysis of the lipids in the outer leaflet by phospholipase A₂. We used electrospray ionization mass-spectrometry to determine the transbilayer distribution of phospholipid classes and individual molecular species. In each bacteriophage, PG was enriched in the outer membrane leaflet and PE in the inner one (except for Bam35). Only modest differences in the transbilayer distribution between different molecular species were observed. The effective shape and charge of the phospholipid molecules and lipid–protein interactions are likely to be most important factors driving the asymmetric distribution of phospholipids in the phage membranes. The results of this first systematic study on the phospholipid distribution in bacteriophage membranes will be very helpful when interpreting the accumulating high-resolution data on these organisms.

Keywords: Transbilayer phospholipid distribution; Membrane asymmetry; Bacteriophage; Molecular species; Effective shape; Lipid–protein interaction

Action mechanism of PEGylated magainin 2 analogue peptide by Yuichi Imura; Minoru Nishida; Katsumi Matsuzaki (pp. 2578-2585).

PEGylation is frequently used to improve the efficacy of protein and peptide drugs. Recently, we investigated its effects on the action mechanism of the cyclic β-sheet antimicrobial peptide tachyplesin I isolated from Tachypleus tridentatus [Y. Imura, M. Nishida, Y. Ogawa, Y. Takakura, K. Matsuzaki, Action Mechanism of Tachyplesin I and Effects of PEGylation, Biochim. Biophys. Acta 1768 (2007) 1160–1169]. PEGylation did not change the basic mechanism behind the membrane-permeabilizing effect of the peptide on liposomes, however, it decreased the antimicrobial activity and cytotoxicity. To obtain further information on the effects of PEGylation on the activities of antimicrobial peptides, we designed another structurally different PEGylated antimicrobial peptide (PEG-F5W, E19Q-magainin 2-amide) based on the α-helical peptide magainin 2 isolated from the African clawed frog Xenopus laevis. The PEGylated peptide induced the leakage of calcein from egg yolkl-α-phosphatidylglycerol/egg yolkl-α-phosphatidylcholine large unilamellar vesicles, however, the activity was weaker than that of the control peptides. The PEGylated peptide induced lipid flip-flop coupled to the leakage and was translocated into the inner leaflet of the bilayer, indicating that PEGylation did not alter the basic mechanism of membrane permeabilization of the parent peptide. The cytotoxicity of the non-PEGylated peptides was nullified by PEGylation. At the same time, the antimicrobial activity was weakened only by 4 fold. The effects of PEGylation on the activity of magainin were compared with those for tachyplesin.

Keywords: Abbreviations; Ac-E19Q-MGa; Ac-F5W, E19Q-magainin 2-amide; AMEM; Alpha modification of Eagle's medium; C; ₆; -NBD-PC; 1-palmitoyl-2-[6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]caproyl]-; l; -α-phosphatidylcholine; CD; circular dichroism; CFU; colony forming unit; CHO; Chinese Hamster Ovary; FBS; fetal bovine serum; Fmoc; 9-fluorenylmethoxycarbonyl; HPLC; high-performance liquid chromatography; L/P; lipid-to-peptide molar ratio; LUVs; large unilamellar vesicles; MG; F5W-magainin 2; MIC; minimal inhibitory concentration; MLVs; multilamellar vesicles; NAPB; 10 mM sodium phosphate/100 mM NaCl, pH 7.4 buffer; ONPG; o; -nitrophenyl-β-; d; -galactoside; PC; egg yolk; l; -α-phosphatidylcholine; PEG; polyethyleneglycol; PEG-E19Q-MGa; PEG-F5W, E19Q-magainin 2-amide; PG; l; -α-phosphatidyl-; dl; -glycerol enzymatically converted from PC; SUVs; small unilamellar vesicles; TSB; tryptic soy broth; WST-1; 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-; 2H; -tetrazolium, monosodium saltAntimicrobial peptide; Magainin 2; PEGylation; Lipid flip-flop; Peptide–lipid interaction; Inner membrane permeabilization

How lipids influence the mode of action of membrane-active peptides by E. Sevcsik; G. Pabst; A. Jilek; K. Lohner (pp. 2586-2595).

The human, multifunctional peptide LL-37 causes membrane disruption by distinctly different mechanisms strongly dependent on the nature of the membrane lipid composition, varying not only with lipid headgroup charge but also with hydrocarbon chain length. Specifically, LL-37 induces a peptide-associated quasi-interdigitated phase in negatively charged phosphatidylglycerol (PG) model membranes, where the hydrocarbon chains are shielded from water by the peptide. In turn, LL-37 leads to a disintegration of the lamellar organization of zwitterionic dipalmitoyl-phosphatidylcholine (DPPC) into disk-like micelles. Interestingly, interdigitation was also observed for the longer-chain C18 and C20 PCs. This dual behavior of LL-37 can be attributed to a balance between electrostatic interactions reflected in different penetration depths of the peptide and hydrocarbon chain length. Thus, our observations indicate that there is a tight coupling between the peptide properties and those of the lipid bilayer, which needs to be considered in studies of lipid/peptide interaction. Very similar effects were also observed for melittin and the frog skin peptide PGLa. Therefore, we propose a phase diagram showing different lipid/peptide arrangements as a function of hydrocarbon chain length and LL-37 concentration and suggest that this phase diagram is generally applicable to membrane-active peptides localized parallel to the membrane surface.

