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BBA - Biomembranes (v.1768, #9)

Editorial Board (pp. ii).

The gramicidin ion channel: A model membrane protein by Devaki A. Kelkar; Amitabha Chattopadhyay (pp. 2011-2025).

The linear peptide gramicidin forms prototypical ion channels specific for monovalent cations and has been extensively used to study the organization, dynamics and function of membrane-spanning channels. In recent times, the availability of crystal structures of complex ion channels has challenged the role of gramicidin as a model membrane protein and ion channel. This review focuses on the suitability of gramicidin as a model membrane protein in general, and the information gained from gramicidin to understand lipid–protein interactions in particular. Special emphasis is given to the role and orientation of tryptophan residues in channel structure and function and recent spectroscopic approaches that have highlighted the organization and dynamics of the channel in membrane and membrane-mimetic media.

Keywords: Abbreviations; DMPC; 1,2-dimyristoyl-; sn; -glycero-3-phosphocholine; DPhPC; 1,2-diphytanoyl-; sn; -glycero-3-phosphocholine; DOPC; 1,2 dioleoyl-; sn; -glycero-3-phosphocholine; DOPS; 1,2-dioleoyl-; sn; -glycero-3-phosphoserine; GMO; glycerylmonoolein; LPC; lysophosphatidylcholine; REES; red edge excitation shift; SDS; sodium dodecyl sulfateGramicidin; Ion Channel; Tryptophan; Membrane Interface; Lipid–protein interaction

Membrane fragmentation by an amyloidogenic fragment of human Islet Amyloid Polypeptide detected by solid-state NMR spectroscopy of membrane nanotubes by Jeffrey R. Brender; Ulrich H.N. Dürr; Deborah Heyl; Mahender B. Budarapu; Ayyalusamy Ramamoorthy (pp. 2026-2029).

A key factor in the development of Type II diabetes is the loss of insulin producing pancreatic β-cells. The amyloidogenic human Islet Amyloid Polypeptide (hIAPP also known as human amylin) is believed to play a crucial role in this biological process. Previous studies have shown that hIAPP forms small aggregates that kill β-cells by disrupting the cellular membrane. In this study, we report membrane fragmentation by hIAPP using solid-state NMR experiments on nanotube arrays of anodic aluminum oxide containing aligned phospholipid membranes. In a narrow concentration range of hIAPP, an isotropic³¹P chemical shift signal indicative of the peptide-induced membrane fragmentation was detected. Solid-state NMR results suggest that membrane fragmentation is related to peptide aggregation as the presence of Congo Red, an inhibitor of amyloid formation, prevented membrane fragmentation and the non-amyloidogenic rat-IAPP did not cause membrane fragmentation. The disappearance of membrane fragmentation at higher concentrations of hIAPP suggests an alternate kinetic pathway to fibril formation in which membrane fragmentation is inhibited.

Keywords: Islet Amyloid Polypeptide; Amylin; Solid-Stat NMR; Membrane; Nanotubes

Protein kinase C dependent inhibition of the heteromeric Kir4.1–Kir5.1 channel by Asheebo Rojas; Ningren Cui; Junda Su; Liang Yang; Jean-Pierre Muhumuza; Chun Jiang (pp. 2030-2042).

Heteromultimerization of Kir4.1 and Kir5.1 leads to a channel with distinct functional properties. The heteromeric Kir4.1–Kir5.1 channel is expressed in the eye, kidney and brainstem and has CO₂/pH sensitivity in the physiological range, suggesting a candidate molecule for the regulation of K⁺ homeostasis and central CO₂ chemoreception. It is known that K⁺ transport in renal epithelium and brainstem CO₂ chemosensitivity are subject to modulation by hormones and neurotransmitters that activate distinct intracellular signaling pathways. If the Kir4.1–Kir5.1 channel is involved in pH-dependent regulation of cellular functions, it may also be regulated by some of the intracellular signaling systems. Therefore, we undertook studies to determine whether PKC modulates the heteromeric Kir4.1–Kir5.1 channel. The channel expressed using a Kir4.1–Kir5.1 tandem dimer construct was inhibited by the PKC activator PMA in a dose-dependent manner. The channel inhibition was produced via reduction of the P_open. The effect of PMA was abolished by specific PKC inhibitors. In contrast, exposure of oocytes to forskolin (a PKA activator) had no significant effect on Kir4.1–Kir5.1 currents. The channel inhibition appeared to be independent of PIP₂ depletion and PKC-dependent internalization. Several consensus sequences of potential PKC phosphorylation sites were identified in the Kir4.1 and Kir5.1 subunits by sequence scan. Although the C-terminal peptides of both Kir4.1 and Kir5.1 were phosphorylated in vitro, site-directed mutagenesis of individual residues failed to reveal the PKC phosphorylation sites suggesting that the channel may have multiple phosphorylation sites. Taken together, these results suggest that the Kir4.1–Kir5.1 but not the homomeric Kir4.1 channel is strongly inhibited by PKC activation.

Keywords: Kir4.1–Kir5.1; Protein kinase C; Phosphorylation; Endocytosis

Corneal endothelium transports fluid in the absence of net solute transport by Friedrich P.J. Diecke; Li Ma; Pavel Iserovich; Jorge Fischbarg (pp. 2043-2048).

The corneal endothelium transports fluid from the corneal stroma to the aqueous humor, thus maintaining stromal transparency by keeping it relatively dehydrated. This fluid transport mechanism is thought to be driven by the transcellular transports of HCO₃⁻ and Cl⁻ in the same direction, from stroma to aqueous. In parallel to these anion movements, for electroneutrality, there are paracellular Na⁺ and transcellular K⁺ transports in the same direction. The resulting net flow of solute might generate local osmotic gradients that drive fluid transport. However, there are reports that some 50% residual fluid transport remains in nominally HCO₃⁻ free solutions. We have examined the driving force for this residual fluid transport. We confirm that in nominally HCO₃⁻ free solutions, 48% of control fluid transport remains. When in addition Cl⁻ channels are inhibited, 30% of control fluid movement still remains. Addition of a carbonic anhydrase inhibitor has no further effect. These manipulations combined inhibit the transcellular transport of all anions, without which there cannot be any net transport of solute and consequently no local osmotic gradients, yet there is residual fluid movement. Only the further addition of benzamil, an inhibitor of epithelial Na⁺ channels, abolishes fluid transport completely. Our data are inconsistent with transcellular local osmosis and instead support the paradigm of paracellular fluid transport driven by electro-osmotic coupling.

Structural divergence among cannabinoids influences membrane dynamics: A²H Solid-State NMR analysis by Elvis K. Tiburu; Caroline E. Bass; Jochem O. Struppe; Gary A. Lorigan; Shalom Avraham; Hava Karsenty Avraham (pp. 2049-2059).

Cannabinoids are compounds that can modulate neuronal functions and immune responses via their activity at the CB₁ receptor. We used²H NMR order parameters and relaxation rate determination to delineate the behavior of magnetically aligned phospholipid bilayers in the presence of several structurally distinct cannabinoid ligands. THC (Δ⁹-Tetrahydrocannabinol) and WIN-55,212-2 were found to lower the phase transition temperature of the DMPC and to destabilize their acyl chains leading to a lower average S_CD (≈0.13), while methanandamide and CP-55,940 exhibited unusual properties within the lipid bilayer resulting in a greater average S_CD (≈0.14) at the top of the phospholipid upper chain. The CB₁ antagonist AM281 had average S_CD values that were higher than the pure DMPC lipids, indicating a stabilization of the lipid bilayer. R_1Z versus | S_CD|² plots indicated that the membrane fluidity is increased in the presence of THC and WIN-55,212-2. The interaction of CP-55,940 with a variety of zwitterionic and charged membranes was also assessed. The unusual effect of CP-55,940 was present only in bicelles composed of DMPC. These studies strongly suggest that cannabinoid action on the membrane depends upon membrane composition as well as the structure of the cannabinoid ligands.

