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BBA - Biomembranes (v.1798, #3)

Editorial Board (pp. i).

Reevaluation of the phospholipid composition in membranes of adult human lenses by³¹P NMR and MALDI MS by Rosendo Estrada; Andres Puppato; Douglas Borchman; M. Cecilia Yappert (pp. 303-311).

The phospholipid composition of adult human lens membranes differs dramatically from that of any other mammalian membrane. Due to minimal cell turnover, cells in the nucleus of the human lens may be considered as the longest lived cells in our body. This work reassesses previous assignments of phospholipid³¹P NMR resonances in adult human lenses. The new assignments are based not only on chemical shifts but also on temperature coefficients. By addition of known phospholipids and examination by matrix-assisted laser desorption/ionization mass spectrometry, several misassigned resonances have been corrected. The revised composition reveals the possible presence of ceramide-1-phosphate and dihydroceramide-1-phosphate. Among glycerophospholipids, the most abundant one does not correspond to phosphatidylglycerol but may be due to the lysoform of alkyl–acyl analogs of phosphatidylethanolamine. Besides sphingophospholipids, adult human lens membranes contain significant amounts of ether (1- O-alkyl) glycerophospholipids and their corresponding lysoforms.

Keywords: Human lens membrane; Phospholipid; ³¹; P NMR spectroscopy; Temperature coefficient; MALDI MS

¹⁵N Solid-state NMR spectroscopic studies on phospholamban at its phosphorylated form at Ser-16 in aligned phospholipid bilayers by Shidong Chu; Shadi Abu-Baker; Junxia Lu; Gary A. Lorigan (pp. 312-317).

Wild-type phospholamban (WT-PLB) is a pentameric transmembrane protein that regulates the cardiac cycle (contraction and relaxation). From a physiological prospective, unphosphorylated WT-PLB inhibits sarcoplasmic reticulum ATPase activity; whereas, its phosphorylated form relieves the inhibition in a mechanism that is not completely understood. In this study, site-specifically¹⁵N-Ala-11- and¹⁵N-Leu-7-labeled WT-PLB and the corresponding phosphorylated forms (P-PLB) were incorporated into 1,2-dioleoyl- sn-glycero-3-phosphocholine/2-dioleoyl- sn-glycero-3-phosphoethanolamine (DOPC/DOPE) mechanically oriented lipid bilayers. The aligned¹⁵N-labeled Ala-11 and Leu-7 WT-PLB samples show¹⁵N resonance peaks at approximately 71ppm and 75ppm, respectively, while the corresponding phosphorylated forms P-PLB show¹⁵N peaks at 92ppm and 99ppm, respectively. These¹⁵N chemical shift changes upon phosphorylation are significant and in agreement with previous reports, which indicate that phosphorylation of WT-PLB at Ser-16 alters the structural properties of the cytoplasmic domain with respect to the lipid bilayers.

Keywords: Abbreviations; CSA; chemical shift anisotropy; CP-MAS; Cross-polarization magic angle spinning; DOPC; 1,2-dioleoyl-; sn; -glycero-3-phosphocholine; DOPE; 1,2-dioleoyl-; sn; -glycero-3-phosphoethanolamine; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycerophosphocholine; POPE; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphoethanolamine; P-PLB; phosphorylated phospholamban; WT-PLB; wild-type phospholamban; SERCA; sarco (endo) plasmic reticulum calcium ATPase; MLVs; multilamellar vesiclesPhospholamban; Protein phosphorylation; Solid-state NMR; Mechanically oriented bilayer

Dynamic and energetic mechanisms for the distinct permeation rate in AQP1 and AQP0 by Hu Qiu; Shaojie Ma; Rong Shen; Wanlin Guo (pp. 318-326).

Despite sharing overall sequence and structural similarities, water channel aquaporin 0 (AQP0) transports water more slowly than other aquaporins. Using molecular dynamics simulations of AQP0 and AQP1, we find that there is a sudden decrease in the distribution profile of water density along the pore of AQP0 in the region of residue Tyr23, which significantly disrupts the single file water chain by forming hydrogen bond with permeating water molecules. Comparisons of free-energy and interaction-energy profiles for water conduction between AQP0 and AQP1 indicate that this interruption of the water chain causes a huge energy barrier opposing water translocation through AQP0. We further show that a mutation of Tyr23 to phenylalanine leads to a 2- to 4-fold enhancement in water permeability of AQP0, from (0.5±0.2)×10−¹⁴ cm³s−¹ to (1.9±0.6)×10−¹⁴ cm³s−¹. Therefore, Tyr23 is a dominate factor leading to the low water permeability in AQP0.

Keywords: Aquaporin; Molecular dynamics; Water conduction; Energetic mechanism

A membranotropic region in the C-terminal domain of Hepatitis C virus protein NS4B by Jaime Guillén; Alejandro González-Álvarez; José Villalaín ⁎ (pp. 327-337).

We have identified a membrane-active region in the HCV NS4B protein by studying membrane rupture induced by a NS4B-derived peptide library on model membranes. This segment corresponds to one of two previously predicted amphipathic helix and define it as a new membrane association domain. We report the binding and interaction with model membranes of a peptide patterned after this segment, peptide NS4B_H2, and show that NS4B_H2 strongly partitions into phospholipid membranes, interacts with them, and is located in a shallow position in the membrane. Furthermore, changes in the primary sequence cause the disruption of the hydrophobicity along the structure and prevent the resulting peptide from interacting with the membrane. Our results suggest that the region where the NS4B_H2 is located might have an essential role in the membrane replication and/or assembly of the viral particle through the modulation of the replication complex. Our findings therefore identify an important region in the HCV NS4B protein which might be implicated in the HCV life cycle and possibly in the formation of the membranous web.

Keywords: Abbreviations; BPI; Bovine brain; l; -α-phosphadidylinositol; BPS; Bovine brain; l; -α-phosphatidylserine; CF; 5-Carboxyfluorescein; Chol; Cholesterol; di-8-ANEPPS; 4-(2-(6-(Dioctylamino)-2-naphthalenyl)-(ethenyl)-1-(3-sulfopropyl)-pyridinium inner salt; DMPC; 1,2-Dimyristoyl-; sn; -glycero-phosphatidylcholine; DMPG; 1,2-Dimyristoyl-; sn; -glycero-phosphatidylglycerol; DMPS; 1,2-Dimyristoyl-; sn; -glycero-3-phosphatidylserine; DPH; 1,6-Diphenyl-1,3,5-hexatriene; DSC; Differential Scanning Calorimetry; EPA; Egg; l; -α-phosphatidic acid; EPC; Egg; l; -α-phosphatidylcholine; EPG; Egg; l; -α-phosphatidylglycerol; ER; Endoplasmic reticulum; FD10/20/70; Fluorescein isothiocyanate dextran with an average molecular weight of 10,000/20,000/70,000; HCV; Hepatitis C virus; LUV; Large unilamellar vesicles; MLV; Multilamellar vesicles; NS; Non-structural protein; SC1/2; Scrambled peptide 1/2; SM; Egg sphingomyelin; SUV; Small unilamellar vesicles; TFE; Trifluoroethanol; T; _m; Temperature of the gel-to-liquid crystalline phase transition; TM; Transmembrane domain; TMA-DPH; 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene; TPE; Egg trans-esterified; l; -α-phosphatidylethanolamineNS4B; HCV; Lipid–peptide interaction; Membranous web

Assessing the uptake kinetics and internalization mechanisms of cell-penetrating peptides using a quenched fluorescence assay by Imre Mäger; Emelía Eiríksdóttir; Kent Langel; Samir EL Andaloussi; Ülo Langel (pp. 338-343).

Cell-penetrating peptides (CPPs) have shown great potency for cargo delivery both in vitro and in vivo. Different biologically relevant molecules need to be delivered into appropriate cellular compartments in order to be active, for instance certain drugs/molecules, e.g. antisense oligonucleotides, peptides, and cytotoxic agents require delivery into the cytoplasm. Assessing uptake mechanisms of CPPs can help to develop novel and more potent cellular delivery vectors, especially in cases when reaching a specific intracellular target requires involvement of a specific internalization pathway. Here we measure the overall uptake kinetics, with emphasis on cytoplasmic delivery, of three cell-penetrating peptides M918, TP10 and pVec using a quenched fluorescence assay. We show that both the uptake levels and kinetic constants depend on the endocytosis inhibitors used in the experiments. In addition, in some cases only the internalization rate is affected by the endocytosis inhibitors while the total uptake level is not and vice versa, which emphasizes importance of kinetic studies when assessing the uptake mechanisms of CPPs. Also, there seems to be a correlation between lower total cellular uptake and higher first-order rate constants. Furthermore, this may indicate simultaneous involvement of different endocytic pathways with different efficacies in the internalization process, as hypothesized but not shown earlier in an uptake kinetics assay.

Keywords: Cell-penetrating peptide; Uptake kinetic; Uptake mechanism; Endocytosis inhibitor

Structural characterization of the transmembrane proximal region of the hepatitis C virus E1 glycoprotein by Roberta Spadaccini; Gerardino D'Errico; Viviana D'Alessio; Eugenio Notomista; Alessia Bianchi; Marcello Merola; Delia Picone (pp. 344-353).

A detailed knowledge of the mechanism of virus entry represents one of the most promising approaches to develop new therapeutic strategies. However, viral fusion is a very complex process involving fusion glycoproteins present on the viral envelope. In the two hepatitis C virus envelope proteins, E1 and E2, several membranotropic regions with a potential role in the fusion process have been identified. Among these, we have selected the 314–342 E1 region. Circular Dichroism data indicate that the peptide exhibits a clear propensity to adopt a helical folding in different membrane mimicking media, such as mixtures of water with fluorinated alcohols and phospholipids, with a slight preference for negative charged bilayers. The 3D structure of E1_314–342 peptide, calculated by 2D-NMR in a low-polarity environment, consists of two helical stretches encompassing residues 319–323 and 329–338 respectively. The peptide, presenting a largely apolar character, interacts with liposomes, as indicated by fluorescence and electron spin resonance spectra. The strength of the interaction and the deepness of peptide insertion in the phospholipid membrane are modulated by the bilayer composition, the interaction with anionic phospholipids being among the strongest ever observed. The presence of cholesterol also affects the peptide–bilayer interaction, favoring the peptide positioning close to the bilayer surface. Overall, the experimental data support the idea that this region of E1 might be involved in membrane destabilization and viral fusion; therefore it may represent a good target to develop anti-viral molecules.

