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BBA - Biomembranes (v.1818, #5)

Editorial Board (pp. i).

Comparing ion conductance recordings of synthetic lipid bilayers with cell membranes containing TRP channels by Katrine R. Laub; Katja Witschas; Andreas Blicher; Søren B. Madsen; Luckhoff Andreas Lückhoff; Thomas Heimburg (pp. 1123-1134).

In this article we compare electrical conductance events from single channel recordings of three TRP channel proteins (TRPA1, TRPM2 and TRPM8) expressed in human embryonic kidney cells with channel events recorded on synthetic lipid membranes close to melting transitions. Ion channels from the TRP family are involved in a variety of sensory processes including thermo- and mechano-reception. Synthetic lipid membranes close to phase transitions display channel-like events that respond to stimuli related to changes in intensive thermodynamic variables such as pressure and temperature. TRP channel activity is characterized by typical patterns of current events dependent on the type of protein expressed. Synthetic lipid bilayers show a wide spectrum of electrical phenomena that are considered typical for the activity of protein ion channels. We find unitary currents, burst behavior, flickering, multistep-conductances, and spikes behavior in both preparations. Moreover, we report conductances and lifetimes for lipid channels as described for protein channels. Non-linear and asymmetric current–voltage relationships are seen in both systems. Without further knowledge of the recording conditions, no easy decision can be made whether short current traces originate from a channel protein or from a pure lipid membrane.Display Omitted► We compare electrical recordings from lipid bilayers with those from TRP channels. ► Artificial membranes display conductance events similar to those of TRP channels. ► We find bursts, flickering, multiple steps, and spikes in both preparations. ► The time scale and conductance in both preparations are of similar order. ► Conduction in artificial membranes is facilitated by the melting transition.

Keywords: TRP channel; TRPA1; TRPM2; TRPM8; Lipid pore; Phase transition

A calcium-permeable non-selective cation channel in the thick ascending limb apical membrane of the mouse kidney by Romain Guinamard; Marc Paulais; Stéphane Lourdel; Jacques Teulon (pp. 1135-1141).

Non-selective cation channels have been described in the basolateral membrane of the renal tubule, but little is known about functional channels on the apical side. Apical membranes of microdissected fragments of mouse cortical thick ascending limbs were searched for ion channels using the cell-free configuration of the patch-clamp technique. A cation channel with a linear current–voltage relationship (19pS) that was permeable both to monovalent cations [P_NH4(1.7)>P_Na (1.0)=P_K (1.0)] and to Ca²⁺ (P_Ca/P_Na≈0.3) was detected. Unlike the basolateral TRPM4 Ca²⁺-impermeable non-selective cation channel, this non-selective cation channel was insensitive to internal Ca²⁺, pH and ATP. The channel was already active after patch excision, and its activity increased after reduced pressure was applied via the pipette. External gadolinium (10−⁵M) decreased the channel-open probability by 70% in outside-out patches, whereas external amiloride (10−⁴M) had no effect. Internal flufenamic acid (10−⁴M) inhibited the channel in inside-out patches. Its properties suggest that the current might be supported by the TRPM7 protein that is expressed in the loop of Henle. The conduction properties of the channel suggest that it could be involved in Ca²⁺ signaling.► A cation channel is present in the apical membrane of the loop of Henle in mouse. ► The channel is permeable to divalent and monovalent cations like the TRPM7 channel. ► The channel provides a trans-cellular pathway for calcium in the loop of Henle.

Keywords: Abbreviations; CTAL; cortical thick ascending limb; E; _r; reversal potential; FA; flufenamic acid; IMCD; inner medullary collecting duct; NSC channel; non-selective cation channel; NSC; _Ca; channel; calcium-activated non-selective cation channel; P; _o; open probability; TRP; transient receptor potentialCation channel; Calcium; Kidney; Epithelium; TRPM7

Magnetically oriented dodecylphosphocholine bicelles for solid-state NMR structure analysis by Olga V. Nolandt; Torsten H. Walther; Stephan L. Grage; Anne S. Ulrich (pp. 1142-1147).

A mixture of 1,2-dimyristoyl- sn-glycero-3-phosphocholine (DMPC) with the short-chain detergent n-dodecylphosphocholine (DPC) is introduced here as a new membrane-mimetic bicelle system for solid-state NMR structure analysis of membrane proteins in oriented samples. Magnetically aligned DMPC/DPC bicelles are stable over a range of concentrations, with an optimum lipid ratio of q=3:1, and they can be flipped with lanthanide ions. The advantage of DMPC/DPC over established bicelle systems lies in the possibility to use one and the same detergent for purification and NMR analysis of the membrane protein, without any need for detergent exchange. Furthermore, the same batch of protein can be studied in both micelles and bicelles, using liquid-state and solid-state NMR, respectively. The applicability of the DMPC/DPC bicelles is demonstrated here with the¹⁵N-labeled transmembrane protein TatA.►Novel bicelle system for solid-state NMR structure analysis of membrane proteins. ►Purification and NMR study in bicelles is possible in the same detergent DPC ►Successful application to SAMPI4 spectra of the transmembrane protein TatA.

Keywords: Abbreviations; Tat; Twin-arginine translocation; DMPC; 1,2-dimyristoyl-; sn; -glycero-3-phosphocholine; DMPG; 1,2-dimyristoyl-; sn; -glycero-3-[phospho-; rac; -(1-glycerol)]; DH(7)PC; 1,2-diheptanoyl-; sn; -glycero-3-phosphocholine; DHPC; 1,2-dihexanoyl-; sn; -glycero-3-phosphocholine; DPC; n-dodecylphosphocholine; 6-O-PC; 1,2-di-O-hexyl-; sn; -glycero-3-phosphocholine; CHAPSO; 3-(cholamidopropyl) dimethylammonio-2-hydroxyl-1-propanesulfonate; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; TBBPC; 1-myristoyl-2-[4-(4-biphenyl) butanoyl]-; sn; -glycero-3-phosphocholineDMPC/DPC bicelles; Twin arginine translocation; Membrane protein; Solid-state NMR; PISEMA; Dodecylphosphocholine

pH-dependent channel gating in connexin26 hemichannels involves conformational changes in N-terminus by Xia Wang; Xue Xu; Ming Ma; Wei Zhou; Yonghua Wang; Ling Yang (pp. 1148-1157).

Connexin (Cx) hemichannels controlling an exchange of ions and metabolites between the cytoplasm and extracellular milieu can be modulated by the variation of intracellular pH during physiological and pathological conditions. To address the mechanism by which the pH exerts its effect on hemichannels, we have performed two 100-ns molecular dynamics simulations of the Cx26 channel in both acidic and neutral states. The results show that: 1) transmembrane domains undergo clockwise motions around the pore axis under both acidic and neutral conditions, while extracellular segments keep stable. 2) Under neutral condition, Cx26 has a tightly closed configuration that occurs through the assembly of N-terminal helix (NTH) region. This shows a constriction formed by the interhelical interactions of Asp2 and Met1 from neighboring NTH, which shapes the narrowest segment (pore radius<2Å) of the pore, preventing the passage of ions from the extracellular side. This indicates that Asp2 may act as a channel gate. 3) Under the acidic condition, the constriction is relieved by the protonation of Asp2 causing interruption of interhelical interactions, Cx26 has a flexibly opening pore (pore radius>4.5Å) around NTH region, allowing the passage of chloride ions unimpeded by the side-chain Asp2. While in the extracellular part two chloride ions interact with the side-chain Lys41 from three subunits. Finally, we provide a plausible mechanism of pH-dependent gating of hemichannel that involves protonation of the aspartic residues, suggesting that the pH sensitivity of hemichannel permeability is a sophisticated mechanism for cell regulating ion permeation.► We performed MD simulations of the Cx26 channel in different pH states. ► Cx26 has a tightly closed configuration under neutral condition, while a flexible open configuration under acidic condition. ► We provide a plausible mechanism of pH-dependent gating of hemichannel.

Keywords: Molecular dynamic simulation; Connexin26; pH; Channel gating

The transmembrane domain of caveolin-1 exhibits a helix–break–helix structure by Jinwoo Lee; Kerney Jebrell Glover (pp. 1158-1164).

Caveolin is an integral membrane protein that is found in high abundance in caveolae. Both the N- and C- termini lie on the same side of the membrane, and the transmembrane domain has been postulated to form an unusual intra-membrane horseshoe configuration. To probe the structure of the transmembrane domain, we have prepared a construct of caveolin-1 that encompasses residues 96–136 (the entire intact transmembrane domain). Caveolin-1(96–136) was over-expressed and isotopically labeled in E. coli, purified to homogeneity, and incorporated into lyso-myristoylphosphatidylglycerol micelles. Circular dichroism and NMR spectroscopy reveal that the transmembrane domain of caveolin-1 is primarily α-helical (57–65%). Furthermore, chemical shift indexing reveals that the transmembrane domain has a helix–break–helix structure which could be critical for the formation of the intra-membrane horseshoe conformation predicted for caveolin-1. The break in the helix spans residues 108 to 110, and alanine scanning mutagenesis was carried out to probe the structural significance of these residues. Our results indicate that mutation of glycine 108 to alanine does not disrupt the structure, but mutation of isoleucine 109 and proline 110 to alanine dramatically alters the helix–break–helix structure. To explore the structural determinants further, additional mutagenesis was performed. Glycine 108 can be substituted with other small side chain amino acids (i.e. alanine), leucine 109 can be substituted with other β-branched amino acids (i.e. valine), and proline 110 cannot be substituted without disrupting the helix–break–helix structure.► Caveolin-1 transmembrane domain displays a helix–break–helix structure. ► Transmembrane domain of caveolin-1 is highly helical. ► Residues 108–110 (G, I, P) are critical amino acids for the break between helices.