Keywords: Abbreviations; DMPC; dimyristoyl-phosphatidylcholine; DMPG; dimyristoyl-phosphatidylglycerol; DPPC; dipalmitoyl-phosphatidylcholine; DPPG; dipalmitoyl-phosphatidylglycerol; DSPC; distearoyl-phosphatidylcholine; DSPG; distearoyl-phosphatidylglycerol; DAPC; diarachidoyl-phosphatidylcholine; DSA; doxyl stearic acid; SWAXS; small- and wide-angle X-ray scattering; DSC; differential scanning calorimetry; ESR; electron spin resonanceLL-37; Antimicrobial peptides; Interdigitated bilayer; Micellar disks

Domain formation by a Rhodococcus sp. biosurfactant trehalose lipid incorporated into phosphatidylcholine membranes by Francisco J. Aranda; José A. Teruel; María J. Espuny; Ana Marqués; Ángeles Manresa; Elisa Palacios-Lidón; Antonio Ortiz (pp. 2596-2604).

The study of the interaction of biosurfactants with biological membranes is of great interest in order to gain insight into the molecular mechanisms of their biological actions. In this work we report on the interaction of a bacterial trehalose lipid produced by Rhodococcus sp. with phosphatidylcholine membranes. Differential scanning calorimetry measurements show a good miscibility of the glycolipid in the gel state and immiscibility in the fluid state, suggesting domain formation. These domains have been visualized and characterized, for the first time, by scanning force microscopy. Incorporation of trehalose lipid into phosphatidylcholine membranes produces a small shift of the antisymmetric stretching band toward higher wavenumbers, as shown by FTIR, which indicates a weak increase in fluidity. The CO stretching band shows that incorporation of trehalose lipid increases the proportion of the dehydrated component in mixtures with the three phospholipids at temperatures below and above the gel to liquid–crystalline phase transition. This dehydration effect is also supported by data on the phospholipid PO stretching bands. Small-angle X-ray diffraction measurements show that in the samples containing trehalose lipid the interlamellar repeat distance is larger than in those of pure phospholipids. These results are discussed within the frame of trehalose lipid domain formation, trehalose lipid/phospholipid interactions and its relevance to membrane-related biological actions.

Keywords: Biosurfactant; Trehalose lipid; Phospholipid membrane; Membrane domain

Discriminating healthy from tumor and necrosis tissue in rat brain tissue samples by Raman spectral imaging by Nadia Amharref; Abdelilah Beljebbar; Sylvain Dukic; Lydie Venteo; Laurence Schneider; Michel Pluot; Michel Manfait (pp. 2605-2615).

The purpose of this study was to investigate molecular changes associated with glioma tissues by Raman microspectroscopy in order to develop its use in clinical practice. Spectroscopic markers obtained from C6 glioma tissues were compared to conventional histological and histochemical techniques. Cholesterol and phospholipid contents were highest in corpus callosum and decreased gradually towards the cortex surface as well as in the tumor. Two different necrotic areas have been identified: a fully necrotic zone characterized by the presence of plasma proteins and a peri-necrotic area with a high lipid content. This result was confirmed by Nile Red staining. Additionally, one structure was detected in the periphery of the tumor. Invisible with histopathological hematoxylin and eosin staining, it was revealed by immunohistochemical Ki-67 and MT1-MMP staining used to visualize the proliferative and invasive activities of glioma, respectively. Hierarchical cluster analysis on the only cluster averaged spectra showed a clear distinction between normal, tumoral, necrotic and edematous tissues. Raman microspectroscopy can discriminate between healthy and tumoral brain tissue and yield spectroscopic markers associated with the proliferative and invasive properties of glioblastoma. Development of in vivo Raman spectroscopy could thus accurately define tumor margins, identify tumor remnants, and help in the development of novel therapies for glioblastoma.

Keywords: Raman imaging; Glioma; Invasion; Necrosis; Brain structure; Edema

Interactions of amphotericin B derivatives with lipid membranes—A molecular dynamics study by Jacek Czub; Edward Borowski; Maciej Baginski (pp. 2616-2626).