Keywords: Abbreviations; HEPES; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; NMR; nuclear magnetic resonance; DMPC; 1,2-dimyristoyl-; sn; -glycero-3-phosphocholine; DMPE; 1,2-dimyristoyl-; sn; -glycero-3-phosphoethanolamine; DMPS; 1,2-dimyristoyl-; sn; -glycero-3-[Phospho-; l; -serine]; DMPG; 1,2-dimyristoyl-; sn; -glycero-3-[Phospho-rac(1-glycerol); DHPC; 1,2-dihexanoyl-; sn; -glycero-3-phosphocholine; BicPC; DMPC/DHPC bicelle; BicPE; DMPE/DMPC/DHPC bicelle; BicPG; DMPG/DMPC/DHPC bicelle; BicPS; DMPS/DMPC/DHPC bicelle; R; _1Z; relaxation rate; T; _1Z; relaxation time; CP-55,940; (−)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-; trans; -4-(3-hydroxypropyl)cyclohexanol; WIN-55,212-2; (; R; )-(+)-[2,3-Dihydro-5-methyl-3-(4-morpholinyl methyl)pyrrolo[1,2,3 ]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone; THC; Δ; ⁹; -Tetrahydrocannabinol; AM281; N-(morpholin-4-yl)-1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-1H-pyrazole-3-carboxamideCannabinoid; NMR; Order parameter; DMPC; Solid-state

Differential effects of human SP-A1 and SP-A2 variants on phospholipid monolayers containing surfactant protein B by Guirong Wang; Svetla Taneva; Kevin M.W. Keough; Joanna Floros (pp. 2060-2069).

Surfactant protein A (SP-A), the most abundant protein in the lung alveolar surface, has multiple activities, including surfactant-related functions. SP-A is required for the formation of tubular myelin and the lung surface film. The human SP-A locus consists of two functional SP-A genes, SP-A1 and SP-A2, with a number of alleles characterized for each gene. We have found that the human in vitro expressed variants, SP-A1 (6A²) and SP-A2 (1A⁰), and the coexpressed SP-A1/SP-A2 (6A²/1A⁰) protein have a differential influence on the organization of phospholipid monolayers containing surfactant protein B (SP-B). Lipid films containing SP-B and SP-A2 (1A⁰) showed surface features similar to those observed in lipid films with SP-B and native human SP-A. Fluorescence images revealed the presence of characteristic fluorescent probe-excluding clusters coexisting with the traditional lipid liquid-expanded and liquid-condensed phase. Images of the films containing SP-B and SP-A1 (6A²) showed different distribution of the proteins. The morphology of lipid films containing SP-B and the coexpressed SP-A1/SP-A2 (6A²/1A⁰) combined features of the individual films containing the SP-A1 or SP-A2 variant. The results indicate that human SP-A1 and SP-A2 variants exhibit differential effects on characteristics of phospholipid monolayers containing SP-B. This may differentially impact surface film activity.

Keywords: Abbreviations; DPPC; 1,2-dipalmitoyl-; sn; -3-phosphocholine; CHO; Chinese Hamster Ovary (CHO)-K1 cell line; LE phase; liquid-expanded phase; LC phase; liquid-condensed phase; PG; l; -α-phosphatidylglycerol (from egg PC, sodium salt); SP-A; surfactant protein A; SP-B; surfactant protein BGenetic variant; Interaction of surfactant protein and lipid; Organization of phospholipid monolayer; Surfactant protien A (SP-A); Surfactant protien B (SP-B)

In vivo relevance of Mrp2-mediated biliary excretion of the Amanita mushroom toxin demethylphalloin by Olga Gavrilova; Joachim Geyer; Ernst Petzinger (pp. 2070-2077).

To determine which efflux carriers are involved in hepatic phalloidin elimination, hepatobiliary [³H]-demethylphalloin (DMP) excretion was studied in normal Wistar rats and in Mrp2 deficient TR(−) Wistar rats as well as in normal wild-type FVB mice, Mdr1a,b(−/−) knockout mice, and Bcrp1(−/−) knockout mice by in situ bile duct/gallbladder cannulation. A subtoxic dose of 0.03 mg DMP/kg b.w. was used, which did not induce cholestasis in any tested animal. Excretion of DMP into bile was not altered in Mdr1a,b(−/−) mice or in Bcrp1(−/−) mice compared with wild-type FVB mice. Whereas 17.6% of the applied dose was excreted into bile of normal Wistar rats, hepatobiliary excretion decreased to 7.9% in TR(−) rats within 2 h after intravenous application. This decrease was not due to reduced cellular DMP uptake, as shown by normal expression of Oatp1b2 in livers of TR(−) rats and functional DMP uptake into isolated TR(−) rat hepatocytes. Tissue concentrations of phalloidin were also not altered in any of the transgenic mice. Interestingly, the decrease of biliary DMP excretion in the TR(−) rats was not followed by any increase of phalloidin accumulation in the liver but yielded a compensatory excretion of the toxin into urine, indicating that hepatocytes of TR(−) rats expelled phalloidin back into blood circulation.

Keywords: Abbreviations; DIDS; diisothiocyanostilbenedisulfonat; DMP; Demethylphalloin; OATP/Oatp; human/rat Organic anion transporting polypeptide; Oct; Organic cation transporter; Oat; Organic anion transporter; MRP/Mrp; human/rat multidrug resistance-associated protein; Mdr1; mouse multidrug resistance protein; Bcrp; mouse breast cancer resistance protein; Bsep; Bile salt export pump; TR(−); transport-deficient rat

A novel method to quantify H⁺-ATPase-dependent Na⁺ transport across plasma membrane vesicles by Yongqing Yang; Lei Hu; Xuemei Chen; Eric A. Ottow; Andrea Polle; Xiangning Jiang (pp. 2078-2088).

To prevent sodium toxicity in plants, Na⁺ is excluded from the cytosol to the apoplast or the vacuole by Na⁺/H⁺ antiporters. The secondary active transport of Na⁺ to apoplast against its electrochemical gradient is driven by plasma membrane H⁺-ATPases that hydrolyze ATP and pump H⁺ across the plasma membrane. Current methods to determine Na⁺ flux rely either on the use of Na-isotopes (²²Na) which require special working permission or sophisticated equipment or on indirect methods estimating changes in the H⁺ gradient due to H⁺-ATPase in the presence or absence of Na⁺ by pH-sensitive probes. To date, there are no methods that can directly quantify H⁺-ATPase-dependent Na⁺ transport in plasma membrane vesicles. We developed a method to measure bidirectional H⁺-ATPase-dependent Na⁺ transport in isolated membrane vesicle systems using atomic absorption spectrometry (AAS). The experiments were performed using plasma membrane-enriched vesicles isolated by aqueous two-phase partitioning from leaves of Populus tomentosa. Since most of the plasma membrane vesicles have a sealed right-side-out orientation after repeated aqueous two-phase partitioning, the ATP-binding sites of H⁺-ATPases are exposed towards inner side. Leaky vesicles were preloaded with Na⁺ sealed for the study of H⁺-ATPase-dependent Na⁺ transport. Our data implicate that Na⁺ movement across vesicle membranes is highly dependent on H⁺-ATPase activity requiring ATP and Mg²⁺ and displays optimum rates of 2.50 μM Na⁺ mg−¹ membrane protein min−¹ at pH 6.5 and 25 °C. In this study, for the first time, we establish new protocols for the preparation of sealed preloaded right-side-out vesicles for the study of H⁺-ATPase-dependent Na⁺ transport. The results demonstrate that the Na⁺ content of various types of plasma membrane vesicle can be directly quantified by AAS, and the results measured using AAS method were consistent with those determined by the previous established fluorescence probe method. The method is a convenient system for the study of bidirectional H⁺-ATPase-dependent Na⁺ transport with membrane vesicles.

