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BBA - Biomembranes (v.1828, #2)

Editorial Board (pp. i).

Prostaglandins, not the leukotrienes, regulate Cl⁻/HCO₃⁻ exchange (DRA, SLC26A3) in villus cells in the chronically inflamed rabbit ileum by Palanikumar Manoharan; Steven Coon; Walter Baseler; Shanmuga Sundaram; Ramesh Kekuda; Uma Sundaram (pp. 179-186).

Previously studies have demonstrated that Cl⁻/HCO₃⁻ exchange was inhibited during chronic intestinal inflammation secondary to decrease in the affinity of the exchanger for Cl⁻ rather than the number of transporters. Arachidonic acid metabolites (AAM) are elevated in the mucosa of the chronically inflamed small intestine. However, their role in the alteration of Cl⁻/HCO₃⁻ during chronic enteritis was unknown. Inhibition of AAM formation with arachidonyl trifluoro methylketone (ATMK) in chronically inflamed rabbit intestine reversed the diminished Cl⁻/HCO₃⁻ exchange activity. Kinetics studies showed that the reversal was secondary to restoration of the altered affinity of transporter. Downstream regulation of Cl⁻/HCO₃⁻ inhibition by AAM was determined to be by the cyclooxygenase pathway since only inhibition of cyclooxygenase with piroxicam treatment reversed the inhibited Cl⁻/HCO₃⁻ exchange. Further, DRA was shown to be the primary Cl⁻/HCO₃⁻ exchanger in villus cells. Kinetics and molecular studies indicated that the mechanism of inhibition of Cl⁻/HCO₃⁻ exchange by cyclooxygenase pathway metabolites was secondary to diminished affinity of the transporter for Cl⁻ without a change in DRA BBM expression. Thus our data indicated that cyclooxygenase pathway metabolites mediate the inhibition of DRA during chronic intestinal inflammation.Display Omitted► DRA mediates the Cl¯/HCO3¯ exchange activity in rabbit ileum. ► In chronic enteritis DRA is inhibited via diminished affinity of transporter for Cl. ► DRA inhibition is reversed when arachidonic acid formation is prevented. ► Specific regulation of DRA inhibition during chronic enteritis is by prostaglandin.

Keywords: Inflammatory bowel disease; Down regulated in adenoma; Cyclooxygenase; Lipoxygenase Arachidonic acid metabolites; Regulation of Cl; ⁻; /HCO; ₃; ⁻; exchange; Piroxicam

Existence of a robust haloacid transport system in a Burkholderia species bacterium by Xianbin Su; Jimmy S.H. Tsang (pp. 187-192).

Bacterium Burkholderia sp. MBA4 can utilize haloacids as the sole carbon and energy source for growth. We have previously reported that a haloacid operon, encoding for a dehalogenase (Deh4a) and an associated permease (Deh4p), was responsible for the transformation and uptake of haloacids in MBA4. A disruption of deh4p in MBA4 caused a decrease in monochloroacetate (MCA) uptake, confirming its role as a haloacid transporter. However, this disruptant retained 68% of its MCA-uptake activity indicating the possibility of an alternative system. In this study, we report the identification of a second MCA-inducible haloacid transporter (Dehp2) in MBA4. Its function was confirmed by gene disruption and heterologous expression in Escherichia coli. A dehp2⁻ mutant has 30% less, and an E. coli expressing Dehp2 has 40% more, of wildtype MCA-uptake activity. Quantitative RT-PCR illustrated that the minor loss of MCA-uptake activity in single disruptants of deh4p and dehp2 was partly due to a compensatory expression of the alternative gene. Competition assay and kinetics study revealed that Deh4p has a higher affinity for MCA while Dehp2 prefers chloropropionate. A deh4p⁻ dehp2⁻ double mutant retained 36% of MCA-uptake activity, indicating a robustness of the haloacid uptake systems. The MCA uptake activities mediated by Deh4p, Dehp2 and the uncharacterized system were completely abolished by protonophore carbonyl cyanide 3-chlorophenylhydrazone, suggesting that transmembrane electrochemical gradient is the driving force for MCA uptake.Display Omitted► A second haloacid transporter Dehp2 was identified in Burkholderia sp. MBA4. ► Deh4p and Dehp2 are major haloacid transporters in MBA4. ► Deh4p and Dehp2 have different substrate specificities. ► Transmembrane electrochemical gradient is the driving force for MCA transport.

Keywords: Abbreviations; MBA4; Burkholderia; sp. MBA4; MCA; monochloroacetate; HAAs; haloacetic acids; 2MCPA; 2-Monochloropropinate; CCCP; carbonyl cyanide 3-chlorophenylhydrazone; MFS; Major Facilitator Superfamily; RT-PCR; reverse-transcriptase PCR; BLAST; Basic Local Alignment Search ToolHaloacid; Transporter; Burkholderia; Electrochemical gradient

Detergent-labile, supramolecular assemblies of KcsA: Relative abundance and interactions involved by A. Marcela Giudici; M. Luisa Molina; José L. Ayala; Estefanía Montoya; M. Lourdes Renart; Fernandez Asia M. Fernández; José A. Encinar; Antonio V. Ferrer-Montiel; José A. Poveda; Gonzalez-Ros José M. González-Ros (pp. 193-200).

In this work, we illustrate the ability of the prokaryotic potassium channel KcsA to assemble into a variety of supramolecular clusters of defined sizes containing the tetrameric KcsA as the repeating unit. Such clusters, particularly the larger ones, are markedly detergent-labile and thus, disassemble readily upon exposure to the detergents commonly used in protein purification or conventional electrophoresis analysis. This is a reversible process, as cluster re-assembly occurs upon detergent removal and without the need of added membrane lipids. Interestingly, the dimeric ensemble between two tetrameric KcsA molecules are quite resistant to detergent disassembly to individual KcsA tetramers and along with the latter, are likely the basic building blocks through which the larger clusters are organized.As to the proteins domains involved in clustering, we have observed disassembly of KcsA clusters by SDS-like alkyl sulfates. As these amphiphiles bind to inter-subunit, “non-annular” sites on the protein, these observations suggest that such sites also mediate channel–channel interactions leading to cluster assembly.Display Omitted► The KcsA channel is assembled into a variety of well-defined supramolecular clusters. ► KcsA clusters disassemble readily and reversibly upon detergent exposure. ► Non-annular binding sites on the protein mediate interactions leading to clustering.

Keywords: Abbreviations; KcsA; potassium channel from; Streptomyces lividans; DDM; dodecyl; β; -; d; -maltoside; SDS-PAGE; polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate; PFO; perfluoro-octanoic acid; BN-PAGE; blue native polyacrylamide gel electrophoresis; T species; individual KcsA tetrameric channels; nT species; supramolecular assemblies containing n number of clustered KcsA tetrameric channelsIon channels supramolecular assembly; Protein clusters; Detergent stability; Crosslinking; Blue native PAGE; Membrane proteins “non-annular” sites

Fsy1, the sole hexose-proton transporter characterized in Saccharomyces yeasts, exhibits a variable fructose:H⁺ stoichiometry by Jorge Anjos; Helena Rodrigues de Sousa; Christophe Roca; Cassio Fernanda Cássio; Marijke Luttik; Jack T. Pronk; Madalena Salema-Oom; Goncalves Paula Gonçalves (pp. 201-207).

In the model yeast Saccharomyces cerevisiae, hexose uptake is mediated exclusively by a family of facilitators (Hxt, hexose transporters). Some other Saccharomyces species (e.g. Saccharomyces bayanus and Saccharomyces pastorianus) possess, in addition, a specific fructose transporter (Fsy1, fructose symporter) that has been previously described to function as a proton symporter. In the present work, we compared growth of a yeast strain in which FSY1 occurs naturally in anaerobic, fructose- and glucose-limited chemostat cultures. Especially at low specific growth rates, fructose-proton symport was shown to have a strong impact on the biomass yield on sugar. We subsequently employed energized hybrid plasma membrane vesicles to confirm previous observations concerning the mode of operation and specificity of Fsy1 mediated transport. Surprisingly, these experiments suggested that the carrier exhibits an unusual fructose:H⁺ stoichiometry of 1:2. This energetically expensive mode of operation was also found consistently in vivo, in shake flask and in chemostat cultures, and both when Fsy1 is the sole transporter and when the Hxt carriers are present. However, it is observed only when Fsy1 is operating at higher glycolytic fluxes, a situation that is normally prevented by downregulation of the gene. Taken together, our results suggest the possibility that fructose symport with more than one proton may constitute an energetically unfavorable mode of operation of the Fsy1 transporter that, in growing cultures, is prevented by transcriptional regulation.Display Omitted► Operation of Fsy1 is a considerable energetic burden in anaerobic, fructose grown cultures. ► Experiments with hybrid vesicles confirm substrate specificity and proton symport by Fsy1. ► A fructose:H⁺ stoichiometry of 1:2 was found in hybrid vesicles. ► In vivo fructose:H⁺ stoichiometry is variable and correlated with glycolytic flux.

Keywords: Abbreviations; MFS; major facilitator superfamily; Hxt; hexose transporters; Fsy1; fructose symporter; PMF; proton motive force; CL–PMV; cytochrome; c; oxidase liposome–plasma membrane vesicles; FCCP; Carbonyl cyanide p-[trifluoromethoxyl]-phenyl-hydrazone; TMPD; NNN; ′; N; ′-tetramethyl-p-phenylenediamine; TPP; tetraphenylphosphoniumHybrid vesicles; Fructose transport; Saccharomyces; Symport stoichiometry

Cytochrome c produces pores in cardiolipin-containing planar bilayer lipid membranes in the presence of hydrogen peroxide by M.N. Puchkov; R.A. Vassarais; E.A. Korepanova; A.N. Osipov (pp. 208-212).

Interaction of cytochrome c with cardiolipin in the presence of hydrogen peroxide induces peroxidase activity in cytochrome c and the ability to oxidize membrane lipids. These cytochrome c properties play a substantial role in the cytochrome c-mediated apoptotic reactions. In the present study the electric properties (specific capacitance and integral conductance) of the cardiolipin-containing asolectin planar bilayer lipid membranes (pBLM) in the presence of cytochrome c and hydrogen peroxide were studied. Cytochrome c interaction with cardiolipin-containing pBLM in the presence of hydrogen peroxide resulted in the dramatic increase of the conductance, pore production, their growth up to 3.5nm diameter and subsequent membrane destruction. In the absence of hydrogen peroxide cytochrome c demonstrated almost no effect on the membrane capacitance and conductance. The data obtained prove the pivotal role of cytochrome c and membrane lipids in the permeabilization of pBLM. Correlation of apoptotic reactions and cytochrome c-mediated membrane permeability is discussed.Display Omitted► The complex of cytochrome c with cardiolipin exhibits peroxidase activity. ► Membrane lipids are oxidized when hydrogen peroxide added to this system. ► Cytochrome c and lipids interaction was studied on planar membranes. ► During their interaction an increase of the ionic conductance was found. ► Calculated diameter of the pores comparable to diameter of the cytochrome c

Keywords: Cytochrome c; Cardiolipin; Hydrogen peroxide; Peroxidase activity; Membrane permeability

The molecular organization of prenylated flavonoid xanthohumol in DPPC multibilayers: X-ray diffraction and FTIR spectroscopic studies by Marta Arczewska; Kaminski Daniel M. Kamiński; Gorecka Ewa Górecka; Damian Pociecha; Roj Edward Rój; Sławinska-Brych Adrianna Sławińska-Brych; Gagos Mariusz Gagoś (pp. 213-222).

Xanthohumol (XN) is the major prenylated flavonoid found in hop resin. It has attracted considerable attention in recent years due to its wide spectrum of biological activities and the beneficial effect on human health. Since lipid membrane is first target for biologically active compounds, we decided to investigate the influence of XN on the dipalmitoylphosphatidylcholine (DPPC) multibilayers. Interactions of XN with DPPC were investigated as a function of temperature and its concentration by using X-ray diffraction and the ATR-FTIR spectroscopy techniques. The aim of understanding the mechanisms of molecular interactions between XN and DPPC was to indicate the localization of the XN with respect to the membrane and the type of interaction with phospholipids. The results revealed that XN changes the physical properties of the DPPC multibilayers in the form of dry film. A new complex formation between XN and DPPC is reported. The detailed analysis of refraction effect indicates the changes in electron density ratio between hydrophobic and hydrophilic zones of lipid at phase transition. This is in compliance with reported changes in FTIR spectra where at pretransition XN moves from interface region between polar heads to the neighborhood of phosphate groups.Display Omitted► The study of XN with DPPC by FTIR and X-ray diffraction techniques ► XN changes the physical properties of the DPPC. ► XN interacts with both the hydrophobic and hydrophilic parts of DPPC.

Keywords: DPPC multibilayers; Xanthohumol; Prenylated chalcone; ATR-FTIR; X-ray diffraction

Antimicrobial and cell-penetrating properties of penetratin analogs: Effect of sequence and secondary structure by Jesper Søborg Bahnsen; Henrik Franzyk; Anne Sandberg-Schaal; Hanne Mørck Nielsen (pp. 223-232).

Cell-penetrating peptides (CPPs) and antimicrobial peptides (AMPs) show great potential as drug delivery vectors and new antibiotic drug entities, respectively. The current study deals with the properties of a variety of peptide analogs derived from the well-known CPP penetratin as well as octaarginine and different Tat sequences. The effects of peptide length, guanidinium content, and sequence of non-cationic residues were assessed in mammalian and bacterial cells. The arginine (Arg) content in the penetratin analogs was found to influence eukaryotic cell uptake efficiency, antimicrobial activity towards both Gram-positive and Gram-negative bacteria as well as eukaryotic cell viability. All examined analogs retained the ability to cross eukaryotic membranes giving rise to a distribution within the vacuolar apparatus. Interestingly, a series of shuffled analogs of penetratin with the cationic residues in conserved positions, attain the same α-helical conformation as native penetratin in the presence of cholesterol-containing liposomes, while conformational differences were observed in the presence of highly anionic liposomes. While the antibacterial effect of the two groups of peptides was similar, the eukaryotic cellular uptake of the shuffled analogs was noticeably lower than for native penetratin. Moreover, a point substitution of Met to Leu in native penetratin had no influence on eukaryotic cellular uptake and antimicrobial effect, and only a minor effect on cytotoxicity, in contrast to the fact that the same substitution in the shuffled analog gave rise to reduced eukaryotic cellular uptake while increasing the antibacterial effect and cytotoxicity.Display Omitted► Penetratin and penetratin derived analogs inhibit growth of E. coli and S. aureus. ► High arginine content increases eukaryotic cellular uptake and antibacterial effect. ► The influence of Met to Leu substitution depends on the peptide sequence. ► PenShuf is α-helical in the presence of cholesterol-containing and weakly anionic liposomes.

Keywords: Abbreviations; CD; circular dichroism; CF(5); 5-carboxyfluorescein; CF; 5(6)-carboxyfluorescein; EMEM; Eagle's minimal essential medium; FBS; fetal bovine serum; HBSS; Hanks balanced salt solution; HEPES; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; hRBC; human red blood cell; MIC; minimal inhibitory concentration; PBS; phosphate buffered saline; PDI; polydispersity index; POPC; palmitoyl-oleoyl phosphatidylcholine; POPG; palmitoyl-oleoyl phosphatidylglycerol; TI; therapeutic indexAntimicrobial peptide; Cell-penetrating peptide; Protein transduction domain; Intracellular delivery; Membrane activity; Secondary structure

Function of plastochromanol and other biological prenyllipids in the inhibition of lipid peroxidation—A comparative study in model systems by Beatrycze Nowicka; Jolanta Gruszka; Jerzy Kruk (pp. 233-240).