Keywords: Abbreviations; CD; circular dichroism; CHOL; cholesterol; COSY; correlated spectroscopy; DLPC; dilauroyl phosphatidylcholine; DLPG; dilauroyl phosphatidylglycerol; DOPC; dioleoyl phosphatidylcholine; DOPG; dioleoyl phosphatidylglycerol; ESR; electron spin resonance; HCV; hepatitis C virus; HFIP; hexafluoroisopropanol; MLV; multilamellar vesicles; NMR; nuclear magnetic resonance; NOESY; nuclear Overhauser effect spectroscopy; n; -PCSL; spin-labeled phosphatidylcholine; SDS; sodium dodecyl sulfate; SUV; small unilammelar vesicle; TFE; trifluoroethanol; TM; transmembrane region of the viral glycoprotein; TOCSY; total correlation spectroscopyHCV; NMR; ESR; Pretransmembrane region; E1 glycoprotein

The stimulatory effect of angiotensin II on Na⁺-ATPase activity involves sequential activation of phospholipases and sustained PKC activity by Aloa Machado De Souza; Thaís Louise Gurjão de Carvalho; Lucienne da Silva Lara; Elaine Gomes-Quintana; Anibal Gil Lopes; Celso Caruso-Neves (pp. 354-359).

Angiotensin II (Ang II) stimulates the proximal tubule Na⁺-ATPase through the AT₁ receptor/phosphoinositide phospholipase Cβ (PI-PLCβ)/protein kinase C (PKC) pathway. However, this pathway alone does not explain the sustained effect of Ang II on Na⁺-ATPase activity for 30 min. The aim of the present work was to elucidate the molecular mechanisms involved in the sustained effect of Ang II on Na⁺-ATPase activity. Ang II induced fast and correlated activation of Na⁺-ATPase and PKC activities with the maximal effect (115%) observed at 1 min and sustained for 30 min, indicating a pivotal role of PKC in the modulation of Na⁺-ATPase by Ang II. We observed that the sustained activation of PKC by Ang II depended on the sequential activation of phospholipase D and Ca²⁺-insensitive phospholipase A₂, forming phosphatidic acid and lysophosphatidic acid, respectively. The results indicate that PKC could be the final target and an integrator molecule of different signaling pathways triggered by Ang II, which could explain the sustained activation of Na⁺-ATPase by Ang II.

Keywords: Angiotensin II; Phospholipase D; Phospholipase A; ₂; Protein kinase C; Renal epithelium; Cellular signaling

Na⁺-ATPase in spontaneous hypertensive rats: Possible AT₁ receptor target in the development of hypertension by Eugênio P. Queiroz-Madeira; Lucienne S. Lara; Mira Wengert; Sharon S. Landgraf; Janaína D. Líbano-Soares; Gisele Zapata-Sudo; Roberto T. Sudo; Christina M. Takiya; Elaine Gomes-Quintana; Anibal G. Lopes; Celso Caruso-Neves ⁎ (pp. 360-366).

Clinical and experimental data show an increase in sodium reabsorption on the proximal tubule (PT) in essential hypertension. It is well known that there is a link between essential hypertension and renal angiotensin II (Ang II). The present study was designed to examine ouabain-insensitive Na⁺-ATPase activity and its regulation by Ang II in spontaneously hypertensive rats (SHR). We observed that Na⁺-ATPase activity was enhanced in 14-week-old but not in 6-week-old SHR. The addition of Ang II from 10−¹² to 10−⁶ mol/L decreased the enzyme activity in SHR to a level similar to that obtained in WKY. The Ang II inhibitory effect was completely reversed by a specific antagonist of AT₂ receptor, PD123319 (10−⁸ mol/L) indicating that a system leading to activation of the enzyme in SHR is inhibited by AT₂-mediated Ang II. Treatment of SHR with losartan for 10 weeks (weeks 4–14) prevents the increase in Na⁺-ATPase activity observed in 14-week-old SHR. These results indicate a correlation between AT₁ receptor activation in SHR and increased ouabain-insensitive Na⁺-ATPase activity. Our results open new possibilities towards our understanding of the pathophysiological mechanisms involved in the increased sodium reabsorption in PT found in essential hypertension.

Keywords: Renal sodium excretion; Cell signaling; Angiotensin receptor; Second sodium pump

N-glycans modulate K_v1.5 gating but have no effect on K_v1.4 gating by Tara A. Schwetz; Sarah A. Norring; Eric S. Bennett (pp. 367-375).

Nerve and muscle action potential repolarization are produced and modulated by the regulated expression and activity of several types of voltage-gated K⁺ (K_v) channels. Here, we show that sialylated N-glycans uniquely impact gating of a mammalian Shaker family K_v channel isoform, K_v1.5, but have no effect on gating of a second Shaker isoform, K_v1.4. Each isoform contains one potential N-glycosylation site located along the S1–S2 linker; immunoblot analyses verified that K_v1.4 and K_v1.5 were N-glycosylated. The conductance–voltage (G–V) relationships and channel activation rates for two glycosylation-site deficient K_v1.5 mutants, K_v1.5^N290Q and K_v1.5^S292A, and for wild-type K_v1.5 expressed under conditions of reduced sialylation, were each shifted linearly by a depolarizing ∼18 mV compared to wild-type K_v1.5 activation. External divalent cation screening experiments suggested that K_v1.5 sialic acids contribute to an external surface potential that modulates K_v1.5 activation. Channel availability was unaffected by changes in K_v1.5 glycosylation or sialylation. The data indicate that sialic acid residues attached to N-glycans act through electrostatic mechanisms to modulate K_v1.5 activation. The sialic acids fully account for effects of N-glycans on K_v1.5 gating. Conversely, K_v1.4 gating was unaffected by changes in channel sialylation or following mutagenesis to remove the N-glycosylation site. Each phenomenon is unique for K_v1 channel isoforms, indicating that sialylated N-glycans modulate gating of homologous K_v1 channels through isoform-specific mechanisms. Such modulation is relevant to changes in action potential repolarization that occur as ion channel expression and glycosylation are regulated.

Keywords: Kv1; Sialic acid; Glycosylation; Potassium channel; Ion channel gating

A calorimetric and spectroscopic comparison of the effects of ergosterol and cholesterol on the thermotropic phase behavior and organization of dipalmitoylphosphatidylcholine bilayer membranes by David A. Mannock; Ruthven N.A.H. Lewis; Ronald N. McElhaney (pp. 376-388).

We performed comparative DSC and FTIR spectroscopic measurements of the effects of cholesterol (Chol) and ergosterol (Erg) on the thermotropic phase behavior and organization of DPPC bilayers. Ergosterol is the major sterol in the biological membranes of yeasts, fungi and many protozoa. It differs from Chol in having two additional double bonds, one in the steroid nucleus at C7–8 and another in the alkyl chain at C22–23. Erg also has an additional methyl group in the alkyl chain at C24. Our DSC studies indicate that the incorporation of Erg is more effective than Chol is in reducing the enthalpy of the pretransition. At lower concentrations Erg is also more effective than Chol in reducing the enthalpies of both the sharp and broad components of main phase transition. However, at sterol concentrations from 30 to 50 mol%, Erg is generally less effective at reducing the enthalpy of the broad components and does not completely abolish the cooperative hydrocarbon chain-melting phase transition at 50 mol%, as does Chol. Nevertheless, in this higher ergosterol concentration range, there is no evidence of the formation of ergosterol crystallites. Our FTIR spectroscopic studies demonstrate that Erg incorporation produces a similar ordering of liquid-crystalline DPPC bilayers as does Chol, but an increased degree of hydrogen bonding of the fatty acyl carbonyl groups in the glycerol backbone region of the DPPC bilayer. These and other results indicate that Erg is less miscible in DPPC bilayers at higher concentrations than is Chol. Finally, we provide a tentative molecular explanation for the comparative experimental and computation results obtained for Erg and Chol in phospholipid bilayers, emphasizing the dynamic conformational differences between these two sterols.

Keywords: Abbreviations; PC; phosphatidylcholine; DPPC; dipalmitoylphosphatidylcholine; DMPC; dimyristoylphosphatidylcholine; POPC; 1-palmitoyl-2-oleoyl-phosphatidylcholine; DEPC; dielaidoylphosphatidylcholine; SpM; sphingomyelin; Chol; cholesterol; Erg; ergosterol; Lano; lanosterol; DSC; differential scanning calorimetry; FTIR; Fourier transform infrared; NMR; nuclear magnetic resonance; T; _p; the pretransition temperature maximum; T; _m; the main transition temperature maximum; Δ; H; the transition enthalpy; ΔT; _½; the width of the phase transition at half height, inversely related to the cooperativity of the phase transition; L; _β′; and L; _β; lamellar gel phases with tilted and untilted hydrocarbon chains, respectively; P; _β′; rippled gel phase with tilted hydrocarbon chains; L; _α; or L; _d; lamellar liquid-crystalline or liquid-disordered phase; L; _O; lamellar liquid-ordered phase; Erg-BC1 and Erg-BC2; first and second broad endotherm components of the ergosterol-DPPC DSC thermogramsErgosterol; Cholesterol; Dipalmitoylphosphatidylcholine; Thermotropic phase behavior; Sterol-lipid interactions; Lipid bilayer; Lipid membranes; Differential scanning calorimetry; Fourier transform infrared spectroscopy

Cell membrane extensions, generated by mechanical constraint, are associated with a sustained lipid raft patching and an increased cell signaling by Romain M. Larive; Laurent Baisamy; Serge Urbach; Peter Coopman; Nadir Bettache (pp. 389-400).