Keywords: Abbreviations; SR; sealed right-side-out; LR; leaked right-side-out; SI; sealed inside-out; LI; leaked inside-out; SRP; sealed preloaded-right-side-outPlasma membrane vesicles; H; ⁺; -ATPase; Na; ⁺; transport; AAS; Populus tomentosa

The relationship between the binding to and permeabilization of phospholipid bilayer membranes by GS14 dK₄, a designed analog of the antimicrobial peptide gramicidin S by Thomas Abraham; Seema Marwaha; Daniel M. Kobewka; Ruthven N.A.H. Lewis; Elmar J. Prenner; Robert S. Hodges; Ronald N. McElhaney (pp. 2089-2098).

The cationic β-sheet cyclic tetradecapeptide cyclo[VKLdKVdYPLKVKLdYP] (GS14 dK₄) is a diastereomeric lysine ring-size analog of the potent naturally occurring antimicrobial peptide gramicidin S (GS) which exhibits enhanced antimicrobial but markedly reduced hemolytic activity compared to GS itself. We have previously studied the binding of GS14 dK₄ to various phospholipid bilayer model membranes using isothermal titration calorimetry [Abraham, T. et al. (2005) Biochemistry 44, 2103–2112]. In the present study, we compare the ability of GS14 dK₄ to bind to and disrupt these same phospholipid model membranes by employing a fluorescent dye leakage assay to determine the ability of this peptide to permeabilize large unilamellar vesicles. We find that in general, the ability of GS14 dK₄ to bind to and to permeabilize phospholipid bilayers of different compositions are not well correlated. In particular, the binding affinity of GS14 dK₄ varies markedly with the charge and to some extent with the polar headgroup structure of the phospholipid and with the cholesterol content of the model membrane. Specifically, this peptide binds much more tightly to anionic than to zwitterionic phospholipids and much less tightly to cholesterol-containing than to cholesterol-free model membranes. In addition, the maximum extent of binding of GS14 dK₄ can also vary considerably with phospholipid composition in a parallel fashion. In contrast, the ability of this peptide to permeabilize phospholipid vesicles is only weakly dependent on phospholipid charge, polar headgroup structure or cholesterol content. We provide tentative explanations for the observed lack of a correlation between the affinity and extent of GS14 dK₄ binding to, and degree of disruption of the structure and integrity of, phospholipid bilayers membranes. We also present evidence that the lack of correlation between these two parameters may be a general phenomenon among antimicrobial peptides. Finally, we demonstrate that the affinity of binding of GS14dK4 to various phospholipid bilayer membranes is much more strongly correlated with the antimicrobial and hemolytic activities of this peptide than with its effect on the rate and extent of dye leakage in these model membrane systems.

Keywords: Abbreviations; AMP; antimicrobial peptide; GS; Gramicidin S, cyclo[VOL; d; FPVOL; d; FP] (the amino acid immediately after; d; is the; d; -enantiomer); GS10; cyclo[VKLdYPVKLdYP]; GS14; gramicidin analogue, cyclo[VKLKV; d; YPLKVKL; d; YP]; GS14; d; K; ₄; gramicidin analogue, cyclo[VKL; d; KV; d; YPLKVKL; d; YP]; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphorylcholine; POPG; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-[phospho-; rac; -(glycerol)] (sodium salt); POPS; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-[phosphoserine] (sodium salt); ITC; isothermal titration calorimetry; LUV; large unilamellar vesicle; K; _c; equilibrium binding constant; B; _max; degree of binding (fraction of bound peptide per mole of total phospholipid); LD; ₅₀; peptide concentration required to induce 50% dye releaseGramicidin S; Antimicrobial peptide; Phospholipid bilayer; Phospholipid membrane; Lipid–peptide interaction; Peptide binding; Membrane permeabilization; Isothermal titration calorimetry; Dye leakage

The role of an intracellular cysteine stretch in the sorting of the type II Na/phosphate cotransporter by Gavin S. McHaffie; Chris Graham; Beate Kohl; Ursula Strunck-Warnecke; Andreas Werner (pp. 2099-2106).

The type II Na/phosphate cotransporters (NaPi-II) are critical for the control of plasma phosphate levels in vertebrates. NaPi-IIb mediates phosphate uptake from the small intestine followed by glomerular filtration and selective reabsorption from the renal proximal tubule by NaPi-IIa and NaPi-IIc. A C-terminal stretch of cysteine residues represents the hallmark of the NaPi-IIb isoforms. This motif is well conserved among NaPi-IIb type transporters but not found in other membrane proteins. To investigate the role of this motif we analyzed NaPi-II constructs in transiently and stably transfected MDCK cells. This cell line targets the NaPi-IIb isoforms from flounder and mouse to the apical membrane whereas the mouse IIa isoform shows no plasma membrane preference. Different parts of mouse NaPi-IIa and NaPi-IIb C-termini were fused to GFP-tagged flounder NaPi-II. The constructs showed strong staining of the plasma membrane with NaPi-IIb related constructs sorted predominantly apically, the IIa constructs localized apically and basolaterally with slight intracellular retention. When the cysteine stretch was inserted into the NaPi-IIa C-terminus, the construct was retained in a cytoplasmic compartment. 2-bromopalmitate, a specific palmitoylation inhibitor, released the transporter to apical and basolateral membranes. The drug also leads to a redistribution of the NaPi-IIb construct to both plasma membrane compartments. Immunoprecipitation of tagged NaPi-II constructs from [³H]-palmitate labeled MDCK cells indicated that the cysteine stretch is palmitoylated. Our results suggest that the modified cysteine motif prevents the constructs from basolateral sorting. Additional sorting determinants located downstream of the cysteine stretch may release the cargo to the apical compartment.

Keywords: Na/phosphate cotransporter; MDCK cells; Intracellular sorting; Palmitoylation

Prediction of HIV-1 entry inhibitors neomycin–arginine conjugates interaction with the CD4-gp120 binding site by molecular modeling and multistep docking procedure by Alexander Berchanski; Aviva Lapidot (pp. 2107-2119).

Developing of multi-target HIV-1 entry inhibitors represents an important avenue of drug therapy. Two such inhibitors are hexa-arginine-neomycin-conjugate (NeoR6) and nona-d-arginine-neomycin-conjugate (Neo-r9). Our findings that NeoR6-resistant mutations appear in the gp120 constant regions; and NeoR6 is not CCR5 antagonist, but inhibits CXCR4 and CCR5 HIV-1 using isolates, led us to suggest that NeoR6 may inhibit HIV-1 entry by interfering with the CD4-gp120 binding. To support this notion, we constructed a homology model of unliganded HIV-1_IIIB gp120 and docked NeoR6 and Neo-r9 to it, using a multistep docking procedure: geometric–electrostatic docking by MolFit; flexible ligand docking by Autodock3 and final refinement of the obtained complexes by Discover3. Binding free energies were calculated by MM-PBSA methodology. The model predicts competitive inhibition of CD4-gp120 binding by NeoR6 and Neo-r9. We determined plausible binding sites between constructed CD4-bound gp120 trimer and homology modeled membranal CXCR4, and tested NeoR6 and Neo-r9 interfering with this interaction. These models support our notion that another mechanism of anti-HIV-1 activity of NeoR6 is inhibition of gp120-CXCR4 binding. These structural models and interaction of NeoR6 and Neo-r9 with gp120 and CXCR4 provide a powerful approach for structural based drug design for selective targeting of HIV-1 entry and/or for inhibition of other retroviruses with similar mechanism of entry.