Lipid peroxidation is an oxidation reaction leading to the generation of lipid hydroperoxides. Here we present comparative data on the inhibition of lipid peroxidation by a variety of biological prenyllipids in liposomes prepared from natural lipid membranes. Lipid peroxidation was initiated by hydrophilic and hydrophobic azo initiators, as well as by singlet oxygen generated via photosensitized reaction of hydrophobic zinc tetraphenylporphine. When lipid peroxidation was initiated in the water phase, tocopherols and plastochromanol-8 were more effective than prenylquinols, such as plastoquinol-9, ubiquinol-10 or α-tocopherolquinol. However, if the peroxidation was initiated within the hydrophobic interior of liposome membranes, long-chain prenyllipids, such as plastoquinol-9 and plastochromanol-8, were considerably more active than tocopherols in the inhibition of the reaction. In the latter system, tocopherols showed even prooxidant activity. The prooxidant activity of α-tocopherol was prevented by plastoquinol, suggesting the reduction of α-tocopheroxyl radical by the quinol. All the investigated prenyllipids were able to inhibit singlet oxygen-mediated lipid peroxidation but the most active were prenylquinols in this respect. Among all the prenyllipids investigated, plastochromanol-8 was the most versatile antioxidant in the inhibition of lipid peroxidation initiated by the three different methods.► Comparative data on the inhibition of lipid peroxidation by biological prenyllipids are presented. ► When peroxidation was initiated in the water phase, chromanols were the most effective. ► When peroxidation was initiated in the hydrophobic membrane core, long-chain prenyllipids were the most active. ► During singlet oxygen-mediated lipid peroxidation, prenylquinols were the most active antioxidants.

Keywords: Abbreviations; AAPH; 2,2′-azobis(2-amidinopropane)-dihydrochloride; AMVN; 2,2′-azobis(2,4-dimethylvaleronitrile); α-Toc; α-tocopherol; α-TQ; α-tocopherolquinone; α-TQH; ₂; α-tocopherolquinol; γ-T3; γ-tocotrienol; γ-Toc; γ-tocopherol; PC-8; plastochromanol; PC-OH; hydroxy-plastochromanol; PQ-9; plastoquinone-9; PQ-C; plastoquinone-C; PQH; ₂; -9; plastoquinol-9; LOOH; lipid hydroperoxide; ROS; reactive oxygen species; SOSG; Singlet Oxygen Sensor Green; UQ-10; ubiquinone-10; UQH; ₂; -10; ubiquinol-10; ZnTPP; zinc 5,10,15,20-tetraphenyl-21H,23H-porphineLipid peroxidation; Plastochromanol; Plastoquinol; Tocopherol; Singlet oxygen

Membrane fluidity and activity of membrane ATPases in human erythrocytes under the influence of polyhydroxylated fullerene by Jacek Grebowski; Anita Krokosz; Mieczyslaw Puchala (pp. 241-248).

The influence of fullerenol on the activities of human erythrocyte membrane ATPases and the fluidity of the plasma membrane as well as the possibility of fullerenol incorporation into the plasma membrane were investigated. Fullerenol at concentrations up to 150μg/mL induced statistically significant decreases in the anisotropy of 1-anilino-8-naphthalene sulfonate (ANS) (14%), N,N,N-trimethyl-4-(6-phenyl-1,3,5,-hexatrien-1-yl)phenylammonium p-toluenesulfonate (TMA-DPH) (7.5%) and 1,6-diphenyl-1,3,5-hexatriene (DPH) (9.5%) after a 1-hour incubation at 37°C. The effect disappeared for ANS and TMA-DPH, but not for DPH, after washing out the fullerenol. Incubation of erythrocyte membranes with fullerenol led to decreases in the activities of Na⁺,K⁺-ATPase (to 23% of the control value), Ca2⁺-ATPase (to 16% of control) and Mg2⁺-ATPase (to 22% of control). Washing out the fullerenol lessened the inhibition of the Na⁺,K⁺-ATPase (37% of control) and Ca2⁺-ATPase (23.5% of control); however, it did not influence Mg2⁺-ATPase activity. Furthermore, fullerenol could associate with erythrocyte plasma membranes.Our results suggest that fullerenol associates primarily with the surface of the plasma membrane; however, it can also migrate deeper inside the membrane. Moreover, fullerenol influences membrane ATPases so that it may modulate ion transport across membranes.Display Omitted► Fullerenol C₆₀(OH)~₃₀ decreases the fluidity of the erythrocyte plasma membrane. ► Fullerenol binds mostly to the surface of the erythrocyte membrane. ► Fullerenol can incorporate into the hydrophobic region of the erythrocyte membrane. ► The activity of membrane ATPases was decreased irreversibly by fullerenol. ► The direct and indirect mechanisms of fullerenol binding for membrane ATPases are proposed.

Keywords: Fullerenol; Erythrocyte; Fluidity; ATPase; Enzyme inhibition

Plantaricin A, a cationic peptide produced by Lactobacillus plantarum, permeabilizes eukaryotic cell membranes by a mechanism dependent on negative surface charge linked to glycosylated membrane proteins by Sverre L. Sand; Jon Nissen-Meyer; Olav Sand; Trude M. Haug (pp. 249-259).

Lactobacillus plantarum C11 releases plantaricin A (PlnA), a cationic peptide pheromone that has a membrane-permeabilizing, antimicrobial effect. We have previously shown that PlnA may also permeabilize eukaryotic cells, with a potency that differs between cell types. It is generally assumed that cationic antimicrobial peptides exert their effects through electrostatic attraction to negatively charged phospholipids in the membrane. The aim of the present study was to investigate if removal of the negative charge linked to glycosylated proteins at the cell surface reduces the permeabilizing potency of PlnA. The effects of PlnA were tested on clonal rat anterior pituitary cells (GH₄ cells) using patch clamp and microfluorometric techniques. In physiological extracellular solution, GH₄ cells are highly sensitive to PlnA, but the sensitivity was dramatically reduced in solutions that partly neutralize the negative surface charge of the cells, in agreement with the notion that electrostatic interactions are probably important for the PlnA effects. Trypsination of cells prior to PlnA exposure also rendered the cells less sensitive to the peptide, suggesting that negative charges linked to membrane proteins are involved in the permeabilizing action. Finally, pre-exposure of cells to a mixture of enzymes that split carbohydrate residues from the backbone of glycosylated proteins also impeded the PlnA-induced membrane permeabilization. We conclude that electrostatic attraction between PlnA and glycosylated membrane proteins is probably an essential first step before PlnA can interact with membrane phospholipids. Deviating glycosylation patterns may contribute to the variation in PlnA sensitivity of different cell types, including cancerous cells and their normal counterparts.Display Omitted► A study of eukaryotic membrane permeabilization by an antimicrobial peptide. ► The permeabilization depends on negative surface charge. ► Trypsination of cells prior to PlnA exposure impedes the permeabilization. ► Removal of carbohydrate residues from membrane proteins impedes permeabilization. ► The permeabilization depends on electrostatic attraction to glycosylated proteins.

Keywords: Antimicrobial peptide; Electrophysiology; Ca; 2; ⁺; imaging; Electrostatic attraction; Cancer cell; Cell lysis

Membrane composition influences the topology bias of bacterial integral membrane proteins by Denice C. Bay; Raymond J. Turner (pp. 260-270).

Small multidrug resistance (SMR) protein family members confer bacterial resistance to toxic antiseptics and are believed to function as dual topology oligomers. If dual topology is essential for SMR activity, then the topology bias should change as bacterial membrane lipid compositions alter to maintain a “neutral” topology bias. To test this hypothesis, a bioinformatic analysis of bacterial SMR protein sequences was performed to determine a membrane protein topology based on charged amino acid residues within loops, and termini regions according to the positive inside rule. Three bacterial lipid membrane parameters were examined, providing the proportion of polar lipid head group charges at the membrane surface (PLH), the relative hydrophobic fatty acid length (FAL), and the proportion of fatty acid unsaturation (FAU). Our analysis indicates that individual SMR pairs, and to a lesser extent SMR singleton topology biases, are significantly correlated to increasing PLH, FAL and FAU differences validating the hypothesis. Correlations between the topology biases of SMR proteins identified in Gram+ compared to Gram− species and each lipid parameter demonstrated a linear inverse relationship.Display Omitted► Mean SMR topology bias values differ between bacterial Gram+ and Gram− phyla. ► Polar lipid head group charges (PLH) inversely correlate to SMR topology biases. ► SMR singleton topology biases show opposite correlations to PLH by Gram type. ► In addition to PLH, acyl chain length and unsaturation correlate with SMR topology. ► SMR topology biases using both +/− charged amino acids improve lipid correlations.

Keywords: Lipid composition; Membrane protein folding; Positive inside rule; Dual topology; Small multidrug resistance (SMR) proteins EmrE; Paired SMR

Insight into the antimicrobial activities of coprisin isolated from the dung beetle, Copris tripartitus, revealed by structure–activity relationships by Eunjung Lee; Jin-Kyoung Kim; Soyoung Shin; Ki-Woong Jeong; Areum Shin; Juneyoung Lee; Dong Gun Lee; Jae-Sam Hwang; Yangmee Kim (pp. 271-283).

The novel 43-residue, insect defensin-like peptide coprisin, isolated from the dung beetle, Copris tripartitus, is a potent antibiotic with bacterial cell selectivity, exhibiting antimicrobial activities against Gram-positive and Gram-negative bacteria without exerting hemolytic activity against human erythrocytes. Tests against Staphylococcus aureus using fluorescent dye leakage and depolarization measurements showed that coprisin targets the bacterial cell membrane. To understand structure–activity relationships, we determined the three-dimensional structure of coprisin in aqueous solution by nuclear magnetic resonance spectroscopy, which showed that coprisin has an amphipathic α-helical structure from Ala¹⁹ to Arg²⁸, and β-sheets from Gly³¹ to Gln³⁵ and Val³⁸ to Arg⁴². Coprisin has electropositive regions formed by Arg²⁸, Lys²⁹, Lys³⁰, and Arg⁴² and ITC results proved that coprisin and LPS have electrostatically driven interactions. Using measurements of nitric oxide release and inflammatory cytokine production, we provide the first verification of the anti-inflammatory activity and associated mechanism of an insect defensin, demonstrating that the anti-inflammatory actions of the defensin-like peptide, coprisin, are initiated by suppressing the binding of LPS to toll-like receptor 4, and subsequently inhibiting the phosphorylation of p38 mitogen-activated protein kinase and nuclear translocation of NF-kB. In conclusion, we have demonstrated that an amphipathic helix and an electropositive surface in coprisin may play important roles in its effective interaction with bacterial cell membranes and, ultimately, in its high antibacterial activity and potent anti-inflammatory activity. In addition to elucidating the antimicrobial action of coprisin, this work may provide insight into the mechanism of action of insect defense systems.Display Omitted► Insect defensin, coprisin is a 43-residue antimicrobial peptide isolated from the dung beetle. ► Coprisin shows antibacterial and anti-inflammatory activity with bacterial cell selectivity. ► An amphipathic helix and electropositive surface may play important role in its antimicrobial activity. ► We provided the first verification of the anti-inflammatory activity and associated mechanism of coprisin.

Keywords: Abbreviations; AMP; antimicrobial peptide; BSA; bovine serum albumin; CCARM; Culture Collection of Antibiotic-Resistant Microbe; CCS; combined consensus scale; CD; circular dichroism; CH; cholesterol; CSI; chemical shift index; diSC3-5; 3,3′-dipropylthiadicarbo-cyanine iodide; DMEM; Dulbecco's Modified Eagle Medium; DMSO; dimethyl sulfoxide; DPC; dodecylphosphocholine; DQF-COSY; double-quantum-filtered correlation; ECL; enhanced chemiluminescence; ELISA; enzyme-linked immunosorbent assay; ERKs; extracellular signal-regulated kinases; FBS; fetal bovine serum; FITC; fluorescein isothiocyanate; Fmoc; fluorenylmethoxycarbonyl; GAPDH; glyceraldehyde 3-phosphate dehydrogenase; GM; geometric mean; HBD; human β-defensin; HNP; human neutrophil peptide; HPLC; high-performance liquid chromatography; hRBCs; human red blood cells; IFN-γ; interferon-γ; IL-1β; interleukin-1β; IgG; immunoglobulin G; iNOS; inducible nitric oxide synthase; ITC; isothermal titration calorimetric; JNKs; c-Jun N-terminal kinases; KCTC; Korean Collection for Type Cultures; LPS; lipopolysaccharide; LUV; large unilamellar vesicle; MALDI-TOF MS; matrix-assisted laser-desorption ionization-time-of-fight mass spectrometry; MAPK; mitogen-activated protein kinase; MD-2; myeloid differentiation factor 2; MDREC; multidrug-resistant; Escherichia coli; MDRST; multidrug-resistant; Salmonella typhimurium; MHC; minimum hemolytic concentration; MIC; minimum inhibitory concentration; mMIP-2; murine macrophage inflammatory protein-2; MRSA; methicillin-resistant; Staphylococcus aureus; mTNF-α; mouse tumor necrosis factor-α; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MyD88; myeloid differentiation primary response gene (88); NF-κB; nuclear factor κ-light chain-enhancer of activated B cells; NMR; nuclear magnetic resonance; NO; nitric oxide; NOE; nuclear Overhauser effect; NOESY; nuclear Overhauser effect spectroscopy; ODS; octadecyl silica; PBS; phosphate-buffered saline; PBST; PBS/0.1% Tween-20; PC; l; -α-phosphatidylcholine; PE; l; -α-phosphatidylethanolamine; PG; l; -α-phosphatidylglycerol; PGN; peptidoglycan; p/l ratio; peptide/lipid ratio; PVDF; polyvinylidene fluoride; phospho-p38 MAPK; phosphorylated p38 mitogen-activated protein kinase; RPMI; Roswell Park Memorial Institute; RT; room temperature; RT-PCR; reverse transcription-polymerase chain reaction; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; STAT; signal transducer and activator of transcription; TBS; Tris-buffered saline; TFE; 2,2,2-trifluoroethanol; TIRAP/MAL; toll-interleukin 1 receptor domain-containing adaptor protein/MyD88 adapter-like; TLR4; toll-like receptor 4; TMB; 3,3′,5,5′-tetramethylbenzidine; TOCSY; total correlation spectroscopy; TRIF; TIP domain-containing adapter protein inducing IFN-βInsect defensin; Antimicrobial peptide; Anti-inflammatory activity; NMR; Structure

Conformational dynamics and membrane interactions of the E. coli outer membrane protein FecA: A molecular dynamics simulation study by Thomas J. Piggot; Daniel A. Holdbrook; Syma Khalid (pp. 284-293).

The TonB-dependent transporters mediate high-affinity binding and active transport of a variety of substrates across the outer membrane of Escherichia coli. The substrates transported by these proteins are large, scarce nutrients that are unable to gain entry into the cell by passive diffusion across the complex, asymmetric bilayer that constitutes the outer membrane. Experimental studies have identified loop regions that are essential for the correct functioning of these proteins. A number of these loops have been implicated in ligand binding. We report the first simulations of an E. coli outer membrane protein in an asymmetric model membrane that incorporates lipopolysaccharide (LPS) molecules. Comparative simulations of the apo and holo forms of the TonB-dependent transporter FecA in different membrane models enable us to identify the nature of the LPS–protein interactions and determine how these interactions impact upon the conformational dynamics of this protein. In particular, our simulations provide molecular-level insights into the influence of the environment and ligand on the dynamics of the functionally important loops of FecA. In addition, we provide insights into the nature of the protein–ligand interactions and ligand induced conformational change in FecA.Display Omitted► Molecular dynamics simulations of the TonB dependent transporter, FecA ► A complex model of the E. coli outer membrane is used. ► Simulations show that LPS–FecA interactions affect the dynamics of the protein. ► Simulations reveal conformational rearrangements of the protein interior. ► The dynamic response of the loops to the presence of ligands is shown.

Keywords: Membrane protein; Molecular dynamics; Simulation; Lipopolysaccharide; E. coli; TonB

Uptake, efflux, and mass transfer coefficient of fluorescent PAMAM dendrimers into pancreatic cancer cells by Armin W. Opitz; Kirk J. Czymmek; Eric Wickstrom; Norman J. Wagner (pp. 294-301).

