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BBA - Biomembranes (v.1768, #11)

Editorial Board (pp. ii).

Stratum corneum hydration: Phase transformations and mobility in stratum corneum, extracted lipids and isolated corneocytes by C.L. Silva; D. Topgaard; V. Kocherbitov; J.J.S. Sousa; A.A.C.C. Pais; E. Sparr (pp. 2647-2659).

The outermost layer of skin, stratum corneum (SC), functions as the major barrier to diffusion. SC has the architecture of dead keratin filled cells embedded in a lipid matrix. This work presents a detailed study of the hydration process in extracted SC lipids, isolated corneocytes and intact SC. Using isothermal sorption microcalorimetry and relaxation and wideline¹H NMR, we study these systems at varying degrees of hydration/relative humidities (RH) at 25 °C. The basic findings are (i) there is a substantial swelling both of SC lipids, the corneocytes and the intact SC at high RH. At low RHs corneocytes take up more water than SC lipids do, while at high RHs swelling of SC lipids is more pronounced than that of corneocytes. (ii) Lipids in a fluid state are present in both extracted SC lipids and in the intact SC. (iii) The fraction of fluid lipids is lower at 1.4% water content than at 15% but remains virtually constant as the water content is further increased. (iv) Three exothermic phase transitions are detected in the SC lipids at RH=91–94%, and we speculate that the lipid re-organization is responsible for the hydration-induced variations in SC permeability. (v) The hydration causes swelling in the corneocytes, while it does not affect the mobility of solid components (keratin filaments).

Keywords: SC; Sorption microcalorimetry; Wideline NMR; Phase behavior; Molecular mobility; Lipid; Corneocyte

Sorting signal of Escherichia coli OmpA is modified by oligo-(R)-3-hydroxybutyrate by Mo Xian; Michelle M. Fuerst; Yuri Shabalin; Rosetta N. Reusch (pp. 2660-2666).

Escherichia coli outer membrane protein A (OmpA) is a well-established model for the study of membrane assembly. Previous studies have shown that the essential sequence for outer membrane localization, known as the sorting signal, is contained in a segment of the eighth β-strand, residues 163–171. Sequential digestion of OmpA, purified from outer membranes or inclusion bodies with cyanogen bromide and Staphylococcus aureus GluC, yielded peptides 162–174(LSLGVSYRFGQGE). Western blot and chemical assays indicated that the peptide was covalently modified by oligo-(R)-3-hydroxybutyrate (cOHB), a flexible, amphipathic oligoester. MALDI/MS was consistent with modification of peptides 162–174 by up to ten R-3-hydroxybutyrate (HB) residues. Western blot analysis of mutants of the peptide, using anti-OHB IgG, indicated that cOHB modification was not inhibited by the single mutations S163G, S167G, Y168F, R169N or R169D; however, cOHB was not detected on peptides containing the double mutations S163G:S167G S163G:V166G, L162G:S167G, and L164G:S167G. MALDI/MS/MS of double mutant S163G:S167G confirmed the absence of cOHB-modification. The results suggest that cOHB may be attached to one or both serines, and point to the importance of the flanking hydrophobic residues. Modification by cOHB may play a role in outer membrane targeting and assembly of OmpA.

Keywords: OmpA; cOHB; Outer membrane sorting; Protein modification; Amphipathic polymer

Antimicrobial activity of histidine-rich peptides is dependent on acidic conditions by Lukasz Kacprzyk; Victoria Rydengård; Matthias Mörgelin; Mina Davoudi; Mukesh Pasupuleti; Martin Malmsten; Artur Schmidtchen (pp. 2667-2680).

Synthetic peptides composed of multiples of the consensus heparin-binding Cardin and Weintraub sequences AKKARA and ARKKAAKA are antimicrobial. Replacement of lysine and arginine by histidine in these peptides completely abrogates their antimicrobial and heparin-binding activities at neutral pH. However, the antibacterial activity against Gram-negative ( Escherichia coli, Pseudomonas aeruginosa) and Gram-positive bacteria ( Bacillus subtilis and Staphylococcus aureus) as well as the fungus Candida albicans, was restored at acidic conditions (pH 5.5). Fluorescence microscopy and FACS analysis showed that the binding of the histidine-rich peptides to E. coli and Candida was significantly enhanced at pH 5.5. Likewise, fluorescence studies for assessment of membrane permeation as well as electron microscopy analysis of peptide-treated bacteria, paired with studies of peptide effects on liposomes, demonstrated that the peptides induce membrane lysis only at acidic pH. No discernible hemolysis was noted for the histidine-rich peptides. Similar pH-dependent antimicrobial activities were demonstrated for peptides derived from histidine-rich and heparin-binding regions of human kininogen and histidine-rich glycoprotein. The results demonstrate that the presence of an acidic environment is an important regulator of the activity of histidine-rich antimicrobial peptides.

Keywords: Abbreviations; AMP; antimicrobial peptide; GAG; glycosaminoglycan; hCAP-18; human cationic antimicrobial protein 18; HMWK; High-molecular weight kininogen; MALDI-TOF; matrix-assisted laser desorption/ ionization-time of flight; MES; 2-(morpholino)-ethanesulfonic acid; PIPES; piperazine-1,4-bis(2-ethanesulfonic acid); TR; Texas red; Tris; trishydroxymethylaminomethaneAntimicrobial peptide; Heparin-binding; Histidine-rich glycoprotein; pH

Two distinct mechanisms of vesicle-to-micelle and micelle-to-vesicle transition are mediated by the packing parameter of phospholipid–detergent systems by Marc C.A. Stuart; Egbert J. Boekema (pp. 2681-2689).

The detergent solubilization and reformation of phospholipid vesicles was studied for various detergents. Two distinct mechanisms of vesicle-to-micelle and micelle-to-vesicle transition were observed by turbidimetry and cryo-electron microscopy. The first mechanism involves fast solubilization of phospholipids and occurs via open vesicular intermediates. The reverse process, micelle-to-vesicle transition, mimics the vesicle-to-micelle transition. In the second mechanism the solubilization is a slow process that proceeds via micelles that pinch off from closed vesicles. During vesicle reformation, the micelle-to-vesicle transition, a large number of densely packed multilamellar vesicles are formed. The route used, for solubilization and reformation, by a given detergent–phospholipid combination is critically dependent on the overall packing parameter of the detergent-saturated phospholipid membranes. By a change of the overall packing parameter the solubilization and or reformation mechanism could be changed. All five detergents tested fit within the proposed model. With two detergents the mechanism could be changed by changing the phospholipid composition or the medium conditions.

Keywords: Phospholipid; Detergent; Solubilization; Micelle; Vesicle; Cryo-electron microscopy

Cloning and functional characterization of human SMCT2 (SLC5A12) and expression pattern of the transporter in kidney by E. Gopal; N.S. Umapathy; P.M. Martin; S. Ananth; J.P. Gnana-Prakasam; H. Becker; C.A. Wagner; V. Ganapathy; P.D. Prasad (pp. 2690-2697).