Platelet activation triggers an imbalance in plasma membrane phospholipids by a specific aminophospholipid outflux, resulting in filopodia formation. Similarly, the addition of a phospholipid excess in the outer leaflet of the plasma membrane induces cellular extensions and actin polymerization. The implication of membrane microdomains in sustaining these mechanical constraints remains, however, unknown and was investigated in human platelets and mouse fibroblasts. The disruption of lipid rafts by cholesterol depletion prevents actin polymerization and formation of cellular extensions. Phospholipid excess triggers raft patching underneath the cell extensions, recruitment of protein raft markers and increase of tyrosine phosphorylation of raft proteins. Using a mass spectrometric analysis of isolated platelet rafts, we identified tyrosine kinases and proteins implicated in the formation of cell membrane extensions, cell adhesion and motility. They are recruited to rafts in response to a mechanical constraint. Taken together, our results demonstrate that exogenous phospholipid addition causes a modulation of the lateral plasma membrane organization and an activation of the cell signaling triggering actin remodeling and the formation of cellular protrusions. Raft disruption abolishes these processes, demonstrating that their integrity is crucial for cell shape changes in response to a mechanical constraint on plasma membrane.

Keywords: Abbreviations; PS; phosphatidylserine; PC; phosphatidylcholine; PE; phosphatidylethanolamine; PL; phospholipids; MβCD; methyl beta cyclodextrin; DRMs; detergent-resistant membranesLipid rafts; Platelets; Phospholipids; Filopodia; Actin; Mechanical constraint; Cell signaling

Interaction with membrane mimics of transmembrane fragments 16 and 17 from the human multidrug resistance ABC transporter 1 (hMRP1/ABCC1) and two of their tryptophan variants by Béatrice de Foresta; Michel Vincent; Jacques Gallay; Manuel Garrigos (pp. 401-414).

The human multidrug resistance-associated protein 1 (hMRP1/ABCC1) belongs to the ATP-binding cassette transporter superfamily. Together with P-glycoprotein (ABCB1) and the breast cancer resistance protein (BCRP/ABCG2), hMRP1 confers resistance to a large number of structurally diverse drugs. The current topological model of hMRP1 includes two cytosolic nucleotide-binding domains and 17 putative transmembrane (TM) helices forming three membrane-spanning domains. Mutagenesis and labeling studies have shown TM16 and TM17 to be important for function. We characterized the insertion of the TM16 fragment into dodecylphosphocholine (DPC) or n-dodecyl-β-d-maltoside (DM) micelles as membrane mimics and extended our previous work on TM17 (Vincent et al., 2007, Biochim. Biophys. Acta 1768, 538). We synthesized TM16 and TM17, with the Trp residues, W1198 in TM16 and W1246 in TM17, acting as an intrinsic fluorescent probe, and TM16 and TM17 Trp variants, to probe different positions in the peptide sequence. We assessed the interaction of peptides with membrane mimics by evaluating the increase in fluorescence intensity resulting from such interactions. In all micelle-bound peptides, the tryptophan residue appeared to be located, on average, in the head group micelle region, as shown by its fluorescence spectrum. Each tryptophan residue was partially accessible to both acrylamide and the brominated acyl chains of two DM analogs, as shown by fluorescence quenching. Tryptophan fluorescence lifetimes were found to depend on the position of the tryptophan residue in the various peptides, probably reflecting differences in local structures. Far UV CD spectra showed that TM16 contained significant β-strand structures. Together with the high Trp correlation times, the presence of these structures suggests that TM16 self-association may occur at the interface. In conclusion, this experimental study suggests an interfacial location for both TM16 and TM17 in membrane mimics. In terms of overall hMRP1 structure, the experimentally demonstrated amphipathic properties of these TM are consistent with a role in the lining of an at least partly hydrophilic transport pore, as suggested by the currently accepted structural model, the final structure being modified by interaction with other TM helices.

Keywords: Abbreviations; hMRP1 (or ABCC1); human multidrug resistance protein 1; BCRP (or ABCG2); breast cancer resistance protein; LTC; ₄; cysteinyl leukotriene C; ₄; E; ₂; 17βG; estradiol 17-(β-; d; -glucuronide); GSH; reduced glutathione; DM; n; -dodecyl-β-; d; -maltoside; BrDM; 7, 8-dibromododecylmaltoside; BrUM; 10, 11-dibromoundecanoylmaltoside; DPC; dodecylphosphocholine; cmc; critical micellar concentration; NATA; N; -acetyltryptophanamide; TOE; tryptophan octyl ester; DMSO; dimethylsulfoxide; TFE; trifluoroethanol; TFA; trifluoroacetic acid; MSD; membrane-spanning domain; TM; transmembrane fragment; NBD; nucleotide-binding domain; MEM; maximum entropy method; CD; circular dichroism; FWHM; full-width at half-maximum; P3; K; ₂; WL; ₉; AL; ₉; K; ₂; A; P5; K; ₂; CLWL; ₇; AL; ₉; K; ₂; A; P7; K; ₂; CL; ₃; WL; ₅; AL; ₉; K; ₂; A; P9; K; ₂; CL; ₅; WL; ₃; AL; ₉; K; ₂; A; P11; K; ₂; CL; ₇; WLAL; ₉; K; ₂; A; P13; K; ₂; CL; ₉; WL; ₉; K; ₂; A; TM16; A; ₁₁₉₅; NRWLAVRLECVGNCIVLFAALFAV; ₁₂₁₉; mTM16; A; ₁₁₉₅; NRWLAVRLESVGNSIVLFAALFAV; ₁₂₁₉; W19-mTM16; A; ₁₁₉₅; NRYLAVRLESVGNSIVLWAALFAV; ₁₂₁₉; TM17; A; ₁₂₂₇; GLVGLSVSYSLQVTTYLNWLVRMS; ₁₂₅₁; mTM17; K; ₁₂₂₇; GLVGLSVSYSLQVTTYLNWLVRMS; ₁₂₅₁; W10-mTM17; K; ₁₂₂₇; GLVGLSVSWSLQVTTYLNYLVRMS; ₁₂₅₁hMRP1 (ABCC1); Multidrug resistance; TM fragments; Steady-state and time-resolved fluorescence; Brominated detergents; Dodecylmaltoside; Dodecylphosphocholine micelles

Extracellular Mg²⁺ regulates the tight junctional localization of claudin-16 mediated by ERK-dependent phosphorylation by Akira Ikari; Keishi Kinjo; Kosuke Atomi; Yohei Sasaki; Yasuhiro Yamazaki; Junko Sugatani (pp. 415-421).

Claudin-16 is involved in the paracellular reabsorption of Mg²⁺ in the thick ascending limb of Henle. Little is known about the mechanism regulating the tight junctional localization of claudin-16. Here, we examined the effect of Mg²⁺ deprivation on the distribution and function of claudin-16 using Madin-Darby canine kidney (MDCK) cells expressing FLAG-tagged claudin-16. Mg²⁺ deprivation inhibited the localization of claudin-16 at tight junctions, but did not affect the localization of other claudins. Re-addition of Mg²⁺ induced the tight junctional localization of claudin-16, which was inhibited by U0126, a MEK inhibitor. Transepithelial permeability to Mg²⁺ was also inhibited by U0126. The phosphorylation of ERK was reduced by Mg²⁺ deprivation, and recovered by re-addition of Mg²⁺. These results suggest that the MEK/ERK-dependent phosphorylation of claudin-16 affects the tight junctional localization and function of claudin-16. Mg²⁺ deprivation decreased the phosphothreonine levels of claudin-16. The phosphothreonine levels of T225A and T233A claudin-16 were decreased in the presence of Mg²⁺ and these mutants were widely distributed in the plasma membrane. Furthermore, TER and transepithelial Mg²⁺ permeability were decreased in the mutants. We suggest that the tight junctional localization of claudin-16 requires a physiological Mg²⁺ concentration and the phosphorylation of threonine residues via a MEK/ERK-dependent pathway.

Keywords: Abbreviations; H-89; N; -[2-(; p; -bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride; MDCK; Madin-Darby canine kidney; PKA; protein kinase A; TAL; thick ascending limb of Henle; TER; transepithelial electrical resistanceClaudin-16; ERK; Phosphorylation

Interactions at the bilayer interface and receptor site induced by the novel synthetic pyrrolidinone analog MMK3 by C. Fotakis; S. Gega; E. Siapi; C. Potamitis; K. Viras; P. Moutevelis-Minakakis; C.G. Kokotos; S. Durdagi; S. Golic Grdadolnik; B. Sartori; M. Rappolt; T. Mavromoustakos (pp. 422-432).

This work presents a thorough investigation of the interaction of the novel synthetic pyrrolidinone analog MMK3 with the model membrane system of dipalmitoylphosphatidylcholine (DPPC) and the receptor active site. MMK3 has been designed to exert antihypertensive activity by functioning as an antagonist of the angiotensin II receptor of subtype 1 (AT₁). Its low energy conformers were characterized by 2D rotating-frame Overhauser effect spectroscopy (ROESY) in combination with molecular dynamics (MD) simulations. Docking study of MMK3 shows that it fits to the AT₁ receptor as SARTANs, however, its biological activity appears to be lower. Thus, differential scanning calorimetry (DSC), Raman spectroscopy and small angle X-ray scattering (SAXS) experiments on the interaction of MMK3 with DPPC bilayers were carried out and results demonstrate that the drug is well incorporated into the membrane leaflets and furthermore causes partial bilayer interdigitation, although less effective than SARTANs. Thus, it appears that the nature of the bilayer matrix and the stereoelectronic active site requirements of the receptor are responsible for the low bioactivity of MMK3.

Keywords: Partial interdigitation; Lipid bilayer; MMK3; AT1 receptor; Dipalmitoylphosphatidylcholine

Design of peptide-targeted liposomes containing nucleic acids by Adriana O. Santos; Lígia C. Gomes da Silva; Luís M. Bimbo; Maria C. Pedroso de Lima; Sérgio Simões; João N. Moreira (pp. 433-441).