Targeted delivery of imaging agents to cells can be optimized with the understanding of uptake and efflux rates. Cellular uptake of macromolecules is studied frequently with fluorescent probes. We hypothesized that the internalization and efflux of fluorescently labeled macromolecules into and out of mammalian cells could be quantified by confocal microscopy to determine the rate of uptake and efflux, from which the mass transfer coefficient is calculated. The cellular influx and efflux of a third generation poly(amido amine) (PAMAM) dendrimer labeled with an Alexa Fluor 555 dye was measured in Capan-1 pancreatic cancer cells using confocal fluorescence microscopy. The Capan-1 cells were also labeled with 5-chloromethylfluorescein diacetate (CMFDA) green cell tracker dye to delineate cellular boundaries. A dilution curve of the fluorescently labeled PAMAM dendrimer enabled quantification of the concentration of dendrimer in the cell. A simple mass transfer model described the uptake and efflux behavior of the PAMAM dendrimer. The effective mass transfer coefficient was found to be 0.054±0.043μm/min, which corresponds to a rate constant of 0.035±0.023min−¹ for uptake of the PAMAM dendrimer into the Capan-1 cells. The effective mass transfer coefficient was shown to predict the efflux behavior of the PAMAM dendrimer from the cell if the fraction of labeled dendrimer undergoing non-specific binding is accounted for. This work introduces a novel method to quantify the mass transfer behavior of fluorescently labeled macromolecules into mammalian cells.Display Omitted► Developed method to quantitatively determine uptake of fluorescent molecules into mammalian cells ► Mass transfer model can describe uptake behavior. ► Efflux behavior can be described with mass transfer model when accounting for non-specific binding inside cell.

Keywords: Confocal microscopy; Drug delivery; Internalization; Efflux; PAMAM dendrimer; Mass transfer coefficient

Mutual inhibition through hybrid oligomer formation of daptomycin and the semisynthetic lipopeptide antibiotic CB-182,462 by TianHua Zhang; Jawad K. Muraih; Evan Mintzer; Nasim Tishbi; Celine Desert; Jared Silverman; Scott Taylor; Michael Palmer (pp. 302-308).

Daptomycin is a clinically important lipopeptide antibiotic that kills Gram-positive bacteria through membrane depolarization. Its activity requires calcium and the presence of phosphatidylglycerol in the target membrane. Calcium and phosphatidylglycerol also promote the formation of daptomycin oligomers, which have been assumed but not proven to be required for the bactericidal effect. Daptomycin shares substantial structural similarity with another lipopeptide antibiotic, A54145; the two have identical amino acid residues in 5 out of 13 positions and similar ones in 4 more positions. We here examined whether these conserved residues are sufficient for oligomer formation. To this end, we used fluorescence energy transfer and excimer fluorescence to detect hybrid oligomers of daptomycin and CB-182,462, a semisynthetic derivative of A54145. Mixtures of the two compounds indeed produced hybrid oligomers, but at the same time displayed a significantly less than additive antibacterial activity against Bacillus subtilis. The existence of functionally impaired oligomers indicates that oligomer formation is indeed important for antibacterial function. However, it also shows that oligomerization is not sufficient; once formed, the oligomers must take another step in order to acquire antibacterial activity. Thus, the amino acid residues shared between daptomycin and CB-182,462 suffice for formation of the oligomer, but not for its subsequent activation.Display Omitted► Daptomycin and the related lipopeptide CB-182,462 form hybrid oligomers. ► Hybrid oligomer formation is accompanied by mutual inhibition. ► Oligomer formation is required but not sufficient for bactericidal activity.

Keywords: Daptomycin; A54145; Lipopeptide antibiotics; Oligomerization; Fluorescence

Nanoencapsulation of quercetin and resveratrol into elastic liposomes by Pabyton G. Cadena; Marcela A. Pereira; Rafaela B.S. Cordeiro; Isabella M.F. Cavalcanti; Benício Barros Neto; Maria do Carmo C.B. Pimentel; José Luiz Lima Filho; Valdinete L. Silva; Santos-Magalhaes Nereide S. Santos-Magalhães (pp. 309-316).

Based on the fact that quercetin (QUE) and resveratrol (RES) induce a synergic inhibition of the adipogenesis and increase apoptosis in adipocytes, and that sodium deoxycholate (SDC) has necrotic effects, the nanoencapsulation of QUE and RES into SDC-elastic liposomes is proposed as a new approach for dissolving the subcutaneous fat. The concentration of constituents and the effect of the drug incorporation into cyclodextrin inclusion complexes on the stability of QUE/RES-loaded liposomes were studied. The best liposomal formulation reduced the use of phosphatidylcholine and cholesterol in 17.7% and 68.4%, respectively. Liposomes presented a mean diameter of 149nm with a polydispersion index of 0.3. The zeta potential of liposomes was slightly negative (−13.3mV) due to the presence of SDC in the phospholipid bilayer. Encapsulation efficiency of QUE and RES into liposomes was almost 97%. To summarize, QUE/RES-loaded elastic liposomes are stable and suitable for subcutaneous injection, thereby providing a new strategy for reducing subcutaneous fat.Display Omitted► Coencapsulated quercetin/resveratrol-loaded elastic liposomes were developed. ► Elastic liposomes are proposed as a new approach for subcutaneous fat dissolution. ► High drug encapsulation efficiency into elastic liposomes was obtained. ► The SEM analysis showed well dispersed vesicles without aggregation. ► Liposomes should be advantageous in the treatment of lipid accumulation diseases.

Keywords: Quercetin; Resveratrol; Sodium deoxycholate; Elastic liposome; Experimental design; Nanotechnology

Phospholipidomics reveals differences in glycerophosphoserine profiles of hypothermically stored red blood cells and microvesicles by Beatriz Bicalho; Jelena L. Holovati,; Jason P. Acker (pp. 317-326).

During their normal in vivo life cycle erythrocytes (red blood cells, RBCs) undergo biochemical changes leading to membrane microvesiculation and shedding. RBC microvesiculation also occurs in vitro under conditions of blood bank storage, so microvesicles (MVs) accumulate in the storage (preservation) medium over storage time. Considerable effort has been put into gaining a mechanistic understanding of the RBC microvesiculation process, as this is crucial to better understand RBC biology in disease and in health. Additionally, MVs accumulated in stored RBCs have been implicated in transfusion adverse inflammatory reactions, with chloroform extractable compounds, thus lipophilic, known to trigger the effect. However, because thin layer chromatography resolution of RBC and MV lipids has always enabled one to conclude high compositional similarities, in depth analysis of MV lipids has not been extensively pursued. Here we present an orbitrap mass spectrometry (MS) approach to compare the phospholipid composition of RBCs and MVs from leukoreduced, hypothermically (2–6°C) stored RBC units. We used shotgun MS analysis and electrospray ionization (ESI) intra-source separation, and demonstrated high similarity of compositional profiles, except for glycerophosphoserines (PS). Contrasting abundances of PS 38:4 and PS 38:1 characterized MV and RBC profiles and suggested that storage-associated microvesiculation possibly involves shedding of specific membrane rafts. This finding indicates that phospholipidomics could likely contribute to a better understanding of the RBC microvesiculation process.Display Omitted► First mass spectrometry report showing the phospholipid composition of stored red blood cell microvesicles. ► Stored red blood cells and microvesicles were compositionally different in regard to glycerophosphoserines. ► Results suggest that glycerophosphoserines are organized in membrane rafts.

Keywords: Stored RBC; Microvesicles; Phospholipids; Orbitrap; Shotgun mass spectrometry

How the amyloid- β peptide and membranes affect each other: An extensive simulation study by Chetan Poojari; Andreas Kukol; Birgit Strodel (pp. 327-339).

The etiology of Alzheimer's disease is thought to be linked to interactions between amyloid- β (A β) and neural cell membranes, causing membrane disruption and increased ion conductance. The effects of A β on lipid behavior have been characterized experimentally, but structural and causal details are lacking. We used atomistic molecular dynamics simulations totaling over 6μs in simulation time to investigate the behavior of A β₄₂ in zwitterionic and anionic lipid bilayers. We simulated transmembrane β-sheets (monomer and tetramer) resulting from a global optimization study and a helical structure obtained from an NMR study. In all simulations A β₄₂ remained embedded in the bilayer. It was found that the surface charge and the lipid tail type are determinants for transmembrane stability of A β₄₂ with zwitterionic surfaces and unsaturated lipids promoting stability. From the considered structures, the β-sheet tetramer is most stable as a result of interpeptide interactions. We performed an in-depth analysis of the translocation of water in the A β₄₂-bilayer systems. We observed that this process is generally fast (within a few nanoseconds) yet generally slower than in the peptide-free bilayers. It is mainly governed by the lipid type, simulation temperature and A β₄₂ conformation. The rate limiting step is the permeation through the hydrophobic core, where interactions between A β₄₂ and permeating H₂O molecules slow the translocation process. The β-sheet tetramer allows more water molecules to pass through the bilayer compared to monomeric A β, allowing us to conclude that the experimentally observed permeabilization of membranes must be due to membrane-bound A β oligomers, and not monomers.Display Omitted► We model various A β structures in membranes on the sub-microsecond time scale. ► A β always remains embedded in the bilayer and enables water translocation. ► Zwitterionic headgroups, unsaturated lipids, and A β oligomerization add to A β stability. ► Water permeation depends on the lipid type and the A β structure. ► Transmembrane A β oligomerization increases water permeability.

Keywords: Amyloid-beta peptide; Phospholipid membranes; Molecular simulations; Water permeation; Alzheimer's disease

The function of the ATP-binding cassette (ABC) transporter ABCB1 is not susceptible to actin disruption by Peter Meszaros; Ina Hummel; Karin Klappe; Oana Draghiciu; Dick Hoekstra; Jan W. Kok (pp. 340-351).

Previously we have shown that the activity of the multidrug transporter ABCC1 (multidrug resistance protein 1), and its localization in lipid rafts, depends on cortical actin (Hummel I, Klappe K, Ercan C, Kok JW. Mol. Pharm. 2011 79, 229–40). Here we show that the efflux activity of the ATP-binding cassette (ABC) family member ABCB1 (P-glycoprotein), did not depend on actin, neither in ABCB1 over expressing murine National Institutes of Health (NIH) 3T3 MDR1 G185 cells nor in human SK-N-FI cells, which endogenously express ABCB1. Disruption of the actin cytoskeleton, upon treatment of the cells with latrunculin B or cytochalasin D, caused severe changes in cell and membrane morphology, and concomitant changes in the subcellular distribution of ABCB1, as revealed by confocal laser scanning and electron microscopy. Nevertheless, irrespective of actin perturbation, the cell surface pool of ABCB1 remained unaltered. In NIH 3T3 MDR1 G185 cells, ABCB1 is partly localized in detergent-free lipid rafts, which partitioned in two different density gradient regions, both enriched in cholesterol and sphingolipids. Interestingly, disruption of the actin cytoskeleton did not change the density gradient distribution of ABCB1. Our data demonstrate that the functioning of ABCB1 as an efflux pump does not depend on actin, which is due to its distribution in both cell surface-localized non-raft membrane areas and lipid raft domains, which do not depend on actin stabilization.Membrane localizations of ABCB1 compared to other ABC transporters and their interactions with the actin cytoskeleton.Five possible membrane pools of an ABC transporter are depicted: 1) non-raft associated and non-actin associated, 2) raft-associated, but non-actin associated, 3) raft-associated and indirectly linked to actin, 4) raft-associated and directly linked to actin, and 5) non-raft associated, but directly linked to actin. PM = plasma membrane and I = integral membrane protein. ABCB1 (P-glycoprotein) appears to be only in pools 1 and 2, while ABCC1 (multidrug resistance protein 1) is also found in pool 3 and ABCC2 (multidrug resistance protein 2) in pool 4; the latter 2 pools are stabilized by actin.Display Omitted► Actin disruption causes membrane redistribution of ABCB1 (P-glycoprotein). ► This does not lead to diminished ABCB1 efflux function. ► Actin disruption does not change the cell surface pool of ABCB1. ► Actin disruption does not affect the extent of lipid raft association of ABCB1. ► ABCB1 function remains normal because its membrane environment does not change.

Keywords: Abbreviations; ABC; ATP-binding cassette; ABCB1; P-glycoprotein; ABCC1; multidrug resistance-protein 1; AM; acetoxymethyl ester; Cav-1; caveolin-1; CSA; cyclosporin A; DRM; detergent-resistant membrane; ERM; ezrin, radixin and moesin; FCS; fetal calf serum; HBSS; Hank's balanced salt solution; LC-ESI–MS/MS; Liquid chromatography-electrospray ionization tandem mass spectrometry; MAF; multidrug activity factor; MDR; multidrug resistance; NIH; National Institutes of Health; PBS; phosphate buffered saline; Rho-GDI; Rho GDP-dissociation inhibitor protein; TCA; trichloroacetic acidABC-transporter; P-glycoprotein; ABCB1; Cytoskeleton; Actin; Lipid raft

Interactions of DMPC and DMPC/gemini liposomes with the cell membrane investigated by electrorotation by R. Cosimati; G.L. Milardi; C. Bombelli; A. Bonincontro; F. Bordi; G. Mancini; G. Risuleo (pp. 352-356).

The electrorotation technique was utilized to investigate the interactions between a mouse fibroblast cell line and zwitterionic liposomes formed by a natural phospholipid or cationic liposomes formulated with the same phospholipid and a cationic gemini surfactant. The application of this technique allowed an accurate characterization of the passive dielectric behavior of the plasma membrane by the determination of its specific capacitance and conductance. Changes of these parameters, upon interaction with the liposomes, are related to variations in the structure and or in the transport properties of the membrane. Cells were exposed to both types of liposomes for 1 or 4h. Electrorotation data show a dramatic reduction of the dielectric parameters of the plasma membrane after one hour treatment. After 4h of treatment the effects are still observed only in the case of the cationic liposomes. Surprisingly, these same treatments did not cause a relevant biological damage as assessed by standard viability tests. A detailed discussion to rationalize this phenomenon is presented.R. Cosimati et al. interactions of DMPC and DMPC/gemini liposomes with the cell plasma membrane investigated by electrorotation.Display Omitted► Plasma-membrane specific capacitance and conductance evaluated by electrorotation. ► Interaction with liposomes determines structure and/or transport variations. ► The effect is transient or persistent depending on the liposome species. ► Both liposome species do not exert relevant effects on the cell survival.

Keywords: Abbreviations; DDS; drug delivery systems; DMPC; 1,2-dimyristoyl-; sn; -phosphatidyl-choline; Ge-1; DMPC–gemini (2S,3S)-2,3-dimethoxy-1,4-bis(N,N-dimethylamine)-butane; PBS; phosphate-buffered-saline solutionLiposomes; Cell survival; Plasma membrane; Electrorotation; Gemini surfactant

Effect of oxidative stress on plasma membrane fluidity of THP-1 induced macrophages by Carlos de la Haba; José R. Palacio; Martinez Paz Martínez; Antoni Morros (pp. 357-364).

Plasma membrane is one of the preferential targets of reactive oxygen species which cause lipid peroxidation. This process modifies membrane properties such as membrane fluidity, a very important physical feature known to modulate membrane protein localization and function. The aim of this study is to evaluate the effect of oxidative stress on plasma membrane fluidity regionalization of single living THP-1 macrophages. These cells were oxidized with H₂O₂ at different concentrations, and plasma membrane fluidity was analyzed by two-photon microscopy in combination with the environment-sensitive probe Laurdan. Results show a significant H₂O₂ concentration dependent increase in the frequency of rigid lipid regions, mainly attributable to lipid rafts, at the expense of the intermediate fluidity regions. A novel statistical analysis evaluated changes in size and number of lipid raft domains under oxidative stress conditions, as lipid rafts are platforms aiding cell signaling and are thought to have relevant roles in macrophage functions. It is shown that H₂O₂ causes an increase in the number, but not the size, of raft domains. As macrophages are highly resistant to H₂O₂, these new raft domains might be involved in cell survival pathways.Display Omitted► Oxidative stress causes an increase in macrophage plasma membrane rigidity. ► H₂O₂ causes an increase in the number, but not the size, of raft domains. ► Macrophage is highly resistant to H₂O₂ as viability, in our conditions, is above 80%. ► New raft domains in macrophages might be involved in cell survival pathways.