Recently, we cloned two Na⁺-coupled lactate transporters from mouse kidney, a high-affinity transporter (SMCT1 or slc5a8) and a low-affinity transporter (SMCT2 or slc5a12). Here we report on the cloning and functional characterization of human SMCT2 (SLC5A12) and compare the immunolocalization patterns of slc5a12 and slc5a8 in mouse kidney. The human SMCT2 cDNA codes for a protein consisting of 618 amino acids. When expressed in mammalian cells or Xenopus oocytes, human SMCT2 mediates Na⁺-coupled transport of lactate, pyruvate and nicotinate. The affinities of the transporter for these substrates are lower than those reported for human SMCT1. Several non-steroidal anti-inflammatory drugs inhibit human SMCT2-mediated nicotinate transport, suggesting that NSAIDs interact with the transporter as they do with human SMCT1. Immunofluorescence microscopy of mouse kidney sections with an antibody specific for SMCT2 shows that the transporter is expressed predominantly in the cortex. Similar studies with an anti-SMCT1 antibody demonstrate that SMCT1 is also expressed mostly in the cortex. Dual-labeling of SMCT1 and SMCT2 with 4F2hc (CD98), a marker for basolateral membrane of proximal tubular cells in the S1 and S2 segments of the nephron, shows that both SMCT1 and SMCT2 are expressed in the apical membrane of the tubular cells. These studies also show that while SMCT2 is broadly expressed along the entire length of the proximal tubule (S1/S2/S3 segments), the expression of SMCT1 is mostly limited to the S3 segment. These studies suggest that the low-affinity transporter SMCT2 initiates lactate absorption in the early parts of the proximal tubule followed by the participation of the high-affinity transporter SMCT1 in the latter parts of the proximal tubule.

Keywords: Kidney; Proximal tubule; Lactate reabsorption; Nicotinate; Immunolocalization

Membrane cholesterol selectively modulates the activity of the human ABCG2 multidrug transporter by Ágnes Telbisz; Marianna Müller; Csilla Özvegy-Laczka; László Homolya; Lajos Szente; András Váradi; Balázs Sarkadi (pp. 2698-2713).

The human ABCG2 multidrug transporter provides protection against numerous toxic compounds and causes multidrug resistance in cancer. Here we examined the effects of changes in membrane cholesterol on the function of this protein. Human ABCG2 was expressed in mammalian and in Sf9 insect cells, and membrane cholesterol depletion or enrichment was achieved by preincubation with beta cyclodextrin or its cholesterol-loaded form. We found that mild cholesterol depletion of intact mammalian cells inhibited ABCG2-dependent dye and drug extrusion in a reversible fashion, while the membrane localization of the transporter protein was unchanged. Cholesterol enrichment of cholesterol-poor Sf9 cell membrane vesicles greatly increased ABCG2-driven substrate uptake, substrate-stimulated ATPase activity, as well as the formation of a catalytic cycle intermediate (nucleotide trapping). Interestingly, modulation of membrane cholesterol did not significantly affect the function of the R482G or R482T substrate mutant ABCG2 variants, or that of the MDR1 transporter. The selective, major effect of membrane cholesterol on the wild-type ABCG2 suggests a regulation of the activity of this multidrug transporter during processing or in membrane micro-domain interactions. The experimental recognition of physiological and pharmacological substrates of ABCG2, as well as the fight against cancer multidrug resistance may be facilitated by demonstrating the key role of membrane cholesterol in this transport activity.

Keywords: Abbreviations; ABC transporters; ATP binding cassette transporters; CD; cyclodextrin; C-CD; cyclodextrin loaded with cholesterol; S-CD; cyclodextrin loaded with sitosterol; EKI; EKI-785 tyrosine kinase inhibitor; ESG; estradiol 17-beta glucuronide; E3S; estrone 3-sulfate; MDR1; multidrug resistance protein 1; MRP1; multidrug resistance associated protein 1; MTX; methotrexate; PheA; Pheophorbide A; R123; Rhodamine 123ABC transporters; ABCG2 transport activity; Multidrug resistance; Membrane cholesterol; ATPase activity

Ca²⁺-independent effects of BAPTA and EGTA on single-channel Cl⁻ currents in brown adipocytes by Victor Sabanov; Jan Nedergaard (pp. 2714-2725).

The Cl⁻ channels of brown adipocytes electrophysiologically resemble outwardly rectifying Cl⁻ channels (ORCC). To study tentative Ca²⁺ regulation of these channels, we attempted to control Ca²⁺ levels at the cytoplasmic side of the inside-out membrane patches with Ca²⁺-chelating agents. However, we found that the commonly used Ca²⁺-chelators EGTA and BAPTA by themselves influenced the Cl⁻ channel currents, unrelated to their calcium chelating effects. Consequently, in this report we delineate effects of Ca²⁺-chelators (acting from the cytoplasmic side) on the single Cl⁻ channel currents in patch-clamp experiments. Using fixed (1–2 mM) concentrations of chelators, two types of Cl⁻ channels were identified, as discriminated by their reaction to the Ca²⁺-chelators and by their conductance: true-blockage channels (31 pS) and quasi-blockage channels (52 pS). In true-blockage channels, EGTA and BAPTA inhibited channel activity in a classical flickery type manner. In quasi-blockage channels, chelators significantly shortened the duration of individual openings, as in a flickering block, but the overall channel activity tended to increase. This dual effect of mean open time decrease accompanied by a tendency of open probability to increase we termed a quasi-blockage. Despite the complications due to the chelators as such, we could detect a moderate inhibitory effect of Ca²⁺. The anionic classical Cl⁻ channel blockers DIDS and SITS could mimic the true/quasi blockage of EGTA and BAPTA. It was concluded that at least in this experimental system, standard techniques for Ca²⁺ level control in themselves could fundamentally affect the behaviour of Cl⁻ channels.

Keywords: Chloride channel; Calcium; Calcium chelator; EGTA; BAPTA; Single channel kinetics; DIDS; SITS

Permeation of a β-heptapeptide derivative across phospholipid bilayers by Toshinori Shimanouchi; Peter Walde; James Gardiner; Yogesh R. Mahajan; Dieter Seebach; Anita Thomae; Stefanie D. Krämer; Matthias Voser; Ryoichi Kuboi (pp. 2726-2736).