Keywords: Hexa-arginine-neomycin-conjugate (NeoR6); poly-arginine-neomycin-conjugate (Neo-r9); Molecular modeling and docking; HIV-1 attachment inhibitor; HIV-1 gp120; CD4/CXCR4

Trehalose preserves DDA/TDB liposomes and their adjuvant effect during freeze-drying by Dennis Christensen; Camilla Foged; Ida Rosenkrands; Hanne Mørck Nielsen; Peter Andersen; Else Marie Agger (pp. 2120-2129).

Disaccharides are well-known reagents to protect biostructures like proteins and phospholipid-based liposomes during freezing and drying. We have investigated the ability of the two disaccharides trehalose and sucrose to stabilize a novel, non-phospholipid-based liposomal adjuvant composed of the cationic dimethyldioctadecylammonium (DDA) and trehalose 6,6′-dibehenate (TDB) upon freeze-drying. The liposomes were freeze-dried using a human dose concentration containing 2.5 mg/ml DDA and 0.5 mg/ml TDB with varying concentrations of the two sugars. The influence on particle size upon rehydration was investigated using photon correlation spectroscopy (PCS) and the gel to fluid phase transition was examined by differential scanning calorimetry (DSC). Data revealed that concentrations above 211 mM trehalose protected and preserved DDA/TDB during freeze-drying, and the liposomes were readily rehydrated. Sucrose was less efficient as a stabilizer and had to be used in concentrations above 396 mM in order to obtain the same effect. Immunization of mice with the tuberculosis vaccine candidate Ag85B-ESAT-6 in combination with the trehalose stabilized adjuvant showed that freeze-dried DDA/TDB liposomes retained their ability to stimulate both a strong cell-mediated immune response and an antibody response. These findings show that trehalose at isotonic concentrations protects cationic DDA/TDB-liposomes during freeze-drying. Since this is not the case for liposomes based on DDA solely, we suggest that the protection is facilitated via direct interaction with the headgroup of TDB and a kosmotropic effect, whereas direct interaction with DDA plays a minor role.

Keywords: Liposome; Freeze-drying; Adjuvant; Vaccine; Dimethyldioctadecylammonium; trehalose 6,6-dibehenate; Sugar

Dynamics and heterogeneity of bovine hippocampal membranes: Role of cholesterol and proteins by Soumi Mukherjee; Mamata Kombrabail; G. Krishnamoorthy; Amitabha Chattopadhyay (pp. 2130-2144).

The structural and dynamic consequence of alterations in membrane lipid composition (specifically cholesterol) in neuronal membranes is poorly understood. Previous work from our laboratory has established bovine hippocampal membranes as a convenient natural source for studying neuronal receptors. In this paper, we have explored the role of cholesterol and proteins in the dynamics and heterogeneity of bovine hippocampal membranes using fluorescence lifetime distribution analysis of the environment-sensitive fluorescent probe Nile Red incorporated into such membranes by the maximum entropy method (MEM), and time-resolved fluorescence anisotropy measurements. The peak position and the width of the lifetime distribution of Nile Red show a progressive reduction with increasing cholesterol depletion from native hippocampal membranes indicating that the extent of heterogeneity decreases with decrease in membrane cholesterol content. This is accompanied by a concomitant decrease of the fluorescence anisotropy and rotational correlation time. Our results point out that the microenvironment experienced by Nile Red is relatively insensitive to the presence of proteins in hippocampal membranes. Interestingly, Nile Red lifetime distribution in liposomes of lipid extracts is similar to that of native membranes indicating that proteins do not contribute significantly to the high level of heterogeneity observed in native membranes. These results could be relevant in understanding the neuronal diseases characterized by defective membrane lipid metabolism.

Keywords: Abbreviations; BCA; Bicinchoninic acid; DMPC; 1,2-dimyristoyl-; sn; -glycero-3-phosphocholine; DOPC; dioleoyl-; sn; -glycero-3-phosphocholine; FWHM; full width at half maximum; MβCD; methyl-β-cyclodextrin; MEM; maximum entropy method; MLV; multilamellar vesicle; Nile Red; 9-diethylamino-5H-benzo[α]phenoxazine-5-one; PMSF; phenylmethylsulfonyl fluoride; TCSPC; time correlated single photon countingHippocampal membrane; Cholesterol; Fluorescence lifetime distribution; Maximum entropy method; Time-resolved fluorescence anisotropy

Interactions of Plasmodium falciparum erythrocyte membrane protein 3 with the red blood cell membrane skeleton by Karena L. Waller; Lisa M. Stubberfield; Valentina Dubljevic; Wataru Nunomura; Xuili An; Anthony J. Mason; Narla Mohandas; Brian M. Cooke; Ross L. Coppel (pp. 2145-2156).

Plasmodium falciparum parasites express and traffick numerous proteins into the red blood cell (RBC), where some associate specifically with the membrane skeleton. Importantly, these interactions underlie the major alterations to the modified structural and functional properties of the parasite-infected RBC. P. falciparum Erythrocyte Membrane Protein 3 (PfEMP3) is one such parasite protein that is found in association with the membrane skeleton. Using recombinant PfEMP3 proteins in vitro, we have identified the region of PfEMP3 that binds to the RBC membrane skeleton, specifically to spectrin and actin. Kinetic studies revealed that residues 38–97 of PfEMP3 bound to purified spectrin with moderately high affinity ( K D_(kin)=8.5×10−⁸ M). Subsequent deletion mapping analysis further defined the binding domain to a 14-residue sequence (IFEIRLKRSLAQVL; K D_(kin)=3.8×10−⁷ M). Interestingly, this same domain also bound to F-actin in a specific and saturable manner. These interactions are of physiological relevance as evidenced by the binding of this region to the membrane skeleton of inside-out RBCs and when introduced into resealed RBCs. Identification of a 14-residue region of PfEMP3 that binds to both spectrin and actin provides insight into the potential function of PfEMP3 in P. falciparum-infected RBCs.

Keywords: Abbreviations; RBC; Red Blood Cell; IRBC; Infected Red Blood Cell; PfEMP3; Plasmodium falciparum; Erythrocyte Membrane Protein 3; K; D; _(kin); and; K; D; _(Scat); dissociation constants determined by kinetic and Scatchard Analysis, respectively; GPC and GPD; Glycophorin C and D, respectively; PfEMP1; Plasmodium falciparum; Erythrocyte Membrane Protein 1; KAHRP; Knob Associated Histidine Rich Protein; MESA; Mature Parasite-infected Erythrocyte Surface Antigen; RESA; Ring-infected Erythrocyte Surface Antigen; αR4; α-spectrin repeat region 4; IOV; Inside-out Vesicle; PCR; Polymerase Chain Reaction; MBP; Maltose Binding Protein; PBS; Phosphate-buffered Saline; IB; IOV Incubation Buffer; BSA; Bovine Serum Albumin; PVDF; Polyscreen® polyvinylidene difluoride; TX100; Triton X-100 Plasmodium falciparum; Protein interaction; Malaria; PfEMP3; Spectrin; Actin; Red blood cell

Glycocardiolipin modulates the surface interaction of the proton pumped by bacteriorhodopsin in purple membrane preparations by Angela Corcelli; Simona Lobasso; Matilde Sublimi Saponetti; Andreas Leopold; Norbert A. Dencher (pp. 2157-2163).