Keywords: Abbreviations; LMPG; lyso-myristoylphosphatidylglycerol; NMR; nuclear magnetic resonance spectroscopy; CD; circular dichroism spectroscopy; CSI; chemical shift indexing; HSQC; heteronuclear single quantum coherence; TROSY; transverse relaxation optimized spectroscopy; Cav1; _96-136; caveolin-1(96–136)Caveolin; NMR spectroscopy; Circular dichroism spectroscopy; Membrane protein; Caveolae; Chemical shift indexing

Orientation and depth of surfactant protein B C-terminal helix in lung surfactant bilayers by Philippe Bertani; Verica Vidovic; Tran-chin Yang; Jennifer Rendell; Larry M. Gordon; Alan J. Waring; Burkhard Bechinger; Valerie Booth (pp. 1165-1172).

SP-B_CTERM is a cationic amphipathic helical peptide and functional fragment composed of residues 63 to 78 of surfactant protein B (SP-B). Static oriented and magic angle spinning solid state NMR, along with molecular dynamics simulation was used to investigate its structure, orientation, and depth in lipid bilayers of several compositions, namely POPC, DPPC, DPPC/POPC/POPG, and bovine lung surfactant extract (BLES). In all lipid environments the peptide was oriented parallel to the membrane surface. While maintaining this approximately planar orientation, SP-B_CTERM exhibited a flexible topology controlled by subtle variations in lipid composition. SP-B_CTERM-induced lipid realignment and/or conformational changes at the level of the head group were observed using³¹P solid-state NMR spectroscopy. Measurements of the depth of SP-B_CTERM indicated the peptide center positions ~8Å more deeply than the phosphate headgroups, a topology that may allow the peptide to promote functional lipid structures without causing micellization upon compression.Display Omitted► First direct observation of SP-B_CTERM's planar orientation in bilayers. ► SP-B_CTERM's topology is flexible and is controlled by variations in lipid composition. ► SP-B_CTERM's positions at a depth of ~8Å deeper than the phosphate headgroups. ► SP-B_CTERM likely induces a modest positive curvature without causing micellularization.

Keywords: Abbreviations; RDS; Respiratory Distress Syndrome; ARDS; Acute Respiratory Distress Syndrome; SP-B; Surfactant Protein B; SDS; sodium dodecyl sulfate; BLES; bovine lipid extract surfactant; MD; molecular dynamics; Fmoc; O-fluorenylmethyl-oxycarbonyl; r.h.; relative humidity; POPC; 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; POPG; 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol; DPPC; Dipalmitoylphosphatidylcholine; TFE; Trifluoroethanol; NMR; nuclear magnetic resonance; TRIS; Tris (Hydroxymethyl) Aminomethane; CSA; chemical shift anisotropyPulmonary surfactant; SP-B; Acute Respiratory Distress Syndrome; Surface-active peptide; Membrane

The amphipathic helix of an enzyme that regulates phosphatidylcholine synthesis remodels membranes into highly curved nanotubules by Svetla G. Taneva; Joseph M.C. Lee; Rosemary B. Cornell (pp. 1173-1186).

CTP:phosphocholine cytidylyltransferase (CCT) is an amphitropic protein regulating phosphatidylcholine synthesis. Lipid-induced folding of its amphipathic helical (AH) membrane-binding domain activates the enzyme. In this study we examined the membrane deforming property of CCT in vitro by monitoring conversion of vesicles to tubules, using transmission electron microscopy. Vesicle tubulation was proportional to the membrane density of CCT and proceeded either as growth from a pre-formed surface bud, or as a global transformation of roughly spherical vesicles into progressively thinner tubules. The tubulation pathway depended on the lipid compositional heterogeneity of the vesicles, with heterogeneous mixtures supporting the bud-extension pathway. Co-existence of vesicles alongside thick and thin tubules suggested that CCT can discriminate between flat membrane surfaces and those with emerging curvature, binding preferentially to the latter. Thin tubules had a limiting diameter of ~12nm, likely representing bilayer cylinders with a very high density of 1 CCT/50 lipids. The AH segment was necessary and sufficient for tubulation. AH regions from diverse CCT sources, including C. elegans, had tubulation activity that correlated with α-helical length. The AH motifs in CCT and the Parkinson's-related protein, α-synuclein, have similar features, however the CCT AH was more effective in its membrane remodeling function. That CCT can deform vesicles of physiologically relevant composition suggests that CCT binding to membranes may initiate deformations required for organelle morphogenesis and at the same time stimulate synthesis of the PC required for the development of these regions.Display Omitted► Membrane binding activates CCT and also locally remodels the bilayer. ► The CCT amphipathic helix is necessary and sufficient for lipid vesicle tubulation. ► Nanotubes 11–12nm in diameter can form with a density of 1 CCT/50 lipids. ► Pathways for nanotube generation are variable, and depend on vesicle lipid composition. ► CCT tubulation efficiency scales with amphipathic helix length, not binding affinity.

Keywords: Abbreviations; CCT; CTP:phosphocholine cytidylyltransferase; AH; amphipathic helix; NLS; nuclear localization sequence; DMPC; 1,2-dimyristoyl-; sn; -glycerophosphocholine; DMPC; 1,2-dimyristoyl-; sn; -glycerophosphoglycerol (sodium salt); DAG; diacylglycerol; POPS; 1-palmytoyl-2-oleoyl-sn-glycero-3-[phospho-; l; -serine] (sodium salt); POPE; 1-palmitoyl-2-oleoyl-sn-glycero-3-phospholethanolamine; PI; bovine liver phosphatidyl inositol; SM; egg sphingomyelin; DTT; 1,4-dithio-; d; -threitol; SUVs; sonicated unilamellar vesicles; MLVs; multilamellar vesicles; LUVs; large unilamellar vesicles; SLVs; sucrose-loaded vesicles; NE; nuclear envelope; PM; phosphomimic; NE mix; LUVs composed of egg PC/POPE/PI /POPS/SM (55/20/10/10/5), PBS, phosphate buffered saline; TEM; transmission electron microscopyCTP:phosphocholine cytidylyltransferase; Membrane curvature; Vesicle tubulation; Electron microscopy

Cysteines control the N- and C-linker-dependent gating of KCNH1 potassium channels by Nirakar Sahoo; Schonherr Roland Schönherr; Toshinori Hoshi; Stefan H. Heinemann (pp. 1187-1195).

KCNH1 (EAG1) is a member of the Kv family of voltage-gated potassium channels. However, KCNH1 channels also show some amino-acid sequence similarity to cyclic-nucleotide-regulated channels: they harbor an N-terminal PAS domain, a C-terminal cyclic nucleotide binding homology domain (cNBHD), and N- and C-terminal binding sites for calmodulin. Another notable feature is the channels' high sensitivity toward oxidative modification. Using human KCNH1 expressed in Xenopus oocytes and HEK 293 cells we investigated how oxidative modification alters channel function. Intracellular application of H₂O₂ or cysteine-specific modifiers potently inhibited KCNH1 channels in two phases. Our systematic cysteine mutagenesis study showed that the rapid and dominant phase was attributed to a right-shift in the voltage dependence of activation, caused by chemical modification of residues C145 and C214. The slow component depended on the C-terminal residues C532 and C562. The cysteine pairs are situated at structural elements linking the transmembrane S1 segment with the PAS domain (N-linker) and the transmembrane channel gate S6 with the cNBH domain (C-linker), respectively. The functional state of KCNH1 channels is determined by the oxidative status of these linkers that provide an additional dimension of channel regulation.► KCNH1 potassium channels are exceptionally sensitive to oxidative modification. ► Thiol-dependent modification of channel gating occurs in two different ways. ► C145/C214 in the N-linker are responsible for fast modification. ► C532/C562 in the C-linker are responsible for slow modification. ► Redox regulation determines information flow from the cytosolic termini to the channel's gate.

Keywords: Abbreviations; EAG; ether à go-go; DTNB; 5, 5′-dithio-bis[2-nitrobenzoic acid]); DTNP; 2,2′-dithio-bis [5-nitropyridine]; DTT; dithiothreitol; MTSET; [2-(trimethylammonium)ethyl] methane thiosulfonate bromide; MTSES; 2-sulfonatoethyl methanethiosulfonate sodiumPotassium channel; Reactive oxygen species; Oxidation; Sulfhydryl modification; Ether à go-go; Gating

Investigation into the role of phosphatidylserine in modifying the susceptibility of human lymphocytes to secretory phospholipase A₂ using cells deficient in the expression of scramblase by Jennifer Nelson; Lyndee L. Francom; Lynn Anderson; Kelly Damm; Ryan Baker; Joseph Chen; Sarah Franklin; Amy Hamaker; Izadora Izidoro; Eric Moss; Mikayla Orton; Evan Stevens; Celestine Yeung; Allan M. Judd; John D. Bell (pp. 1196-1204).

Normal human lymphocytes resisted the hydrolytic action of secretory phospholipase A₂ but became susceptible to the enzyme following treatment with a calcium ionophore, ionomycin. To test the hypothesis that this susceptibility requires exposure of the anionic lipid phosphatidylserine on the external face of the cell membrane, experiments were repeated with a human Burkitt's lymphoma cell line (Raji cells). In contrast to normal lymphocytes or S49 mouse lymphoma cells, most of the Raji cells (83%) did not translocate phosphatidylserine to the cell surface upon treatment with ionomycin. Those few that did display exposed phosphatidylserine were hydrolyzed immediately upon addition of phospholipase A₂. Interestingly, the remaining cells were also completely susceptible to the enzyme but were hydrolyzed at a slower rate and after a latency of about 100s. In contradistinction to the defect in phosphatidylserine translocation, Raji cells did display other physical membrane changes upon ionomycin treatment that may be relevant to hydrolysis by phospholipase A₂. These changes were detected by merocyanine 540 and trimethylammonium diphenylhexatriene fluorescence and were common among normal lymphocytes, S49 cells, and Raji cells. The levels of these latter effects corresponded well with the relative rates of hydrolysis among the three cell lines. These results suggested that while phosphatidylserine enhances the rate of cell membrane hydrolysis by secretory phospholipase A₂, it is not an absolute requirement. Other physical properties such as membrane order contribute to the level of membrane susceptibility to the enzyme independent of phosphatidylserine.► Phosphatidylserine exposure is not an absolute requirement for membrane hydrolysis by secretory phospholipase A₂. ► Dying lymphoma cells are more susceptible to hydrolysis by secretory phospholipase A₂ than their normal human counterparts. ► Hydrolysis of cell membranes correlates best with physical membrane changes that occur during ionomycin-induced cell death.