Based on a number of experiments it is concluded that the fluorescein labeled β-heptapeptide fluoresceinyl-NH-CS- (S)- β³hAla- (S)- β³hArg- (R)- β³hLeu- (S)- β³hPhe- (S)- β³hAla- (S)- β³hAla- (S)- β³hLys-OH translocates across lipid vesicle bilayers formed from DOPC (1,2-dioleoyl- sn-glycero-3-phosphocholine). The conclusion is based on the following observations: (i) addition of the peptide to the vicinity of micrometer-sized giant vesicles leads to an accumulation of the peptide inside the vesicles; (ii) if the peptide is injected inside individual giant vesicles, it is released from the vesicles in a time dependent manner; (iii) if the peptide is encapsulated within sub-micrometer-sized large unilamellar vesicles, it is released from the vesicles as a function of time; (iv) if the peptide is submitted to immobilized liposome chromatography, the peptide is retained by the immobilized DOPC vesicles. Furthermore, the addition of the peptide to calcein-containing DOPC vesicles does not lead to significant calcein leakage and vesicle fusion is not observed. The finding that derivatives of the β-heptapeptide (S)- β³hAla- (S)- β³hArg- (R)- β³hLeu- (S)- β³hPhe- (S)- β³hAla- (S)- β³hAla- (S)- β³hLys-OH can translocate across phospholipid bilayers is supported by independent measurements using Tb³⁺-containing large unilamellar vesicles prepared from egg phosphatidylcholine and wheat germ phosphatidylinositol (molar ratio of 9:1) and a corresponding peptide that is labeled with dipicolinic acid instead of fluorescein. The experiments show that this dipicolinic acid labeled β-heptapeptide derivative also permeates across phospholipid bilayers. The possible mechanism of the translocation of the particular β-heptapeptide derivatives across the membrane of phospholipid vesicles is discussed within the frame of the current understanding of the permeation of certain oligopeptides across simple phospholipid bilayers.

Keywords: Abbreviations; DNS-PE; N; -[[5-(dimethylamino)naphthyl]-1-sulfonyl]dipalmitoyl-; l; -α-phosphatidylethanolamine; PC; phosphatidylcholine; PE; phosphatidylethanolamine; PG; phosphatidylglycerol; PI; phosphatidylinositol; LPC; lysophosphatidylcholine; CL; cardiolipin; SM; sphingomyelin; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; DOPC; 1,2-dioleoyl-; sn; -glycero-3-phospholcholine; DPPC; 1,2-dipalmitoyl-; sn; -glycero-3-phospholcholine; POPG; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-[phospho-; rac; -(1-glycerol)]; SUVs; small unilamellar vesicles; LUVs; large unilamellar vesicles; GUVs; giant unilamellar vesiclesPermeability; Cell penetrating peptide; Liposome; Vesicle

Complexation of integral membrane proteins by phosphorylcholine-based amphipols by C. Diab; C. Tribet; Y. Gohon; J.-L. Popot; F.M. Winnik (pp. 2737-2747).

Amphiphilic macromolecules, known as amphipols, have emerged as promising candidates to replace conventional detergents for handling integral membrane proteins in water due to the enhanced stability of protein/amphipol complexes as compared to protein/detergent complexes. The limited portfolio of amphipols currently available prompted us to develop amphipols bearing phosphorylcholine-based units (PC). Unlike carboxylated polymers, PC-amphipols remain soluble in aqueous media under conditions of low pH, high salt concentration, or in the presence of divalent ions. The solubilizing properties of four PC-amphipols were assessed in the case of two membrane proteins, cytochrome b₆ f and bacteriorhodopsin. The protein/PC-amphipol complexes had a low dispersity in size, as determined by rate zonal ultracentrifugation. Short PC-amphipols (< M>≈22 kDa) of low dispersity in length, containing ∼30 mol% octyl side groups, ∼35 mol% PC-groups, and ∼35 mol% isopropyl side groups, appeared best suited to form stable complexes, preserving the native state of BR over periods of several days. BR/PC-amphipol complexes remained soluble in aqueous media at pH≥5, as well as in the presence of 1 M NaCl or 12 mM calcium ions. Results from isothermal titration calorimetry indicated that the energetics of the conversion of BR/detergent complexes into BR/amphipol complexes are similar for PC-amphipols and carboxylated amphiphols.

Keywords: Amphipols; Integral membrane proteins; Phosphorylcholine; ITC; Amphiphilic macromolecules

Cellular uptake and subcellular distribution of chlorin e6 as functions of pH and interactions with membranes and lipoproteins by Halina Mojzisova; Stéphanie Bonneau; Christine Vever-Bizet; Daniel Brault (pp. 2748-2756).

The uptake and more importantly the subcellular distribution of photosensitizers are major determinants of their efficacy. In this paper, the cellular internalization of chlorin e6 (Ce6), a photosensitizer bearing three carboxylic chains, is considered with emphasize on pH effects. Small unilamellar vesicles are used as models to investigate the dynamics of interactions of Ce6 with membranes. The entrance and exit steps from the outer lipid hemileaflet are very fast (∼ms). A slow transfer of Ce6 through the membrane was observed only for thin bilayers made of dimyristoleoyl-phosphatidylcholine. Ce6 did not permeate through bilayers consisting of longer phospholipids more representative of biological membranes. These results along with previous data on the interactions of Ce6 with low-density lipoproteins (LDL) are correlated with cellular studies. After 15 min incubation of HS68 human fibroblasts with Ce6, fluorescence microscopy revealed labeling of the plasma membrane and cytosolic vesicles different from lysosomes. When vectorized by LDL, Ce6 was mainly localized in lysosomes but absent from the plasma membrane. Internalization of LDL bound photosensitizer via ApoB/E receptor mediated pathway was demonstrated by overexpression experiments. A pH decrease from 7.4 to 6.9 did not affect the intracellular distribution of Ce6, but significantly increased its overall cellular uptake.

Keywords: Photosensitizer; Cellular uptake; Subcellular localization; pH; Lipoprotein; Model membranes

The geometry of the ionic chànnel lumen formed by α-latroinsectotoxin from black widow spider venom in the bilayer lipid membranes by Oleg Ya. Shatursky; Tatyana M. Volkova; Nina H. Himmelreich; Eugene V. Grishin (pp. 2757-2763).

The dependence of single channel conductance formed by α-latroinsectotoxin (α-LIT) from black widow spider venom in the planar phospholipid membrane on the hydrodynamic radii of different nonelectrolytes allowed to determine the geometry of α-LIT water lumen. It was found that the cis- and trans-entrances of α-LIT channel had the same effective radii of 0.55–0.58 nm. Relatively small conductance of α-LIT channel (23.5 +3.7 pS) in a symmetrical membrane bathing solution of 100 mM KCl (pH 7.4) may result from the constriction inside the channel with apparent radius of 0.37 nm located 32.5% of channel length away from the cis-entrance.

Keywords: Lipid bilayer; α-latroinsectotoxin; α-latrotoxin; Ion channel geometryAbbreviations; (α-LIT); α-Latroinsectotoxin; (α-LT); α-Latrotoxin; (BLM); Bilayer lipid membrane; (NEs); Nonelectrolytes; (PC); Phosphatidylcholine; (PEGs); Polyethylene glycols

Phase studies of model biomembranes: Complex behavior of DSPC/DOPC/Cholesterol by Jiang Zhao; Jing Wu; Frederick A. Heberle; Thalia T. Mills; Paul Klawitter; Grace Huang; Greg Costanza; Gerald W. Feigenson (pp. 2764-2776).