Anticancer systemic gene silencing therapy has been so far limited by the inexistence of adequate carrier systems that ultimately provide an efficient intracellular delivery into target tumor cells. In this respect, one promising strategy involves the covalent attachment of internalizing-targeting ligands at the extremity of PEG chains grafted onto liposomes. Therefore, the present work aims at designing targeted liposomes containing nucleic acids, with small size, high encapsulation efficiency and able to be actively internalized by SCLC cells, using a hexapeptide (antagonist G) as a targeting ligand. For this purpose, the effect of the liposomal preparation method, loading material (ODN versus siRNA) and peptide-coupling procedure (direct coupling versus post-insertion) on each of the above-mentioned parameters was assessed. Post-insertion of DSPE-PEG-antagonist G conjugates into preformed liposomes herein named as stabilized lipid particles, resulted in targeted vesicles with a mean size of about 130 nm, encapsulation efficiency close to 100%, and a loading capacity of approximately 5 nmol siRNA/μmol of total lipid. In addition, the developed targeted vesicles showed increased internalization in SCLC cells, as well as in other tumor cells and HMEC-1 microvascular endothelial cells. The improved cellular association, however, did not correlate with enhanced downregulation of the target protein (Bcl-2) in SCLC cells. These results indicate that additional improvements need to be performed in the future, namely by ameliorating the access of the nucleic acids to the cytoplasm of the tumor cells following receptor-mediated endocytosis.

Keywords: Abbreviations; ODN; oligodeoxynucleotide; SCLC; small cell lung cancer; siRNA; small interfering RNA; PEG; poly(ethylene glycol); CCL; coated cationic liposomes; SALP; stabilized antisense lipid particles; SNALP; stabilized nucleic acid lipid particles; EGF; epidermal growth factor; PFA; paraformaldehyde; NaN; ₃; sodium azide; AD; actinomycin D; 7-AAD; 7-aminoactinomycin D; BSA; bovine serum albumin; C12E8; octaethylene glycol monododecyl ether; HSPC; hydrogenated soy phosphatidylcholine; DSPC; 1,2-distearoyl-; sn; -glycero-3-phosphocholine; CerC; ₁₆; -PEG; N-palmitoyl-sphingosine-1-succinyl(methoxypolyethylene glycol); ₂₀₀₀; DSPE-PEG; N-palmitoyl-sphingosine-1-[succinyl(polyethylene glycol)]; ₂₀₀₀; DSPE-PEG-Mal; 1,2-distearoyl-; sn; -glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)]; ₂₀₀₀; rhodamine-PE; L-α-phosphatidylethanolamine-N-(lissamine rhodamine B sulfonyl); DOTAP; 1,2-dioleyl-3-trimethylammonium-propane; DODAP; 1,2-dioleyl-3-dimethylammonium-propane; MES; 2-(n-morpholino)ethanesulfonic acid; Tris; 2-amino-2-(hydroxymethyl)propane-1,3-diol; HEPES; 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid; CHOL; cholesterol; MBS; MES buffered saline; HBS; HEPES buffered saline; SLP; stabilized lipid particles; SLP; ^ODN; SLP encapsulating ODN; SLP; ^siRNA; SLP encapsulating siRNALiposomes; Antagonist G; Targeted delivery; Oligodeoxynucleotide; siRNA; Cancer

Oxidatively modified fatty acyl chain determines physicochemical properties of aggregates of oxidized phospholipids by Abhay H. Pande; Subhabrata Kar; Rajan K. Tripathy (pp. 442-452).

In vivo oxidation of glycerophospholipid generates a variety of products including truncated oxidized phospholipids (tOx-PLs). The fatty acyl chains at the sn-2 position of tOx-PLs are shorter in length than the parent non-oxidized phospholipids and contain a polar functional group(s) at the end. The effect of oxidatively modified sn-2 fatty acyl chain on the physicochemical properties of tOx-PLs aggregates has not been addressed in detail, although there are few reports that modified fatty acyl chain primarily determines the biological activities of tOx-PLs. In this study we have compared the properties of four closely related tOx-PLs which differ only in the type of modified fatty acyl chain present at the sn-2 position: 1-palmitoyl-2-azelaoyl- sn-glycero-3-phosphocholine (PazePC), 1-palmitoyl-2-(9′-oxo-nonanoyl)- sn-glycero-3-phosphocholine (PoxnoPC), 1-palmitoyl-2-glutaroyl- sn-glycero-3-phosphocholine (PGPC), and 1-palmitoyl-2-(5′-oxo-valeroyl)- sn-glycero-3-phosphocholine (POVPC). Aggregates of individual tOx-PL in aqueous solution were characterized by fluorescence spectroscopy, size exclusion chromatography, native polyacrylamide and agarose gel electrophoresis. The data suggest that aggregates of four closely related tOx-PLs form micelle-like particles of considerably different properties. Our result provides first direct evidence that because of the specific chemical composition of the sn-2 fatty acyl chain aggregates of particular tOx-PL possess a distinctive set of physicochemical properties.

Keywords: Abbreviations; ANS; 8-anilinonaphthalene-1-sulfonate; CMC; critical micellar concentration; DPH; 1,6-diphenyl-1,3,5-hexatriene; Laurdan; 2-dimethylamino-(6-lauroyl)-naphthalene; NBD-DMPG; 7-nitrobenz-2-oxa-1,3-diazol-4-yl labeled DMPG; PazePC; 1-palmitoyl-2-azelaoyl-; sn; -glycero-3-phosphocholine; PoxnoPC; 1-palmitoyl-2-(9′-oxo-nonanoyl)-; sn; -glycero-3-phosphocholine; PGPC; 1-palmitoyl-2-glutaroyl-; sn; -glycero-3-phosphocholine; POVPC; 1-palmitoyl-2-(5′-oxo-valeroyl)-; sn; -glycero-3-phosphocholine; tOx-PLs; oxidized phospholipidsCritical micelle concentration; Microfluidity; Micropolarity; Anisotropy; Generalized polarization; Stokes diameter; Surface potential

Characterization of membrane properties of inositol phosphorylceramide by Anders Björkbom; Henna Ohvo-Rekilä; Pasi Kankaanpää; Thomas K.M. Nyholm; Bodil Westerlund; J. Peter Slotte (pp. 453-460).

Inositol phosphorylceramides (IPCs) are a class of anionic sphingolipids with a single inositol-phosphate head group coupled to ceramide. IPCs and more complex glycosylated IPCs have been identified in fungi, plants and protozoa but not in mammals. IPCs have also been identified in detergent resistant membranes in several organisms. Here we report on the membrane properties of the saturated N-palmitoyl-IPC (P-IPC) in one component bilayers as well as in complex bilayers together with 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine and cholesterol. The membrane properties of P-IPC were shown to be affected by calcium. According to anisotropy changes reported by DPH, the gel-to-liquid transition temperature ( T_m) of P-IPC was 48 °C. Addition of 5 mM CaCl₂ during vesicle preparation markedly increased the T_m (65 °C). According to fluorescence quenching experiments in complex lipid mixtures, P-IPC formed sterol containing domains in an otherwise fluid environment. The P-IPC containing domains melted at a lower temperature and appeared to contain less sterol as compared to domains containing N-palmitoyl-sphingomyelin. Calcium further reduced the sterol content of the ordered domains and also increased the thermal stability of the domains. Calcium also induced vesicle aggregation of unilamellar vesicles containing P-IPC, as was observed by 4D confocal microscopy and dynamic light scattering. We believe that IPCs and the calcium induced effects could be important in numerous membrane associated cellular processes such as membrane fusion and in membrane raft linked processes.

Keywords: Abbreviations; 7SLPC; 1-palmitoyl-2-stearoyl-(7-doxyl)-; sn; -glycero-3-phosphocholine; CTL; cholesta-5,7,9(11)-trien-3-beta-ol; DPH; 1,6-diphenyl-1,3,5-hexatriene; DPPG; 1,2-dipalmitoyl-; sn; -glycero-3-phosphoglycerol; IPC; inositol-phosphorylceramide; PI; L-α-phosphatidylinostol (Liver, Bovine); P-IPC; d; -erythro-; N; -palmitoyl-inositol-phosphorylceramide; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; PSM; d; -; erythro; -; N; -palmitoyl-sphingomyelin; T; _m; transition temperature between gel and fluid phasesSphingolipid; Ordered domain; Calcium effect; Vesicle aggregation; Cholesterol

Structural and functional changes in a synthetic S5 segment of KvLQT1 channel as a result of a conserved amino acid substitution that occurs in LQT1 syndrome of human by Richa Verma; Jimut Kanti Ghosh (pp. 461-470).

Mutations in various voltage gated cardiac ion channels are the cause of different forms of long QT syndrome (LQTS), which is an inherited arrhythmic disorder marked as a prolonged QT interval on electrocardiogram. Of these LQTS1 is associated with mutations in the gene encoding KCNQ1 (KvLQT1) channel. One responsible mutation, G269S, in the S5 segment of KvLQT1, that affects the proper expression and function of channel protein leads to LQTS1. Our objective was to study how G269S mutation interferes with the structure and function of a synthetic S5 segment of KvLQT1 channel. One wild type 22-residue peptide and another mutant peptide of the same length with G269S mutation, derived from the S5 segment were synthesized and labeled with fluorescent probes. The mutant peptide exhibited lower affinity towards phospholipid vesicles as compared to the wild type peptide and showed impaired assembly and localization onto the lipid vesicles as evidenced by membrane-binding, energy transfer and proteolytic cleavage experiments. Loss in the helical content of S5 mutant peptide in membrane-mimetic environments was observed. Furthermore, it was observed that G269S mutation significantly inhibited the ability of S5 peptide to permeabilize the lipid vesicles. The present studies show the basis of change in function of the selected S5 segment as a result of G269S mutation which is associated with LQT1 syndrome. We speculate that the structural and functional changes related to the glycine to serine amino acid substitution in the S5 segment may also influence the activity of the whole KvLQT1 channel.