Keywords: Secretory phospholipase A2; Phosphatidylserine exposure; Biophysical membrane change; Hydrolysis; Merocyanine 540; Trimethylammonium diphenylhexatriene

Membrane models of E. coli containing cyclic moieties in the aliphatic lipid chain by Kunal R. Pandit; Jeffery B. Klauda (pp. 1205-1210).

Nearly all molecular dynamics simulations of bacterial membranes simplify the lipid bilayer by composing it of only one or two lipids. Previous attempts of developing a model E. coli membrane have used only 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphoethanolamine (POPE) and (1-palmitoyl-2-oleoyl- sn-glycero-3-phosphoglycerol) POPG lipids. However, an important constituent of bacterial membranes are lipids containing a cyclopropane ring within the acyl chain. We have developed a complex membrane that more accurately reflects the diverse population of lipids within E. coli cytoplasmic membranes, including lipids with a cyclic moiety. Differences between the deuterium order profile of cyclic lipids and monounsaturated lipids are observed. Furthermore, the inclusion of the cyclopropane ring decreases the surface density of the bilayer and produces a more rigid membrane as compared to POPE/POPG membranes. Additionally, the diverse acyl chain length creates a thinner bilayer which matches the hydrophobic thickness of E. coli transmembrane proteins better than the POPE/POPG bilayer. We believe that the complex lipid bilayer more accurately describes a bacterial membrane and suggest the use of it in molecular dynamic simulations rather than simple POPE/POPG membranes.Display Omitted ► E. coli membranes consist of lipids with a cyclopropane moiety in the acyl-chain. ► New CHARMM lipid force field parameters are developed for these lipids. ► Hydrophobic thickness is underestimated without these cyclic-containing lipids. ► Hydrophobic matching of E. coli. transmembrane proteins is best with our model.

Keywords: E. coli; membranes; Hydrophobic mismatch; Molecular simulations; Force field development

Probing the interaction between heparan sulfate proteoglycan with biologically relevant molecules in mimetic models for cell membranes: A Langmuir film study by Luciano Caseli; Renan Pelluzzi Cavalheiro; Helena B. Nader; Carla Cristina Lopes (pp. 1211-1217).

Investigating the role of proteoglycans associated to cell membranes is fundamental to comprehend biochemical process that occurs at the level of membrane surfaces. In this paper, we exploit syndecan-4, a heparan sulfate proteoglycan obtained from cell cultures, in lipid Langmuir monolayers at the air–water interface. The monolayer served as a model for half a membrane, and the molecular interactions involved could be evaluated with tensiometry and vibrational spectroscopy techniques. Polarization–modulation infrared reflection–absorption spectroscopy (PM-IRRAS) employed in a constant surface pressure regime showed that the main chemical groups for syndecan-4 were present at the air–water interface. Subsequent monolayer decompression and compression showed surface pressure-area isotherms with a large expansion for the lipid monolayers interacting with the cell culture reported to over-express syndecan-4, which was also an indication that the proteoglycan was inserted in the lipid monolayer. The introduction of biological molecules with affinity for syndecam-4, such as growth factors, which present a key role in biochemical process of cell signaling, changed the surface properties of the hybrid film, leading to a model, by which the growth factor binds to the sulfate groups present in the heparan sulfate chains. The polypeptide moiety of syndecan-4 responds to this interaction changing its conformation, which leads to lipid film relaxation and further monolayer condensation.Display Omitted► Syndecan-4 was incorporated in lipid monolayer. ► Cell culture that over-expresses syndecan-4 expands lipid monolayers. ► Growth factor interacts with sulfate in heparan chains. ► This interaction relaxes the DPPC monolayer. ► The monolayer becomes more condensed.

Keywords: Abbreviations; PM-IRRAS; polarization–modulation infrared reflection–absorption spectroscopy; HSPGs; heparan sulfate proteoglycans; EGF; epidermal growth factor; EC; endothelial cell; EJ-; ras; EC; Endothelial cell transfected with EJ-ras oncogene; shRNA-Syn4-EC; EC transfected with plasmid vector for the expression of RNA interference to syndecan-4; DPPC; dipalmitoylphosphatidylcholineHeparan sulfate proteoglycan; Syndecan-4; Molecular recognizing; Langmuir monolayer

Molar concentrations of sorbitol and polyethylene glycol inhibit the Plasmodium aquaglyceroporin but not that of E. coli: Involvement of the channel vestibules by Jie Song; Abdulnasser Almasalmeh; Dawid Krenc; Eric Beitz (pp. 1218-1224).

The aquaglyceroporins of Escherichia coli, EcGlpF, and of Plasmodium falciparum, PfAQP, are probably the best characterized members of the solute-conducting aquaporin (AQP) subfamily. Their crystal structures have been elucidated and numerous experimental and theoretical analyses have been conducted. However, opposing reports on their rates of water permeability require clarification. Hence, we expressed EcGlpF and PfAQP in yeast, prepared protoplasts, and compared water and glycerol permeability of both aquaglyceroporins in the presence of different osmolytes, i.e. sucrose, sorbitol, PEG300, and glycerol. We found that water permeability of PfAQP strongly depends on the external osmolyte, with full inhibition by sorbitol, and increasing water permeability when glycerol, PEG300, and sucrose were used. EcGlpF expression did not enhance water permeability over that of non-expressing control protoplasts regardless of the osmolyte. Glycerol permeability of PfAQP was also inhibited by sorbitol, but to a smaller extent, whereas EcGlpF conducted glycerol independently of the osmolyte. Mixtures of glycerol and urea passed PfAQP equally well under isosmotic conditions, whereas under hypertonic conditions in a countercurrent with water, glycerol was clearly preferred over urea. We conclude that PfAQP has high and EcGlpF low water permeability, and explain the inhibiting effect of sorbitol on PfAQP by its binding to the extracellular vestibule. The preference for glycerol under hypertonic conditions implies that in a physiological setting, PfAQP mainly acts as a water/glycerol channel rather than a urea facilitator.Display Omitted ► Plasmodium falciparum PfAQP conducts water, E. coli EcGlpF does not. ► Sorbitol inhibits PfAQP water and glycerol permeability but not that of EcGlpF. ► The extracellular vestibule of PfAQP is a possible binding site for sorbitol. ► PfAQP prefers glycerol when exposed to mixed hypertonic glycerol–urea gradients.

Keywords: Aquaporin; Aquaglyceroporin; Plasmodium falciparum; E. coli; Sorbitol; Sucrose

A modified squeeze-out mechanism for generating high surface pressures with pulmonary surfactant by Eleonora Keating; Yi Y. Zuo; Seyed M. Tadayyon; Nils O. Petersen; Fred Possmayer; Ruud A.W. Veldhuizen (pp. 1225-1234).

The exact mechanism by which pulmonary surfactant films reach the very low surface tensions required to stabilize the alveoli at end expiration remains uncertain. We utilized the nanoscale sensitivity of atomic force microscopy (AFM) to examine phospholipid (PL) phase transition and multilayer formation for two Langmuir–Blodgett (LB) systems: a simple 3 PL surfactant-like mixture and the more complex bovine lipid extract surfactant (BLES). AFM height images demonstrated that both systems develop two types of liquid condensed (LC) domains (micro- and nano-sized) within a liquid expanded phase (LE). The 3 PL mixture failed to form significant multilayers at high surface pressure (π while BLES forms an extensive network of multilayer structures containing up to three bilayers. A close examination of the progression of multilayer formation reveals that multilayers start to form at the edge of the solid-like LC domains and also in the fluid-like LE phase. We used the elemental analysis capability of time-of-flight secondary ion mass spectrometry (ToF-SIMS) to show that multilayer structures are enriched in unsaturated PLs while the saturated PLs are concentrated in the remaining interfacial monolayer. This supports a modified squeeze-out model where film compression results in the hydrophobic surfactant protein-dependent formation of unsaturated PL-rich multilayers which remain functionally associated with a monolayer enriched in disaturated PL species. This allows the surface film to attain low surface tensions during compression and maintain values near equilibrium during expansion.► Direct evidence for a modified squeeze-out mechanism for pulmonary surfactant. ► AFM height images showing monolayer–multilayer transition and progression of multilayer structures. ► Multilayer structures form in LE phase and at edge of LC domains. ► ToF-SIMS data showing unsaturated PL in multilayer structures at high surface pressure. ► ToF-SIMS data showing saturated PL in remaining monolayer at high surface pressure.

Keywords: Pulmonary surfactant; Adsorption pores; Modified squeeze-out model; AFM; ToF-SIMS; LB films

Bacillus cereus can attack the cell membranes of the alga Chara corallina by means of HlyII by Anatoly A. Kataev; Zhanna I. Andreeva-Kovalevskaya; Alexander S. Solonin; Vadim I. Ternovsky (pp. 1235-1241).

We studied the influence of Bacillus cereus bacteria on cells of the freshwater alga Chara corallina. These bacteria and recombinant Bacillus subtilis strains are capable of producing the secreted toxin HlyII, which changes the electrophysiological parameters of the algal electrically excitable plasma membrane by forming pores. Cooperative incubation of bacterial cells, which carry active hlyII gene, and Chara corallina cells caused a decrease in the resting potential ( V_m) and plasma membrane resistance ( R_m) of algal cells. The efficiency of each strain was commensurable with its ability to produce HlyII. Purified hemolysin II caused a similar effect on V_m and R_m of intact and perfused cells. This protein changed the kinetics and magnitude of transient voltage-dependent calcium and calcium-activated chloride currents owing to the formation of additional Ca2⁺-permeable pores in algal cell membrane. Occurrence of the cellulose cell wall with pores 2.1 to 4.6nm in diameter suggests that HlyII molecules reach the plasma membrane surface strictly as monomers.►The influence of Bacillus cereus and Bacillus subtilis on cells of Chara corallina. ►Bacterium decreased the resting potential and plasma membrane resistance of algal cells. ►The efficiency of each strain was commensurable with its ability to produce HlyII. ►HlyII changed the kinetics and magnitude of transient calcium and chloride currents. ►HlyII-toxin formed additional Ca2⁺-permeable pores in algal cell membrane.