We have undertaken a series of experiments to examine the behavior of individual components of cell membranes. Here we report an initial stage of these experiments, in which the properties of a chemically simple lipid mixture are carefully mapped onto a phase diagram. Four different experimental methods were used to establish the phase behavior of the 3-component mixture DSPC/DOPC/chol: (1) confocal fluorescence microscopy observation of giant unilamellar vesicles, GUVs; (2) FRET from perylene to C20:0-DiI; (3) fluorescence of dilute dyes C18:2-DiO and C20:0-DiI; and (4) wide angle X-ray diffraction. This particular 3-component mixture was chosen, in part, for a high level of immiscibility of the components in order to facilitate solving the phase behavior at all compositions. At 23 °C, a large fraction of the possible compositions for this mixture give rise to a solid phase. A region of 3-phase coexistence of {Lα+Lβ+Lo} was detected and defined based on a combination of fluorescence microscopy of GUVs, FRET, and dilute C20:0-DiI fluorescence. At very low cholesterol concentrations, the solid phase is the tilted-chain phase Lβ′. Most of the phase boundaries have been determined to be within a few percent of the composition. Measurements of the perturbations of the boundaries of this accurate phase diagram could serve as a means to understand the behaviors of a range of added lipids and proteins.

Keywords: Abbreviations; DSPC; 1,2-distearoyl-; sn; -glycero-3-phosphocholine; DOPC; 1,2-dioleoyl-; sn; -glycero-3-phosphocholine; DSPG; 1,2-distearoyl-; sn; -glycero-3-[phospho-; rac; -(1-glycerol)] (sodium salt); DOPG; 1,2-dioleoyl-; sn; -glycero-3-[phospho-; rac; -(1-glycerol)] (sodium salt); TLC; thin-layer chromatography; GUV; giant unilamellar vesicle; RSE; rapid solvent exchange; FRET; fluorescence resonance energy transfer; RRE; region of reduced efficiency; REE; region of enhanced efficiency; (16:0,Bodipy)-PC; 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-; s; -indacene-3-pentanoyl)-1-hexadecanoyl-; sn; -glycero-3-phosphocholine; C20:0-DiI; 1,1′-dieicosanyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate; C18:2-DiO; 3,3′-dilinoleyloxacarbocyanine perchlorate; WAXS; Wide-angle X-ray scattering; MC; Monte CarloTernary phase diagram; Bilayer phase separation; Liquid-disordered phase; Liquid-ordered phase; Lipid rafts

Phase studies of model biomembranes: Macroscopic coexistence of Lα+Lβ, with light-induced coexistence of Lα+Lo Phases by Jiang Zhao; Jing Wu; Huilin Shao; Fanrong Kong; Nieraj Jain; Geoffrey Hunt; Gerald Feigenson (pp. 2777-2786).

Phase diagrams of 3-component lipid bilayer mixtures containing cholesterol reveal major differences among the different types of lipids. Here we report that mixtures of cholesterol together with POPC and a high-melting temperature PC or sphingomyelin show different phase behavior from similar mixtures that contain DOPC or di-phytanoyl-PC instead of POPC. In particular, only one region of macroscopic phase coexistence occurs with POPC, a region of coexisting liquid disordered and solid phases, {Lα+Lβ}. Fluorescence microscopy imaging is useful for these studies, but is subject to artifactual light-induced domain formation, as reported by Ayuyan and Cohen [A.G. Ayuyan, F.S. Cohen, Lipid peroxides promote large rafts: Effects of excitation of probes in fluorescence microscopy and electrochemical reactions during vesicle formation, Biophys. J. 91 (2006) 2172-2183.]. This artifact can be attenuated by decreased illumination and low dye concentration. The use of the free radical scavenger n-propyl gallate can reduce the artifact, but this molecule enters the bilayer and itself perturbs the phase behavior. We suggest that the light-induced domain separation artifact might actually arise from pre-existing lipid clusters that are induced to coalesce, and therefore indicates highly nonrandom mixing of the lipid components.

Keywords: Abbreviations; DPPC; 1,2-Dipalmitoyl-; sn; -Glycero-3-Phosphocholine; DSPC; 1,2-Distearoyl-; sn; -Glycero-3-Phosphocholine; DOPC; 1,2-Dioleoyl-; sn; -Glycero-3-Phosphocholine; POPC; 1-Palmitoyl-2-Oleoyl-; sn; -Glycero-3-Phosphocholine; SOPC; 1-Stearoyl-2-Oleoyl-; sn; -Glycero-3-Phosphocholine; DLPC; 1,2-Dilauroyl-; sn; -Glycero-3-Phosphocholine; DSPG; 1,2-Distearoyl-; sn; -Glycero-3-[Phospho-; rac; -(1-glycerol)] (Sodium Salt); DOPG; 1,2-Dioleoyl-; sn; -Glycero-3-[Phospho-; rac; -(1-glycerol)] (Sodium Salt); SM; sphingomyelin; NPG; n-propyl gallate; TLC; thin-layer chromatography; GUV; giant unilamellar vesicle; RSE; rapid solvent exchange; FRET; fluorescence resonance energy transfer; (16:0,Bodipy-PC); 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-; s; -indacene-3-pentanoyl)-1-hexadecanoyl-; sn; -glycero-3-phosphocholine; C20:0-DiI; 1,1′-dieicosanyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate; TR-DHPE; Texas Red 1,2-dihexadecanoyl-; sn; -glycero-3-phosphoethanolamine, triethylammonium saltPhoto-induced; Lipid peroxidation; Liquid-disordered phase; Liquid-ordered phase; Antioxidant; Phase boundary shift

Interactions of the Australian tree frog antimicrobial peptides aurein 1.2, citropin 1.1 and maculatin 1.1 with lipid model membranes: Differential scanning calorimetric and Fourier transform infrared spectroscopic studies by Gordon W.J. Seto; Seema Marwaha; Daniel M. Kobewka; Ruthven N.A.H. Lewis; Frances Separovic; Ronald N. McElhaney (pp. 2787-2800).

The interactions of the antimicrobial peptides aurein 1.2, citropin 1.1 and maculatin 1.1 with dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG) and dimyristoylphosphatidylethanolamine (DMPE) were studied by differential scanning calorimetry (DSC) and Fourier-transform infrared (FTIR) spectroscopy. The effects of these peptides on the thermotropic phase behavior of DMPC and DMPG are qualitatively similar and manifested by the suppression of the pretransition, and by peptide concentration-dependent decreases in the temperature, cooperativity and enthalpy of the gel/liquid–crystalline phase transition. However, at all peptide concentrations, anionic DMPG bilayers are more strongly perturbed than zwitterionic DMPC bilayers, consistent with membrane surface charge being an important aspect of the interactions of these peptides with phospholipids. However, at all peptide concentrations, the perturbation of the thermotropic phase behavior of zwitterionic DMPE bilayers is weak and discernable only when samples are exposed to high temperatures. FTIR spectroscopy indicates that these peptides are unstructured in aqueous solution and that they fold into α-helices when incorporated into lipid membranes. All three peptides undergo rapid and extensive H–D exchange when incorporated into D₂O-hydrated phospholipid bilayers, suggesting that they are located in solvent-accessible environments, most probably in the polar/apolar interfacial regions of phospholipid bilayers. The perturbation of model lipid membranes by these peptides decreases in magnitude in the order maculatin 1.1>aurein 1.2>citropin 1.1, whereas the capacity to inhibit Acholeplasma laidlawii B growth decreases in the order maculatin 1.1>aurein 1.2≅citropin 1.1. The higher efficacy of maculatin 1.1 in disrupting model and biological membranes can be rationalized by its larger size and higher net charge. However, despite its smaller size and lower net charge, aurein 1.2 is more disruptive of model lipid membranes than citropin 1.1 and exhibits comparable antimicrobial activity, probably because aurein 1.2 has a higher propensity for partitioning into phospholipid membranes.