Keywords: Abbreviations; CD; circular dichroism; Fmoc; N; -(9-fluorenyl)methoxycarbonyl; HPLC; high performance liquid chromatography; Kv; voltage gated potassium channels; LQTS; long QT syndrome; NBD; 7-nitrobenz-2-oxa-1,3-diazole; PBS; phosphate buffered saline (pH 7.4); PC; phosphatidylcholine; PG; phosphatidylglycerol; Rho; tetramethylrhodamine; LUVs; large unilamellar vesicles; TFE; trifluoroethanol; TM; transmembraneLong QT syndrome; LQTS1 associated mutation; Voltage gated potassium channel; Cardiac voltage gated potassium channel—KvLQT1; Peptide–membrane interaction; Assembly of synthetic S5; Pore forming activity of S5 peptide in phospholipid vesicle

Sphingomyelin-rich domains are sites of lysenin oligomerization: Implications for raft studies by Magdalena Kulma; Monika Hereć; Wojciech Grudziński; Gregor Anderluh; Wiesław I. Gruszecki; Katarzyna Kwiatkowska; Andrzej Sobota (pp. 471-481).

Lysenin is a self-assembling, pore-forming toxin which specifically recognizes sphingomyelin. Mutation of tryptophan 20 abolishes lysenin oligomerization and cytolytic activity. We studied the interaction of lysenin WT and W20A with sphingomyelin in membranes of various lipid compositions which, according to atomic force microscopy studies, generated either homo- or heterogeneous sphingomyelin distribution. Liposomes composed of SM/DOPC, SM/DOPC/cholesterol and SM/DPPC/cholesterol could bind the highest amounts of GST-lysenin WT, as shown by surface plasmon resonance analysis. These lipid compositions enhanced the release of carboxyfluorescein from liposomes induced by lysenin WT, pointing to the importance of heterogeneous sphingomyelin distribution for lysenin WT binding and oligomerization. Lysenin W20A bound more weakly to sphingomyelin-containing liposomes than did lysenin WT. The same amounts of lysenin W20A bound to sphingomyelin mixed with either DOPC or DPPC, indicating that the binding was not affected by sphingomyelin distribution in the membranes. The mutant lysenin had a limited ability to penetrate hydrophobic region of the membrane as indicated by measurements of surface pressure changes. When applied to detect sphingomyelin on the cell surface, lysenin W20A formed large conglomerates on the membrane, different from small and regular clusters of lysenin WT. Only lysenin WT recognized sphingomyelin pool affected by formation of raft-based signaling platforms. During fractionation of Triton X-100 cell lysates, SDS-resistant oligomers of lysenin WT associated with membrane fragments insoluble in Triton X-100 while monomers of lysenin W20A partitioned to Triton X-100-soluble membrane fractions. Altogether, the data suggest that oligomerization of lysenin WT is a prerequisite for its docking in raft-related domains.

Keywords: Raft; Pore-forming toxin; Sphingomyelin; Plasma membrane; Protein oligomerization

Assessing the size, stability, and utility of isotropically tumbling bicelle systems for structural biology by Hongwei Wu; Kai Su; Xudong Guan; M. Elizabeth Sublette; Ruth E. Stark (pp. 482-488).

Aqueous phospholipid mixtures that form bilayered micelles (bicelles) have gained wide use by molecular biophysicists during the past 20 years for spectroscopic studies of membrane-bound peptides and structural refinement of soluble protein structures. Nonetheless, the utility of bicelle systems may be compromised by considerations of cost, chemical stability, and preservation of the bicelle aggregate organization under a broad range of temperature, concentration, pH, and ionic strength conditions. In the current work,³¹P nuclear magnetic resonance (NMR) and atomic force microscopy (AFM) have been used to monitor the size and morphology of isotropically tumbling small bicelles formed by mixtures of 1,2-dimyristoyl- sn-glycero-3-phosphocholine (DMPC) or 1,2-di-O-tetradecyl- sn-glycero-3-phosphocholine (DIOMPC) with either 1,2-dihexanoyl- sn-glycero-3-phosphocholine (DHPC) or 1,2-di-O-hexyl- sn-glycero-3-phosphocholine (DIOHPC), testing their tolerance of variations in commonly used experimental conditions.¹H-¹⁵N 2D NMR has been used to demonstrate the usefulness of the robust DMPC–DIOHPC system for conformational studies of a fatty acid-binding protein that shuttles small ligands to and from biological membranes.

Keywords: Bicelle; Phospholipid; Membrane mimetic; NMR; ³¹; P NMR; 2D NMR; AFM

Restoring the activity of serum-inhibited bovine lung extract surfactant (BLES) using cationic additives by Edgar J. Acosta; Zdenka Policova; Simon Lee; Andrew Dang; Michael L. Hair; A. Wilhelm Neumann (pp. 489-497).

In this work four cationic additives were used to improve the surface activity of lung surfactants, particularly in the presence of bovine serum that was used as a model surfactant inhibitor. Two of those additives were chitosan in its soluble hydrochloride form with average molecular weights of 113kDa and 213kDa. The other two additives were cationic peptides, polylysine 50kDa and polymyxin B. These additives were added to bovine lipid extract surfactant (BLES) and the optimal additive–surfactant ratio was determined based on the minimum surface tension upon dynamic compression, carried out in a constrained sessile drop (CSD) device in the presence of 50μl/ml serum. At the optimal ratio all the BLES-additive mixtures were able to achieve desirable minimum surface tensions. The optimal additive–surfactant ratios for the chitosan chlorides are consistent with a previously proposed patch model for the binding of the anionic lipids in BLES to the positive charges in chitosan. For the peptides, the optimal binding ratios were consistent with ratios established previously for the binding of these peptides to monolayers of anionic lipids. The optimal formulation containing these peptides were able to reach low minimum surface tension in systems containing 500μl/ml of serum, matching the effectiveness of a lung surfactant extract that had not undergone post-separation processes and therefore contained all its proteins and lipids (complete lung surfactant).

Keywords: Surfactant; Dysfunction; Cationic peptide; Surface tension; Inhibition

Micron-scale phase segregation in lipid monolayers induced by myelin basic protein in the presence of a cholesterol analog by C.M. Rosetti; B. Maggio; N. Wilke (pp. 498-505).

It was previously shown that myelin basic protein (MBP) can induce phase segregation in whole myelin monolayers and myelin lipid films, which leads to the accumulation of proteins into a separate phase, segregated from a cholesterol-enriched lipid phase. In this work we investigated some factors regulating the phase segregation induced by MBP using fluorescent microscopy of monolayers formed with binary and ternary lipid mixtures of dihydrocholesterol (a less-oxidable cholesterol analog) and phospholipids. The influence of the addition of salts to the subphase and of varying the lipid composition was analyzed. Our results show that MBP can induce a dihydrocholesterol-dependent segregation of phases that can be further regulated by the electrolyte concentration in the subphase and the composition (type and proportion) of non-sterol lipids. In this way, changes of the lipid composition of the film or the ionic strength in the aqueous media modify the local surface density of MBP and the properties (phase state and composition) of the protein environment.

Keywords: Myelin monolayer; Myelin basic protein; Cholesterol; Phase diagram; Phase segregation

Differential effects of conjugated linoleic acid isomers on the biophysical and biochemical properties of model membranes by Papasani V. Subbaiah; Debajit Sircar; Buzulagu Aizezi; Evan Mintzer (pp. 506-514).

Conjugated linoleic acids (CLA) are known to exert several isomer-specific biological effects, but their mechanisms of action are unclear. In order to determine whether the physicochemical effects of CLA on membranes play a role in their isomer-specific effects, we synthesized phosphatidylcholines (PCs) with 16:0 at sn-1 position and one of four CLA isomers ( trans 10 cis 12 (A), trans 9 trans 11 (B), cis 9 trans 11 (C), and cis 9 cis 11 (D)) at sn-2, and determined their biophysical properties in monolayers and bilayers. The surface areas of the PCs with the two natural CLA (A and C) were similar at all pressures, but they differed significantly in the presence of cholesterol, with PC-A condensing more than PC-C. Liposomes of PC-A similarly showed increased binding of cholesterol compared to PC-C liposomes. PC-A liposomes were less permeable to carboxyfluorescein compared to PC-C liposomes. The PC with two trans double bonds (B) showed the highest affinity to cholesterol and lowest permeability. The two natural CLA-PCs (A and C) stimulated lecithin-cholesterol acyltransferase activity by 2-fold, whereas the unnatural CLA-PCs (B and D) were inhibitory. These results suggest that the differences in the biophysical properties of CLA isomers A and C may partly contribute to the known differences in their biological effects.

Keywords: Conjugated linoleic acid; Phosphatidylcholine; Monolayer property; Membrane fluidity; Cholesterol-binding; Membrane permeability

P-glycoprotein substrate transport assessed by comparing cellular and vesicular ATPase activity by Pierluigi Nervi; Xiaochun Li-Blatter; Päivi Äänismaa; Anna Seelig (pp. 515-525).

We compared the P-glycoprotein ATPase activity in inside–out plasma membrane vesicles and living NIH-MDR1-G185 cells with the aim to detect substrate transport. To this purpose we used six substrates which differ significantly in their passive influx through the plasma membrane. In cells, the cytosolic membrane leaflet harboring the substrate binding site of P-glycoprotein has to be approached by passive diffusion through the lipid membrane, whereas in inside–out plasma membrane vesicles, it is accessible directly from the aqueous phase. Compounds exhibiting fast passive influx compared to active efflux by P-glycoprotein induced similar ATPase activity profiles in cells and inside–out plasma membrane vesicles, because their concentrations in the cytosolic leaflets were similar. Compounds exhibiting similar influx as efflux induced in contrast different ATPase activity profiles in cells and inside–out vesicles. Their concentration was significantly lower in the cytosolic leaflet of cells than in the cytosolic leaflet of inside-out membrane vesicles, indicating that P-glycoprotein could cope with passive influx. P-glycoprotein thus transported all compounds at a rate proportional to ATP hydrolysis (i.e. all compounds were substrates). However, it prevented substrate entry into the cytosol only if passive influx of substrates across the lipid bilayer was in a similar range as active efflux.

Keywords: Abbreviations; ABC; ATP binding cassette transporter; ATP; adenosine triphosphate; ECAR; extracellular acidification rate; MDR; multidrug resistance; Pgp; human P-glycoprotein-ATPase (MDR1, ABCB1)ATPase activity; Extracellular acidification rate; Passive diffusion; Influx; P-glycoprotein substrate; Active efflux

Multimeric forms of the small multidrug resistance protein EmrE in anionic detergent by Denice C. Bay; R. Arief Budiman; Mu-Ping Nieh; Raymond J. Turner (pp. 526-535).