Keywords: Pore-forming toxin HlyII; Bacillus cereus; Recombinant; Bacillus subtilis; strain; Chara corallina; Electrically excitable membrane; Calcium channel; Chloride channel

Hydrophobic mismatch of mobile transmembrane helices: Merging theory and experiments by Erik Strandberg; Esteban-Martin Santi Esteban-Martín; Anne S. Ulrich; Jesús Salgado (pp. 1242-1249).

Hydrophobic mismatch still represents a puzzle for transmembrane peptides, despite the apparent simplicity of this concept and its demonstrated validity in natural membranes. Using a wealth of available experimental^²H NMR data, we provide here a comprehensive explanation of the orientation and dynamics of model peptides in lipid bilayers, which shows how they can adapt to membranes of different thickness. The orientational adjustment of transmembrane α-helices can be understood as the result of a competition between the thermodynamically unfavorable lipid repacking associated with peptide tilting and the optimization of peptide/membrane hydrophobic coupling. In the positive mismatch regime (long-peptide/thin-membrane) the helices adapt mainly via changing their tilt angle, as expected from simple geometrical predictions. However, the adaptation mechanism varies with the peptide sequence in the flanking regions, suggesting additional effects that modulate hydrophobic coupling. These originate from re-adjustments of the peptide hydrophobic length and they depend on the hydrophobicity of the flanking region, the strength of interfacial anchoring, the structural flexibility of anchoring side-chains and the presence of alternative anchoring residues.Display Omitted►^²H NMR data from transmembrane peptides are analyzed considering explicit dynamics. ► Tilt angles are calculated in lipid system-peptides at different hydrophobic mismatch. ► The mismatch behavior is highly dependent on flanking residues of the peptides. ► The tilt of WW-flanked peptides follows a simple cosine rule. ► KK-flanked cases deviate from cosine dependence due to change of length with mismatch.

Keywords: Orientation of transmembrane peptides; Dynamics of transmembrane peptides; Peptide tilt angle; Solid-state; ²; H NMR; WALP peptide family; XWALP peptide family

Structural determinants of the specificity of a membrane binding domain of the scaffold protein Ste5 of budding yeast: Implications in signaling by the scaffold protein in MAPK pathway by Anirban Bhunia; Harini Mohanram; Surajit Bhattacharjya (pp. 1250-1260).

In the mitogen activated protein kinase (MAPK) cascades of budding yeast, the scaffold protein Ste5 is recruited to the plasma membrane to transmit pheromone induced signal. A region or domain of Ste5 i.e. residues P44-R67, referred here as Ste5PM24, has been known to be involved in direct interactions with the membrane. In order to gain structural insights into membrane interactions of Ste5, here, we have investigated structures and interactions of two synthetic peptide fragments of Ste5, Ste5PM24, and a hyperactive mutant, Ste5PM24LM, by NMR, ITC, and fluorescence spectroscopy, with lipid membranes. We observed that Ste5PM24 predominantly interacted only with the anionic lipid vesicles. By contrast, Ste5PM24LM exhibited binding with negatively charged as well as zwitterionic or mixed lipid vesicles. Binding of Ste5 peptides with the negatively charged lipid vesicles were primarily driven by hydrophobic interactions. NMR studies revealed that Ste5PM24 assumes dynamic or transient conformations in zwitterionic dodecylphosphocholine (DPC) micelles. By contrast, NMR structure, obtained in anionic sodium dodecyl sulphate (SDS), demonstrated amphipathic helical conformations for the central segment of Ste5PM24. The hydrophobic surface of the helix was found to be buried inside the micelles. Taken together, these results provide important insights toward the structure and specificity determinants of the scaffold protein interactions with the plasma membrane.Display Omitted► Solution structure of membrane binding domain of scaffold protein Ste5 in SDS micelles. ► Structure formation is specific to negatively charged lipids. ► Structure shows critical packing among hydrophobic residues.

Keywords: Abbreviations; NMR; Nuclear Magnetic Resonance; MAPK; mitogen activated protein kinase; SDS; sodiumdodecylsulphate; POPG; 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-sn-glycerol); DMPG; 1,2-Dimyristoyl-; sn; -glycero-3-Phospho-rac-(1-glycerol); DMPC; (1,2-dimyristoyl-sn-glycero-3-phosphocholine); NOESY; nuclear Overhauser effect spectroscopy; ITC; isothermal titration calorimetryScaffold protein; NMR; MAPK; Pheromone response; Yeast; Signaling

NMR structures of the transmembrane domains of the α4β2 nAChR by Vasyl Bondarenko; David Mowrey; Tommy Tillman; Tanxing Cui; Lu Tian Liu; Yan Xu; Pei Tang (pp. 1261-1268).

The α4β2 nicotinic acetylcholine receptor (nAChR) is the predominant heteromeric subtype of nAChRs in the brain, which has been implicated in numerous neurological conditions. The structural information specifically for the α4β2 and other neuronal nAChRs is presently limited. In this study, we determined structures of the transmembrane (TM) domains of the α4 and β2 subunits in lauryldimethylamine-oxide (LDAO) micelles using solution NMR spectroscopy. NMR experiments and size exclusion chromatography-multi-angle light scattering (SEC-MALS) analysis demonstrated that the TM domains of α4 and β2 interacted with each other and spontaneously formed pentameric assemblies in the LDAO micelles. The Na⁺ flux assay revealed that α4β2 formed Na⁺ permeable channels in lipid vesicles. Efflux of Na⁺ through the α4β2 channels reduced intra-vesicle Sodium Green™ fluorescence in a time-dependent manner that was not observed in vesicles without incorporating α4β2. The study provides structural insight into the TM domains of the α4β2 nAChR. It offers a valuable structural framework for rationalizing extensive biochemical data collected previously on the α4β2 nAChR and for designing new therapeutic modulators.► The structures of the α4β2 TM domains are solved for the first time in micelles. ► The α4β2 TM domains alone can form pentameric channels permeable to Na⁺. ► The reported methods are useful for structural studies of other membrane proteins. ► The study offers a framework for rationalizing previous data collected on α4β2. ► The structures are valuable for designing new therapeutic modulators of nAChRs.

Keywords: Nicotinic acetylcholine receptor; nAChR; α4β2; NMR; Ion channel structure; Cys-loop receptor

Na,K-ATPase activity modulates Src activation: A role for ATP/ADP ratio by Karl M. Weigand; Herman G.P. Swarts; Natalya U. Fedosova; Frans G.M. Russel; Jan B. Koenderink (pp. 1269-1273).

Digitalis-like compounds (DLCs), specific inhibitors of Na,K-ATPase, are implicated in cellular signaling. Exposure of cell cultures to ouabain, a well-known DLC, leads to up- or down regulation of various processes and involves activation of Src kinase. Since Na,K-ATPase is the only known target for DLC binding an in vitro experimental setup using highly purified Na,K-ATPase from pig kidney and commercially available recombinant Src was used to investigate the mechanism of coupling between the Na,K-ATPase and Src. Digoxin was used as a representative DLC for inhibition of Na,K-ATPase. The activation of Src kinase was measured as the degree of its autophosphorylation. It was observed that in addition to digoxin, Src activation was dependent on concentrations of other specific ligands of Na,K-ATPase: Na⁺, K⁺, vanadate, ATP and ADP. The magnitude of the steady-state ATPase activity therefore seemed to affect Src activation. Further experiments with an ATP regenerating system showed that the ATP/ADP ratio determined the extent of Src activation. Thus, our model system which represents the proposed very proximal part of the Na,K-ATPase-Src signaling cascade, shows that Src kinase activity is regulated by both ATP and ADP concentrations and provides no evidence for a direct interaction between Na,K-ATPase and Src.►Characterization of the Na,K-ATPase signaling complex in vitro. ►Inhibition of Na,K-ATPase activity increases levels of phosphorylated Src protein. ►High ATP and low ADP concentrations are required for phosphorylation of Src. ►Na,K-ATPase activity and Src phosphorylation are linked to each other in signaling.

Keywords: Na,K-ATPase; Src; Ouabain; Digoxin; Digitalis-like compound

Charge distribution and imperfect amphipathicity affect pore formation by antimicrobial peptides by Maja Mihajlovic; Themis Lazaridis (pp. 1274-1283).

Antimicrobial peptides often permeabilize biological membranes via a pore mechanism. Two pore types have been proposed: toroidal, where the pore is partly lined by lipid, and barrel-stave, where a cylindrical pore is completely lined by peptides. What drives the preference of antimicrobial peptides for a certain pore type is not yet fully understood. According to neutron scattering and oriented circular dichroism, melittin and MG-H2 induce toroidal pores whereas alamethicin forms barrel-stave pores. In previous work we found that indeed melittin seems to favor toroidal pores whereas alamethicin favors cylindrical pores. Here we designed mutants of these two peptides and the magainin analog MG-H2, aimed to probe how the distribution of charges along the helix and its imperfectly amphipathic structure influence pore formation. Molecular dynamics (MD) simulations of the peptides in a pre-formed cylindrical pore have been performed. The duration of the simulations was 136ns to 216ns. We found that a melittin mutant with lysine 7 neutralized favors cylindrical pores whereas a MG-H2 mutant with lysines in the N-terminal half of these peptides neutralized and an alamethicin mutant with a positive charge at the position 7 form semitoroidal pores. These results suggest that charged residues within the N-terminal half are important for toroidal pore formation. Toroidal pores produced by MG-H2 are more disordered than the melittin pores, likely because of the charged residues located in the middle of the MG-H2 helix (K11 and K14). Imperfect amphipathicity of melittin seems to play a role in its preference for toroidal pores since the substitutions of charged residues located within the nonpolar face by hydrophobic residues suppress evolution of a toroidal pore. The mutations change the position of lysine 7 near the N-terminus, relative to the lower leaflet headgroups. The MD simulations also show that the melittin P14A mutant forms a toroidal pore, but its configuration diverges from that of melittin and it is probably metastable.► MD simulations of melittin, alamethicin, magainin mutants in pre-formed cylindrical pores. ► Charged residues in the N-terminal half are crucial for toroidal pore formation. ► Charge in the middle of the helix produces more disordered toroidal pores. ► Imperfect amphipathicity plays a role in toroidal pore formation. ► Melittin P14A mutant exhibits distinct behavior.