Keywords: Abbreviations; AMP; antimicrobial peptide; DSC; differential scanning calorimetry; FTIR; Fourier-transform infrared; PC; phosphatidylcholine; PE; phosphatidylethanolamine; PG; phosphatidylglycerol; NMR; nuclear magnetic resonance; POPC; 1-palmitoyl, 2-oleoyl phosphatidylcholine; POPG; 1-palmitoyl, 2-oleoyl phosphatidylglycerol; DMPC; dimyristoylphosphatidylcholine; DMPG; dimyristoylphosphatidylglycerol; DMPE; dimyristoylphosphatidylethanolamine; DOPE; dioleoylphosphatidylethanolamine; DOPG; dioleoylphosphatidylglycerol; HPLC; high performance liquid chromatography; MLV; multilamellar vesicles; LD; ₅₀; concentration required for 50% growth inhibition; MIC; minimum inhibitory concentrationLipid–peptide interaction; Antimicrobial peptide; Aurein 1.2; Citropin 1.1; Maculation 1.1; Lipid bilayer membrane; DSC; FTIR spectroscopy

An Hg-sensitive channel mediates the diffusional component of glucose transport in olive cells by Carlos Conde; Paulo Silva; Alice Agasse; Rui M. Tavares; Serge Delrot; Hernâni Gerós (pp. 2801-2811).

In several organisms solute transport is mediated by the simultaneous operation of saturable and non-saturable (diffusion-like) uptake, but often the nature of the diffusive component remains elusive. The present work investigates the nature of the diffusive glucose transport in Olea europaea cell cultures. In this system, glucose uptake is mediated by a glucose-repressible, H⁺-dependent active saturable transport system that is superimposed on a diffusional component. The latter represents the major mode of uptake when high external glucose concentrations are provided. In glucose-sufficient cells, initial velocities ofd- andl-[U-¹⁴C]glucose uptake were equal and obeyed linear concentration dependence up to 100 mM sugar. In sugar starved cells, where glucose transport is mediated by the saturable system, countertransport of the sugar pairs 3- O-methyl-d-glucose/d-[U-¹⁴C]glucose and 3- O-methyl-d-glucose/3- O-methyl-d-[U-¹⁴C]glucose was demonstrated. This countertransport was completely absent in glucose-sufficient cells, indicating that linear glucose uptake is not mediated by a typical sugar permease. The endocytic inhibitors wortmannin-A and NH₄Cl inhibited neither the linear component ofd- andl-glucose uptake nor the absorption of the nonmetabolizable glucose analog 3- O-methyl-d-[U-¹⁴C]glucose, thus excluding the involvement of endocytic mediated glucose uptake. Furthermore, the formation of endocytic vesicles assessed with the marker FM1–43 proceeded at a very slow rate. Activation energies for glucose transport in glucose sufficient cells and plasma membrane vesicles were 7 and 4 kcal mol−¹, respectively, lower than the value estimated for diffusion of glucose through the lipid bilayer of phosphatidylethanolamine liposomes (12 kcal mol−¹). Mercury chloride inhibited both the linear component of sugar uptake in sugar sufficient cells and plasma membrane vesicles, and the incorporation of the fluorescent glucose analog 2-NBDG, suggesting protein-mediated transport. Diffusive uptake of glucose was inhibited by a drop in cytosolic pH and stimulated by the protein kinase inhibitor staurosporine. The data demonstrate that the low-affinity, high-capacity, diffusional component of glucose uptake occurs through a channel-like structure whose transport capacity may be regulated by intracellular protonation and phosphorylation/dephosphorylation.

Keywords: Diffusive uptake; Glucose uptake; Olea europaea; Proteinaceous channel; Biphasic kinetics

Behaviour of bacterial division protein FtsZ under a monolayer with phospholipid domains by Céline Lafontaine; Jean-Marc Valleton; Nicole Orange; Vic Norris; Eugenia Mileykovskaya; Stéphane Alexandre (pp. 2812-2821).

Assembly of the tubulin-like protein FtsZ at or near the cytoplasmic membrane is one of the earliest steps in division of bacteria such as Escherichia coli. Exactly what constitutes the site at which FtsZ acts is less clear. To investigate the influence of the membrane phospholipids on FtsZ localization and assembly, we have elaborated with the Langmuir technique a two-lipid monolayer made of dilauryl-phosphatidylethanolamine (DLPE) and dipalmitoyl-phosphatidylglycerol (DPPG). This monolayer comprised stable condensed domains in an expanded continuous phase. In the presence of GTP, FtsZ assembly disrupts the condensed domains within 5 min. After several hours, with or without GTP, FtsZ assembled into large aggregates at the domain interface. We suggest that the GTP-induced polymerization of FtsZ is coupled to the association of FtsZ protofilaments with domain interfaces.

Keywords: FtsZ; Lipid domain; Membrane model; Monolayer; Interface; Cell division

Ciprofloxacin interactions with bacterial protein OmpF: Modelling of FRET from a multi-tryptophan protein trimer by Fábio Fernandes; Patrícia Neves; Paula Gameiro; Luís M.S. Loura; Manuel Prieto (pp. 2822-2830).

The outer membrane protein F (OmpF) is known to play an important role in the uptake of fluoroquinolone antibiotics by bacteria. In this study, the degree of binding of the fluoroquinolone antibiotic ciprofloxacin to OmpF in a lipid membrane environment is quantified using a methodology based on Förster resonance energy transfer (FRET). Analysis of the fluorescence quenching of OmpF is complex as each OmpF monomer presents two tryptophans at different positions, thus sensing two different distributions of acceptors in the bilayer plane. Specific FRET formalisms were derived accounting for the different energy transfer contributions to quenching of each type of tryptophan of OmpF, allowing the recovery of upper and lower boundaries for the ciprofloxacin-OmpF binding constant ( K_B). log ( K_B) was found to lie in the range 3.15–3.62 or 3.58–4.00 depending on the location for the ciprofloxacin binding site assumed in the FRET modelling, closer to the centre or to the periphery of the OmpF trimer, respectively. This methodology is suitable for the analysis of FRET data obtained with similar protein systems and can be readily adapted to different geometries.

Keywords: Abbreviations; CP; Ciprofloxacin; FRET; Förster resonance energy transfer; oPOE; n-octylpolyoxyethylene; OmpF; Outer Membrane protein F; DMPC; Dimyristoyl-; l; -α-phosphatidylcholine; Trp; tryptophan; CMC; critical micellar concentrationFluorescence; Fluoroquinolones; Porin; Membrane protein; Membrane model system

A multidomain outer membrane protein from Pasteurella multocida: Modelling and simulation studies of PmOmpA by Timothy Carpenter; Syma Khalid; Mark S.P. Sansom (pp. 2831-2840).