Escherichia coli multidrug resistance protein E (EmrE) is a four transmembrane α-helix protein, and a member of the small multidrug resistance protein family that confers resistance to a broad range of quaternary cation compounds (QCC) via proton motive force. The multimeric states of EmrE protein during transport or ligand binding are variable and specific to the conditions of study. To explore EmrE multimerization further, EmrE extracted from E. coli membranes was solubilized in anionic detergent, sodium dodecyl sulphate (SDS), at varying protein concentrations. At low concentrations (≤1μM) in SDS–EmrE is monomeric, but upon increasing EmrE concentration, a variety of multimeric states can be observed by SDS-Tricine polyacrylamide gel electrophoresis (PAGE). Addition of the (QCC), tetraphenyl phosphonium (TPP), to SDS–EmrE samples enhanced EmrE multimer formation using SDS-Tricine PAGE. The relative shapes of EmrE multimers in SDS with or without TPP addition were determined by small angle neutron scattering (SANS) analysis and revealed that EmrE dimers altered in conformation depending on the SDS concentration. SANS analysis also revealed that relative shapes of larger EmrE multimers (≥100nm sizes) altered in the presence of TPP. Circular dichroism spectropolarimetry displayed no differences in secondary structure under the conditions studied. Fluorescence spectroscopy of SDS–EmrE protein demonstrated that aromatic residues, Trp and Tyr, are more susceptible to SDS concentration than TPP addition, but both residues exhibit enhanced quenching at high ligand concentrations. Hence, EmrE forms various multimers in SDS that are influenced by detergent concentration and TPP substrate addition.

Keywords: Small multidrug resistance protein (SMR); Small angle neutron scattering (SANS); SDS-Tricine PAGE; EmrE; Quaternary ammonium compound (QAC); Multimerization; Fluorescence

Surface adsorption of protein corona controls the cell internalization mechanism of DC-Chol–DOPE/DNA lipoplexes in serum by Giulio Caracciolo; Luciano Callipo; Sofia Candeloro De Sanctis; Chiara Cavaliere; Daniela Pozzi; Aldo Laganà (pp. 536-543).

Serum has often been reported as a barrier to efficient lipid-mediated transfection. Here we found that the transfection efficiency of DC-Chol–DOPE/DNA lipoplexes increases in serum. To provide insight into the mechanism of lipoplex-serum interaction, several state-of-the-art methodologies have been applied. The nanostructure of DC-Chol–DOPE/DNA lipoplexes was found to be serum-resistant as revealed by high resolution synchrotron small angle X-ray scattering, while dynamic light scattering measurements showed a marked size increase of complexes. The structural stability of DC-Chol–DOPE/DNA lipoplexes was confirmed by electrophoresis on agarose gel demonstrating that plasmid DNA remained well protected by lipids. Proteomics experiments showed that serum proteins competed for the cationic surface of lipid membranes leading to the formation of a rich a ‘protein corona’. Combining structural results with proteomics findings, we suggest that such a protein corona can promote large aggregation of intact lipoplexes. According to a recently proposed size-dependent mechanism of lipoplex entry within cells, protein corona-induced formation of large aggregates most likely results in a switch from a clathrin-dependent to caveolae-mediated entry pathway into the cells which is likely to be responsible for the observed transfection efficiency boost. As a consequence, we suggest that surface adsorption of protein corona can have a high biological impact on serum-resistant cationic formulations for in vitro and in vivo lipid-mediated gene delivery applications.

Keywords: Cationic liposome; DNA; Lipoplex; SAXS; Protein corona; Cell transfection

The membrane insertion of helical antimicrobial peptides from the N-terminus of Helicobacter pylori ribosomal protein L1 by Tzong-Hsien Lee; Kristopher N. Hall; Marcus J. Swann; Jonathan F. Popplewell; Sharon Unabia; Yoonkyung Park; Kyung-Soo Hahm; Marie-Isabel Aguilar (pp. 544-557).

The interaction of two helical antimicrobial peptides, HPA3 and HPA3P with planar supported lipid membranes was quantitatively analysed using two complementary optical biosensors. The peptides are analogues of Hp(2–20) derived from the N-terminus of Helicobacter pylori ribosomal protein L1 (RpL1). The binding of these two peptide analogues to zwitterionic dimyristoyl-phosphatidylcholine (DMPC) and negatively charged membranes composed of DMPC/dimyristoylphosphatidylglycerol (DMPG) (4:1) was determined using surface plasmon resonance (SPR) and dual polarisation interferometry (DPI). Using SPR analysis, it was shown that the proline substitution in HPA3P resulted in much lower binding for both zwitterionic and anionic membranes than HPA3. Structural changes in the planar DMPC and DMPC/DMPG (4:1) bilayers induced by the binding of both Hp(2–20) analogues were then resolved in real-time with DPI. The overall process of peptide-induced changes in membrane structure was analysed by the real-time changes in bound peptide mass as a function of bilayer birefringence. The insertion of both HPA3 and HPA3P into the supported lipid bilayers resulted in a decrease in birefringence with increasing amounts of bound peptide which reflects a decrease in the order of the bilayer. The binding of HPA3 to each membrane was associated with a higher level of bound peptide and greater membrane lipid disordering and a faster and higher degree of insertion into the membrane than HPA3P. Furthermore, the binding of both HPA3 and HPA3P to negatively charged DMPC/DMPG bilayers also leads to a greater disruption of the lipid ordering. These results demonstrate the geometrical changes in the membrane upon peptide insertion and the extent of membrane structural changes can be obtained quantitatively. Moreover, monitoring the effect of peptides on a structurally characterised bilayer has provided further insight into the role of membrane structure changes in the molecular basis of peptide selectivity and activity and may assist in defining the mode of antimicrobial action.

Keywords: Antimicrobial peptide; Hp(2–20); Surface plasmon resonance; Dual polarisation interferometry; Supported lipid bilayer; Birefringence

NBD-cholesterol probes to track cholesterol distribution in model membranes by Daniel M. Carter Ramirez; William W. Ogilvie; Linda J. Johnston (pp. 558-568).

A series of cholesterol (Chol) probes with NBD and Dansyl fluorophores attached to the 3-hydroxyl position via carbamate linkers has been designed and synthesized and their ability to mimic the behavior of natural cholesterol in bilayer membranes has been examined. Fluorescence spectroscopy data indicate that the NBD-labeled lipids are located in the polar headgroup region of the bilayer with their position varying with the method of fluorophore attachment and the linker length. The partitioning of the Chol probes between liquid-ordered (L_o) and liquid-disordered (L_o) phases in supported bilayers prepared from ternary lipid mixtures of DOPC, Chol and either egg sphingomyelin or DPPC was examined by fluorescence microscopy. The carbamate-linked NBD-Chols show a stronger preference for partitioning into L_o domains than does a structurally similar probe with an ester linkage, indicating the importance of careful optimization of probe and linker to provide the best Chol mimic. Comparison of the partitioning of NBD probes to literature data for native Chol indicates that the probes reproduce well the modest enrichment of Chol in L_o domains as well as the ceramide-induced displacement of Chol. One NBD probe was used to follow the dynamic redistribution of Chol in phase separated membranes in response to in situ ceramide generation. This provides the first direct optical visualization of Chol redistribution during enzymatic ceramide generation and allows the assignment of new bilayer regions that exclude dye and have high lateral adhesion to ceramide-rich regions.

Keywords: Cholesterol; Atomic force microscopy; Supported lipid bilayer; Fluorescence; Ceramide

Regulation by CRAMP of the responses of murine peritoneal macrophages to extracellular ATP by Michèle Seil; Elie Kabré; Carole Nagant; Michel Vandenbranden; Unai Fontanils; Aida Marino; Stéphanie Pochet; Jean-Paul Dehaye (pp. 569-578).

Peritoneal macrophages were isolated from wild type (WT) mice and from mice invalidated for the P2X₇ receptor (KO) which had been pretreated with thioglycolate. In cells from WT mice, 1 mM ATP increased the intracellular concentration of calcium ([Ca²⁺]_i), the uptake of ethidium bromide, the production of reactive oxygen species (ROS), the secretion of IL-1β, the release of oleic acid and of lactate dehydrogenase; it decreased the intracellular concentration of potassium ([K⁺]_i). In KO mice, ATP transiently increased the [Ca²⁺]_i confirming that the P2X₇ receptor is a major receptor of peritoneal macrophages. WKYMVm, an agonist of receptors for formylated peptides (FPR) also increased the [Ca²⁺]_i in murine macrophages. The slight increase of the [Ca²⁺]_i was strongly potentiated by ivermectin confirming the expression of functional P2X₄ receptors by murine peritoneal macrophages. CRAMP, the unique antimicrobial peptide derived from cathelin in mouse inhibited all the responses coupled to P2X₇ receptors in macrophages from WT mice. Agonists for FPR had no effect on the increase of the [Ca²⁺]_i in response to ATP. CRAMP had no effect on the increase of the [Ca²⁺]_i evoked by a combination of ATP and ivermectin in macrophages from P2X₇-KO mice.In summary CRAMP inhibits the responses secondary to the activation of the murine P2X₇ receptors expressed by peritoneal macrophages. This inhibition is not mediated by FPR receptors and is specific since CRAMP has no effect on the response coupled to P2X₄ receptors. It can thus be concluded that the interaction between P2X₇ receptors and cathelin-derived antimicrobial peptides is species-specific, in some cases (man) positive in others (mouse) negative.