Keywords: Abbreviations; MD; molecular dynamics; AMT; alamethicin; MLT; melittin; AMP; antimicrobial peptide; DMPC; 1,2-dimyristoyl-; sn; -glycero-3-phosphocholine; DPPC; 1,2-dipalmitoyl-; sn; -glycero-3-phosphocholine; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; P/L; peptide-to-lipid ratioMolecular dynamics simulation; Antimicrobial peptide; Charge distribution; Imperfect amphipathicity; Melittin; Magainin MG-H2

A novel leaflet-selective fluorescence labeling technique reveals differences between inner and outer leaflets at high bilayer curvature by Salvatore Chiantia; Andrey S. Klymchenko; Erwin London (pp. 1284-1290).

Understanding the differences in the physical properties of the inner and outer leaflet of membranes and how the leaflets are coupled to each other requires methods that can selectively label both the outer and inner leaflets. In this report we introduce a combined chromatography/cyclodextrin method for selective labeling of the inner leaflet. Combining this method with selective labeling of the outer leaflet, we are able to show that there is a distinct difference in polar headgroup physical properties of the inner and outer leaflet headgroups in small unilamellar vesicles composed of a wide variety of phosphatidylcholines and a phosphaticylcholine/sphingomyelin mixture. It appears that the inner leaflet headgroups are more tightly packed than those of the outer leaflet. This differential packing disappears when vesicle size increases, showing that it is a consequence of membrane curvature. Differential packing is also reduced as acyl chain length is decreased. In the future, selective leaflet labeling is likely to be a powerful tool for investigating the properties of asymmetric lipid vesicles.► We introduce a method to label the inner or outer leaflet of lipid vesicles. ► In SUV, the outer leaflet headgroups are less tightly packed and more hydrated. ► In the inner leaflet, headgroups are more tightly packed and less hydrated. ► The differences between the two leaflets vanish when vesicles size is increased. ► The differences are also small when lipids with short acyl chains are used.

Keywords: Abbreviations; 6-NBD-PC; 2-(6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine; bSM; porcine brain sphingomyelin; DLS; dynamic light scattering; DOPC; 1,2-dioleoyl-; sn; -glycero-3-phosphocholine; mβCD; methyl-beta-cyclodextrin; NBD; nitrobenzoxadiazole; NBD-DPPE; N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine triethylammonium salt; NR12S; N-[3-[[9-(diethylamino)-5-oxo-5H-benzo[a]phenoxazin-2-yl]oxy]propyl]-N-methyl-N-(3-sulfopropyl)-1-dodecanaminium; PBS; phosphate buffered saline; PC; phosphatidylcholine; SOPC; 1-stearoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; SUV; small unilamellar vesicles; T; _m; mixing temperature; TMA-DPH; 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene; p; -toluenesulfonateInner leaflet; Outer leaflet; Asymmetric membrane; Fluorescence; Hydration; Curvature

Ceramide channels: Influence of molecular structure on channel formation in membranes by Meenu N. Perera; Vidyaramanan Ganesan; Leah J. Siskind; Zdzislaw M. Szulc; Jacek Bielawski; Alicja Bielawska; Robert Bittman; Marco Colombini (pp. 1291-1301).

The sphingolipid, ceramide, self-assembles in the mitochondrial outer membrane (MOM), forming large channels capable of translocating proteins. These channels are believed to be involved in protein release from mitochondria, a key decision-making step in cell death. Synthetic analogs of ceramide, bearing modifications in each of the major structural features of ceramide were used to probe the molecular basis for the stability of ceramide channels. Channel stability and mitochondrial permeabilization were disrupted by methylation of the C1-hydroxyl group whereas modifications of the C3 allylic hydroxyl group were well tolerated. A change in chirality at C2 that would influence the orientation of the C1-hydroxyl group resulted in a strong reduction of channel-forming ability. Similarly, methylation of the amide nitrogen is also detrimental to channel formation. Many changes in the degree, location and nature of the unsaturation of ceramide had little effect on mitochondrial permeabilization. Competition experiments between ceramide and analogs resulted in synergy with structures compatible with the ceramide channel model and antagonism with incompatible structures. The results are consistent with ceramide channels being highly organized structures, stabilized by specific inter-molecular interactions, similar to the interactions responsible for protein folding.► Ceramides self-assemble forming membrane channels capable of protein translocation. ► The use of ceramide analogs identified regions critical for channel formation. ► Experiments were performed on isolated mitochondria, liposomes, planar membranes. ► The stereochemistry at C2 of the sphingoid base was particularly critical. ► Competition experiments revealed synergy and antagonism between ceramide and analogs.

Keywords: Abbreviations; d; -e-; d; -; erythro; -; l; -e-; l; -; erythro; -; C; ₈; -Cer; D-e-C; ₈; -ceramide; C; ₁₆; -Cer; D-e-C; ₁₆; -ceramide; C; _18:1; -Cer; D-e-C; _18:1; -ceramide; DNP; 2,4-dinitrophenol; BSA; fatty acid depleted bovine serum albumin; MOM; mitochondrial outer membrane; DPX; p-xylene-bis-pyridinium bromideCeramide; Analogs; Channels; Mitochondria; Permeabilization; Pore

Interactions of the antifungal mycosubtilin with ergosterol-containing interfacial monolayers by Mehmet Nail Nasir; Françoise Besson (pp. 1302-1308).

Mycosubtilin, an antimicrobial lipopeptide produced by Bacillus subtilis, is characterized by strong antifungal activities. The molecular mechanisms of its biological activities on the membranes of the sensitive yeasts or fungi have not yet been clearly elucidated. Our purpose was to mimic the mycosubtilin interactions with these membranes using various Langmuir monolayers. Since the major sterol of yeasts or fungi is ergosterol, the interactions of mycosubtilin with monolayers constituted by ergosterol, DPPC/ergosterol or DPPC/sphingomyelin/ergosterol were examined at different initial surface pressures (Πi). Plotting the mycosubtilin-induced surface pressure increases versus Πi allowed to determine that the exclusion pressures of mycosubtilin from these different monolayers is higher than the surface prevailing within the biological membranes. However, this behavior was lost when mycosubtilin was interacting with ergosteryl acetate-containing monolayers. This suggests the involvement of the sterol alcohol group in the mycosubtilin interactions within membranes. Furthermore, the behavior of mycosubtilin with stigmasterol, similar to that observed with ergosterol, differs from that previously observed with cholesterol, suggesting a role of the alkyl side chain of the sterols. The adsorption of mycosubtilin to ergosterol monolayers induced changes in the lipopeptide orientation at the air–water interface as revealed by polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS). Moreover, imaging the air–water interface by Brewster angle microscopy (BAM) indicates that mycosubtilin induced changes in the organization and morphology of monolayers containing pure ergosterol with the appearance of small condensed dots, suggesting again that the target of mycosubtilin might be the ergosterol present in the membranes of the sensitive yeasts or fungi.► Mycosubtilin is a natural antifungal lipopeptide produced by B. subtilis. ► Mycosubtilin adsorption to different lipid monolayers is surface pressure-dependent. ► Mycosubtilin interacts preferentially with pure ergosterol monolayers.

Keywords: Abbreviations; BAM; Brewster angle microscopy; Chol; cholesterol; DPPC; dipalmitoylphosphatidylcholine; Erg; ergosterol; GL; gray level; Пex; exclusion pressures; Π; _i; initial surface pressure; OS; obturation speed; PM-IRRAS; polarization modulation infrared reflection absorption spectroscopyLipopeptide; Iturin; Bacillus subtilis; BAM; Ergosterol; PM-IRRAS

Fatty acid composition of membrane bilayers: Importance of diet polyunsaturated fat balance by Sarah K. Abbott; Paul L. Else; Taleitha A. Atkins; A.J. Hulbert (pp. 1309-1317).

In one of the most extensive analyses to date we show that the balance of diet n−3 and n−6 polyunsaturated fatty acids (PUFA) is the most important determinant of membrane composition in the rat under ‘normal’ conditions. Young adult male Sprague–Dawley rats were fed one of twelve moderate-fat diets (25% of total energy) for 8weeks. Diets differed only in fatty acid (FA) profiles, with saturate (SFA) content ranging 8–88% of total FAs, monounsaturate (MUFA) 6–65%, total PUFA 4–81%, n−6 PUFA 3–70% and n−3 PUFA 1–70%. Diet PUFA included only essential FAs 18:2n−6 and 18:3n−3. Balance between n−3 and n−6 PUFA is defined as the PUFA balance (n−3 PUFA as % of total PUFA) and ranged 1–86% in the diets. FA composition was measured for brain, heart, liver, skeletal muscle, erythrocytes and plasma phospholipids, as well as adipose tissue and plasma triglycerides. The conformer–regulator model was used (slope=1 indicates membrane composition completely conforming to diet). Extensive changes in diet SFA, MUFA and PUFA had minimal effect on membranes (average slopes 0.01, 0.07, 0.07 respectively), but considerable influence on adipose tissue and plasma triglycerides (average slopes 0.27, 0.53, 0.47 respectively). Diet balance between n−3 and n−6 PUFA had a biphasic influence on membrane composition. When n−3 PUFA<10% of total PUFA, membrane composition completely conformed to diet (average slope 0.95), while diet PUFA balance>10% had little influence (average slope 0.19). The modern human diet has an average PUFA balance ~10% and this will likely have significant health implications.► An extensive study of influence of diet fat on membrane composition in rat tissues. ► Membrane SFA, MUFA and PUFA homeostatically regulated irrespective of diet variation. ► Adipose tissue and plasma triglyceride composition are highly responsive to diet. ► Membranes respond biphasically to balance between diet n−3 and n−6 PUFA. ► Important health implications for diets with n−3 PUFA<10% total PUFA.