PmOmpA is a two-domain outer membrane protein from Pasteurella multocida. The N-terminal domain of PmOmpA is a homologue of the transmembrane β-barrel domain of OmpA from Escherichia coli, whilst the C-terminal domain of PmOmpA is a homologue of the extra-membrane Neisseria meningitidis RmpM C-terminal domain. This enables a model of a complete two domain PmOmpA to be constructed and its conformational dynamics explored via MD simulations of the protein embedded within two different phospholipid bilayers (DMPC and DMPE). The conformational stability of the transmembrane β-barrel is similar to that of a homology model of OprF from Pseudomonas aeruginosa in bilayer simulations. There is a degree of water penetration into the interior of the β-barrel, suggestive of a possible transmembrane pore. Although the PmOmpA model is stable over 20 ns simulations, retaining its secondary structure and fold integrity throughout, substantial flexibility is observed in a short linker region between the N- and the C-terminal domains. At low ionic strength, the C-terminal domain moves to interact electrostatically with the lipid bilayer headgroups. This study demonstrates that computational approaches may be applied to more complex, multi-domain outer membrane proteins, rather than just to transmembrane β-barrels, opening the possibility of in silico proteomics approaches to such proteins.

Keywords: Outer membrane protein; OmpA; Molecular dynamics; Homology model; RmpM; OprF

Ontogeny up-regulates renal Na⁺/Cl⁻/creatine transporter in rat by M. García-Delgado; P. García-Miranda; M.J. Peral; M.L. Calonge; A.A. Ilundáin (pp. 2841-2848).

Creatine plays a role in energy storage and transport/shuttle of high-energy phosphate in heart, brain, retina, testis and skeletal muscle. These tissues take creatine from the plasma via a 2Na⁺/1Cl⁻/1creatine cotransporter (CRT). We have previously demonstrated that renal apical membrane presents a 2Na⁺/1Cl⁻/1creatine cotransport activity. The goal of this study was to determine whether this transporter is ontogenically regulated. Na⁺/Cl⁻/creatine transport activity was evaluated by measuring [¹⁴C]-creatine uptake into renal brush-border membrane vesicles. CRT mRNA expression was measured by Northern and real-time PCR assays. E20 foetuses, newborn, suckling, weaning and adult (2- and 8-month-old) Wistar rats were used. The results revealed that neither the vesicular volume, the binding of creatine to the brush-border membrane vesicles, nor the purity of the brush-border membrane vesicle preparations was affected by maturation. Fetal and neonatal kidneys contained a creatine transporter that was qualitatively indistinguishable from that in the adult: it was concentrative, Na⁺- and Cl⁻-dependent, electrogenic and inhibited by guanidinopropionic acid. Maturation increased this transport activity by increasing the maximal rate of transport ( V_max) without significantly changing the apparent K_m. Northern analysis revealed two transcripts for CRT of 2.7 kb and 4.2 kb in all the ages tested. Northern and real-time PCR assays showed that, as seen with NaCl-dependent creatine transport activity, maturation increased CRT mRNA expression. This study reports for the first time that: (i) an apical renal Na⁺/Cl⁻/creatine cotransporter is already active in rat foetuses and (ii) development regulates Na⁺/Cl⁻/creatine cotransport activity by increasing the density and/or turnover of the transporters.

Keywords: Development; Kidney; Epithelia; Creatine

Isc1 regulates sphingolipid metabolism in yeast mitochondria by Hiroshi Kitagaki; L. Ashley Cowart; Nabil Matmati; Silvia Vaena de Avalos; Sergei A. Novgorodov; Youssef H. Zeidan; Jacek Bielawski; Lina M. Obeid; Yusuf A. Hannun (pp. 2849-2861).

The Saccharomyces cerevisiae inositol sphingolipid phospholipase C (Isc1p), a homolog of mammalian neutral sphingomyelinases, hydrolyzes complex sphingolipids to produce ceramide in vitro. Epitope-tagged Isc1p associates with the mitochondria in the post-diauxic phase of yeast growth. In this report, the mitochondrial localization of Isc1p and its role in regulating sphingolipid metabolism were investigated. First, endogenous Isc1p activity was enriched in highly purified mitochondria, and western blots using highly purified mitochondrial membrane fractions demonstrated that epitope-tagged Isc1p localized to the outer mitochondrial membrane as an integral membrane protein. Next, LC/MS was employed to determine the sphingolipid composition of highly purified mitochondria which were found to be significantly enriched in α-hydroxylated phytoceramides (21.7 fold) relative to the whole cell. Mitochondria, on the other hand, were significantly depleted in sphingoid bases. Compared to the parental strain, mitochondria from isc1Δ in the post-diauxic phase showed drastic reduction in the levels of α-hydroxylated phytoceramide (93.1% loss compared to WT mitochondria with only 2.58 fold enrichment in mitochondria compared to whole cell). Functionally, isc1Δ showed a higher rate of respiratory-deficient cells after incubation at high temperature and was more sensitive to hydrogen peroxide and ethidium bromide, indicating that isc1Δ exhibits defects related to mitochondrial function. These results suggest that Isc1p generates ceramide in mitochondria, and the generated ceramide contributes to the normal function of mitochondria. This study provides a first insight into the specific composition of ceramides in mitochondria.

Keywords: Abbreviations; IPC; inositol phosphorylceramide; MIPC; mannosylinositol phosphorylceramide; M(IP); ₂; C; mannosyldiinositol phosphorylceramide; YPD; yeast/peptone/dextrose; YPGE; yeast/peptone/glycerol/ethanol; SEM; standard error of mean; ER; endoplasmic reticulum; IS; internal standard; a-HO-; α-hydroxylated; Cer; ceramide; DHSph; dihydrosphingosine; Phyto-Sph; phytosphingosine; dhCn-Cer; dihydroceramide with Cn fatty acid; phytoCn-Cer; phytoceramide with Cn fatty acidMitochondria; Sphingolipid; Sphingomyelinase; Lipidomics; ISC1; Yeast

The γ-core motif correlates with antimicrobial activity in cysteine-containing kaliocin-1 originating from transferrins by Nannette Y. Yount; María T. Andrés; José F. Fierro; Michael R. Yeaman (pp. 2862-2872).

Kaliocin-1 is a 31-residue peptide derived from human lactoferrin, and with antimicrobial properties that recapitulate those of its 611 amino acid parent holoprotein. As kaliocin-1 is a cysteine-stabilized peptide, it was of interest to determine whether it contained a multidimensional γ-core signature recently identified as common to virtually all classes of disulfide-stabilized antimicrobial peptides. Importantly, sequence and structural analyses identified an iteration of this multidimensional antimicrobial signature in kaliocin-1. Further, the γ-core motif was found to be highly conserved in the transferrin family of proteins across the phylogenetic spectrum. Previous studies suggested that the mechanism by which kaliocin-1 exerts anti-candidal efficacy depends on mitochondrial perturbation without cell membrane permeabilization. Interestingly, results of a yeast two-hybrid screening analysis identified an interaction between kaliocin-1 and mitochondrial initiation factor 2 in a Saccharomyces cerevisiae model system. Taken together, these data extend the repertoire of antimicrobial peptides that contain γ-core motifs, and suggest that the motif is conserved within large native as well as antimicrobial peptide subcomponents of transferrin family proteins. Finally, these results substantiate the hypothesis that antimicrobial activity associated with host defense effector proteins containing a γ-core motif may correspond to targets common to fungal mitochondria or their bacterial ancestors.