Keywords: Abbreviations; a.f.u.; arbitrary fluorescence units; ATR-FTIR; attenuated total reflection–Fourier transform infrared spectroscopy; BSA; bovine serum albumin; CRAMP; cathelin-related antimicrobial peptide; DCFH-DA; 2′,7′-dichlorodihydrofluorescein diacetate; EGTA; ethylene glycol-; bis; -(β-aminoethyl ether)-; N; ,; N; ,; N; ',; N; '-tetraacetic acid; FBS; fetal bovine serum; FPR; receptors for formylated peptides; HEPES; N; -[2-hydroxyethyl] piperazine-; N; '-[2-ethanesulfonic acid]; KO mice; knockout mice; ROS; reactive oxygen species; WT mice; wild-type miceAntimicrobial peptide; Purinergic receptor; P2X; ₇; -KO mice; Ivermectin

Role of membranotropic sequences from herpes simplex virus type I glycoproteins B and H in the fusion process by Stefania Galdiero; Annarita Falanga; Giuseppe Vitiello; Mariateresa Vitiello; Carlo Pedone; Gerardino D'Errico; Massimiliano Galdiero (pp. 579-591).

The entry of enveloped viruses involves attachment followed by close apposition of the viral and plasma membranes. Then, either on the cell surface or in an endocytotic vesicle, the two membranes fuse by an energetically unfavourable process requiring the destabilisation of membrane microenvironment in order to release the viral nucleocapsid into the cytoplasm. The core fusion machinery, conserved throughout the herpesvirus family, involves glycoprotein B (gB) and the non-covalently associated complex of glycoproteins H and L (gH/gL). Both gB and gH possess several hydrophobic domains necessary for efficient induction of fusion, and synthetic peptides corresponding to these regions are able to associate to membranes and induce fusion of artificial liposomes. Here, we describe the first application of surface plasmon resonance (SPR) to the study of the interaction of viral membranotropic peptides with model membranes in order to enhance our molecular understanding of the mechanism of membrane fusion. SPR spectroscopy data are supported by tryptophan fluorescence, circular dichroism and electron spin resonance spectroscopy (ESR). We selected peptides from gB and gH and also analysed the behaviour of HIV gp41 fusion peptide and the cationic antimicrobial peptide melittin. The combined results of SPR and ESR showed a marked difference between the mode of action of the HSV peptides and the HIV fusion peptide compared to melittin, suggesting that viral-derived membrane interacting peptides all act via a similar mechanism, which is substantially different from that of the non-cell selective lytic peptide melittin.

Keywords: Fusion peptide; Membrane; Virus; Surface plasmon resonance; Glycoprotein

Palmitoylation of R-Ras by human DHHC19, a palmitoyl transferase with a CaaX box by Florian Baumgart; María Corral-Escariz; Perez-Gil Jesús Pérez-Gil; Rodriguez-Crespo Ignacio Rodríguez-Crespo (pp. 592-604).

Mammalian proteins that contain an aspartate-histidine-histidine-cysteine-(DHHC) motif have been recently identified as a group of membrane-associated palmitoyl acyltransferases (PATs). Among the several protein substrates known to become palmitoylated by DHHC PATs are small GTPases prenylated at their carboxy-terminal end, such as H-Ras or N-Ras, eNOS, kinases myristoylated at their N-terminal end, such as Lck, and many transmembrane proteins and channels. We have focused our studies on the product of the human gene DHHC19, a putative palmitoyl transferase that, interestingly, displays a conserved CaaX box at its carboxy-terminal end. We show herein that the amino acid sequence present at the carboxy-terminus of DHHC19 is able to exclude a green fluorescent protein (GFP) reporter from the nucleus and direct it towards perinuclear regions. Transfection of full-length DHHC19 in COS7 cells reveals a perinuclear distribution, in analogy to other palmitoyl transferases, with a strong colocalization with the trans-Golgi markers Gal-T and TGN38. We have tested several small GTPases that are known to be palmitoylated as possible substrates of DHHC19. Although DHHC19 failed to increase the palmitoylation of H-Ras, N-Ras, K-Ras4A, RhoB or Rap2 it increased the palmitoylation of R-Ras approximately two-fold. The increased palmitoylation of R-Ras cotransfected with DHHC19 is accompanied by an augmented association with membranes as well as with rafts/caveolae. Finally, using both wild-type and an activated GTP bound form of R-Ras (G38V), we also show that the increased palmitoylation of R-Ras due to DHHC19 coexpression is accompanied by an enhanced viability of the transfected cells.

Keywords: Abbreviations; DHHC; Asp-His-His-Cys motif; PAT; palmitoyl acyltransferase; GFP; green fluorescent protein; HPLC; high pressure liquid chromatography; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromidePalmitoyl transferases; Palmitoylation; DHHC proteins; Small GTPases; R-Ras

Strong oligomerization behavior of PDGFβ receptor transmembrane domain and its regulation by the juxtamembrane regions by Joanne Oates; Gavin King; Ann M. Dixon (pp. 605-615).

The platelet-derived growth factor β-receptor (PDGFβR) represents an important subclass of receptor tyrosine kinase (RTK) thought to be activated by ligand-induced dimerization. Interestingly, the receptor is also activated by the bovine papillomavirus E5 oncoprotein, an interaction involving the transmembrane domains of both proteins and resulting in constitutive downstream signalling. This unique mode of activation along with emerging data for other RTKs raises important questions about the role of the PDGFβR transmembrane domain in signalling. To address this, we have investigated the murine PDGFβR transmembrane and juxtamembrane domains. We show for the first time the strong oligomerization behavior of PDGFβR transmembrane domain, forming dimers and trimers in natural membranes and detergents; and that these self-interactions are mediated by a leucine-zipper-like motif. The juxtamembrane regions are found to regulate these helix–helix interactions and select specifically for dimer formation. These data provide evidence that PDGFβR is able to form ligand-independent dimers, supporting similar observations in a number of other RTK's. A point mutant in the PDGFβR juxtamembrane domain previously shown to cause receptor activation was studied and yielded no change in oligomerization or folding, suggesting (in-line with observations of the c-Kit receptor) that it may moderate interactions with other regions of PDGFβR.

Keywords: Platelet-derived growth factor receptor; Transmembrane domain; Juxtamembrane domain; Analytical ultracentrifugation; TOXCAT

Role of guanidinium group in the insertion ofl-arginine in DMPE and DMPC lipid interphases by Ana Bouchet; Fabiana Lairion; E. Aníbal Disalvo (pp. 616-623).

l-Arginine (Arg) is a positively charged amino acid constituent of peptides and proteins, participating in diverse mechanisms of protein–membrane interaction. The effect of Arg on phosphatidylcholine (PC) membranes has been previously related to water structure changes and to the presence of water defects in the hydrocarbon region. However, no information is available with regard to phosphatidylethanolamine (PE), another important component of lipid membranes. For this reason, the aim of this study is to determine the effect of Arg on DMPE membranes and partially methylated PEs in comparison to DMPC. The adsorption of the amino acid onto the lipid membranes was followed by determining the changes in the surface potential as a function of the bulk amino acid concentrations. The effects of Arg on the surface properties were also measured by changes in the surface pressure and the dipole potential. The onset of the transition temperature was measured with a fluorophore anchored at the membrane interphase. The results provide a new insight on amino acid—PE interactions, which can be ascribed to specific perturbations in the head group region induced by the guanidinium residue.

Keywords: l; -Arginine; Lipid membrane; DMPE; DMPC; Guanidinium group; Surface potential; Dipole potential; Interfacial anisotropy

Selective localization of phosphatidylcholine-derived signaling in detergent-resistant membranes from synaptic endings by M.V. Mateos; G.A. Salvador; N.M. Giusto (pp. 624-636).

Detergent-resistant membranes (DRMs) are a class of specialized microdomains that compartmentalize several signal transduction processes. In this work, DRMs were isolated from cerebral cortex synaptic endings (Syn) on the basis of their relative insolubility in cold Triton X-100 (1%). The lipid composition and marker protein content were analyzed in DRMs obtained from adult and aged animals. Both DRM preparations were enriched in Caveolin, Flotillin-1 and c-Src and also presented significantly higher sphingomyelin (SM) and cholesterol content than purified Syn. Total phospholipid-fatty acid composition presented an increase in 16:0 (35%), and a decrease in 20:4n-6 (67%) and 22:6n-3 (68%) content in DRM from adults when compared to entire synaptic endings. A more dramatic decrease was observed in the 20:4n-6 and 22:6n-3 content in DRMs from aged animals (80%) with respect to the results found in adults. The coexistence of phosphatidylcholine-specific-phospholipase C (PC-PLC) and phospholipase D (PLD) in Syn was previously reported. The presence of these signaling pathways was also investigated in DRMs isolated from adult and aged rats. Both PC-PLC and PLD pathways generate the lipid messenger diacylglycerol (DAG) by catalyzing PC hydrolysis. PC-PLC and PLD1 localization were increased in the DRM fraction. The increase in DAG generation (60%) in the presence of ethanol, confirmed that PC-PLC was also activated when compartmentalized in DRMs. Conversely, PLD2 was excluded from the DRM fraction. Our results show an age-related differential fatty acid composition and a selective localization of PC-derived signaling in synaptic DRMs obtained from adult and aged rats.

Keywords: Detergent-resistant membrane; Synaptic ending; Aging; PC-PLC; PLD; DAG

Light harvesting, energy transfer and electron cycling of a native photosynthetic membrane adsorbed onto a gold surface by Gerhard J. Magis; Mart-Jan den Hollander; Willem G. Onderwaater; John D. Olsen; C. Neil Hunter; Thijs J. Aartsma; Raoul N. Frese (pp. 637-645).

Photosynthetic membranes comprise a network of light harvesting and reaction center pigment–protein complexes responsible for the primary photoconversion reactions: light absorption, energy transfer and electron cycling. The structural organization of membranes of the purple bacterial species Rb. sphaeroides has been elucidated in most detail by means of polarized light spectroscopy and atomic force microscopy. Here we report a functional characterization of native and untreated membranes of the same species adsorbed onto a gold surface. Employing fluorescence confocal spectroscopy and light-induced electrochemistry we show that adsorbed membranes maintain their energy and electron transferring functionality. Gold-adsorbed membranes are shown to generate a steady high photocurrent of 10μA/cm² for several minutes and to maintain activity for up to three days while continuously illuminated. The surface-adsorbed membranes exhibit a remarkable functionality under aerobic conditions, even when exposed to light intensities well above that of direct solar irradiation. The component at the interface of light harvesting and electron cycling, the LH1 complex, displays exceptional stability, likely contributing to the robustness of the membranes. Peripheral light harvesting LH2 complexes show a light intensity dependent decoupling from photoconversion. LH2 can act as a reversible switch at low-light, an increased emitter at medium light and photobleaches at high light.