Keywords: Omega; −; 3; Omega; −; 6; Membrane; Phospholipids; Triglyceride; PUFA balance

Spectroscopic analysis of small multidrug resistance protein EmrE in the presence of various quaternary cation compounds by Denice C. Bay; Raymond J. Turner (pp. 1318-1331).

Escherichia coli EmrE protein is the archetypical member of the small multidrug resistance protein family in bacteria and confers host resistance to a wide assortment of toxic quaternary cation compounds by secondary active efflux. This protein can form a variety of multimers under various membrane mimetic conditions, and the consensus of most biochemical and biophysical studies indicate that the active form is a dimer. The purpose of this study is to characterize the conformation of organically extracted detergent solubilized EmrE protein known to predominate as monomer yet demonstrates ligand binding ability. Active site EmrE-E14 replacements were also examined as functionally inactive controls for this study. EmrE was solubilized in detergents, sodium dodecyl sulfate (SDS) and dodecyl maltoside (DDM), and protein conformation was examined in the presence of four known quaternary cation compound (QCC) substrates, tetraphenyl phosphonium (TPP), methyl viologen, cetylpyridinium, and ethidium. SDS-Tricine PAGE analysis of both detergent solubilized proteins revealed that DDM-EmrE preparations enhanced the formation of dimer (and in some cases trimer) forms in the presence of all four QCC above 25 QCC:1 EmrE molar ratios. Examination of EmrE and its active site variant tertiary structures in DDM by circular dichroism spectropolarimetry, intrinsic Trp fluorescence quenching and second order derivative ultraviolet absorbance revealed that the variant fails to bind TPP but interacts with all other compounds. The results of this study show that monomeric detergent solubilized EmrE is capable of forming multimeric complexes that are enhanced by chemically diverse QCCs.

Keywords: Small multidrug resistance protein (SMR); Quaternary cation compound (QCC); Tetraphenyl phosphonium (TPP); Cetylpyridinium (CTP); Ethidium (ET); Methyl viologen (MV)

Control of pH responsive peptide self-association during endocytosis is required for effective gene transfer by Valentina Iacobucci; Francesca Di Giuseppe; Tam T. Bui; Louic S. Vermeer; Jayneil Patel; Daniel Scherman; Antoine Kichler; Alex F. Drake; A. James Mason (pp. 1332-1341).

Cationic amphipathic histidine rich peptides demonstrate differential nucleic acid binding capabilities at neutral and acidic pH and adopt conformations at acidic pH that enable interaction with endosomal membranes, their subsequent disordering and facilitate entry of cargo to the cell cytosol. To better understand the relative contributions of each stage in the process and consequently the structural requirements of pH responsive peptides for optimal nucleic acid transfer, we used biophysical methods to dissect the series of events that occur during endosomal acidification. Far-UV circular dichroism was used to characterise the solution conformation of a series of peptides, containing either four or six histidine residues, designed to respond at differing pH while a novel application of near-UV circular dichroism was used to determine the binding affinities of the peptides for both DNA and siRNA. The peptide induced disordering of neutral and anionic membranes was investigated using²H solid-state NMR. While each of these parameters models key stages in the nucleic acid delivery process and all were affected by increasing the histidine content of the peptide, the effect of a more acidic pH response on peptide self-association was most notable and identified as the most important barrier to further enhancing nucleic acid delivery. Further, the results indicate that Coulombic interactions between the histidine residues modulate protonation and subsequent conformational transitions required for peptide mediated gene transfer activity and are an important factor to consider in future peptide design.► We characterise the pH responsive behaviour of nucleic acid delivery peptides. ► We determine their pH response in solution, bound to DNA/RNA cargo or membranes. ► All peptides bound to DNA/RNA or membranes respond at pH found in endocytosis. ► The most effective peptides avoid self‐association in solution during endocytosis. ► The peptide pH response in solution determines nucleic acid transfer efficacy.

Keywords: pH responsive peptides; Endocytosis; Gene delivery; DNA binding; Circular dichroism

The nitrite transport protein NirC from Salmonella typhimurium is a nitrite/proton antiporter by Adriana Rycovska; Lina Hatahet; Klaus Fendler; Hartmut Michel (pp. 1342-1350).

In anaerobically grown bacteria, transport of nitrite is catalyzed by an integral membrane protein of the form ate–nitrite transporter family, NirC, which in Salmonella typhimurium plays a critical role in intracellular virulence. We present a functional characterization of the S. typhimurium nitrite transporter StmNirC in native membrane vesicles as well as purified and reconstituted into proteoliposomes. Using an electrophysiological technique based on solid supported membranes, we show nitrite induced translocation of negative charges into proteoliposomes reconstituted with purified StmNirC. These data demonstrate the electrogenicity of StmNirC and its substrate specificity for nitrite. Monitoring changes in ΔpH on everted membrane vesicles containing overexpressed StmNirC using acridine orange as a pH indicator we demonstrate that StmNirC acts as a secondary active transporter. It promotes low affinity transport of nitrite coupled to H⁺ antiport with a pH independent profile in the pH range from 6 to 8. In addition to nitrite also nitrate is transported by StmNirC, but with reduced flux and complete absence of proton antiport activity. Taken together, these data suggest a bispecific anion selectivity of StmNirC with an ion specific transport mode. This may play a role in regulating nitrite transport under physiological conditions.► Functional assays in natural membranes and with purified protein in proteoliposomes. ► Transport is electrogenic. ► StmNirC is a nitrite/proton antiporter. ► Indications for uncoupled nitrate transport.

Keywords: Abbreviations; His-tag; polyhistidine tag; IC; ₅₀; half maximum inhibitory concentration; IPTG; isopropyl-β-; d; -thiogalactoside; LB; Luria-Bertani growth media; DDM; n; -dodecyl-β-; d; -maltoside; Ni-NTA; nickel-nitrilotriacetic acid; ORF; open reading frame; SDS-PAGE; sodium dodecyl sulfate polyacrylamide electrophoresis; SSM; solid supported membranes; TM; transmembrane helix; TEV; Tobacco Etch Virus; BTS; 1,3-Bis[tris(hydroxymethyl) methylamino] propane; S. typhimurium; Salmonella typhimuriumNitrite transport; Dequenching assay; Acridine orange; Solid supported membrane; Transport mechanism

Efficiency of detergents at maintaining membrane protein structures in their biologically relevant forms by David V. Tulumello; Charles M. Deber (pp. 1351-1358).

High-resolution structural analysis of membrane proteins by X-ray crystallography or solution NMR spectroscopy often requires their solubilization in the membrane-mimetic environments of detergents. Yet the choice of a detergent suitable for a given study remains largely empirical. In the present work, we considered the micelle-crystallized structures of lactose permease (LacY), the sodium/galactose symporter (vSGLT), the vitamin B₁₂ transporter (BtuCD), and the arginine/agmatine antiporter (AdiC). Representative transmembrane (TM) segments were selected from these proteins based on their relative contact(s) with water, lipid, and/or within the protein, and were synthesized as Lys-tagged peptides. Each peptide was studied by circular dichroism and fluorescence spectroscopy in water, and in the presence of the detergents sodium dodecylsulfate (SDS, anionic); n-dodecyl phosphatidylcholine (DPC, zwitterionic); n-dodecyl-β-d-maltoside (DDM, neutral); and n-octyl-β-d-glucoside (OG, neutral, varying acyl tail length). We found that (i) the secondary structures of the TM segments were statistically indistinguishable in the four detergents studied; and (ii) a strong correlation exists between the extent of helical structure of each individual TM segment in detergents with its helicity level as it exists in the full-length protein, indicating that helix adoption is fundamentally the same in both environments. The denaturing properties of so-called ‘harsh’ detergents may thus largely be due to their interactions with non-membranous regions of proteins. Given the consistency of structural features observed for each TM segment in a variety of micellar media, the overall results suggest that the structure likely corresponds to its relevant biological form in the intact protein in its native lipid bilayer environment.Display Omitted► The choice of detergent for membrane protein characterization is largely empirical. ► TM domain structure may be classified according to local environment. ► The choice of detergent does not affect secondary structure of TM segments. ► Native secondary structure is preserved in detergents. ► Denaturation is largely due to interactions with non-membranous regions of proteins.

Keywords: Abbreviations; TM; transmembrane; SDS; sodium dodecylsulfate; PBD; Protein Data Bank; CD; circular dichroism; DPC; n; -dodecyl phosphatidylcholine; DDM; n; -dodecyl-β-; d; -maltoside; OG; n; -octyl-β-; d; -glucoside; TFA; trifluoroacetic acid; MRE; mean residue ellipticityMembrane protein folding; Transmembrane domain classification; Detergent–protein interaction; Transmembrane peptide secondary structure; Membrane protein denaturation

Molecular events associated with Macrovipera lebetina obtusa and Montivipera raddei venom intoxication and condition of biomembranes by Naira M. Ayvazyan; Naira A. Zaqaryan; Narine A. Ghazaryan (pp. 1359-1364).

Studies on the interaction of snake venom and organized lipid interfaces have been conducted using a variety of systems, including BLMs, SUVs and GUVs. The present study was undertaken to elucidate how the plastic properties (namely, its microviscosity, thickness, permeability) of model membranes from native lipids of different tissues of rats change in the course of Macrovipera lebetina obtusa (MLO), Montivipera raddei (MR) and Naja kaouthia (NK) venoms processing. The presence of viper venom in organism leads to increasing of the electrical resistance of BLMs from liver and muscle lipids approximately on a sequence, while the BLMs from brain lipids have not shown noticeable differences of plastic properties compared to the control. Giant unilamellar vesicles (GUVs) with a mean diameter of 30μm have a minimum curvature and mimic cell membranes in this respect. Snake venom was added to the sample chamber before the vesicles were formed. The membrane fluorescence probes, ANS and pyrene, were used to assess the state of the membrane and specifically mark the phospholipid domains. Fluorescent spectra were acquired on a Varian fluorometer instrument. ANS and pyrene allow us to quantify the fluidity changes in the membrane by measuring of the fluorescence intensity. The presence of viper venom in GUVs media reveals a noticeable decreasing of membrane fluidity compared to the control, while the binding of fluorophores with GUVs modified by venom leads to the appearance of channel activity. These studies also emphasize the importance of a membrane surface curvature for its interaction with enzymatic components of venom.Display Omitted► Membrane bilayer condition abnormalities following Macrovipera lebetina obtusa and Montivipera raddei snakes envenomation. ► Viper venoms disturb the local packing structure of lipids and fluidity of bilayer. ► The binding of viper venom proteins to lipid bilayer is strictly curvature-dependent. ► Viper venoms demonstrate rather surface activity during BLM-peptide interaction.