Glycocardiolipin is an archaeal analogue of mitochondrial cardiolipin, having an extraordinary affinity for bacteriorhodopsin, the photoactivated proton pump in the purple membrane of Halobacterium salinarum. Here purple membranes have been isolated by osmotic shock from either cells or envelopes of Hbt. salinarum. We show that purple membranes isolated from envelopes have a lower content of glycocardiolipin than standard purple membranes isolated from cells. The properties of bacteriorhodopsin in the two different purple membrane preparations are compared; although some differences in the absorption spectrum and the kinetic of the dark adaptation process are present, the reduction of native membrane glycocardiolipin content does not significantly affect the photocycle (M-intermediate rise and decay) as well as proton pumping of bacteriorhodopsin. However, interaction of the pumped proton with the membrane surface and its equilibration with the aqueous bulk phase are altered.

Keywords: Archaeal cardiolipin; Bacteriorhodopsin; Proton pumping; Photocycle; Proton transfer

Evolutionary origins of members of a superfamily of integral membrane cytochrome c biogenesis proteins by Jong-Hoon Lee; Edgar M. Harvat; Julie M. Stevens; Stuart J. Ferguson; Milton H. Saier Jr. (pp. 2164-2181).

We have analyzed the relationships of homologues of the Escherichia coli CcmC protein for probable topological features and evolutionary relationships. We present bioinformatic evidence suggesting that the integral membrane proteins CcmC ( E. coli; cytochrome c biogenesis System I), CcmF ( E. coli; cytochrome c biogenesis System I) and ResC ( Bacillus subtilis; cytochrome c biogenesis System II) are all related. Though the molecular functions of these proteins have not been fully described, they appear to be involved in the provision of heme to c-type cytochromes, and so we have named them the putative Heme Handling Protein (HHP) family (TC #9.B.14). Members of this family exhibit 6, 8, 10, 11, 13 or 15 putative transmembrane segments (TMSs). We show that intragenic triplication of a 2 TMS element gave rise to a protein with a 6 TMS topology, exemplified by CcmC. This basic 6 TMS unit then gave rise to two distinct types of proteins with 8 TMSs, exemplified by ResC and the archaeal CcmC, and these further underwent fusional or insertional events yielding proteins with 10, 11 and 13 TMSs (ResC homologues) as well as 15 TMSs (CcmF homologues). Specific evolutionary pathways taken are proposed. This work provides the first evidence for the pathway of appearance of distantly related proteins required for post-translational maturation of c-type cytochromes in bacteria, plants, protozoans and archaea.

Keywords: Abbreviations; TMS; Transmembrane Segments; HHP; Heme Handling Protein; Ccm; C; ytochrome; c m; aturation; CCHL; C; ytochrome; c H; eme; L; yase; MC; Mitochondrial CarrierTransport; Protein evolution; Transporter topology; Gene duplication; Gene fusion; Gene deletion

Fluorescence probe partitioning between L_o/L_d phases in lipid membranes by Tobias Baumgart; Geoff Hunt; Elaine R. Farkas; Watt W. Webb; Gerald W. Feigenson (pp. 2182-2194).

Fluorescence microscopy imaging is an important technique for studying lipid membranes and is increasingly being used for examining lipid bilayer membranes, especially those showing macroscopic coexisting domains. Lipid phase coexistence is a phenomenon of potential biological significance. The identification of lipid membrane heterogeneity by fluorescence microscopy relies on membrane markers with well-defined partitioning behavior. While the partitioning of fluorophores between gel and liquid-disordered phases has been extensively characterized, the same is not true for coexisting liquid phases. We have used fluorescence microscopy imaging to examine a large variety of lipid membrane markers for their liquid phase partitioning in membranes with various lipid compositions. Most fluorescent lipid analogs are found to partition strongly into the liquid-disordered (L_d) phase. In contrast, some fluorescent polycyclic aromatic hydrocarbons with a flat ring system were found to partition equally, but others partition preferentially into liquid-ordered (L_o) phases. We have found these fluorescent markers effective for identification of coexisting macroscopic membrane phases in ternary lipid systems composed of phospholipids and cholesterol.

Keywords: Liquid ordered; Liquid disordered; Fluorescence microscopy; Probe partitioning; Liposome; Vesicle

Modification of membranes by quercetin, a naturally occurring flavonoid, via its incorporation in the polar head group by Bożena Pawlikowska-Pawlęga; Wiesław Ignacy Gruszecki; Lucjan Misiak; Roman Paduch; Tomasz Piersiak; Barbara Zarzyka; Jarosław Pawelec; Antoni Gawron (pp. 2195-2204).

Quercetin is a naturally occurring flavonoid that has a lot of beneficial properties to human health. In this report, using the spin label technique, the influence of quercetin on the fluidity of multilamellar DPPC liposomes was studied. The polarity of the environment preferred by quercetin was also examined by determining the dependence of the position of electronic absorption maxima on dielectric properties of different environments. Autofluorescence of quercetin was also used to examine its distribution in cells. An additional aim of the study was to find how quercetin presence affects human skin fibroblasts. The results showed that incorporation of quercetin at physiological pH into DPPC liposomes caused changes in the partition coefficient of the Tempo spin label between water and polar head group phases. By determining the electronic absorption maxima, we observed that the chromophore of quercetin is localized in the polar head region. Fluorescence microscopy of HSF cells showed quercetin presence in the membrane, cytoplasm and inside the nucleus. Ultrastructural observation revealed some changes, especially in membranous structures, after flavonol treatment. From the results we have concluded that quercetin present in the membrane and other structures can cause changes within cells crucial for its pharmacological activity.

Keywords: Quercetin; DPPC liposome; EPR; Fluorescence microscopy; Electron microscopy

Effect of ceramide N-acyl chain and polar headgroup structure on the properties of ordered lipid domains (lipid rafts) by Megha; Peter Sawatzki; Thomas Kolter; Robert Bittman; Erwin London (pp. 2205-2212).

Ceramides are sphingolipids that greatly stabilize ordered membrane domains (lipid rafts), and displace cholesterol from them. Ceramide-rich rafts have been implicated in diverse biological processes. Because ceramide analogues have been useful for probing the biological function of ceramide, and may have biomedical applications, it is important to characterize how ceramide structure affects membrane properties, including lipid raft stability and composition. In this report, fluorescence quenching assays were used to evaluate the effect of analogues of ceramide with different N-acyl chains or different sphingoid backbones on raft stability and sterol content. The effect of replacing 18 mol% of sphingomyelin (SM) with ceramide in vesicles composed of a 1:1 (mol:mol) mixture of SM and dioleoylphosphatidylcholine (DOPC), with or without 25 mol% sterol, was examined. In the absence of sterol, the thermal stability of the SM-rich ordered domains increased with ceramide N-acyl chain length in the order C2:0∼C6:0∼C8:0N-acyl chain length increased in the order C8:0∼C6:0N-lauroyl- and N-palmitoylsphingosine analogues containing altered structures in or near the polar portion of the sphingoid base. In almost all cases, the analogues stabilized rafts to about the same degree as a normal ceramide containing the same acyl chain. The only exception was N-palmitoyl-4D-ribophytosphingosine, which was very strongly raft-stabilizing. We conclude that variations in sphingoid base structure induce only insignificant changes in raft properties. N-Lauroyl and N-palmitoylsphingosine and their analogues displaced sterol from rafts to a significant degree. Both C12:0 and C16:0 analogues of ceramide may be good mimics of natural ceramide, and useful for cellular studies in which maintenance of the normal physical properties of ceramide are important.

Keywords: Sphingolipid; Fluorescence quenching; Cholesterol; Lipid phase; Tempo; Sphingomyelin; Ceramide

The phenyltetraene lysophospholipid analog PTE-ET-18-OMe as a fluorescent anisotropy probe of liquid ordered membrane domains (lipid rafts) and ceramide-rich membrane domains by Omar Bakht; Javier Delgado; Francisco Amat-Guerri; A. Ulises Acuña; Erwin London (pp. 2213-2221).