Keywords: Photosynthetic membrane; Rhodobacter; sphaeroides; Supramolecular organization; Confocal spectroscopy; Light-induced electrochemistry; Energy transfer; Electron transfer; Light-harvesting complex; Reaction center complex

Nanosized self-emulsifying lipid vesicles of diacylglycerol-PEG lipid conjugates: Biophysical characterization and inclusion of lipophilic dietary supplements by Rumiana Koynova; Mariana Tihova (pp. 646-653).

Hydrated diacylglycerol-PEG lipid conjugates, glyceryl dioleate-PEG12 (GDO-PEG12) and glyceryl dipalmitate-PEG23 (GDP-PEG23), spontaneously form uni- or oligolamellar liposomes in their liquid crystalline phase, in distinct difference from the PEGylated phospholipids which form micelles. GDP-PEG23 exhibits peculiar hysteretic phase behavior and can arrange into a long-living hexagonal phase at ambient and physiological temperatures. Liposomes of GDO-PEG12 and its mixture with soy lecithin exchange lipids with the membranes much more actively than common lecithin liposomes; such an active lipid exchange might facilitate the discharging of the liposome cargo upon uptake and internalization, and can thus be important in drug delivery applications. Diacylglycerol-PEG lipid liposome formulations can encapsulate up to 20–30wt.% lipophilic dietary supplements such as fish oil, coenzyme Q10, and vitamins D and E. The encapsulation is feasible by way of dry mixing, avoiding the use of organic solvent.

Keywords: Liposome; Nanoparticle; Diacylglycerol-PEG lipid; Hexagonal phase; Lipophilic dietary supplement; Encapsulation

Role of loop 9 on the function of neuronal nicotinic receptors by Manuel Criado; Mar Castillo; José Mulet; Francisco Sala; Salvador Sala (pp. 654-659).

We have studied the role of loop 9 in the function of neuronal nicotinic receptors. By systematically mutating the residues in the loop we have determined that the most important amino acids determining the coupling of binding to gating are the ones closer to the transmembrane region. Single mutations at location E173 in homomeric α7 receptors destroyed their function by completely abolishing the current while preserving the expression at the membrane. In contrast, heteromeric receptor α3β4 with the same mutations retained some function. We conclude that loop 9 has a different role in the function of homomeric and heteromeric receptors.

Keywords: Abbreviations; L9; loop 9; ACh; acetylcholine; nAChR; nicotinic acetylcholine receptor; α-Bgt; α-bungarotoxin; WT; wild-typeAcetylcholine receptor; Loop 9; Homomer; Heteromer

Membrane charge dependent states of the β-amyloid fragment Aβ (16–35) with differently charged micelle aggregates by Manuela Grimaldi; Mario Scrima; Cinzia Esposito; Giuseppe Vitiello; Anna Ramunno; Vittorio Limongelli; Gerardino D'Errico; Ettore Novellino; Anna Maria D'Ursi (pp. 660-671).

A β (16–35) is the hydrophobic central core of β-amyloid peptide, the main component of plaques found in the brain tissue of Alzheimer's disease patients. Depending on the conditions present, β-amyloid peptides undergo a conformational transition from random coil or α-helical monomers, to highly toxic β-sheet oligomers and aggregate fibrils. The behavior of β-amyloid peptide at plasma membrane level has been extensively investigated, and membrane charge has been proved to be a key factor modulating its conformational properties. In the present work we probed the conformational behavior of A β (16–35) in response to negative charge modifications of the micelle surface. CD and NMR conformational analyses were performed in negatively charged pure SDS micelles and in zwitterionic DPC micelles “doped” with small amounts of SDS. To analyze the tendency of A β (16–35) to interact with these micellar systems, we performed EPR experiments on three spin-labeled analogues of A β (16–35), bearing the methyl 3-(2,2,5,5-tetramethyl-1-oxypyrrolinyl) methanethiolsulfonate spin label at the N-terminus, in the middle of the sequence and at the C-terminus, respectively. Our conformational data show that, by varying the negative charge of the membrane, A β (16–35) undergoes a conformational transition from a soluble helical–kink–helical structure, to a U-turn shaped conformation that resembles protofibril models.

Keywords: Amyloid peptide; NMR spectroscopy; Micelle solution; EPR spectroscopy; Spin-labeled peptideAbbreviations; Aβ; amyloid; β; peptide; APP; amyloid precursor proteins; Boc; tert; -butyloxycarbonyl; CD; circular dichroism; CMC; critical micellar concentration; DCM; dichloromethane; DMF; N; ,; N; -dimethylformamide; DPC; dodecylphosphocholine; DQF-COSY; double quantum filtered correlation spectroscopy; 5-DSA; 5-doxylstearic acid; 16-DSA; 16-doxylstearic acid; EPR; electron paramagnetic resonance; ESI-MS; electrospray/ionization mass spectra; EtOAc; Ethyl acetate; Fmoc; 9-fluorenylmethyloxycarbonyl; HOBt; N; -hydroxybenzotriazole; HPLC; high performance liquid chromatography; LiDS; lithium dodecyl sulfate; SLMTS; spin-label methyl 3-(2,2,5,5-tetramethyl-1-oxypyrrolinyl) methanethiolsulfonate; MTSL; methyl 3-(2,2,5,5-tetramethyl-1-oxypyrrolinyl) methanethiolsulfonate label; MD; Molecular dynamics; NMM; N; -methylmorpholine; NOE; nuclear Overhauser effect; NOESY; NOE Spectroscopy; RMSD; root mean square deviation; RP-HPLC; reversed phase HPLC; Rt; retention time; SDS; sodium dodecyl sulphate; SPPS; solid phase peptide synthesis; THF; Tetrahydrofuran; TBTU; 2-(1; H; -benzotriazol-1-yl)-1,1,3,3,-tetramethyluronium tetrafluoroborate; TLC; thin-layer chromatography; TFA; trifluoroacetic acid; TEA; triethylamine; TOCSY; Total Correlated Spectroscopy

Rapid screening of membrane topology of secondary transport proteins by Ramon ter Horst; Juke S. Lolkema (pp. 672-680).

Limited experimental data may be very useful to discriminate between membrane topology models of membrane proteins derived from different methods. A membrane topology screening method is proposed by which the cellular disposition of three positions in a membrane protein are determined, the N- and the C-termini and a position in the middle of the protein. The method involves amplification of the encoding genes or gene fragments by PCR, rapid cloning in dedicated vectors by ligation independent cloning, and determination of the cellular disposition of the three sites using conventional techniques. The N-terminus was determined by labeling with a fluorescent probe, the central position and the C-terminus by the reporter fusion technique using alkaline phosphatase (PhoA) and green fluorescence protein (GFP) as reporters. The method was evaluated using 16 transporter proteins of known function from four different structural classes. For 13 proteins a complete set of three localizations was obtained. The experimental data was used to discriminate between membrane topology models predicted by TMHMM, a widely used predictor using the amino acid sequence as input and by MemGen that uses hydropathy profile alignment and known 3D structures or existing models. It follows that in those cases where the models from the two methods were similar, the models were consistent with the experimental data. In those cases where the models differed, the MemGen model agreed with the experimental data. Three more recent predictors, MEMSAT3, OCTOPUS and TOPCONS showed a significantly higher consistency with the experimental data than observed with TMHMM.

Keywords: Abbreviations; LIC; ligation independent cloning; GFP; green fluorescent protein; FM; fluorescein-5-maleimide; TMS; transmembrane segmentMembrane topology; Structural class; Topology prediction; MemGen; Transport protein; Reporter fusion

Mitochondria isolated in nearly isotonic KCl buffer: Focus on cardiolipin and organelle morphology by Angela Corcelli; Matilde Sublimi Saponetti; Patrizia Zaccagnino; Patrizia Lopalco; Maria Mastrodonato; Giuseppa E. Liquori; Michele Lorusso (pp. 681-687).

Rat liver mitochondria were isolated in parallel in two different isolation buffers: a standard buffer containing mannitol/sucrose and a nearly physiological KCl based solution. The two different organelle preparations were comparatively characterized by respiratory activity, heme content, microsomal and Golgi contamination, electron microscopy and lipid analyses. The substitution of saccharides with KCl in the isolation buffer does not induce the formation of mitoplasts or disruption of mitochondria. Mitochondria isolated in KCl buffer are coupled and able to maintain a stable transmembrane charge separation. A number of biochemical and functional differences between the two organelle preparations are described; in particular KCl mitochondria exhibit lower cardiolipin content and smaller intracristal compartments in comparison with the standard mitochondrial preparation.

Keywords: Mitochondrion; KCl based isolation buffer; Mitochondrial lipids; Cardiolipin; Cristae morphology

Liquid-ordered phases induced by cholesterol: A compendium of binary phase diagrams by Derek Marsh (pp. 688-699).

Mixtures of phospholipids with cholesterol are able to form liquid-ordered phases that are characterised by short-range orientational order and long-range translational disorder. These L_o-phases are distinct from the liquid-disordered, fluid L_α-phases and the solid-ordered, gel L_β-phases that are assumed by the phospholipids alone. The liquid-ordered phase can produce spatially separated in-plane fluid domains, which, in the form of lipid rafts, are thought to act as platforms for signalling and membrane sorting in cells. The areas of domain formation are defined by the regions of phase coexistence in the phase diagrams for the binary mixtures of lipid with cholesterol. In this paper, the available binary phase diagrams of lipid–cholesterol mixtures are all collected together. It is found that there is not complete agreement between different determinations of the phase diagrams for the same binary mixture. This can be attributed to the indirect methods largely used to establish the phase boundaries. Intercomparison of the various data sets allows critical assessment of which phase boundaries are rigorously established from direct evidence for phase coexistence.

Keywords: Membrane domain; Lateral phase separation; Lipid phase diagram; Cholesterol; Lipid raft

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BBA - Biomembranes (v.1798, #3)

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BBA - Biomembranes (v.1798, #3)