Keywords: Abbreviations; AAPH; 2,2′‐azobis(2‐amidinopropane) dihydrochloride; DMSO; dimethylsulphoxide; DPPC; dipalmitoylphosphatidylcholine; FBS; fetal bovine serum; GP; generalized polarization; GUVs; giant unilamellar vesicles; l; _d; liquid disordered; l; _o; liquid ordered; MTT; 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyltetrazolium bromide; PMA; phorbol‐12‐myristate‐13‐acetate; ROS; reactive oxygen species; THP‐1; human acute monocytic leukemia cell lineOxidative stress; Macrophage; Membrane fluidity; Laurdan; Two-photon microscopy; Lipid raft

Changes of cell electrical parameters induced by electroporation. A dielectrophoresis study by Mihaela G. Moisescu; Mihai Radu; Eugenia Kovacs; Lluis M. Mir; Tudor Savopol (pp. 365-372).

Dielectrophoresis was employed to distinguish the electroporated from non-electroporated cells. It was found that the electric field frequency at which cells change the direction of their movement (the crossover frequency f_CO) is higher when cells are electroporated. The contribution to the cell dielectrophoretic behavior of four electric and geometrical cell parameters was analyzed using a single shell model. f_CO measurements were performed in media with conductivities of 0.001–0.09S/m, on B16F10 cells which were electroporated in a Mannitol solution (0.001S/m), using rectangular or exponential pulses. The control cells' f_CO was found in a domain of 2 to 105kHz, while the electroporated cells' f_CO was in a domain of 5 to 350kHz, depending on the external media conductivities. At exterior conductivities above ~0.02S/m, f_CO of electroporated cells became significantly higher compared to controls. Even though the possible contribution of membrane permittivity to explain the observed f_CO shift toward higher values cannot be excluded, the computations highlight the fact that the variation of cytosol conductivity might be the major contributor to the dielectrophoretic behavior change. Our experimental observations can be described by considering a linear dependence of electroporated cells' cytosol conductivity against external conductivity.Display Omitted► Dielectrophoresis was employed to characterize the cell electroporation. ► Six electric and geometrical cell parameters were analyzed using a single shell model. ► Model indicates cytosol conductivity as major contributor for porated cell behavior. ► Cytosol conductivity is assumed linearly depending on external buffer conductivity.

Keywords: Dielectrophoresis; Electropermeabilization; Cell dielectrical properties; Single shell model; Crossover frequency

Effects of a polar amino acid substitution on helix formation and aggregate size along the detergent-induced peptide folding pathway by Rohan D.A. Alvares; David V. Tulumello; Peter M. Macdonald; Charles M. Deber; R. Scott Prosser (pp. 373-381).

Membrane proteins constitute a significant fraction of the proteome and are important drug targets. While the transmembrane (TM) segments of these proteins are primarily composed of hydrophobic residues, the inclusion of polar residues—either naturally occurring or as a consequence of a disease-related mutation—places a significant folding burden in this environment, potentially impacting bilayer insertion and/or association of neighboring TM helices. Here we investigate the role of an anionic detergent, sodium dodecylsulfate (SDS), and a zwitterionic detergent, dodecylphosphocholine (DPC), in the folding process, and the effects induced by a single polar substitution, on structure and topology of model α-helical TM segments. The peptides, represented by KK-YAAAIAAIAWAXAAIAAAIAA-KKK-NH₂, where X is I or N, are designed with high aqueous solubilities, through poly-lysine tags. Circular dichroism (CD) and NMR were used to monitor peptide secondary structure and diffusional mobility of both peptide and the detergent hosts. For both peptides, SDS binding commenced at a concentration below its CMC, due to Coulombic attraction of anionic SDS to cationic Lys residues. Increasing SDS binding correlated with increasing peptide helicity. Pulsed field gradient (PFG) NMR diffusion measurements revealed that the Asn-containing peptide bound four fewer detergent molecules, corresponding to ca. 20% less SDS than bound by the Ile peptide. Conversely, zwitterionic DPC binding to either peptide was not observed until the DPC concentration approached its CMC. Our findings confirm quantitatively that a single polar residue within a TM segment may have a significant influence on its local membrane environment.Display Omitted► Transmembrane peptide–detergent interactions are studied using CD and NMR. ► Secondary structure and aggregate size of peptide–detergent complexes are obtained. ► Peptides exhibit a detergent-specific sequence of folding and insertion events. ► The number of detergents coating a peptide is obtained by diffusion NMR. ► A single polar substitution results in a 20% reduction in SDS binding.

Keywords: Abbreviations; ANS; 1-anilino-8-naphthalene sulfonate; AI5; model transmembrane peptide without a central polar insert; DPC; n; -dodecyl phosphatidylcholine; I12N; model transmembrane peptide with a central polar insert; TM; transmembrane; SDS; sodium dodecylsulfate; CMC; critical micelle concentration; CD; circular dichroism; PFG (STE); pulsed field gradient (stimulated echo); MRE; mean residue ellipticity; GPCR; G protein-coupled receptorMembrane protein folding; Detergent–protein interaction; Transmembrane peptide NMR; Pulsed field gradient diffusion; Detergent–peptide aggregate size; Membrane protein denaturation

Fusion of gemini based cationic liposomes with cell membrane models: implications for their biological activity by Simone Aleandri; Cecilia Bombelli; Maria Grazia Bonicelli; Federico Bordi; Luisa Giansanti; Giovanna Mancini; Marco Ierino; Simona Sennato (pp. 382-390).

The interaction of neutral and anionic phospholipid liposomes, used as cell models, with cationic liposomes formulated with 1,2-dimyristoyl- sn-glicero-3-phosphocholine and stereomeric cationic gemini surfactants was investigated by differential scanning calorimetry, fluorescence experiments and dynamic laser light scattering. This study was aimed at rationalizing the different biological features shown by liposomes based on different gemini stereoisomers observed in previous investigations. In fact, to correlate the observed biological activity of liposomes with the molecular structure of their components is critical for a rational and systematic approach to the design of new carriers for drug delivery. The obtained results show that the different stereochemistry of the gemini surfactant controls the interaction and the extent of fusion with different cell models.Display Omitted► We investigated the interaction of mixed cationic vesicles with cell models. ► The gemini surfactants in liposomes differ for the stereochemistry of the spacer. ► DSC showed different rates and ways of interaction depending on the gemini. ► Fluorescence experiments showed a high extent of fusion with cell models. ► The stereochemistry of the gemini controls the interaction with biomembranes.

Keywords: Mixed cationic liposomes; Stereochemistry of gemini; DSC; Fluorescence; DLS; Fusion

Stabilization of sphingomyelin interactions by interfacial hydroxyls — A study of phytosphingomyelin properties by Shishir Jaikishan; J. Peter Slotte (pp. 391-397).

D- ribo-phytosphingosines are biologically significant long-chain bases present in various sphingolipids from yeasts, fungi, plants and mammals. In this study we prepared phytopalmitoylsphingomyelin (phytoPSM) analogs based on the D- ribo-phytosphingosine base. The N-linked acyl chains were either 16:0, 2OH(R)16:0 (natural isomer), or 2OH(S)16:0. The gel-phase of phytoPSM was more stable than that of PSM (T_m 48.6°C and 41.0°C, respectively). The gel-liquid crystalline phase transition enthalpies were 9.1±0.4 and 6.1±0.3kcal/mol for phytoPSM and PSM, respectively. An N-linked 2OH(R)16:0 in phytoPSM destabilized the gel phase relative to phytoPSM (by ~+6°C, based on DPH anisotropy measurements), whereas 2OH(S)16:0 in phytoPSM stabilized it (by ~−6°C). All phytoPSM analogs formed sterol-enriched ordered domains in a fluid ternary bilayer, and those containing phytoPSM or 2OH(S)phytoPSM were more thermostable than the domains containing 2OH(R)phytoPSM or PSM. The affinity of cholestatrienol for POPC bilayers containing 20mol% phytoPSM was higher than for comparable bilayers with an equal amount of PSM. The 2-hydroxylated acyl chains in phytoPSM did not markedly alter sterol affinity. We conclude that phytoPSM is a more ordered sphingolipid than PSM, and is fully capable of interacting with cholesterol.Display Omitted► The bilayer membrane properties of phytosphingomyelins were examined. ► The gel-phase of phytoSM was markedly stabilized compared to nonhydroxylated SM. ► Sterols displayed high affinity for bilayers containing phytoSM. ► We speculate that phytoPSM may form highly ordered domains in membranes.

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; K; _x; partitioning coefficient; POPC; 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; SM; sphingomyelin; T; _m; mid temperature of the gel to liquid-crystalline phase transition; T; _1/2; half-width of the gel to liquid-crystalline phase transitionCholesterol; Sphingolipids; Cholestatrienol; Fluorescence quenching; Anisotropy; DSC

NMR resolved multiple anesthetic binding sites in the TM domains of the α4β2 nAChR by Vasyl Bondarenko; David Mowrey; Lu Tian Liu; Yan Xu; Pei Tang (pp. 398-404).

The α4β2 nicotinic acetylcholine receptor (nAChR) has significant roles in nervous system function and disease. It is also a molecular target of general anesthetics. Anesthetics inhibit the α4β2 nAChR at clinically relevant concentrations, but their binding sites in α4β2 remain unclear. The recently determined NMR structures of the α4β2 nAChR transmembrane (TM) domains provide valuable frameworks for identifying the binding sites. In this study, we performed solution NMR experiments on the α4β2 TM domains in the absence and presence of halothane and ketamine. Both anesthetics were found in an intra-subunit cavity near the extracellular end of the β2 transmembrane helices, homologous to a common anesthetic binding site observed in X-ray structures of anesthetic-bound GLIC (Nury et al., [32]). Halothane, but not ketamine, was also found in cavities adjacent to the common anesthetic site at the interface of α4 and β2. In addition, both anesthetics bound to cavities near the ion selectivity filter at the intracellular end of the TM domains. Anesthetic binding induced profound changes in protein conformational exchanges. A number of residues, close to or remote from the binding sites, showed resonance signal splitting from single to double peaks, signifying that anesthetics decreased conformation exchange rates. It was also evident that anesthetics shifted population of two conformations. Altogether, the study comprehensively resolved anesthetic binding sites in the α4β2 nAChR. Furthermore, the study provided compelling experimental evidence of anesthetic-induced changes in protein dynamics, especially near regions of the hydrophobic gate and ion selectivity filter that directly regulate channel functions.Display Omitted► Anesthetics halothane and ketamine bound to multiple sites of the α4β2 nAChR. ► A common intra-subunit anesthetic site was near the extracellular end of the β2 TM. ► Halothane occupied inter-subunit sites near the extracellular end of the TM domain. ► Both drugs bound to intra- and inter-subunit sites near the selectivity filter. ► Anesthetics induced, directly or allosterically, dynamics changes of α4β2.

Keywords: Halothane; Ketamine; General anesthetic; NMR; α4β2 nAChR; Protein dynamic

γ-Hemolysin oligomeric structure and effect of its formation on supported lipid bilayers: An AFM Investigation by Andrea Alessandrini; Gabriella Viero; Mauro Dalla Serra; Prevost Gilles Prévost; Paolo Facci (pp. 405-411).

γ-Hemolysins are bicomponent β-barrel pore forming toxins produced by Staphylococcus aureus as water-soluble monomers, which assemble into oligomeric pores on the surface of lipid bilayers. Here, after investigating the oligomeric structure of γ-hemolysins on supported lipid bilayers (SLBs) by atomic force microscopy (AFM), we studied the effect produced by this toxin on the structure of SLBs. We found that oligomeric structures with different number of monomers can assemble on the lipid bilayer being the octameric form the stablest one. Moreover, in this membrane model we found that γ-hemolysins can form clusters of oligomers inducing a curvature in the lipid bilayer, which could probably enhance the aggressiveness of these toxins at high concentrations.Display Omitted► γ-Hemolysin assembles into oligomeric structures with different number of subunits. ► AFM direct space evidence of γ-hemolysin octamers as the most stable structure ► Clusters of γ-hemolysins can induce a curvature in the hosting lipid bilayer.

Keywords: γ-Hemolysin; Atomic force microscopy; Supported lipid bilayers; Membrane curvature

The role of the helper lipid dioleoylphosphatidylethanolamine (DOPE) for DNA transfection cooperating with a cationic lipid bearing ethylenediamine by Shinichi Mochizuki; Naho Kanegae; Koichi Nishina; Yumi Kamikawa; Kazunori Koiwai; Hiroyasu Masunaga; Kazuo Sakurai (pp. 412-418).

Gene therapy is expected to treat various incurable diseases including viral infections, autoimmune disorders, and cancers. Cationic lipids (CL) have been used as carriers of therapeutic DNAs for gene therapy because they can form a complex with DNA and such a complex can be incorporated into cells and transport the bound DNA to cytosol. The CL/DNA complexes are called lipoplexes and categorized as a non-viral vector. Lipoplexes are often prepared by adding a neutral phospholipid dioleoylphosphatidylethanolamine (DOPE) to CL in order to enhance transfection. However, the role of DOPE is not fully understood. We synthesized a new CL having an ethylenediamine cationic head group, denoted by DA, and found that addition of DOPE to DA achieved a good efficiency, almost in the similar level of commonly used transfection reagent Lipofectamine 2000 (Invitrogen). The composition of DA:DOPE=1:1 showed the highest efficiency. This lipoplex showed structural transition when pH was changed from 7 to 4, corresponding pH lowering in late endosome, while DOPE itself showed structural transition at more basic pH around 8. The present data showed that the DOPE/DA composition determines the structural transition pH and choosing a suitable pH, i.e., a suitable composition, is essential to increase the transfection efficiency.Addition of a neutral phospholipid dioleoylphosphatidylethanolamine (DOPE) to cationic lipid (DA) can achieve a better transfection efficiency than commonly used transfection reagent.Display Omitted► We synthesized a new cationic lipid having an ethylenediamine head group (DA). ► We found that the addition of DOPE to DA achieved a better efficiency. ► DOPE is responsible for the pH responsive structural transition. ► The adding of DA can tune the transition pH well-suited to the pH change. ► This role of DOPE can be generalized for other cationic lipids containing DOPE.

Keywords: Lipoplex; DOPE; pH responsiveness; Structural transition

Characterization of the diffusion of epidermal growth factor receptor clusters by single particle tracking by Mohan Boggara; Krishna Athmakuri; Sunit Srivastava; Richard Cole; Ravi S. Kane (pp. 419-426).

A number of studies have shown that receptors of the epidermal growth factor receptor family (ErbBs) exist as higher-order oligomers (clusters) in cell membranes in addition to their monomeric and dimeric forms. Characterizing the lateral diffusion of such clusters may provide insights into their dynamics and help elucidate their functional relevance. To that end, we used single particle tracking to study the diffusion of clusters of the epidermal growth factor (EGF) receptor (EGFR; ErbB1) containing bound fluorescently-labeled ligand, EGF. EGFR clusters had a median diffusivity of 6.8×10^–11cm²/s and were found to exhibit different modes of transport (immobile, simple, confined, and directed) similar to that previously reported for single EGFR molecules. Disruption of actin filaments increased the median diffusivity of EGFR clusters to 10.3×10^–11cm²/s, while preserving the different modes of diffusion. Interestingly, disruption of microtubules rendered EGFR clusters nearly immobile. Our data suggests that microtubules may play an important role in the diffusion of EGFR clusters either directly or perhaps indirectly via other mechanisms. To our knowledge, this is the first report probing the effect of the cytoskeleton on the diffusion of EGFR clusters in the membranes of live cells.Display Omitted► We characterized the diffusion of EGFR clusters in cell membranes. ► We showed that EGFR clusters exhibit different modes of diffusion. ► We found that the disruption of microtubules makes most of the clusters immobile. ► Our data suggests an important role for microtubules in EGFR cluster diffusion.

Keywords: Receptor; Cluster; EGFR; Cytoskeleton; Diffusion

Protection against cardiac injury by small Ca2⁺-sensitive K⁺ channels identified in guinea pig cardiac inner mitochondrial membrane by David F. Stowe; Ashish K. Gadicherla; Yifan Zhou; Mohammed Aldakkak; Qunli Cheng; Wai-Meng Kwok; Ming Tao Jiang; James S. Heisner; MeiYing Yang; Amadou K.S. Camara (pp. 427-442).