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

Keywords: Fluorescence polarization; Fluorescence anisotropy; Edelfosine; Lipid domain; Lipid microdomain; Energy transfer; FRET; Shingomyelin; Shingolipid

Cell-permeable peptides induce dose- and length-dependent cytotoxic effects by Alessandra K. Cardozo; Valérie Buchillier; Marc Mathieu; Jianhua Chen; Fernanda Ortis; Laurence Ladrière; Nathalie Allaman-Pillet; Olivier Poirot; Stephan Kellenberger; Jacques S. Beckmann; Decio L. Eizirik; Christophe Bonny; Fabienne Maurer (pp. 2222-2234).

We have explored the threshold of tolerance of three unrelated cell types to treatments with potential cytoprotective peptides bound to Tat_48–57 and Antp_43–58 cell-permeable peptide carriers. Both Tat_48–57 and Antp_43–58 are well known for their good efficacy at crossing membranes of different cell types, their overall low toxicity, and their absence of leakage once internalised. Here, we show that concentrations of up to 100 μM of Tat_48–57 were essentially harmless in all cells tested, whereas Antp_43–58 was significantly more toxic. Moreover, all peptides bound to Tat_48–57 and Antp_43–58 triggered significant and length-dependent cytotoxicity when used at concentrations above 10 μM in all but one cell types (208F rat fibroblasts), irrespective of the sequence of the cargo. Absence of cytotoxicity in 208F fibroblasts correlated with poor intracellular peptide uptake, as monitored by confocal laser scanning fluorescence microscopy. Our data further suggest that the onset of cytotoxicity correlates with the activation of two intracellular stress signalling pathways, namely those involving JNK, and to a lesser extent p38 mitogen-activated protein kinases. These responses are of particular concern for cells that are especially sensitive to the activation of stress kinases. Collectively, these results indicate that in order to avoid unwanted and unspecific cytotoxicity, effector molecules bound to Tat_48–57 should be designed with the shortest possible sequence and the highest possible affinity for their binding partners or targets, so that concentrations below 10 μM can be successfully applied to cells without harm. Considering that cytotoxicity associated to Tat_48–57- and Antp_43–58 bound peptide conjugates was not restricted to a particular type of cells, our data provide a general framework for the design of cell-penetrating peptides that may apply to broader uses of intracellular peptide and drug delivery.

Keywords: Cell-penetrating peptide; Tat; Intracellular delivery; Pancreatic beta-cell; HeLa cell

Structural organisation of prolamellar bodies (PLB) isolated from Zea mays. Parallel TEM, SAXS and absorption spectra measurements on samples subjected to freeze–thaw, reduced pH and high-salt perturbation by Eva Selstam; Jenny Schelin; W. Patrick Williams; Anthony P.R. Brain (pp. 2235-2245).

Well-organised PLB gives rise to a X-ray diffraction pattern overlaid by a scattering pattern arising from individual tubules within less well-organised regions of the lattice. TEM and SAXS measurements were used to characterise the structural changes in PLB subjected to perturbation by freeze–thaw, exposure to pH 6.5, or resuspension in high-salt media. Comparison of SAXS patterns measured, before and after structural perturbation allows the separation of the contributions from ordered and disordered PLB. The diffraction pattern is shown to be based on a diamond cubic (Fd3m) lattice of unit cell a=78 nm. Freeze–thaw and high-salt disruption lead to the breakdown of ordered PLB into disordered tubules of similar dimensions to those making up the original PLB lattice. Their scattering patterns suggest that they are approximately 26 nm in diameter with a central lumen about 16 nm in diameter. The tubules formed at pH 6.5 are appreciably narrower, probably reflecting changes in the pattern of ionisation of charged groups at the membrane surface. Absorption spectra of PLB in media containing different concentrations of salts indicated that the structural and spectral changes are related. NADPH, have a significant role in the protection of POR-PChlide₆₅₀ but to have only a relatively small effect on the preservation of PLB organisation indicating that the retention of POR-PChlide₆₅₀ in isolated PLB preparations is a poor guide to their structural integrity.

Keywords: Abbreviations; Chlide; chlorophyllide; PChlide; protochlorophyllide; PLB; prolamellar body; PT; prothylakoid; POR; protochlorophyllide oxidoreductase; POR-PChlide; ₆₄₀; POR-PChlide; ₆₅₀; , spectral forms of; POR-PChlide; complexes with absorption maxima at 640 and 650 nmProtochlorophyllide oxidoreductase; Prolamellar body; Protochlorophyllide; Chlorophyllide; Electron microscopy; X-ray scattering

Membrane fluidity changes in goat sperm induced by cholesterol depletion using beta-cyclodextrin by Mònica Companyó; Antoni Iborra; Joaquim Villaverde; Paz Martínez; Antoni Morros (pp. 2246-2255).

Cholesterol efflux from membranes promotes acrosome reaction in goat spermatozoa. In 1 h of incubation of sperm in the presence of beta-cyclodextrin (βCD), all the interchangeable cholesterol is desorbed from sperm membranes, although acrosome reaction is fully accomplished only after 3–4 h of incubation, as previously published. In the present paper we investigate the effect of cholesterol removal from mature goat spermatozoa on the overall membrane “fluidity” of live cell membranes and of liposomes from sperm lipid extracts. Using steady state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH), we studied the average thermotropic behaviour of membrane lipids, after incubation of live sperm for 1 h in BSA-free medium with the presence/absence of 8 mM β-cyclodextrin, as a cholesterol acceptor. Unimodal and bimodal theoretical sigmoids fitted best to the experimental thermotropic profiles of liposomes and whole cells, respectively. In the case of whole sperm, two phase transitions, attributable to different lipid domains, were clearly separated by using the fitting parameters. After cholesterol removal, important changes in the relative anisotropy range of the two transitions were found, indicating an increase in the “fluidity” of some of the lipid microdomains of sperm membranes. These changes in sperm lipid dynamics are produced before the onset of sperm acrosome reaction.

Keywords: Goat sperm capacitation; Cholesterol efflux; Cyclodextrin; Fluorescence anisotropy; Membrane fluidityAbbreviations; AR; acrosome reaction; βCD; β-cyclodextrin; DPH; 1,6-diphenyl-1,3,5-hexatriene; Δ; H; _vH; van't Hoff enthalpy; M-199; Hanks Salts Base incubation medium; PC; phosphatidylcholine; PE; phosphatidylethanolamine; r; fluorescence anisotropy; SPM; sphingomyeline; TBS; Tris-buffered saline; T; _m; phase transition temperature

Toxicity of fatty acid hydroperoxides towards Yarrowia lipolytica: Implication of their membrane fluidizing action by Hoa Tran Thanh; Laurent Beney; Hélène Simonin; Thi Xuan Sam Nguyen; Patrick Gervais; Jean-Marc Belin; Florence Husson (pp. 2256-2262).

Linoleic acid hydroperoxide (HPOD), substrate of hydroperoxide lyase, an enzyme of the lipoxygenase pathway, can be transformed into many aromatic compounds, the so-called “green notes”. The presence of linoleic acid hydroperoxide in the culture medium of Yarrowia lipolytica, the yeast expressing the cloned hydroperoxide lyase of green bell pepper, undoubtedly exerted an inhibition on the growth and a toxic effect with 90% of yeast cells died after 120 min of exposition in 100 mM HPOD solution. The increase in cell membrane fluidity evaluated by measuring fluorescence generalized polarization with the increasing concentration of HPOD in the medium confirmed the fluidizing action of HPOD on yeast membrane. In addition, we determined by infrared spectroscopy measurement that this compound rapidly diffused into model phospholipids [1, 2-Dimyristoyl-D54- sn-Glycero-3-Phosphocholine (DMPC-D54)] bilayer, modifying their general physical state and their phase transition. In the presence of various concentrations of HPOD, the phase transition of DMPC-D54 occurred with an increase of both the corresponding wave number shift and the temperature range but the phase transition temperature was not modified. These results show that the toxic effects of HPOD on the yeast Yarrowia lipolytica may be initially linked to a strong interaction of this compound with the cell membrane phospholipids and components.