Keywords: Transferrin; Antimicrobial; Peptide; Protein; Structure; Kaliocin

The partition of cholesterol between ordered and fluid bilayers of phosphatidylcholine: A synchrotron X-ray diffraction study by Lin Chen; Zhiwu Yu; Peter J. Quinn (pp. 2873-2881).

The structure and composition of coexisting bilayer phases separated in binary mixtures of dipalmitoylphosphatidylcholine and cholesterol and ternary mixtures of equimolar proportions of dipalmitoyl- and dioleoylphosphatidycholines containing different proportions of cholesterol have been characterized by synchrotron X-ray diffraction methods. The liquid-ordered phase is distinguished from gel and fluid phases by a disordering of the hydrocarbon chains intermediate between the two phases as judged from the wide-angle X-ray scattering profiles. Electron density distribution calculated in coexisting bilayer phases shows that liquid-ordered phase is enriched in dipalmitoylphosphatidylcholine and cholesterol and a higher electron density in the methylene chain region of the bilayer ascribed to the location of the sterol ring of cholesterol. The ratio of the two constituents in the liquid-ordered phase is not constant because the stoichiometry is temperature-dependent as seen by respective changes in bilayer thickness over the range 20° to 36 °C where coexisting phases are observed. Three coexisting phases were deconvolved in the ternary mixture at 20 °C. From an analysis of the ternary mixtures containing mole fractions of cholesterol from 0.09 to 0.15 it was found that the liquid-crystal and gel phases each contained about 10% of the cholesterol molecules and the liquid-ordered phase was comprised of 30% cholesterol molecules.

Keywords: Liquid-ordered phase; Bilayer electron density; Phosphatidylcholine; Cholesterol partition coefficient; Membrane raft

Mutational analysis of potential pore-lining amino acids in TM IV of the Na⁺/H⁺ exchanger by Emily Slepkov; Jie Ding; Jiayi Han; Larry Fliegel (pp. 2882-2889).

The Na⁺/H⁺ exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH by extruding an intracellular H⁺ in exchange for one extracellular Na⁺. In this study we examined the effect of site-specific mutagenesis on the pore-lining amino acid Phe161 and effects of mutagenesis on the charged amino acids Asp159 and Asp172. There was no absolute requirement for a carboxyl side chain at amino acid Asp159 or Asp172. Mutation of Asp159 to Asn or Gln maintained or increased the activity of the protein. Similarly, for Asp172, substitution with a Gln residue maintained activity of the protein, even though substitution with an Asn residue was inhibitory. The Asp172Glu mutant possessed normal activity after correction for its aberrant expression and surface targeting. Replacement of Phe161 with a Leu demonstrated that it was not irreplaceable in NHE1 function. However, the mutation Phe161lys inhibited NHE1 function, while the Phe161Ala mutation caused altered NHE1 targeting and expression levels. Our results show that these three amino acids, while being important in NHE1 function, are not irreplaceable. This study demonstrates that multiple substitutions at a single amino acid residue may be necessary to get a clearer picture membrane protein function.

Keywords: Cation transport; Membrane; Na; ⁺; /H; ⁺; exchanger; pH regulation

Development and characterization of lipidic cochleate containing recombinant factor VIII by Razvan D. Miclea; Prashant R. Varma; Aaron Peng; Sathy V. Balu-Iyer (pp. 2890-2898).

Hemophilia A, a life-threatening bleeding disorder, is caused by deficiency of factor VIII (FVIII). Replacement therapy using rFVIII is the first line therapy for hemophilia A. However, 15–30% of patients develop neutralizing antibody, mainly against the C2, A3 and A2 domains. It has been reported that PS-FVIII complex reduced total and neutralizing anti-rFVIII antibody titers in hemophilia A murine models. Here, we developed FVIII-containing cochleate cylinders, utilizing PS-Ca²⁺ interactions and characterized these particles for optimal in vivo properties using biophysical and biochemical techniques. Approximately 75% of the protein was associated with cochleate cylinders. Sandwich ELISA, acrylamide quenching and enzymatic digestion studies established that rFVIII was shielded from the bulk aqueous phase by the lipidic structures, possibly leading to improved in vivo stability. Freeze–thawing and rate-limiting diffusion studies revealed that small cochleate cylinders with a particle size of 500 nm or less could be generated. The release kinetics and in vivo experiments suggested that there is slow and sustained release of FVIII from the complex upon systemic exposure. In vivo studies using tail clip method indicated that FVIII–cochleate complex is effective and protects hemophilic mice from bleeding. Based on these studies, we speculate that the molecular interaction between FVIII and PS may provide a basis for the design of novel FVIII lipidic structures for delivery applications.

Keywords: Abbreviations; aPTT; activated partial thromboplastin time; FVIII; B domain deleted recombinant factor VIII; BPS; brain phosphatidylserine; BSA; bovine serum albumin; ELISA; enzyme-linked immunosorbent assay; FVIII; factor VIII; PB; phosphate buffer; PBA; phosphate buffer with 1% bovine serum albumin; PBT; Tween 20 containing phosphate buffer; PS; phosphatidylserine; rFVIII; recombinant human factor VIII; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresisCochleate cylinder; B domain deleted recombinant factor VIII; Epitope shielding; Laurdan; Protein formulation; Acrylamide quenching

1-Alkanols and membranes: A story of attraction by Beate Griepernau; Simon Leis; Matthias F. Schneider; Martin Sikor; Daniel Steppich; Rainer A. Böckmann (pp. 2899-2913).

Although 1-alkanols have long been known to act as penetration enhancers and anesthetics, the mode of operation is not yet understood. In this study, long-time molecular dynamics simulations have been performed to investigate the effect of 1-alkanols of various carbon chain lengths onto the structure and dynamics of dimyristoylphosphatidylcholine bilayers. The simulations were complemented by microcalorimetry, continuous bleaching and film balance experiments. In the simulations, all investigated 1-alkanols assembled inside the lipid bilayer within tens of nanoseconds. Their hydroxyl groups bound preferentially to the lipid carbonyl group and the hydrocarbon chains stretched into the hydrophobic core of the bilayer. Both molecular dynamics simulations and experiments showed that all 1-alkanols drastically affected the bilayer properties. Insertion of long-chain 1-alkanols decreased the area per lipid while increasing the thickness of the bilayer and the order of the lipids. The bilayer elasticity was reduced and the diffusive motion of the lipids within the bilayer plane was suppressed. On the other hand, integration of ethanol into the bilayer enlarged the area per lipid. The bilayer became softer and lipid diffusion was enhanced.

Keywords: Molecular dynamics (MD) simulation; DMPC; Alkanol; Anesthetic; Penetration enhancer; Elasticity

Bilayer polarity and its thermal dependency in the ℓ_o and ℓ_d phases of binary phosphatidylcholine/cholesterol mixtures by Dalila Arrais; Jorge Martins (pp. 2914-2922).