We tested if small conductance, Ca2⁺‐sensitive K⁺ channels (SK_Ca) precondition hearts against ischemia reperfusion (IR) injury by improving mitochondrial (m) bioenergetics, if O₂‐derived free radicals are required to initiate protection via SK_Ca channels, and, importantly, if SK_Ca channels are present in cardiac cell inner mitochondrial membrane (IMM). NADH and FAD, superoxide (O₂⁻), and m[Ca2⁺] were measured in guinea pig isolated hearts by fluorescence spectrophotometry. SK_Ca and IK_Ca channel opener DCEBIO (DCEB) was given for 10min and ended 20min before IR. Either TBAP, a dismutator of O^₂⁻, NS8593, an antagonist of SK_Ca isoforms, or other K_Ca and K_ATP channel antagonists, were given before DCEB and before ischemia. DCEB treatment resulted in a 2-fold increase in LV pressure on reperfusion and a 2.5 fold decrease in infarct size vs. non-treated hearts associated with reduced O₂⁻ and m[Ca2⁺], and more normalized NADH and FAD during IR. Only NS8593 and TBAP antagonized protection by DCEB. Localization of SK_Ca channels to mitochondria and IMM was evidenced by a) identification of purified mSK_Ca protein by Western blotting, immuno-histochemical staining, confocal microscopy, and immuno-gold electron microscopy, b) 2-D gel electrophoresis and mass spectroscopy of IMM protein, c) [Ca2⁺]‐dependence of mSK_Ca channels in planar lipid bilayers, and d) matrix K⁺ influx induced by DCEB and blocked by SK_Ca antagonist UCL1684. This study shows that 1) SK_Ca channels are located and functional in IMM, 2) mSK_Ca channel opening by DCEB leads to protection that is O₂⁻ dependent, and 3) protection by DCEB is evident beginning during ischemia.Display Omitted► We identify small Ca2⁺‐sensitive K⁺ channels in guinea pig cardiac mitochondria. ► Identity based on Western blot, confocal microscopy, immune-gold EM, and MS ► Function based on K⁺ flux, Ca2⁺, apamin, and UCL1684‐sensitive conductance ► Channel agonist, DCEBIO protects hearts against IR injury, and antagonist NS8593 blocks protection. ► Mechanism of protection in isolated hearts is mediated by the superoxide radical.

Keywords: Abbreviations; IR; ischemia reperfusion; SK; _Ca; small conductance Ca; 2; ⁺; ‐sensitive K; ⁺; channel; BK; _Ca; big conductance Ca; 2; ⁺; ‐sensitive K; ⁺; channel; K; _ATP; ATP‐sensitive K; ⁺; channel; DCEB; 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one; IMM; inner mitochondrial membrane; TBAP; Mn(III) tetrakis (4-benzoic acid) porphyrin; PPC; pharmacological preconditioning; TRAM; TRAM-34: 1-[(2-chlorophenyl) (diphenyl)methyl]-1; H; -pyrazole; GLIB; glibenclamide; PAX; paxilline; BSA; bovine serum albumin; IEM; immune-electron microscopy; MS; mass spectroscopy; NS8593; N-[(1R)-1,2,3,4-tetrahydro-1-naphthalenyl]-1H-benzimidazol-2-amine hydrochloride; UCL 1684; 6,10-diaza-3(1,3)8,(1,4)-dibenzena-1,5(1,4)-diquinolinacy clodecaphaneCardiac mitochondria; Inner mitochondrial membrane; Cell signaling; Ischemia reperfusion injury; Oxidant stress; Small conductance Ca; 2; ⁺; ‐sensitive K; ⁺; channel

Antimicrobial HPA3NT3 peptide analogs: Placement of aromatic rings and positive charges are key determinants for cell selectivity and mechanism of action by Jong-Kook Lee; Seong-Cheol Park; Kyung-Soo Hahm; Yoonkyung Park (pp. 443-454).

In an earlier study, we determined that HP(2‐20) (residues 2‐20 of parental HP derived from the N-terminus of the Helicobacter pylori ribosomal protein L1) and its analog, HPA3NT3, had potent antimicrobial effects. However, HPA3NT3 also showed undesirable cytotoxicity against HaCaT cells. In the present study, we designed peptide analogs including HPA3NT3-F1A (‐F1A), HPA3NT3-F8A (‐F8A), HPA3NT3-F1AF8A (‐F1AF8A), HPA3NT3-A1 (‐A1) and HPA3NT3-A2 (‐A2) in an effort to investigate the effects of amino acid substitutions in reducing their hydrophobicity or increasing their cationicity, and any resulting effects on their selectivity in their interactions with human cells and pathogens, as well as their mechanism of antimicrobial action. With the exception of HPA3NT3-A1, all of these peptides showed potent antimicrobial activity. Moreover, substitution of Ala for Phe at positions 1 and/or 8 of the HPA3NT3 peptides (‐F1A, -F8A and -F1AF8A) dramatically reduced their cytotoxicity. Thus the cytotoxicity of HPA3NT3 appears to be related to its Phe residues (positions 1 and 8), which strongly interact with sphingomyelin in the mammalian cell membrane. HPA3NT3 exerted its bactericidal effects through membrane permeabilization mediated by pore formation. In contrast, fluorescent dye leakage and nucleic acid gel retardation assays showed that ‐A2 acted by penetrating into the cytoplasm, where it bound to nucleic acids and inhibited protein synthesis. Notably, Staphylococcus aureus did not develop resistance to -A2 as it did with rifampin. These results suggest that the -A2 peptide could potentially serve as an effective antibiotic agent against multidrug-resistant bacterial strains.Placement of aromatic rings and positive charges are key determinants of cell selectivity and the mechanism of action against mammalian cells and human pathogens of antimicrobial HPA3NT3 peptide analogs, Jong-Kook Lee, Seong-Cheol Park, Kyung-Soo Hahm, Yoonkyung Park, BBA-BiomembranesDisplay Omitted► Placement of aromatic rings and positive charges are key determinants of cell selectivity. ► HPA3NT3‐A2 acted by penetrating into the cytoplasm, where it bound to nucleic acids and inhibited protein synthesis. ► HPA3NT3-A2 peptide could potentially serve as an effective antibiotic agent against multidrug-resistant bacterial strains.

Keywords: Abbreviations; Fmoc; 9-fluorenylmethoxycarbonyl; DMF; dimethylformamide; DCM; dichloromethane; hRBCs; human red blood cells; HaCaT; human keratinocyte; CLSM; Confocal laser-scanning microscopy; ATCC; American Type Culture Collection; KCTC; Korean Collection for Type Cultures; CCARM; Collection of Antibiotics Resistance Microbes; CFU; colony forming unit; MIC; minimum inhibitory concentration; TAMRA; tetramethyl rhodamine; SUVs; small unilamellar vesicles; LUVs; large unilamellar vesicles; TEM; transmission electron microscopy; UAC; uranyl acetateDeterminants of cell selectivity; Antimicrobial action; Resistance

Biophysical characterization of a binding site for TLQP-21, a naturally occurring peptide which induces resistance to obesity by V. Cassina; A. Torsello; A. Tempestini; D. Salerno; D. Brogioli; L. Tamiazzo; E. Bresciani; J. Martinez; J.A. Fehrentz; Verdie P. Verdié; R.J. Omeljaniuk; R. Possenti; L. Rizzi; V. Locatelli; F. Mantegazza (pp. 455-460).

Recently, we demonstrated that TLQP-21 triggers lipolysis and induces resistance to obesity by reducing fat accumulation [1]. TLQP-21 is a 21 amino acid peptide cleavage product of the neuroprotein VGF and was first identified in rat brain. Although TLQP-21 biological activity and its molecular signaling is under active investigation, a receptor for TLQP-21 has not yet been characterized. We now demonstrate that TLQP-21 stimulates intracellular calcium mobilization in CHO cells. Furthermore, using Atomic Force Microscopy (AFM), we also provide evidence of TLQP-21 binding-site characteristics in CHO cells. AFM was used in force mapping mode equipped with a cantilever suitably functionalized with TLQP-21. Attraction of this functionalized probe to the cell surface was specific and consistent with the biological activity of TLQP-21; by contrast, there was no attraction of a probe functionalized with biologically inactive analogues. We detected interaction of the peptide with the binding-site by scanning the cell surface with the cantilever tip. The attractive force between TLQP-21 and its binding site was measured, statistically analyzed and quantified at approximately 40 pN on average, indicating a single class of binding sites. Furthermore we observed that the distribution of these binding sites on the surface was relatively uniform.Display Omitted► TLQP-21 stimulates intracellular calcium mobilization in CHO cells. ► We provide evidence of TLQP-21 binding-site characteristics in CHO cells. ► Atomic Force Spectroscopy on living cells. ► Interaction of peptide with its binding-site by functionalized AFM cantilever tip.

Keywords: TLQP-21; VGF; Ligand; –; receptor; Atomic force Microscopy (AFM); CHO; Fluorescence

The effect of extracellular conductivity on electroporation-mediated molecular delivery by Jianbo Li; Wenchang Tan; Miao Yu; Hao Lin (pp. 461-470).

In this work, the effect of extra-cellular conductivity on electroporation-mediated molecular delivery efficiency is investigated. A numerical model combining the Smoluchowski equation for membrane permeabilization and the Nernst–Planck equations for ion transport is used to simulate the evolution of ion concentration spatially and temporally. The results are compared with and used to interpret trends observed from previous experimental measurements. Agreements are found which suggest the critical importance of electrophoretic transport. This mechanism controls delivery efficiency on the quantitative level. Meanwhile, a simple formula is developed to predict the molecular content delivered via electrophoresis. The formula can be used as a compact model which provides good approximation to the full numerical model while avoiding the computational cost.Display Omitted► The effects of electrical conductivity on electroporation-mediated molecular delivery are investigated. ► Conductivity affects delivery by inducing non-uniform electric fields. ► The model predicts an inverse correlation between delivery and extracellular conductivity. ► The model offers a mechanistic interpretation to previous experimental observations. ► A compact formula is developed to predict total molecular delivery.

Keywords: Electroporation; Electropermeabilization; Electrophoresis; Molecular delivery; Electrical conductivity; Field amplified sample stacking

Conductance properties of the inwardly rectifying channel, Kir3.2: Molecular and Brownian dynamics study by Tamsyn A. Hilder; Shin-Ho Chung (pp. 471-478).

Using the recently unveiled crystal structure, and molecular and Brownian dynamics simulations, we elucidate several conductance properties of the inwardly rectifying potassium channel, Kir3.2, which is implicated in cardiac and neurological disorders. We show that the pore is closed by a hydrophobic gating mechanism similar to that observed in Kv1.2. Once open, potassium ions move into, but not out of, the cell. The asymmetrical current–voltage relationship arises from the lack of negatively charged residues at the narrow intracellular mouth of the channel. When four phenylalanine residues guarding the intracellular gate are mutated to glutamate residues, the channel no longer shows inward rectification. Inward rectification is restored in the mutant Kir3.2 when it becomes blocked by intracellular Mg2⁺. Tertiapin, a polypeptide toxin isolated from the honey bee, is known to block several subtypes of the inwardly rectifying channels with differing affinities. We identify critical residues in the toxin and Kir3.2 for the formation of the stable complex. A lysine residue of tertiapin protrudes into the selectivity filter of Kir3.2, while two other basic residues of the toxin form hydrogen bonds with acidic residues located just outside the channel entrance. The depth of the potential of mean force encountered by tertiapin is −16.1kT, thus indicating that the channel will be half-blocked by 0.4μM of the toxin.Display Omitted► Crystal structure is in closed configuration and no conduction is observed. ► Pore is closed by a hydrophobic gating mechanism similar to Kv1.2. ► Open channel conductance compares well to experiment. ► Inward rectification due to no negatively charged residues at intracellular mouth. ► Channel is half blocked by 0.4μM tertiapin.

Keywords: Abbreviations; Kir; Inwardly rectifying potassium channel; GIRK; G protein-gated inwardly rectifying potassium channel; TPN; Tertiapin; MD; molecular dynamics; BD; Brownian dynamicsGIRK2; Kir3.2; Inwardly rectifying potassium channel; Conductance property; Molecular dynamics; Brownian dynamics

A conserved interdomain communication pathway of pseudosymmetrically distributed residues affects substrate specificity of the fungal multidrug transporter Cdr1p by Marcin Kolaczkowski; Sroda-Pomianek Kamila Środa-Pomianek; Anna Kolaczkowska; Krystyna Michalak (pp. 479-490).

Understanding the communication pathways between remote sites in proteins is of key importance for understanding their function and mechanism of action. These remain largely unexplored among the pleiotropic drug resistance (PDR) representatives of the ubiquitous superfamily of ATP-binding cassette (ABC) transporters. To identify functionally coupled residues important for the polyspecific transport by the fungal ABC multidrug transporter Cdr1p a new selection strategy, towards increased resistance to a preferred substrate of the homologous Snq2p, was applied to a library of randomly generated mutants. The single amino acid substitutions, located pseudosymmetrically in each domain of the internally duplicated protein: the H-loop of the N-terminal nucleotide binding domain (NBD1) (C363R) and in the C-terminal NBD2 region preceding Walker A (V885G). The central regions of the first transmembrane helices 1 and 7 of both transmembrane domains were also affected by the G521S/D and A1208V substitutions respectively. Although the mutants were expressed at a similar level and located correctly to the plasma membrane, they selectively affected transport of multiple drugs, including azole antifungals. The synergistic effects of combined mutations on drug resistance, drug dependent ATPase activity and transport support the view inferred from the statistical coupling analysis (SCA) of aminoacid coevolution and mutational analysis of other ABC transporter families that these residues are an important part of the conserved, allosterically coupled interdomain communication network. Our results shed new light on the communication between the pseudosymmetrically arranged domains in a fungal PDR ABC transporter and reveal its profound influence on substrate specificity.Display Omitted► New selection identifies novel residues selectively increasing transport by Cdr1p. ► Mutations cluster at four remote pseudosymmetrical locations of each protein domain. ► Double mutant analysis reveals functional coupling of residues over long distance. ► Statistical coupling analysis indicates their role in interdomain communication. ► The ATPase activity of the quadruple mutant is not coupled to drug transport.

Keywords: Abbreviations; MDR; multidrug resistance; PDR; pleiotropic drug resistance; ABC; ATP-binding cassette; NBD; nucleotide binding domain; TMD; transmembrane domain; TM; transmembrane; SCA; statistical coupling analysis; 4-NQO; 4-nitroquinoline 1-oxide; DiOC; ₆; 3,3′-dihexyloxacarbocyanine iodideABC transporter; Azole antifungal; Mutagenesis; Statistical coupling analysis; Coevolution; Allosteric interaction

Benzoic acid and specific 2-oxo acids activate hepatic efflux of glutamate at OAT2 by Till Pfennig; Beate Herrmann; Tim Bauer; Schomig Edgar Schömig; Grundemann Dirk Gründemann (pp. 491-498).

The liver is the principal source of glutamate in blood plasma. Recently we have discovered that efflux of glutamate from hepatocytes is catalyzed by the transporter OAT2 (human gene symbol SLC22A7). Organic anion transporter 2 (OAT2) is an integral membrane protein of the sinusoidal membrane domain; it is primarily expressed in liver and much less in kidney, both in rats and humans. Many years ago, Häussinger and coworkers have demonstrated in isolated perfused rat liver that benzoic acid or specific 2-oxo acid analogs of amino acids like e.g. 2-oxo-4-methyl-pentanoate (‘2-oxo-leucine’) strongly stimulate release of glutamate (up to 7-fold); ‘2-oxo-valine’ and the corresponding amino acids were without effect. The molecular mechanism of efflux stimulation has remained unclear. In the present study, OAT2 from human and rat were heterologously expressed in 293 cells. Addition of 1mmol/l benzoic acid to the external medium increased OAT2-specific efflux of glutamate up to 20-fold; ‘2-oxo-leucine’ was also effective, but not ‘2-oxo-valine’. Similar effects were seen for efflux of radiolabeled orotic acid. Expression of OAT2 did not increase uptake of benzoic acid; thus, benzoic acid is no substrate, and trans-stimulation can be excluded. Instead, further experiments suggest that increased efflux of glutamate is caused by direct interaction of benzoic acid and specific 2-oxo acids with OAT2. We propose that stimulators bind to a distinct extracellular site and thereby accelerate relocation of the empty substrate binding site to the intracellular face. Increased glutamate efflux at OAT2 could be the main benefit of benzoate treatment in patients with urea cycle defects.Display Omitted► Efflux of glutamate from hepatocytes is catalyzed by the transporter OAT2 ( SLC22A7). ► External benzoic acid increases OAT2-specific efflux of glutamate up to 20-fold. ► 2-oxo-4-methyl-pentanoate (2-oxo-leucine) was also effective, but not 2-oxo-valine. ► Benzoic acid is no substrate of OAT2, and thus trans-stimulation can be excluded. ► Stimulators accelerate relocation of the empty substrate binding site.