Keywords: Yarrowia lipolytica; Membrane fluidity; Infrared spectroscopy; Toxicity; Laurdan

Segment TM7 from the cytoplasmic hemi-channel from V_O-H⁺-V-ATPase includes a flexible region that has a potential role in proton translocation by Afonso M.S. Duarte; Edwin R. de Jong; Rainer Wechselberger; Carlo P.M. van Mierlo; Marcus A. Hemminga / (pp. 2263-2270).

A 900-MHz NMR study is reported of peptide sMTM7 that mimics the cytoplasmic proton hemi-channel domain of the seventh transmembrane segment (TM7) from subunit a of H⁺-V-ATPase from Saccharomyces cerevisiae. The peptide encompasses the amino acid residues known to actively participate in proton translocation. In addition, peptide sMTM7 contains the amino acid residues that upon mutation cause V-ATPase to become resistant against the inhibitor bafilomycin. 2D TOCSY and NOESY¹H–¹H NMR spectra are obtained of sMTM7 dissolved in d₆-DMSO and are used to calculate the three-dimensional structure of the peptide. The NMR-based structures and corresponding dynamical features of peptide sMTM7 show that sMTM7 is composed of two α-helical regions. These regions are separated by a flexible hinge of two residues. The hinge acts as a ball-and-joint socket and both helical segments move independently with respect to one another. This movement in TM7 is suggested to cause the opening and closing of the cytoplasmic proton hemi-channel and enables proton translocation.

Keywords: Abbreviations; CSI; Chemical shift index; DMSO; Dimethylsulfoxide; DSS; Sodium 2,2-dimethyl-2-silapentane; -; 5-sulfonate; HMBC; Heteronuclear multiple bond correlation; HSQC; Heteronuclear single quantum coherence; sMTM7; Peptide mimicking the cytoplasmic side of the seventh transmembrane segment of subunit; a; from V-ATPase; NMR; Nuclear magnetic resonance; NOESY; Nuclear Overhauser enhancement spectroscopy; TM; Transmembrane; RMSD; Root-mean-square deviation; TOCSY; Total correlation spectroscopy; V-ATPase; Vacuolar proton-translocating adenosine triphosphatase; SA; simulated annealingNMR; Transmembrane; V-ATPase subunit; a; Peptide conformation; Proton translocation

A comparative approach towards thylakoid membrane proteome analysis of unicellular green alga Scenedesmus obliquus by Konstantinos Kantzilakis; Michalis Aivaliotis; Christos Kotakis; Fanourios Krasanakis; Apostolos K. Rizos; Kiriakos Kotzabasis; Georgios Tsiotis (pp. 2271-2279).

The chlorophyll ( Chl)-containing membrane protein complexes from the green alga Scenedesmus obliquus have been isolated from the thylakoid membranes by solubilization with dodecyl-β-maltoside and fractionation using a sucrose density gradient. The Chl-containing protein fractions were characterized by absorption spectroscopy, tricine SDS PAGE, BN-PAGE, and dynamic light scattering (DLS). BN-PAGE showed the presence of seven protein complexes with molecular weights in the range of 68, 118, 157, 320, 494, 828 and 955 kDa, respectively. Furthermore, light scattering reveals the simultaneous presence of particles of different sizes in the 3–4 nm and 6.0–7.5 nm range, respectively. The smaller size is related to the hydrodynamic radius of the trimer Light Harvesting Complex (LHCII), whereas the larger size is associated with the presence of photosystem I and photosystem II reaction centers. Additionally, functional information regarding protein–protein interactions was deconvoluted using coupling 2-D BN-PAGE, MALDI-TOF MS and a detailed mapping of S. obliquus photosynthetic proteome of the solubilized thylakoid membranes is therefore presented.

Keywords: Abbreviations; SDS PAGE; sodium dodecyl sulfate polyacrylamide gel electrophoresis; DLS; dynamic light scattering; DM; Dodecyl-β-maltoside; BN; blue native; MALDI-TOF; matrix assisted laser desorption ionisation-time of flight; LC-ESI; liquid chromatography electrospray ionisation; MS; mass spectrometry Scenedesmus obliquus; Light scattering; Blue native; Molecular size; Membrane protein; Photosynthetic apparatus

Transfection efficiency boost by designer multicomponent lipoplexes by Giulio Caracciolo; Daniela Pozzi; Ruggero Caminiti; Cristina Marchini; Maura Montani; Augusto Amici; Heinz Amenitsch (pp. 2280-2292).

Cationic liposome–DNA complexes (lipoplexes) have emerged as leading nonviral gene carriers in worldwide gene therapy clinical trials. Arriving at therapeutic dosages requires the full understanding of the mechanism of transfection. We investigated the correlation between structural evolution of multicomponent lipoplexes when interacting with cellular lipids, the extent of DNA release and the efficiency in transfecting mouse fibroblast (NIH 3T3), ovarian (CHO) and tumoral myofibroblast-like (A17) cell lines. We show, for the first time, that the transfection pattern increases monotonically with the number of lipid components and further demonstrate by means of synchrotron small angle X- ray scattering (SAXS) that structural changes of lipoplexes induced by cellular lipids correlate with the transfection efficiency. Specifically, inefficient lipoplexes either fused too rapidly upon interaction with anionic lipids or, alternatively, are found to be extremely resistant to solubilization. The most efficient lipoplex formulations exhibited an intermediate behaviour. The extent of DNA unbinding (measured by electrophoresis on agarose gel) correlates with structural evolution of the lipoplexes but DNA-release does not scale with the extent of transfection. The general meaning of our results is of broad interest in the field of non-viral gene delivery: rational adjusting of lipoplex composition to generate the proper interaction between lipoplexes and cellular lipids may be the most appropriate strategy in optimizing synthetic lipid transfection agents.

Keywords: Cationic liposome; DNA; Lipoplex; Gene delivery; Transfection efficiency; SAXS

A membrane associated metalloprotease cleaves Cry3Aa Bacillus thuringiensis toxin reducing pore formation in Colorado potato beetle brush border membrane vesicles by C. Rausell; C. Ochoa-Campuzano; A.C. Martínez-Ramírez; A. Bravo; M.D. Real (pp. 2293-2299).

Insect proteases are implicated in Bacillus thuringiensis insecticidal proteins mode of action determining toxin specificity and sensitivity. Few data are available on the involvement of proteases in the later steps of toxicity such as protease interaction with toxin–receptor complexes and the pore formation process. In this study, a Colorado potato beetle (CPB) midgut membrane metalloprotease was found to be involved in the proteolytic processing of Cry3Aa. Interaction of Cry3Aa with BBMV membrane proteases resulted in a distinct pattern of proteolysis. Cleavage was demonstrated to occur in protease accessible regions of domain III and was specifically inhibited by the metalloprotease inhibitors 1,10-phenanthroline and acetohydroxamic acid. Proteolytic inhibition by a peptide representing a segment of proteolysis in domain III and the metalloprotease inhibitor acetohydroxamic acid correlated with increased pore formation, evidencing that Cry3Aa is a specific target of a CPB membrane metalloprotease that degrades potentially active toxin.