Amphotericin B (AmB) is a well-known polyene macrolide antibiotic used to treat systemic fungal infections. AmB targets more efficiently fungal than animal membranes. However, there are only minor differences in the mode of action of AmB against both types of membranes, which is a source of AmB toxicity. In this work, we analyzed interactions of two low toxic derivatives of AmB (SAmE and PAmE), synthesized in our laboratory, with lipid membranes. Molecular dynamics simulations of the lipid bilayers containing ergosterol (fungal cells) or cholesterol (animal cells) and the studied antibiotic molecules were performed to compare the structural and dynamic properties of AmB derivatives and the parent drug inside the membrane. A number of differences was found for AmB and its derivatives' behavior in cholesterol- and ergosterol-containing membranes. We found that PAmE and SAmE can penetrate deeper into the hydrophobic region of the membrane compared to AmB. Modification of the amino and carboxyl group of AmB also resulted in the conformational transition within the antibiotic's polar head. Wobbling dynamics differentiation, depending on the sterol present, was discovered for the AmB derivatives. These differences may be interpreted as molecular factors responsible for the improved selectivity observed macroscopically for the studied AmB derivatives.

Keywords: Amphotericin B; Molecular dynamics; Molecular modeling; Lipid membranes; Cholesterol; Ergosterol

Structure analysis of the protein translocating channel TatA in membranes using a multi-construct approach by Christian Lange; Sonja D. Müller; Torsten H. Walther; Jochen Bürck; Anne S. Ulrich (pp. 2627-2634).

The twin- arginine- translocase (Tat) can transport proteins in their folded state across bacterial or thylakoid membranes. In Bacillus subtilis the Tat-machinery consists of only two integral (inner) membrane proteins, TatA and TatC. Multiple copies of TatA are supposed to form the transmembrane channel, but little structural data is available on this 70-residue component. We used a multi-construct approach for expressing several characteristic fragments of TatA_d, to determine their individual structures and to cross-validate them comprehensively within the architecture of the full-length protein. Here, we report the design, high-yield expression, detergent-aided purification and lipid-reconstitution of five constructs of TatA_d, overcoming difficulties associated with the very different hydrophobicities and sizes of these membrane protein fragments. Circular dichroism (CD) and oriented CD (OCD) were used to determine their respective conformations and alignments in suitable, negatively charged phospholipid bilayers. CD spectroscopy showed an N-terminal α-helix, a central helical stretch, and an unstructured C-terminus, thus proving the existence of these secondary structures in TatA_d for the first time. The OCD spectra demonstrated a transmembrane orientation of the N-terminal α-helix and a surface alignment of the central amphiphilic helix in lipid bilayers, thus supporting the postulated topology model and function of TatA as a transmembrane channel.

Keywords: Twin-arginine-translocation; TatA; Membrane protein; Circular dichroism; Oriented circular dichroism; Bacillus subtilisAbbreviations; Tat; twin-arginine-translocation; CD; circular dichroism; OCD; oriented circular dichroism; NLS; N-lauroylsarcosine; DMPC; dimyristoylphosphatidylcholine; DMPG; dimyristoylphosphatidylglycerol; TMS; transmembrane segment; APH; amphiphilic helix; TatA; _2–70; full-length protein; TatA; _1–27; transmembrane fragment; TatA; _22–70; extramembraneous fragment; TatA; _22–45; central fragment; TatA; _2–45; transmembrane plus central fragment

Oxygen permeability of the lipid bilayer membrane made of calf lens lipids by Justyna Widomska; Marija Raguz; Witold K. Subczynski (pp. 2635-2645).

The oxygen permeability coefficient across the membrane made of the total lipid extract from the plasma membrane of calf lens was estimated from the profile of the oxygen transport parameter (local oxygen diffusion-concentration product) and compared with those estimated for membranes made of an equimolar 1-palmitoyl-2-oleoylphosphatidylcholine/cholesterol (POPC/Chol) mixture and of pure POPC. Profiles of the oxygen transport parameter were obtained by observing the collision of molecular oxygen with nitroxide radical spin labels placed at different depths in the membrane using the saturation-recovery EPR technique and were published by us earlier (J. Widomska, M. Raguz, J. Dillon, E. R. Gaillard, W. K. Subczynski, Biochim. Biophys. Acta. 1768 (2007) 1454–1465). At 35 °C, the estimated oxygen permeability coefficients were 51.3, 49.7, and 157.4 cm/s for lens lipid, POPC/Chol, and POPC membranes, respectively (compared with 53.3 cm/s for a water layer with the same thickness as a membrane). Membrane permeability significantly decreases at lower temperatures. In the lens lipid membrane, resistance to the oxygen transport is located in and near the polar headgroup region of the membrane to the depth of the ninth carbon, which is approximately where the steroid-ring structure of cholesterol reaches into the membrane. In the central region of the membrane, oxygen transport is enhanced, significantly exceeding that in bulk water. It is concluded that the high level of cholesterol in lens lipids is responsible for these unique membrane properties.

Keywords: Oxygen permeation; Lens lipids; Lipid bilayer; Cholesterol; Hydrophobic barrier; Membrane rigidity; EPR

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BBA - Biomembranes (v.1768, #10)