Keywords: Abbreviations; EAAT; excitatory amino acid transporter; GABA; gamma-aminobutyric acid; LC; liquid chromatography; MS; mass spectrometry; OAT; organic anion transporterGlutamate transport; SLC22A7; Benzoic acid; 2-Oxo acid; Liver

Modeling of non-covalent complexes of the cell-penetrating peptide CADY and its siRNA cargo by Jean-Marc Crowet; Laurence Lins; Sébastien Deshayes; Gilles Divita; May Morris; Robert Brasseur; Annick Thomas (pp. 499-509).

CADY is a cell-penetrating peptide spontaneously making non-covalent complexes with Short interfering RNAs (siRNAs) in water. Neither the structure of CADY nor that of the complexes is resolved. We have calculated and analyzed 3D models of CADY and of the non-covalent CADY–siRNA complexes in order to understand their formation and stabilization. Data from the ab initio calculations and molecular dynamics support that, in agreement with the experimental data, CADY is a polymorphic peptide partly helical. Taking into consideration the polymorphism of CADY, we calculated and compared several complexes with peptide/siRNA ratios of up to 40. Four complexes were run by using molecular dynamics. The initial binding of CADYs is essentially due to the electrostatic interactions of the arginines with siRNA phosphates. Due to a repetitive arginine motif (XLWR(K)) in CADY and to the numerous phosphate moieties in the siRNA, CADYs can adopt multiple positions at the siRNA surface leading to numerous possibilities of complexes. Nevertheless, several complex properties are common: an average of 14±1 CADYs is required to saturate a siRNA as compared to the 12±2 CADYs experimentally described. The 40 CADYs/siRNA that is the optimal ratio for vector stability always corresponds to two layers of CADYs per siRNA. When siRNA is covered by the first layer of CADYs, the peptides still bind despite the electrostatic repulsion. The peptide cage is stabilized by hydrophobic CADY–CADY contacts thanks to CADY polymorphism. The analysis demonstrates that the hydrophobicity, the presence of several positive charges and the disorder of CADY are mandatory to make stable the CADY–siRNA complexes.Display Omitted► Models of CADY and CADY–siRNA complexes have been calculated and analyzed. ► Initial binding of CADYs to siRNA is essentially due to electrostatic interactions. ► CADY can adopt multiple positions at the siRNA surface. ► The peptide cage is stabilized by hydrophobic contacts thanks to CADY polymorphism. ► Hydrophobicity, positive charges and polymorphism are mandatory to form complexes.

Keywords: Cell penetrating peptide; siRNA; CADY; Non-covalent complex; Molecular modeling; Molecular dynamics

Lipid peroxidation and water penetration in lipid bilayers: A W-band EPR study by Elena Conte; Francesco Maria Megli; Himanshu Khandelia; Gunnar Jeschke; Enrica Bordignon (pp. 510-517).

Lipid peroxidation plays a key role in the alteration of cell membrane's properties. Here we used as model systems multilamellar vesicles (MLVs) made of the first two products in the oxidative cascade of linoleoyl lecithin, namely 1-palmitoyl-2-(13-hydroperoxy-9,11-octadecanedienoyl)-lecithin (HpPLPC) and 1-palmitoyl-2-(13-hydroxy-9,11-octadecanedienoyl)-lecithin (OHPLPC), exhibiting a hydroperoxide or a hydroxy group at position 13, respectively. The two oxidized lipids were used either pure or in a 1:1 molar ratio mixture with untreated 1-palmitoyl-2-linoleoyl-lecithin (PLPC). The model membranes were doped with spin-labeled lipids to study bilayer alterations by electron paramagnetic resonance (EPR) spectroscopy. Two different spin-labeled lipids were used, bearing the doxyl ring at position (n) 5 or 16: γ-palmitoyl-β-(n-doxylstearoyl)-lecithin (n-DSPPC) and n-doxylstearic acid (n-DSA).Small changes in the acyl chain order in the sub-polar region and at the methyl-terminal induced by lipid peroxidation were detected by X-band EPR. Concomitantly, the polarity and proticity of the membrane bilayer in those regions were investigated at W band in frozen samples. Analysis of the g_xx and A_zz parameters revealed that OHPLPC, but mostly HpPLPC, induced a measurable increase in polarity and H-bonding propensity in the central region of the bilayer. Molecular dynamics simulation performed on 16-DSA in the PLPC–HpPLPC bilayer revealed that water molecules are statistically favored with respect to the hydroperoxide groups to interact with the nitroxide at the methyl-terminal, confirming that the H-bonds experimentally observed are due to increased water penetration in the bilayer. The EPR and MD data on model membranes demonstrate that cell membrane damage by oxidative stress cause alteration of water penetration in the bilayer.Display Omitted► We study lipid peroxidation effects on bilayers properties. ► Model membranes made of PLPC and two oxidized lipids are used. ► Membranes contain spin-labeled lipids to study bilayer alterations by EPR. ► EPR and MD data show that oxidation increases water penetration in membranes.

Keywords: Abbreviations; 5-DSPPC; 1-palmitoyl-2-(5-doxylstearoyl)-lecithin; 16-DSPPC; 1-palmitoyl-2-(16-doxylstearoyl)-lecithin; 5-DSA; 5-doxylstearic acid; 16-DSA; 16-doxylstearic acid; EPR; electron paramagnetic resonance; HpPLPC; 1-palmitoyl-2-(13-hydroperoxy-9,11-octadecanedienoyl)-lecithin; MLV; multilamellar vesicles; OHPLPC; 1-palmitoyl-2-(13-hydroxy-9,11-octadecanedienoyl)-lecithin; PLPC; 1-palmitoyl-2-linoleoyl-lecithin; ROS; reactive oxygen speciesPhospholipid bilayer; Lipoperoxidation; Spin labeling; W-band EPR; Bilayer polarity; Molecular dynamics

FTIR,¹H NMR and EPR spectroscopy studies on the interaction of flavone apigenin with dipalmitoylphosphatidylcholine liposomes by Pawlikowska-Pawlega Bożena Pawlikowska-Pawlęga; Lucjan E. Misiak; Barbara Zarzyka; Roman Paduch; Antoni Gawron; Wiesław I. Gruszecki (pp. 518-527).

Apigenin (5,7,4′-trihydroxyflavone) is a cancer chemopreventive agent and a member of the family of plant flavonoids. Apigenin interaction with liposomes formed with dipalmitoylphosphatidylcholine (DPPC) was investigated by means of FTIR spectroscopy,¹H NMR and EPR techniques. Fluorescent microscopy and electron microscopy were applied to study the apigenin effects on colon myofibroblasts and human skin fibroblasts. The strong rigidifying effect of apigenin with respect to polar head groups was concluded on the basis of the action of the flavone on partition coefficient of Tempo spin label between the water and lipid phases. The ordering effect was also found in hydrophobic region at the depth monitored by 5-SASL and 16-SASL spin labels. The inclusion of apigenin to the membrane restricted the motional freedom of polar head groups lowering penetration of Pr3⁺ ions to the membranes. The¹H NMR technique supported also the restriction of motional freedom of the membrane in the hydrophobic region, especially in the zone of CH₂ groups of alkyl chains. FTIR analysis showed that apigenin incorporates into DPPC liposomes via hydrogen bonding between its own hydroxyl groups and lipid polar head groups in the COPOC segment. It is also very likely that hydroxyl groups of apigenin link with polar groups of DPPC by water bridges. Electron and fluorescence microscopic observations revealed changes in the internal membrane organization of the examined cells. In conclusion, the changes of the structural and dynamic properties of membranes can be crucial for processes involving tumor suppression signal transduction pathways and cell cycle regulation.Display Omitted► Apigenin incorporates into DPPC liposomes and changes their dynamic properties. ► Apigenin preferentially locates in the upper part of the membrane containing chain/glycerol/headgroup. ► Hydrogen bonds with lipid polar heads are formed by hydroxyl and keto groups of apigenin. ► COPOC segment of lipid polar head groups creates hydrogen bonds with apigenin. ► Normal biomembranes are sensitive to apigenin activity.

Keywords: Abbreviations; DPPC; dipalmitoylphosphatidylcholine; EPR spectroscopy; electron paramagnetic resonance spectroscopy; FTIR spectroscopy; Fourier-transform infrared spectroscopy; NMR spectroscopy; nuclear magnetic resonance spectroscopy; L; _α; lamellar liquid-crystalline phase; L; _β; lamellar gel phase; 5-SASL; 2-(3-carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinyloxy free radical; 16-SASL; 2-(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinyloxy free radical; P; _β; rippled gel phase; Tempo; 2,2,6,6-tetramethyl-1-piperidinyloxy free radicalApigenin; DPPC liposome; ¹; H NMR; EPR; FTIR; Electron microscopy

Peptide-induced bilayer thinning structure of unilamellar vesicles and the related binding behavior as revealed by X-ray scattering by Chun-Jen Su; Shiuan-Shiaou Wu; U-Ser Jeng; Ming-Tao Lee; An-Chung Su; Kuei-Fen Liao; Wei-Yu Lin; Yu-Shan Huang; Chun-Yu Chen (pp. 528-534).

We have studied the bilayer thinning structure of unilamellar vesicles (ULV) of a phospholipid 1,2-dierucoyl- sn-glycero-3-phosphocholine (di22:1PC) upon binding of melittin, a water-soluble amphipathic peptide. Successive thinning of the ULV bilayers with increasing peptide concentration was monitored via small-angle X-ray scattering (SAXS). Results suggest that the two leaflets of the ULV of closed bilayers are perturbed and thinned asymmetrically upon free peptide binding, in contrast to the centro-symmetric bilayer thinning of the substrate-oriented multilamellar membranes (MLM) with premixed melittin. Moreover, thinning of the melittin-ULV bilayer associates closely with peptide concentration in solution and saturates at ~4%, compared to the ~8% maximum thinning observed for the correspondingly premixed peptide-MLM bilayers. Linearly scaling the thinning of peptide-ULV bilayers to that of the corresponding peptide-MLM of a calibrated peptide-to-lipid ratio, we have deduced the number of bound peptides on the ULV bilayers as a function of free peptide concentration in solution. The hence derived X-ray-based binding isotherm allows extraction of a low binding constant of melittin to the ULV bilayers, on the basis of surface partition equilibrium and the Gouy–Chapman theory. Moreover, we show that the ULV and MLM bilayers of di22:1PC share a same thinning constant upon binding of a hydrophobic peptide alamethicin; this result supports the linear scaling approach used in the melittin-ULV bilayer thinning for thermodynamic binding parameters of water-soluble peptides.Bilayer thinning behaviors for the melittin-bound, free-floating unilamellar vesicles and the substrate-oriented multilamellar membranes of zwitterionic phospholipids.Display Omitted► Peptide-induced asymmetric bilayer thinning behavior is revealed. ► Small-angle X-ray scattering and X-ray diffraction results are integrated. ► Articulating the membrane thinning structure with peptide binding affinity. ► ULV and MLM bilayers share a same bilayer thinning constant.

Keywords: Peptide; Membrane; Vesicle; X-ray scattering

The melanocortin 4 receptor: Oligomer formation, interaction sites and functional significance by Kathryn L. Chapman; John B.C. Findlay (pp. 535-542).

This study involves the structural and functional properties of the recombinant melanocortin 4 receptor (MC₄R) expressed in the HEK-293 cell line. Using co-immuno-purification approaches, the receptor appears to be an oligomer, which can be crosslinked through disulphide bonds involving a native cysteine residue (84) to give a dimeric species. This position is located near the cytosolic region of transmembrane segment 2 and it is suggested that this is an interacting interface between MC₄R monomers. Using co-expression of the native protein and a C84A mutant, it appears that the receptor also forms higher order oligomers via alternative interfaces. Interestingly, disulphide crosslink formation does not occur if the receptor is uncoupled from its G-protein, even though the oligomeric state is preserved. This suggests that the conformational changes, which occur on activation, affect the TM2 interface. The pharmacology of the agonist, NDP-MSH, indicates that the MC₄R retains high affinity for the ligand in the absence of the G-protein but occupancy for the ligand is increased. The data can be interpreted to suggest that the G-protein exerts a negative allosteric effect on the receptor. Co-expression of one receptor lacking the ability to signal with another, which cannot bind the agonist, restored ligand-dependent activation of the G-protein to situations in which neither receptor on its own could activate the G-protein. Such transactivation suggests meaningful cross talk between the receptor subunits in the oligomeric complex. These studies demonstrate further unique features of the MC₄R.Display Omitted► The MC4R is an oligomer. ► C84 located on TMII can crosslink between dimers when coupled to the G-protein. ► The MC4R retains high affinity for the agonist in the absence of the G-protein. ► Agonist occupancy increases in the absence of G-protein. ► Transactivation studies on the MC4R demonstrated crosstalk in the oligomer.

Keywords: Abbreviations; ACTH; Adrenocorticotropic hormone; AgRP; Agouti-related protein; CRE-Luc cells; cAMP response element luciferase cells; CuP; copper 1–10 phenanthroline; ECL; Extracellular loop; Gα; G-protein α-subunit; G-protein; guanine nucleotide-binding regulatory protein; GPCR; G-protein-coupled receptor; Gpp(NH)p; guanosine-5′-(βγ-imino)triphosphate; GTPγS; Guanosine 5′-[γ-thio]triphosphate; HA epitope; human influenza haemagglutinin; MC; ₄; R; Melanocortin 4 receptor; NDP-MSH; [Nle; ⁴; , D-Phe; ⁷; ] α-MSH; TM; transmembrane segment; POMC; proopiomelanocortinMelanocortin 4 receptor; Oligomerization; Trans-activation; GPCR

Chemical and physical requirements for lipid extraction by bovine binder of sperm BSP1 by Alexandre Therrien; Puttaswamy Manjunath; Michel Lafleur (pp. 543-551).

The bovine seminal plasma contains phosphocholine-binding proteins, which associate to sperm membranes upon ejaculation. These binder-of-sperm (BSP) proteins then induce a phospholipid and cholesterol efflux from these membranes. In this work, we determined physical and chemical parameters controlling this efflux by characterizing the lipid extraction induced by BSP1, the most abundant of BSP protein in bull seminal plasma, from model membranes with different composition. The model membranes were formed from binary mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) with 1-palmitoyl-2-hydroxy- sn-glycero-3-phosphocholine (Lyso-PC), 1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) or cholesterol. The modulation of BSP1-induced lipid extraction from membranes by their chemical composition and their physical properties brings us to propose a 3-step extraction mechanism. First, the protein associates with membranes via specific binding to phosphocholine groups. Second, BSP1 penetrates in the membrane, essentially in the external lipid leaflet. Third, BSP1 molecules solubilize a lipid patch coming essentially from the outer lipid leaflet, without any lipid specificity, to ultimately form small lipid/protein auto-assemblies. The stoichiometry of these complexes corresponds to 10–15 lipids per protein. It is also shown that fluid-phase membranes are more prone to BSP1-induced lipid extraction than gel-phase ones. The inhibition of the lipid extraction in this case appears to be related to the inhibition of the protein penetration in the membrane (step 2) and not to the protein association with PC head groups (step 1). These findings contribute to our understanding of the mechanism by which BSP1 modify the lipid composition of sperm membranes, a key event in sperm capacitation.Display Omitted► A 3-step mechanism for BSP1-induced extraction of lipids from sperm membranes is proposed. ► BSP1 requires 2 phosphatidylcholines to bind sperm membranes ► BSP1-induced lipid extraction is largely non lipid selective. ► The stoichiometry of the BSP1/lipid complexes resulting from the extraction corresponds to 10–15 lipids per protein.