Keywords: BLM; GUV; Electroporation; Snake venomics; Artificial membrane

Novel ether lipid cardiolipins in archaeal membranes of extreme haloalkaliphiles by Roberto Angelini; Paulina Corral; Patrizia Lopalco; Antonio Ventosa; Angela Corcelli (pp. 1365-1373).

The lipidome of two extremely haloalkaliphilic archaea, Natronococcus occultus and Natronococcus amylolyticus, has been examined by means of combined thin-layer chromatography and MALDI-TOF/MS analyses. The detailed investigation of lipid profiles has confirmed the presence of i) ether lipid phosphatidylglycerol and phosphatidylglycerophosphate methyl ester as main lipid components, ii) both C₂₀ and C₂₅ isopranoid chains in the lipid core and yielded new findings on membrane lipids of these unusual organisms. Besides some novel minor or trace phospholipids and glycolipids, data indicate the presence of ether lipid cardiolipin variants constituted by different combinations of C₂₀ and C₂₅ isopranoid chains, never before described in archaea. The role of C₂₅ isopranoid chains in the adaptation to high pH gradients in the presence of very high salt concentrations is discussed.► Membrane lipids of two extremely haloalkaliphilic archaea of Natronococcus genus. ► Identification of ether lipid cardiolipins with C₂₀ and C₂₅ isopranoid chains. ► Evidence for heterogeneity in lipid chains of archaeal cardiolipin. ► MALDI-TOF/MS fingerprints of archaeal cardiolipin variants is provided. ► Structures of novel ether lipid cardiolipins deposited in LipidMaps database.

Keywords: Abbreviations; BPG; bisphosphatidylglycerol (diphytanylglycerol ether analog) or ether lipid cardiolipin; ESI-MS; electrospray ionization mass spectrometry; Hbt. salinarum; Halobacterium salinarum; HPTLC; high-performance thin-layer chromatography; Hrr.; Halorubrum; MALDI-TOF/MS; matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; MS; mass spectrometry; Ncc. occultus; Natronococcus occultus; Ncc. amylolyticus; Natronococcus amylolyticus; NMR; Nuclear Magnetic Resonance; PE; phosphatidylethanolamine (diphytanylglycerol ether analog); PG; phosphatidylglycerol (diphytanylglycerol ether analog); PGP; phosphatidylglycerophosphate (diphytanylglycerol ether analog); PGP-Me; phosphatidylglycerophosphate methyl ester (diphytanylglycerol ether analog); PGS; phosphatidylglycerosulfate (diphytanylglycerol ether analog); PL; phospholipid; PM; purple membranes; S-DGD-5-PA; (2’-sulfo)Man; p; α1-2Glc; p; α1-1-[; sn; -2,3-di-; O; -phytanylglycerol]-6-[phospho-; sn; -2,3-di-; O; -phytanylglycerol]; S-TGD-1; (3’-sulfo)Gal; p; β1-6Man; p; α1-2Glc; p; α1-1-[; sn; -2,3-di-; O; -phytanylglycerol]; S-TGD-1-PA; (3’-sulfo)Gal; p; β1-6Man; p; α1-2Glc; p; α1-1-[; sn; -2,3-di-; O; -phytanylglycerol]-6-[phospho-; sn; -2,3-di-; O; -phytanylglycerol] (Glycocardiolipin); 9-AA; 9-aminoacridineArchaea; Cardiolipin; Ether lipids; Isopranoid chains; MALDI-TOF/MS; Natronococcus

A tetrahedral coordination of Zinc during transmembrane transport by P-type Zn²⁺-ATPases by Daniel Raimunda; Poorna Subramanian; Timothy Stemmler; Arguello José M. Argüello (pp. 1374-1377).

Zn²⁺ is an essential transition metal required in trace amounts by all living organisms. However, metal excess is cytotoxic and leads to cell damage. Cells rely on transmembrane transporters, with the assistance of other proteins, to establish and maintain Zn²⁺ homeostasis. Metal coordination during transport is key to specific transport and unidirectional translocation without the backward release of free metal. The coordination details of Zn²⁺ at the transmembrane metal binding site responsible for transport have now been established. Escherichia coli ZntA is a well-characterized Zn²⁺-ATPase responsible for intracellular Zn²⁺ efflux. A truncated form of the protein lacking regulatory metal sites and retaining the transport site was constructed. Metrical parameters of the metal–ligand coordination geometry for the zinc bound isolated form were characterized using x-ray absorption spectroscopy (XAS). Our data support a nearest neighbor ligand environment of (O/N)₂S₂ that is compatible with the proposed invariant metal coordinating residues present in the transmembrane region. This ligand identification and the calculated bond lengths support a tetrahedral coordination geometry for Zn²⁺ bound to the TM-MBS of P-type ATPase transporters.► Transmembrane transporters contribute to Zn²⁺ homeostasis in cells. ► Metal coordination is key to specific Zn²⁺ transmembrane translocation. ► Zn²⁺ coordination at the P_IB-ATPases transmembrane metal binding site was studied. ► A nearest neighbor ligand environment of (O/N)₂S₂ was found.

Keywords: Abbreviations; TM-MBS; transmembrane metal binding site; N-MBD; N-terminal metal binding domain; TM; transmembrane segment; T-ZntA; truncated ZntA lacking the first 100 amino acids; DDM; dodecyl-β-; d; -maltoside; TEV; Tobacco etch virus; TCEP; tris(2 carboxyethyl)phosphine hydrochloride; AAS; atomic absorption spectroscopy; XAS; x-ray absorption spectroscopy; EXAFS; extended x-ray absorption fine structureZinc; Transport; Transmembrane; Metal coordination; X-ray spectroscopy; EXAFS

The role of sulfatide lipid domains in the membrane pore-forming activity of cobra cardiotoxin by Po-Long Wu; Chang-Ru Chiu; Wei-Ning Huang; Wen-Guey Wu (pp. 1378-1385).

Cobra CTX A3, the major cardiotoxin (CTX) from Naja atra, is a cytotoxic, basic β-sheet polypeptide that is known to induce a transient membrane leakage of cardiomyocytes through a sulfatide-dependent CTX membrane pore formation and internalization mechanism. The molecular specificity of CTX A3-sulfatide interaction at atomic levels has also been shown by both nuclear magnetic resonance (NMR) and X-ray diffraction techniques to reveal a role of CTX-induced sulfatide conformational changes for CTX A3 binding and dimer formation. In this study, we investigate the role of sulfatide lipid domains in CTX pore formation by various biophysical methods, including fluorescence imaging and atomic force microscopy, and suggest an important role of liquid-disordered ( ld) and solid-ordered ( so) phase boundary in lipid domains to facilitate the process. Fluorescence spectroscopic studies on the kinetics of membrane leakage and CTX oligomerization further reveal that, although most CTXs can oligomerize on membranes, only a small fraction of CTXs oligomerizations form leakage pores. We therefore suggest that CTX binding at the boundary between the so and so/ ld phase coexistence sulfatide lipid domains could form effective pores to significantly enhance the CTX-induced membrane leakage of sulfatide-containing phosphatidylcholine vesicles. The model is consistent with our earlier observations that CTX may penetrate and lyse the bilayers into small aggregates at a lipid/protein molar ratio of about 20 in the ripple P_β′ phase of phosphatidylcholine bilayers and suggest a novel mechanism for the synergistic action of cobra secretary phospholipase A2 and CTXs.►Phase boundary in lipid domains to facilitate the leakage process of CTX. ►Only a small fraction of CTX can form pores and induce vesicle leakage. ►CTX binding at the boundary of so and so/ ld phase could form effective pores.

Keywords: Cardiotoxin; Pore; Lipid domain; Sulfatide

Binding of peptides corresponding to the carboxy-terminal region of human-β-defensins-1–3 with model membranes investigated by isothermal titration calorimetry by V. Krishnakumari; R. Nagaraj (pp. 1386-1394).

Human-β-defensins HBD-1–3 are important components of the innate immune system. Synthetic peptides Phd-1–3 with a single disulphide bond, spanning the cationic C-terminal region of HBD-1–3, have antimicrobial activity. The interaction of Phd-1–3 with model membranes was investigated using isothermal titration calorimetry (ITC) and steady-state fluorescence polarization to understand the biophysical basis for the mechanism of antimicrobial action. Calorimetric titration of POPE:POPG (7:3) vesicles with peptides at 25°C and 37°C showed complex profiles with two distinct regions of heat changes. The data indicate binding of Phd-1–3 at 37°C to both negative and zwitterionic lipid vesicles is exothermic with low enthalpy values (ΔH ~−1.3 to −2.8kcal/mol) as compared to amphipathic helical antibacterial peptides. The adsorption of peptides to negatively charged lipid membranes is modulated by electrostatic interactions that are described by surface partition equilibrium model using Gouy–Chapman theory. However, this model could not explain the isotherms of peptide binding to zwitterionic lipid vesicles. Fluorescence polarization of TMA-DPH (1-[4-(trimethylammonio) phenyl]-6-phenyl-1,3,5-hexatriene) and DPH (1,6-diphenyl-1,3,5-hexatriene) located in the head group and acyl chain region respectively, indicates that the peptides interact with interfacial region of negatively charged membranes. Based on the results obtained, we conclude that adsorption of cationic peptides Phd-1–3 on lipid surface do not result in conformational change or pore formation. It is proposed that interaction of Phd-1–3 with the negatively charged lipid head group causes membrane destabilization, which in turn affects the efficient functioning of cytoplasmic membrane proteins in bacteria, resulting in cell death.► Binding of peptides to model membranes was investigated by ITC. ► Binding of peptides to lipid vesicles was analyzed at 37°C. ► Binding to zwitterionic membranes is not explained by Gouy–Chapman theory. ► Binding to negatively charged model membranes does not result in pore formation. ► Membrane destabilization may affect functioning of cytoplasmic membrane proteins.