Keywords: Bacillus thuringiensis; Colorado potato beetle; Insecticidal toxin; Protease; Cry3Aa proteolysis; Pore formation

Cholesterol modulation of membrane resistance to Triton X-100 explored by atomic force microscopy by Karim El Kirat; Sandrine Morandat (pp. 2300-2309).

Biomembranes are not homogeneous, they present a lateral segregation of lipids and proteins which leads to the formation of detergent-resistant domains, also called “rafts”. These rafts are particularly enriched in sphingolipids and cholesterol. Despite the huge body of literature on raft insolubility in non-ionic detergents, the mechanisms governing their resistance at the nanometer scale still remain poorly documented. Herein, we report a real-time atomic force microscopy (AFM) study of model lipid bilayers exposed to Triton X-100 (TX-100) at different concentrations. Different kinds of supported bilayers were prepared with dioleoylphosphatidylcholine (DOPC), sphingomyelin (SM) and cholesterol (Chol). The DOPC/SM 1:1 (mol/mol) membrane served as the non-resistant control, and DOPC/SM/Chol 2:1:1 (mol/mol/mol) corresponded to the raft-mimicking composition. For all the lipid compositions tested, AFM imaging revealed that TX-100 immediately solubilized the DOPC fluid phase leaving resistant patches of membrane. For the DOPC/SM bilayers, the remaining SM-enriched patches were slowly perforated leaving crumbled features reminiscent of the initial domains. For the raft model mixture, no holes appeared in the remaining SM/Chol patches and some erosion occurred. This work provides new, nanoscale information on the biomembranes' resistance to the TX-100-mediated solubilization, and especially about the influence of Chol.

Keywords: Raft; DRM; Real-time AFM; Lipid bilayer; Solubilization; Non-ionic detergent

Membrane curvature stress controls the maximal conversion of violaxanthin to zeaxanthin in the violaxanthin cycle—influence of α-tocopherol, cetylethers, linolenic acid, and temperature by Anna Szilágyi; Marianne Sommarin; Hans-Erik Åkerlund (pp. 2310-2318).

Zeaxanthin, an important component in protection against overexcitation in higher plants, is formed from violaxanthin by the enzyme violaxanthin de-epoxidase. We have investigated factors that may control the maximal degree of conversion in the violaxanthin cycle. The conversion of violaxanthin to zeaxanthin in isolated spinach thylakoids was followed at different temperatures and in the presence of lipid packing modifiers. The maximum degree of conversion was found to be 35%, 70% and 80% at 4 °C, 25 °C and 37 °C respectively. In the presence of membrane modifying agents, known to promote non-lamellar structures (H_II), such as linolenic acid the conversion increased, and the maximal level of violaxanthin de-epoxidation obtained was close to 100%. In contrast, substances promoting lamellar phases (L_α), such as α-tocopherol and 8-cetylether (C₁₆EO₈), only 55% and 35% of the violaxanthin was converted at 25 °C, respectively. The results are interpreted in light of the lipid composition of the thylakoid membrane, and we propose a model where a negative curvature elastic stress in the thylakoid lipid bilayer is required for violaxanthin de-epoxidase activity. In this model zeaxanthin with its longer hydrophobic stretch is proposed to promote lamellar arrangements of the membrane. As a result, zeaxanthin relieves the curvature elastic stress, which in turn leads to inactivation of violaxanthin de-epoxidase.

Keywords: Abbreviations; C; ₁₆; EO; _x; Poly(oxyethylene) hexadecyl ethers and; x; is the number of oxyethylene units in the polar head groups; C; ₁₆; EO; ₈; octaethylene glycol monohexadecyl ether; DEPE; 1,2-dielaidoyl-; sn; -glycero-3-phosphoethanolamine; DGDG; digalactosyldiacylglycerol; MGDG; monogalactosyldiacylglycerol; H; _II; inverted hexagonal phase; HPLC; high performance liquid chromatography; L; _α; lamellar liquid crystal phase; L; _β; lamellar gel phase; MES; 2-(N-morpholino)ethanesulfonic acid; MOPS; 3-(N-morpholino)propanesulfonic acid; NPQ; non-photochemical quenching; PC; phosphatidylcholine; PE; phosphatidylethanolamine; PSII; photosystem II; VDE; violaxanthin de-epoxidase; XC; xanthophyll cycleMembrane curvature stress; Inverted hexagonal phase (H; _II; ); Thylakoid membrane; Violaxanthin de-epoxidase (VDE); Zeaxanthin; Xanthophyll cycle

Interfacial interactions of glutamate, water and ions with carbon nanopore evaluated by molecular dynamics simulations by Sean M. Cory; Yunyun Liu; Mladen I. Glavinović (pp. 2319-2341).

Molecular dynamics simulations were used to assess the transport of glutamate, water and ions (Na⁺ and Cl⁻) in a single wall carbon nanopore. The spatial profiles of Na⁺ and Cl⁻ ions are largely determined by the pore wall charges. Co-ions are repelled whereas the counter-ions are attracted by the pore charges, but this ‘rule’ breaks down when the water concentration is set to a level significantly below that in the physiological bulk solution. In such cases water is less able to counteract the ion–wall interactions (electrostatic or non-electrostatic), co-ions are layered near the counter-ions attracted by the wall charges and are thus layered as counter-ions. Glutamate is concentrated near the pore wall even at physiological water concentration, and irrespective of whether the pore wall is neutral or charged (positively or negatively), and its peak levels are up to 40 times above mean values. The glutamate is thus always layered as a counter-ion. Layering of water near the wall is independent of charges on the pore wall, but its peak levels near the wall are ‘only’ 6–8 times above the pore mean values. However, if the mean concentration of water is significantly below the level in the physiological bulk solution, its layering is enhanced, whereas its concentration in the pore center diminishes to very low levels. Reasons for such a ‘paradoxical’ behavior of molecules (glutamate and water) are that the non-electrostatic interactions are (except at very short distances) attractive, and electrostatic interactions (between the charged atoms of the glutamate or water and the pore wall) are also attractive overall. Repulsive interactions (between equally charged atoms) exist, and they order the molecules near the wall, whereas in the pore center the glutamate (and water) angles are largely randomly distributed, except in the presence of an external electric field. Diffusion of molecules and ions is complex. The translational diffusion is in general both inhomogeneous and anisotropic. Non-electrostatic interactions (ion–wall, glutamate–wall or water–wall) powerfully influence diffusion. In the neutral nanopore the effective axial diffusion constants of glutamate, water and Na⁺ and Cl⁻ ions are all <10% of their values in the bulk, and the electrostatic interactions can reduce them further. Diffusion of molecules and ions is further reduced if the water concentration in the pore is low. Glutamate⁻ is slowed more than water, and ions are reduced the most especially co-ions. In conclusion the interfacial interactions influence the spatial distribution of glutamate, water and ions, and regulate powerfully, in a complex manner and over a very wide range their transport through nanosize pores.

Erratum to “Conformation of a double-membrane-spanning fragment of a G protein-coupled receptor: Effects of hydrophobic environment and pH” [Biochim. Biophys. Acta 1768 (2007) 1199–1210] by Aaron Kerman; V.S. Ananthanarayanan (pp. 2342-2342).

Erratum to “Conformational behavior of oxygenated mycobacterial mycolic acids from Mycobacterium bovis BCG” [Biochim. Biophys. Acta 1768 (2007) 1717–1726] by Masumi Villeneuve; Mizuo Kawai; Motoko Watanabe; Yutaka Aoyagi; Yukio Hitotsuyanagi; Koichi Takeya; Hiroaki Gouda; Shuichi Hirono; David E. Minnikin; Hiroo Nakahara (pp. 2343-2343).

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BBA - Biomembranes (v.1768, #9)