Keywords: Abbreviations; PC; Phosphocholine; PE; Phosphatidylethanolamine; PG; Phosphatidylglycerol; PS; Phosphatidylserine; Chol; Cholesterol; SM; Sphingomyelin; BSP; Binder-of-sperm protein; POPC; 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; Lyso-PC; 1-palmitoyl-2-hydroxy-; sn; -glycero-3phosphocholine; DPPC; 1,2-dipalmitoyl-; sn; -glycero-3-phosphocholine; POPE; 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; POPS; 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine; DMPC; 1,2-dimyristoyl-sn-glycero-3-phosphocholine; DMPG; 1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol); DMPE; 1,2-dimyristoyl-; sn; -glycero-3-phosphoethanolamine; DOPC; 1,2-dioleoyl-; sn; -glycero-3-phosphocholine; Fn2; Fibronectin type II domain; Trp; Tryptophan; Tyr; Tyrosine; MLV; Multilamellar vesicle; SUV; Small unilamellar vesicle; CRAC; Cholesterol recognition amino acid concensusBSP1/PDC-109; Sperm membranes; Lipid extraction; Lipid–protein interactions; liposomes; phospholipids

Membrane docking mode of the C2 domain of PKCε: An infrared spectroscopy and FRET study by Alessio Ausili; Mattias Berglin; Hans Elwing; Egea-Jimenez Antonio L. Egea-Jiménez; Corbalan-Garcia Senena Corbalán-García; Gomez-Fernandez Juan C. Gómez-Fernández (pp. 552-560).

The C2 domain of PKCε binds to negatively charged phospholipids but little is known so far about the docking orientation of this domain when it is bound. By using a FRET assay we have studied the binding of this domain to model membranes. We have also used ATR-Fourier transform infrared spectroscopy with polarized light (ATR-FTIR) to determine the docking mode by calculating the β-sandwich orientation when the domain is bound to different types of model membranes. The vesicle lipid compositions were: POPC/POPE/POPA (22:36:42) imitating the inner leaflet of a plasma membrane, POPC/POPA (50:50) in which POPE has been eliminated with respect to the former composition and POPC/POPE/CL (43:36:21) imitating the inner mitochondrial membrane. Results show that the β-sandwich of the PKCα-C2 domain is inclined at an angle α close to 45° to the membrane normal. Some differences were found with respect to the extent of binding as a function of phospholipid composition and small changes on secondary structure were only evident when the domain was bound to model membranes of POPC/POPA: in this case, the percentage of β-sheet of the C2 domain increases if compared with the secondary structure of the domain in the absence of vesicles. With respect to the β-sandwich orientation, when the domain is bound to POPC/POPE/CL membranes it forms an angle with the normal to the surface of the lipid bilayer (39°) smaller than that one observed when the domain interacts with vesicles of POPC/POPA (49°).Display Omitted► The membrane docking of PKCε to membranes has been studied by using ATR-IR. ► The angle α formed by the protein β-sandwich orientation and the normal to the membrane was measured. ► It amounted to 39° for POPC/POPE/cardiolipin (43:26:31) and 49° for POPC/POPA (50:50).

Keywords: Abbreviations; PKC; protein kinase C; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; POPA; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphate; CL; bovine heart cardiolipin; POPE; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphoethanolamine; ATR-IR; attenuated total internal reflection infrared spectroscopy; DHPE; N-(5-dimethylaminonaphthalene-1-sulfonyl)-1,2-dihexadecanoyl-; sn; -glycero-3-phosphoethanolamineATR-IR; C2 domains; PKCε; Membrane docking

A lipid-dependent link between activity and oligomerization state of the M. tuberculosis SMR protein TBsmr by Mors Karsten Mörs; Ute A. Hellmich; Daniel Basting; Philipp Marchand; Jan Philip Wurm; Winfried Haase; Clemens Glaubitz (pp. 561-567).

TBsmr is a secondary active multidrug transporter from Mycobacterium tuberculosis that transports a plethora of compounds including antibiotics and fluorescent dyes. It belongs to the small multidrug resistance (SMR) superfamily and is structurally and functionally related to E. coli EmrE. Of particular importance is the link between protein function, oligomeric state and lipid composition. By freeze fracture EM, we found three different size distributions in three different lipid environments for TBsmr indicating different oligomeric states. The link of these states with protein activity has been probed by fluorescence spectroscopy revealing significant differences. The drug binding site has been probed further by¹⁹F-MAS NMR through chemical labeling of native cysteine residues showing a water accessible environment in agreement with the alternating access model.Display Omitted► The link between lipid-induced oligomerization and function of the SMR protein TBsmr is investigated. ► Lipid bilayer bulk properties determine TBsmr transport activity and oligomerization. ►¹⁹F Solid State NMR data indicate a water-filled pore for the binding pocket.

Keywords: SMR transporter; EmrE; Lipid; Freeze fracture EM; NMR; Fluorescence

Fluorone dyes have binding sites on both cytoplasmic and extracellular domains of Na,K-ATPase by Havlikova Marika Havlíková; Huliciak Miroslav Huličiak; Václav Bazgier; Karel Berka; Martin Kubala (pp. 568-576).

Combination of fluorescence techniques and molecular docking was used to monitor interaction of Na,K-ATPase and its large cytoplasmic loop connecting fourth and fifth transmembrane helices (C45) with fluorone dyes (i.e. eosin Y, 5(6)-carboxyeosin, rose bengal, fluorescein, and erythrosine B). Our data suggested that there are at least two binding sites for all used fluorone dyes, except of 5(6)-carboxyeosin. The first binding site is located on C45 loop, and it is sensitive to the presence of nucleotide. The other site is located on the extracellular part of the enzyme, and it is sensitive to the presence of Na⁺ or K⁺ ions. The molecular docking revealed that in the open conformation of C45 loop (which is obtained in the presence of ATP) all used fluorone dyes occupy position directly inside the ATP-binding pocket, while in the closed conformation (i.e. in the absence of any ligand) they are located only near the ATP-binding site depending on their different sizes. On the extracellular part of the protein, the molecular docking predicts two possible binding sites with similar binding energy near Asp897(α) or Gln69(β). The former was identified as a part of interaction site between α- and β-subunits, the latter is in contact with conserved FXYD sequence of the γ-subunit. Our findings provide structural explanation for numerous older studies, which were performed with fluorone dyes before the high-resolution structures were known. Further, fluorone dyes seem to be good probes for monitoring of intersubunit interactions influenced by Na⁺ and K⁺ binding.Display Omitted► Monitoring of Na,K-ATPase interactions with fluorone dyes by fluorescence methods ► Two binding sites: cytoplasmic ATP-binding pocket and extracellular part of enzyme ► Fluorone dyes are good probes for monitoring of intersubunit interactions. ► 5(6)-Carboxyeosin is the best probe for examination of the ATP-binding site. ► Fluorescein is the most sensitive probe for monitoring E1/E2 conformational change.

Keywords: Abbreviations; NKA; Na,K-ATPase, sodium pump; SERCA; sarco(endo)plasmic reticulum; C23; cytoplasmic segment connecting the second and third transmembrane helices, other cytoplasmic segments analogously; ATP; adenosine 5′-triphosphate; RB; Rose Bengal; ErB; Erythrosine B; EY; Eosin Y; CE; 5(6)-carboxyeosin; Fl; Fluorescein; PMSF; Phenylmethylsulfonyl Fluoride; IPTG; isopropyl β-; d; -thiogalactoside; DTT; dithiothreitol; SDS-PAGE; sodium dodecyl sulfate polyacrylamide gel electrophoresis; TCSPC; time-correlated single photon counting method; IRF; instrument response function; K; _SV; Stern–Volmer quenching constant; K; _D; dissociation constants; FITC; fluorescein isothiocyanateNa,K-ATPase; C45 loop; Binding sites; Fluorone dyes; Time-resolved fluorescence; Fluorescence quenching

Role of conformational sampling of Ser16 and Thr17-phosphorylated phospholamban in interactions with SERCA by Maryam Sayadi; Michael Feig (pp. 577-585).

Phosphorylation of phospholamban (PLB) at Ser16 and/ or Thr17 is believed to release its inhibitory effect on sarcoplasmic reticulum calcium ATPase. Ser16 phosphorylation of PLB has been suggested to cause a conformational change that alters the interaction between the enzyme and protein. Using computer simulations, the conformational sampling of Ser16 phosphorylated PLB in implicit membrane environment is compared here with the unphosphorylated PLB system to investigate these conformational changes. The results suggest that conformational changes in the cytoplasmic domain of PLB upon phosphorylation at Ser16 increase the likelihood of unfavorable interactions with SERCA in the E2 state prompting a conformational switch of SERCA from E2 to E1. Phosphorylation of PLB at Thr17 on the other hand does not appear to affect interactions with SERCA significantly suggesting that the mechanism of releasing the inhibitory effect is different between Thr17 phosphorylated and Ser16 phosphorylated PLB.Display Omitted► Implicit membrane simulations of phosphorylated phospholamban ► Ser16 and Ser17 phosphorylation leads to significant conformational changes. ► Ser16 phosphorylation appears to be incompatible with binding to SERCA E2 state. ► Thr17 phosphorylation may lead to complete PLB dissociation.

Keywords: SERCA; Calcium pump; Implicit membrane; Heterogeneous dielectric; Molecular dynamics; Replica exchange

Aurein 2.3 functionality is supported by oblique orientated α-helical formation by Manuela Mura; Sarah R. Dennison; Andrei V. Zvelindovsky; David A. Phoenix (pp. 586-594).

In this study, an amphibian antimicrobial peptide, aurein 2.3, was predicted to use oblique orientated α-helix formation in its mechanism of membrane destabilisation. Molecular dynamic (MD) simulations and circular dichroism (CD) experimental data suggested that aurein 2.3 exists in solution as unstructured monomers and folds to form predominantly α-helical structures in the presence of a dimyristoylphosphatidylcholine membrane. MD showed that the peptide was highly surface active, which supported monolayer data where the peptide induced surface pressure changes>34mNm−¹. In the presence of a lipid membrane MD simulations suggested that under hydrophobic mismatch the peptide is seen to insert via oblique orientation with a phenylalanine residue (PHE3) playing a key role in the membrane interaction. There is evidence of snorkelling leucine residues leading to further membrane disruption and supporting the high level of lysis observed using calcein release assays (76%). Simulations performed at higher peptide/lipid ratio show peptide cooperativity is key to increased efficiency leading to pore-formation.Display Omitted► MD simulations were used to study interaction of aurein 2.3 and DMPC lipid bilayer. ► Under hydrophobic mismatch aurein 2.3 inserts via oblique orientation. ► Phenylalanine residue plays a key role in the membrane interaction of aurein 2.3.

Keywords: Abbreviations; α-AMPs; α-helical antimicrobial peptide; CD; circular dichroism; DMPC; dimyristoylphosphatidylcholine; HEPES; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; EDTA; ethylenediaminetetraacetic acid; MD; molecular dynamic; NMR; nuclear magnetic resonance; PBS; phosphate-buffered saline; SAR; structure–activity relationship; SPC; simple point charge; RMSF; root-mean-square fluctuationsAntimicrobial peptide; Langmuir monolayers; Oblique orientated α-helix; Molecular dynamics; Hydrophobic mismatch; Cooperative effects

Insight into the modulation of Shaw2 Kv channels by general anesthetics: Structural and functional studies of S4–S5 linker and S6 C-terminal peptides in micelles by NMR by Jin Zhang; Xiaoguang Qu; Manuel Covarrubias; Markus W. Germann (pp. 595-601).

The modulation of the Drosophila Shaw2 Kv channel by 1-alkanols and inhaled anesthetics is correlated with the involvement of the S4–S5 linker and C-terminus of S6, and consistent with stabilization of the channel's closed state. Structural analysis of peptides from S4–S5 (L45) and S6 (S6c), by nuclear magnetic resonance and circular dichroism spectroscopy supports that an α-helical conformation was adopted by L45, while S6c was only in an unstable/dynamic partially folded α-helix in dodecylphosphocholine micelles. Solvent accessibility and paramagnetic probing of L45 revealed that L45 lies parallel to the surface of micelles with charged and polar residues pointing towards the solution while hydrophobic residues are buried inside the micelles. Chemical shift perturbation introduced by 1-butanol on residues Gln320, Thr321, Phe322 and Arg323 of L45, as well as Thr423 and Gln424 of S6c indicates possible anesthetic binding sites on these two important components in the channel activation apparatus. Diffusion measurements confirmed the association of L45, S6c and 1-butanol with micelles which suggests the capability of 1-butanol to influence a possible interaction of L45 and S6c in the micelle environment.Display Omitted► We investigated the S4–S5 linker (L45) in the 1-alkanol modulation of Shaw2 K⁺ channels. ► NMR and CD support that an α-helical conformation was adopted by L45 in DPC micelles. ► L45 lies parallel to the surface of micelles with charged and polar residues solvent exposed. ► Several exposed residues are sensing the presence of low 1-butanol concentrations. ► The results shed light on the structure and interaction of key components of the Shaw2 Kv channel.

Keywords: Abbreviations; L45; peptide derived from the S4–S5 linker of Shaw2 Kv; S6c; peptide derived from the S6 C-terminus of Shaw2 Kv; VSD; voltage sensing domain; PRE; paramagnetic relaxation enhancement; A; _i; paramagnetic attenuation; DPC; dodecylphosphocholine; Gd-DTPA-BMA; gadolinium-diethylenetriaminepentaacetic acid bismethylamide; TFE; 2,2,2-trifluoroethanol; DMPC; 1,2-dimyristoyl-; sn; -glycero-3-phosphocholine; DHPC; 1,2-dihexanoyl-; sn; -glycero-3-phosphocholine; TTAB; tetradecyltrimethylammonium bromide; TOCSY; total correlation spectroscopy; NOESY; nuclear Overhauser effect spectroscopy; HSQC; heteronuclear single-quantum correlation spectroscopy; DOSY; diffusion-ordered spectroscopyNMR; Membrane peptide structure; Micelle; Potassium channel; DOSY

Myogenic differentiation and lipid-raft composition of L6 skeletal muscle cells are modulated by PUFAs by Anne Briolay; Rami Jaafar; Georges Nemoz; Laurence Bessueille (pp. 602-613).

Lipid composition and fatty acid analysis of the major classes of membrane phospholipids were determined during myogenic differentiation of L6 skeletal muscle cells. The cholesterol to glycerophospholipids ratio decreased during differentiation, both in total (TM) and detergent-resistant membranes (DRM). Analyses of the membrane lipids showed that differentiation had a major impact on the molecular composition of glycerophospholipids. A significant decrease in the concentration of saturated fatty acids was detected in glycerophospholipid classes, and to a lesser extent in sphingolipids, while the concentration of 16:1n-7, 18:1n-7 and 18:1n-9 increased. At the same time, the concentration of long polyunsaturated fatty acid chains decreased in TM and DRM glycerophospholipids, resulting in a lower saturated to unsaturated fatty acid ratio in myotubes as compared to myoblasts. Interestingly, the observed n-3/n-6 ratio was lower in differentiated cell membranes. PUFA supplementation of L6 cells led to an increase in myogenic differentiation correlated to an incorporation of added PUFAs in TM and DRM glycerophospholipids. As expected after n-3 PUFA supplementation, the n-3/n-6 ratio was clearly increased in TM and, surprisingly, this was also the case in isolated DRM. n-3 and n-6 PUFAs significantly and time-dependently increased the phosphorylation of kinase p70S6K1 during myogenic differentiation, revealing the activation of the upstream kinase mTORC1, a major regulator of cell cycle and protein translation. In contrast, PUFAs did not affect the phosphorylation of the kinase Akt, another pivotal regulator of cell metabolism. These results suggest that PUFA supplementation modified the membrane lipid composition and affected the differentiation of L6 cells.► Membrane and DRM of L6 cells modify their lipid composition during differentiation ► PUFAs activate myogenic differentiation by altering lipid and protein compositions ► n-3 or n-6 PUFAs promote an increase of the phosphorylation of p70S6K ► We report that the myogenic differentiation is linked to membrane structural changes

Keywords: Cell membrane lipid; Fatty acid; Membrane microdomains; Myogenic differentiation; p70S6K

A²H solid-state NMR study of the effect of antimicrobial agents on intact Escherichia coli without mutating by Catherine Tardy-Laporte; Alexandre A. Arnold; Bertrand Genard; Romain Gastineau; Morancais Michèle Morançais; Jean-Luc Mouget; Réjean Tremblay; Isabelle Marcotte (pp. 614-622).