Keywords: Human-β-defensin analog; Peptide–membrane interaction; Surface partition equilibrium model; Electrostatic interaction; Peptide conformation; Membrane destabilization

Methylation of ethanolamine groups in phosphoethanolamines is relevant for L-arginine insertion in lipid membranes by Ana Bouchet; Fabiana Lairion; Anibal Disalvo (pp. 1395-1401).

The interaction of L-arginine with membranes composed by phospholipids with different degrees of methylation of the ethanolamine group was studied by means of surface and dipole potentials and surface pressure variations. The subsequent methylation of the amine head group appears to hinder the synergic response of the adsorption observed in phosphatidylethanolamine membranes. The kinetics of the binding process denotes that the methyl groups are relevant in regulating the specific interaction of the amino acid with the interface by hydrogen bonds. This response can be put in correlation with the function of signal transduction assigned previously to methyl lipids [F. Hirata and J. Axelrod, 1980] and appears to be relevant to understand the mechanism of insertion of arginine residues in peptides of biological interest.► H bond formation with the lipid NH groups drives Arg adsorption on DMPE in a 2:1 lipid Arg complex. ► Blocking the NH groups of PE by methylation changes the pattern of adsorption to a 1:1 lipid:Arg ratio as in PCs. ► Similar response is obtained when PE groups are in a PC environment.

Keywords: Arginine; Phosphatidylethanolamine; Phosphatidylethanolamine-methyl derivates; Phosphatidylcholine; Surface pressure; Isotherm adsorption

Comparative molecular dynamics simulations of the antimicrobial peptide CM15 in model lipid bilayers by Yi Wang; Diana E. Schlamadinger; Judy E. Kim; J. Andrew McCammon (pp. 1402-1409).

We report altogether 3-μs molecular dynamics (MD) simulations of the antimicrobial peptide CM15 to systematically investigate its interaction with two model lipid bilayers, pure POPC and mixed POPG:POPC (1:2). Starting with either an α-helical or a random-coil conformation, CM15 is found to insert into both bilayers. Peptide-lipid interaction is stronger with the anionic POPG:POPC than the zwitterionic POPC, which is largely attributed to the electrostatic attraction between CM15 and the negatively charged POPG. Simulations initiated with CM15 as a random coil allowed us to study peptide folding at the lipid-water interface. Interestingly, CM15 folding appears to be faster in POPC than POPG:POPC, which may be explained by a lower activation energy barrier of structural rearrangement in the former system. Our data also suggest that compared with the random-coil conformation, CM15 in a pre-folded α-helix has significantly reduced interactions with the lipids, indicating that peptide initial structures may bias the simulation results considerably on the 100-ns timescale. The implications of this result should be considered when preparing and interpreting future AMP simulations.►We report over 3-μs MD simulations of CM15 with POPC and POPG:POPC. ►CM15 binds and inserts into both bilayers despite its non-hemolytic nature. ►CM15 folding is faster in POPC than POPG:POPC. ►Peptide initial conformation affects simulation results on 100-ns timescale.

Keywords: Antimicrobial peptide; Molecular dynamics; cm15; Selectivity; popc; popg

Ethanol perturbs lipid organization in models of stratum corneum membranes: An investigation combining differential scanning calorimetry, infrared and²H NMR spectroscopy by Sungjong Kwak; Elana Brief; Denis Langlais; Neil Kitson; Michel Lafleur; Jenifer Thewalt (pp. 1410-1419).

Ethanol is used in a variety of topical products. It is known to enhance the permeability of the skin by altering the ability of the stratum corneum (SC) intercellular membranes to form an effective barrier. In addition, ethanol and other alcohols are key components of antiseptic gels currently used for hand wash. Using infrared and deuterium NMR spectroscopy as well as calorimetry, we have investigated the effect of ethanol on a model membrane composed of lipids representing the three classes of SC lipids, an equimolar mixture of N-palmitoylsphingosine (ceramide), palmitic acid and cholesterol. Ethanol is found to influence the membrane in a dose dependent manner, disrupting packing and increasing lipid motion at low concentrations and selectively extracting lipids at moderate concentrations.► We studied the effect of ethanol on the behavior of stratum corneum model membranes. ► Ethanol is found to influence the membrane in a dose dependent manner. ► Ethanol disrupts lipid packing and fluidizes the lipid matrix. ► Ethanol selectively extracts the free fatty acid component from the ensemble.

Keywords: Abbreviations; SC; Stratum corneum; Cer16; N; -palmitoyl; D; -; erythro; -sphingosine; PA; palmitic acid; chol; cholesterol; lo; liquid ordered; Cer16-d; ₃₁; N; -perdeuterated-palmitoyl D-; erythro; -sphingosine; PA-d; ₃₁; perdeuterated palmitic acidEthanol; ²; H NMR; IR; DSC; Ceramide; Stratum corneum

Evaluation of the membrane lipid selectivity of the pea defensin Psd1 by Goncalves Sónia Gonçalves; Alexandre Teixeira; João Abade; Luciano Neves de Medeiros; Eleonora Kurtenbach; Nuno C. Santos (pp. 1420-1426).

Psd1, a 46 amino acid residues defensin isolated from the pea Pisum sativum seeds, exhibits anti-fungal activity by a poorly understood mechanism of action. In this work, the interaction of Psd1 with biomembrane model systems of different lipid compositions was assessed by fluorescence spectroscopy. Partition studies showed a marked lipid selectivity of this antimicrobial peptide (AMP) toward lipid membranes containing ergosterol (the main sterol in fungal membranes) or specific glycosphingolipid components, with partition coefficients ( K_p) reaching uncommonly high values of 10⁶. By the opposite, Psd1 does not partition to cholesterol-enriched lipid bilayers, such as mammalian cell membranes. The Psd1 mutants His36Lys and Gly12Glu present a membrane affinity loss relative to the wild type. Fluorescence quenching data obtained using acrylamide and membrane probes further clarify the mechanism of action of this peptide at the molecular level, pointing out the potential therapeutic use of Psd1 as a natural antimycotic agent.► Psd1 shows lipid selectivity toward membranes containing ergosterol (as fungal membranes). ► Psd1 mutants His36Lys and Gly12Glu present reduced membrane binding relative to wild type. ► Psd1 does not interact with cholesterol-rich membranes, limiting side-effects on mammalian cells. ► Psd1 lipid selectivity confers it a high potential to be applied as antimycotic agent.

Keywords: Ps; d1; Antimicrobial peptide; Defensin; Glycosphingolipids; Ergosterol; Fluorescence spectroscopy

Gross morphological changes in thylakoid membrane structure are associated with photosystem I deletion in Synechocystis sp. PCC 6803 by Allison M.L. van de Meene; William P. Sharp; Jennifer H. McDaniel; Heiner Friedrich; Wim F.J. Vermaas; Robert W. Roberson (pp. 1427-1434).

Cells of Synechocystis sp. PCC 6803 lacking photosystem I (PSI-less) and containing only photosystem II (PSII) or lacking both photosystems I and II (PSI/PSII-less) were compared to wild type (WT) cells to investigate the role of the photosystems in the architecture, structure, and number of thylakoid membranes. All cells were grown at 0.5μmol photons m−²s−¹. The lumen of the thylakoid membranes of the WT cells grown at this low light intensity were inflated compared to cells grown at higher light intensity. Tubular as well as sheet-like thylakoid membranes were found in the PSI-less strain at all stages of development with organized regular arrays of phycobilisomes on the surface of the thylakoid membranes. Tubular structures were also found in the PSI/PSII-less strain, but these were smaller in diameter to those found in the PSI-less strain with what appeared to be a different internal structure and were less common. There were fewer and smaller thylakoid membrane sheets in the double mutant and the phycobilisomes were found on the surface in more disordered arrays. These differences in thylakoid membrane structure most likely reflect the altered composition of photosynthetic particles and distribution of other integral membrane proteins and their interaction with the lipid bilayer. These results suggest an important role for the presence of PSII in the formation of the highly ordered tubular structures.► Deletion of cyanobacterial PSII led to tubes of thylakoids instead of sheets. ► Deletion of PSI and PSII reduced the total number of thylakoid membranes. ► Phycobilisome arrangement was altered in the mutant lacking PSI and PSII.

Keywords: Abbreviations; PSI; Photosystem I; PSII; Photosystem II; FF; freeze fracture; EF; endoplasmic face; PF; protoplasmic face; LHC; light harvesting complexElectron tomography; Photosynthesis; Thylakoid membrane; Photosystem I; Photosystem II; Phycobilisome

Corrigendum to “ATP hydrolysis at one of the two sites in ABC transporters initiates transport related conformational transitions” [Biochim. Biophys. Acta 1808 (2011) 2954–2964] by Gergely Gyimesi; Srinivas Ramachandran; Pradeep Kota; Nikolay V. Dokholyan; Balázs Sarkadi; Hegedus Tamás Hegedűs (pp. 1435-1435).

Corrigendum to “Thermal, dynamic and structural properties of drug AT₁ antagonist olmesartan in lipid bilayers” [Biochim. Biophys. Acta 1808 (2011) 2995–3006] by Dimitrios Ntountaniotis; Gregor Mali; Simona Golic Grdadolnik; Maria Halabalaki; Alexios-Leandros Skaltsounis; Constantinos Potamitis; Eleni Siapi; Petros Chatzigeorgiou; Michael Rappolt; Thomas Mavromoustakos (pp. 1436-1436).

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BBA - Biomembranes (v.1818, #5)