Diverse variations in membrane properties are observed in binary phosphatidylcholine/cholesterol mixtures. These mixtures are nonideal, displaying single or phase coexistence, depending on chemical composition and other thermodynamic parameters. When compared with pure phospholipid bilayers, there are changes in water permeability, bilayer thickness and thermomechanical properties, molecular packing and conformational freedom of phospholipid acyl chains, in internal dipolar potential and in lipid lateral diffusion. Based on the phase diagrams for DMPC/cholesterol and DPPC/cholesterol, we compare the equivalent polarity of pure bilayers with specific compositions of these mixtures, by using the Py empirical scale of polarity. Besides the contrast between pure and mixed lipid bilayers, we find that liquid-ordered (ℓ_o) and liquid-disordered (ℓ_d) phases display significantly different polarities. Moreover, in the ℓ_o phase, the polarities of bilayers and their thermal dependences vary with the chemical composition, showing noteworthy differences for cholesterol proportions at 35, 40, and 45 mol%. At 20 °C, for DMPC/cholesterol at 35 and 45 mol%, the equivalent dielectric constants are 21.8 and 23.8, respectively. Additionally, we illustrate potential implications of polarity in various membrane-based processes and reactions, proposing that for cholesterol containing bilayers, it may also go along with the occurrence of lateral heterogeneity in biological membranes.

Keywords: Abbreviations; DMPC; 1,2-dimiristoyl-; sn; -glycero-3-phosphocholine; DPPC; 1,2-dipalmitoyl-; sn; -glycero-3-phosphocholine; L; _α; liquid–crystalline phase; ℓ; _d; liquid-disordered phase; ℓ; _o; liquid-ordered phase; MLV; multilamellar vesicles; S.D.; standard deviationPyrene; Fluorescence; Ham effect; Py polarity scale; Lipid bilayer; Phospholipid/Cholesterol mixture

Detection of apolipoprotein B100 early conformational changes during oxidation by Anita Kriško; Goran Stjepanović; Greta Pifat; Jean-Marie Ruysschaert; Erik Goormaghtigh (pp. 2923-2930).

Conformational changes of human plasma apolipoprotein B100 (apoB) during oxidative modification of low-density lipoproteins (LDL) have been investigated. Emphasis has been put on the early stages of LDL oxidation and the modification of apoB. We have applied two different modes of LDL oxidation initiation in order to approach the problem from different perspectives. To study conformational changes of the protein and the phospholipids surface monolayer, we have applied attenuated total reflection infrared as well as fluorescence spectroscopy. We have found for the first time that conformational changes of apoB occur even in the earliest stages of oxidation process and that those are located predominantly in the β-sheet regions. The dynamics of changes has also been described and related to different stages of oxidation. After initial increase in particle surface accessibility and mobility, by entering into the propagation phase of oxidation process, LDL surface accessibility and mobility are decreased. Finally, in the decomposition phase of LDL oxidation, as the particle faces large chemical and physical changes, surface mobility and accessibility is increased again. These observations provide new insights into the modifications of LDL particles upon oxidation.

Keywords: LDL; apoB; Oxidation; Infrared spectroscopy; Fluorescence spectroscopy; TMA-DPH

Interaction of the C-terminal domain of Bcl-2 family proteins with model membranes by Alejandro Torrecillas; María M. Martínez-Senac; Alessio Ausili; Senena Corbalán-García; Juan C. Gómez-Fernández (pp. 2931-2939).

Bcl-2 family proteins are involved in the cell homeostasis by regulating programmed cell death. Some of these proteins promote apoptosis, while others inhibit the same process. The C-terminal hydrophobic domain of some of these proteins is predicted to be involved in anchoring them to a variety of cell membranes, such as mitochondrial, endoplasmic reticulum and nuclear membranes. We have used five synthetic peptides imitating the C-terminal domain from both anti-apoptotic (Bcl-2) and pro-apoptotic members (Bak, Bax, and two mutants of this last protein) of this family to study their interaction with model membranes. Some differences were detected in the interaction with these peptides. The addition of all the peptides to large unilamellar vesicles destabilized them and released encapsulated carboxyfluorescein to different degrees, so that fluidity and the increase in negative curvature favoured the extent in the release of carboxyfluorescein. Bcl-2-C and Bax-C peptides produced the highest release levels in most cases, while BaxS184K-C was the least efficient in this respect. These results indicate that these C-terminal domains are able to insert themselves in the membranes, each in a different way that is probably related with their different way which can be related to their differing locations within the cell and their different roles in regulating apoptosis.

Keywords: Abbreviations; CF; 5(6)-carboxyfluorescein; DAG; diacylglycerol obtained from egg yolk phosphatidylcholine; DPH; 1,6-diphenyl-1,3,5-hexatriene; EYPC; egg yolk phosphatidylcholine; LUVs; large unilamellar vesicles; MLVs; multilamellar vesicles; P; polarization of the emitted fluorescence; TFE; 2,2,2-trifluoroethanol; THF; tetrahydrofuranBcl-2; Bax; Bak; Apoptosis; Protein–lipid interaction; Fluorescence

Effect of unsaturated bonds in the sn-2 acyl chain of phosphatidylcholine on the membrane-damaging action of Clostridium perfringens alpha-toxin toward liposomes by Masahiro Nagahama; Akiko Otsuka; Masataka Oda; Rajesh K. Singh; Zyta M. Ziora; Hiroshi Imagawa; Mugio Nishizawa; Jun Sakurai (pp. 2940-2945).

Clostridium perfringens alpha-toxin degrades phosphatidylcholine (PC) in the bilayer of liposomes and destroys the membrane. The effect of the type and position of unsaturation in the fatty acyl chain of PC (18:0/18:1 PC) synthesized on the toxin-induced leakage of carboxyfluorescein (CF) from PC liposomes was examined. Differential scanning calorimetry showed that the phase transition temperature ( T_m) was minimal when the triple bond was positioned at C (9) in the sn-2 acyl chain. The toxin-induced CF leakage decreased with the migration of the bond from C (9) to either end of the acyl chain in PC. The PC containing the cis-double bond had a similar T_m to that with the triple bond, but a lower value than the PC containing the trans-double bond. Furthermore, the toxin-induced leakage from liposomes composed of PC containing the cis-double bond resembled that with PC having the triple bond and was greater than that from liposomes with PC having the trans-double bond. The binding of a H148G mutant to PC liposomes showed a reciprocal relationship in terms of the T_m value of PC containing the triple bond. These results indicate that the toxin-induced membrane damage is closely related to membrane fluidity in liposomes.

Keywords: Abbreviations; DSC; differential scanning calorimetry; T; _m; phase transition temperature; CF; carboxyfluorescein; PC; phosphatidylcholine; PCs; phosphatidylcholines; c; double bond of the; cis; type; t; double bond of the; trans; type; y; triple bond; PLC; phospholipase C; SDC; sodium deoxycholate Clostridium perfringens; Alpha-toxin; Membrane fluidity; Phase transition; Temperature; Liposome

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BBA - Biomembranes (v.1768, #11)