Solid-state nuclear magnetic resonance (NMR) is a useful tool to probe the organization and dynamics of phospholipids in bilayers. The interactions of molecules with membranes are usually studied with model systems; however, the complex composition of biological membranes motivates such investigations on intact cells. We have thus developed a protocol to deuterate membrane phospholipids in Escherichia coli without mutating to facilitate²H solid-state NMR studies on intact bacteria. By exploiting the natural lipid biosynthesis pathway and using perdeuterated palmitic acid, our results show that 76% deuteration of the phospholipid fatty acid chains was attained. To verify the responsiveness of these membrane-deuterated E. coli, the effect of known antimicrobial agents was studied.²H solid-state NMR spectra combined to spectral moment analysis support the insertion of the antibiotic polymyxin B lipid tail in the bacterial membrane. The use of membrane-deuterated bacteria was shown to be important in cases where antibiotic action of molecules relies on the interaction with lipopolysaccharides. This is the case of fullerenol nanoparticles which showed a different effect on intact cells when compared to dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylglycerol membranes. Our results also suggest that membrane rigidification could play a role in the biocide activity of the detergent cetyltrimethyammonium chloride. Finally, the deuterated E. coli were used to verify the potential antibacterial effect of a marennine-like pigment produced by marine microalgae. We were able to detect a different perturbation of the bacteria membranes by intra- and extracellular forms of the pigment, thus providing valuable information on their action mechanism and suggesting structural differences.Display Omitted► A protocol was developed to deuterate membrane phospholipids in E. coli without mutating. ► This protocol facilitates²H solid-state NMR studies on intact bacteria. ► The action mechanism of PxB, fullerenol and CTAC on intact bacteria is studied. ► A different action mechanism is shown for two forms of a microalgal bioactive pigment.

Keywords: Abbreviations; ACP; acyl carrier protein; AMP; antimicrobial peptide; CL; cardiolipin; CTAC; cetyltrimethylammonium chloride; DMPC; dimyristoylphosphatidylcholine; DMPG; dimyristoylphosphatidylglycerol; DMSO; dimethyl sulfoxide; DPC; dodecyl phosphocholine; DOPC; dioleoylphosphatidylcholine; DPPC; dipalmitoylphosphatidylcholine; DPPG; dipalmitoylphosphatidylglycerol; ExP; extracellular form of the marennine-like pigment; InP; intracellular form of the marennine-like pigment; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; FA; fatty acid; FAME; fatty acid methyl ester; LPS; lipopolysaccharide; MeOH; methanol; PA-d; ₃₁; perdeuterated palmitic acid; PC; phosphatidylcholine; PE; phosphatidylethanolamine; PG; phosphatidylglycerol; PxB; polymyxin B; QAC; quaternary ammonium compound; SS-NMR; solid-state nuclear magnetic resonanceMembrane interaction; Polymyxin B; Polyhydroxylated fullerene nanoparticle; Cetyltrimethylammonium chloride; Marennine-like pigment; Nuclear magnetic resonance

Vhc1, a novel transporter belonging to the family of electroneutral cation–Cl⁻ cotransporters, participates in the regulation of cation content and morphology of Saccharomyces cerevisiae vacuoles by Silvia Petrezselyova; Olga Kinclova-Zimmermannova; Hana Sychrova (pp. 623-631).

Cation–Cl⁻ cotransporters (CCCs) are integral membrane proteins which catalyze the coordinated symport of Cl⁻ with Na⁺ and/or K⁺ ions in plant and mammalian cells. Here we describe the first Saccharomyces cerevisiae CCC protein, encoded by the YBR235w open reading frame. Subcellular localization studies showed that this yeast CCC is targeted to the vacuolar membrane. Deletion of the YBR235w gene in a salt-sensitive strain (lacking the plasma-membrane cation exporters) resulted in an increased sensitivity to high KCl, altered vacuolar morphology control and decreased survival upon hyperosmotic shock. In addition, deletion of the YBR235w gene in a mutant strain deficient in K⁺ uptake produced a significant growth advantage over the parental strain under K⁺-limiting conditions, and a hypersensitivity to the exogenous K⁺/H⁺ exchanger nigericin. These results strongly suggest that we have identified a novel yeast vacuolar ion transporter mediating a K⁺–Cl⁻ cotransport and playing a role in vacuolar osmoregulation. Considering its identified function, we propose to refer to the yeast YBR235w gene as VHC1 (vacuolarprotein homologous toCCC family 1).Display Omitted► We describe the first yeast CCC transporter. ► Vhc1 participates in yeast osmotolerance. ► Vhc1 absence affects morphology of vacuoles. ► Vhc1 plays a role in yeast potassium homeostasis.

Keywords: Salt tolerance; Yeast vacuole; Potassium homeostasis; Cation–chloride cotransport

Porter domain opening and closing motions in the multi-drug efflux transporter AcrB by Nadine Fischer; Christian Kandt (pp. 632-641).

Acriflavine resistance protein B acts as the active transporter in the multi-drug efflux pump Acriflavine resistance proteins A / B - Tolerance to colicins protein in Escherichia coli. Within the same reaction cycle intermediate all Acriflavine resistance protein B X-ray structures display highly similar conformations of the substrate-recruiting and transporting porter domain. To assess if this structural homogeneity is an intrinsic feature of Acriflavine resistance protein B or stems from other causes we performed a series of six independent, unbiased 100ns molecular dynamics simulations of membrane-embedded, asymmetric, substrate-free wild type Acriflavine resistance protein B in a 150mM NaCl solution. We find the porter domain more flexible than previously assumed displaying clear opening and closing motions of the proximal binding pocket (L and T-state) and the exit of the drug transport channels (O-intermediate). Concurrently the hydrophobic binding pocket favors a closed conformation in all three protomers. Our findings suggest that the conformational homogeneity seen in the crystal structures is likely an effect of bound but structurally unresolved substrate. Our simulations further imply that each of the known three reaction cycle intermediates occurs in at least two variants, the Thr676 loop independently regulates porter domain access and likely plays a key role in substrate transport. On a 100ns time scale we find no evidence supporting the proposed LLL resting state in the absence of substrate. If the proximal binding pocket dynamics have an inhibiting effect on Acriflavine resistance protein B pump activity lowering the life time of substrate-accessible conformations, the observed dynamics could provide a structural explanation for the Acriflavine resistance protein B activity-enhancing effect of the adaptor protein Acriflavine resistance protein A stabilizing PC1 and PC2 subdomain orientations.Display Omitted► Proximal binding pocket opens and closes in L and T but remains closed in O. ► Hydrophobic binding pocket predominantly closed in all protomers. ► Porter domain exit opens and closes in O but remains closed in L and T. ► Thr676 loop independently regulates substrate accessibility and is likely involved in transport. ► AcrB X-ray structures are likely stabilized by bound but unresolved substrate.

Keywords: Molecular dynamics simulation; Membrane protein; Antibiotics resistance; Proximal binding pocket; Hydrophobic binding pocket; Reaction cycle

Enzyme-catalyzed hydrolysis of the supported phospholipid bilayers studied by atomic force microscopy by HengLiang Wu; Le Yu; Yujin Tong; Aimin Ge; Shuehlin Yau; Masatoshi Osawa; Shen Ye (pp. 642-651).

Atomic force microscopy (AFM) is employed to reveal the morphological changes of the supported phospholipid bilayers hydrolyzed by a phospholipase A₂ (PLA₂) enzyme in a buffer solution at room temperature. Based on the high catalytic selectivity of PLA₂ towardl-enantiomer phospholipids, five kinds of supported bilayers made ofl- andd-dipalmitoylphosphatidylcholines (DPPC), includingl-DPPC (upper leaflet adjacent to solution)/l-DPPC (bottom leaflet) (orl/l in short),l/d,d/l,d/d, and racemicld/ld, were prepared on a mica surface in gel-phase, to explicate the kinetics and mechanism of the enzyme-induced hydrolysis reaction in detail. AFM observations for thel/l bilayer show that the hydrolysis rate forl-DPPC is significantly increased by PLA₂ and most of the hydrolysis products desorb from substrate surface in 40min. Asd-enantiomers are included in the bilayer, the hydrolysis rate is largely decreased in comparison with thel/l bilayer. The time used to hydrolyze the as-prepared bilayers by PLA₂ increases in the sequence ofl/l,l/d,ld/ld, andd/l (d/d is inert to the enzyme action).d-enantiomers in the enantiomer hybrid bilayers remain on the mica surface at the end of the hydrolysis reaction. It was confirmed that the hydrolysis reaction catalyzed by PLA₂ preferentially occurs at the edges of pits or defects on the bilayer surface. The bilayer structures are preserved during the hydrolysis process. Based on these observations, a novel kinetics model is proposed to quantitatively account for the PLA₂-catalyzed hydrolysis of the supported phospholipid bilayers. The model simulation demonstrates that PLA₂ mainly binds with lipids at the perimeter of defects in the upper leaflet and leads to a hydrolysis reaction, yielding species soluble to the solution phase. The lipid molecules underneath subsequently flip up to the upper leaflet to maintain the hydrophilicity of the bilayer structure. Our analysis shows thatd-enantiomers in the hybrid bilayers considerably reduce the hydrolysis rate by its ineffective binding with PLA₂.Display Omitted► Hydrolysis of a DPPC bilayer catalyzed by PLA₂ enzyme is studied by AFM and simulation. ► Hydrolysis products are desorbed while lipid bilayer structures are preserved during the reaction. ► Lipid enantiomers are employed to analyze kinetics and mechanism for the PLA2-catalyzed hydrolysis reaction. ► Enzyme-induced flip-up process plays important roles in the hydrolysis reaction.

Keywords: Phospholipase A; ₂; (PLA; ₂; ); Enzyme reaction; Supported lipid bilayer; Cell membrane; Kinetics and mechanism; Atomic force microscopy (AFM)

The effect ofd-amino acid substitution on the selectivity of temporin L towards target cells: Identification of a potent anti- Candida peptide by Paolo Grieco; Alfonso Carotenuto; Luigia Auriemma; Maria Rosaria Saviello; Pietro Campiglia; Isabel M. Gomez-Monterrey; Ludovica Marcellini; Vincenzo Luca; Donatella Barra; Ettore Novellino; Maria Luisa Mangoni (pp. 652-660).

The frog skin peptide temporin L (TL, 13-residues long) has a wide and potent spectrum of antimicrobial activity, but it is also toxic on mammalian cells at its microbicidal concentrations. Previous studies have indicated that its analogue [Pro³]TL has a slightly reduced hemolytic activity and a stable helical conformation along residues 6–13. Here, to expand our knowledge on the relationship between the extent/position of α-helix in TL and its biological activities, we systematically replaced single amino acids within the α-helical domain of [Pro³]TL with the correspondingd isomers, known as helix breakers. Structure–activity relationship studies of these analogues, by means of CD and NMR spectroscopy analyses as well as antimicrobial and hemolytic assays were performed. Besides increasing our understanding on the structural elements that are responsible for cell selectivity of TL, this study revealed that a singlel tod amino acid substitution can preserve strong anti- Candida activity of [Pro³]TL, without giving a toxic effect towards human cells.Display Omitted► Replacement of single residues with D amino acids. ► Structure–activity relationship studies for the target cell selectivity of temporin L. ► Alpha-helix disruption at both N and C-terminal ends abolishes cytolytic activity. ► Anti-yeast activity is not dependent on peptide's helicity. ► Discovery of a TL analogue endowed with potent anti- Candida activity and devoid of cytotoxic effects.

Keywords: Abbreviations; 1D, and 2D; one and two-dimensional; CD; circular dichroism; Cho; cholesterol; CL; cardiolipin; DCM; dichloromethane; DMEM; Dulbecco's modified Eagle's medium; DIEA; diisopropyl ethylamine; DMF; N; ,; N; -dimethylformamide; DPC; dodecylphosphocholine; DQF-COSY; double quantum filtered correlated spectroscopy; EDTA; ethylenediaminetetraacetic acid; Et; ₃; SiH; triethylsilane; Fmoc; 9-fluorenyl-methoxycarbonyl; HBTU; 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-phosphate; HOBt; N; -hydroxy-benzotriazole; LUV; large unilamellar vesicles; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NMR; nuclear magnetic resonance; NOE; nuclear Overhauser effect; NOESY; nuclear Overhauser enhancement spectroscopy; Pbf; 2,2,4,6,7-pentamethyldihydrobenzo-furan-5-sulfonyl; POPC; 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; POPE; 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; POPG; 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol; RP-HPLC; reversed-phase high performance liquid chromatography; SAR; structure activity relationship; SDS; sodium dodecylsulphate; TL; temporin TL; TOCSY; total correlation spectroscopyAntimicrobial peptide; Temporin L; d; -Amino acid; Peptide–membrane interaction; Drug-resistance; NMR spectroscopy

Thermotropic and structural effects of poly(malic acid) on fully hydrated multilamellar DPPC–water systems by Berenyi Szilvia Berényi; Mihaly Judith Mihály; Kristyan Sándor Kristyán; Lívia Naszályi Nagy; Judit Telegdi; Bota Attila Bóta (pp. 661-669).

The thermotropic and structural effects of low molecular weight poly(malic acid) (PMLA) on fully hydrated multilamellar dipalmitoylphosphatidylcholine (DPPC)–water systems were investigated using differential scanning calorimetry (DSC), small-angle X-ray scattering (SAXS), and freeze-fracture transmission electron microscopy (FFTEM). Systems of 20wt% DPPC concentration and 1 and 5wt% PMLA to lipid ratios were studied. The PMLA derivatives changed the thermal behavior of DPPC significantly and caused a drastic loss in correlation between lamellae in the three characteristic thermotropic states (i.e., in the gel, rippled gel and liquid crystalline phases). In the presence of PBS or NaCl, the perturbation was more moderate. The structural behavior on the atomic level was revealed by FTIR spectroscopy. The molecular interactions between DPPC and PMLA were simulated via modeling its measured infrared spectra, and their peculiar spectral features were interpreted. Through this interpretation, the poly(malic acid) is inferred to attach to the headgroups of the phospholipids through hydrogen bonds between the free hydroxil groups of PMLA and the phosphodiester groups of DPPC.Display Omitted► Multilamellar DPPC liposomes were studied in the presence of poly(malic acid). ► The structural effect of the polymer was characterized by DSC, SAXS and FF-TEM. ► PMLA perturbs the ordered structure of the multilamellar lipid system. ► The molecular interactions were examined by FTIR and MD simulation. ► PMLA attaches to the headgroups of the lipids through hydrogen bonds.

Keywords: Poly(malic-acid); DPPC; SAXS; FTIR; DSC; Model membrane

Role of membrane oxidation in controlling the activity of human group IIa secretory phospholipase A₂ toward apoptotic lymphoma cells by Elizabeth Gibbons; Jennifer Nelson; Lynn Anderson; Kelly Brewer; Stephanie Melchor; Allan M. Judd; John D. Bell (pp. 670-676).

The membranes of healthy lymphocytes normally resist hydrolysis by secretory phospholipase A₂. However, they become susceptible during the process of apoptosis. Previous experiments have demonstrated the importance of certain physical changes to the membrane during cell death such as a reduction in membrane lipid order and exposure of phosphatidylserine on the membrane surface. Nevertheless, those investigations also showed that at least one additional factor was required for rapid hydrolysis by the human group IIa phospholipase isozyme. This study was designed to test the possibility that oxidation of membrane lipids is the additional factor. Flow cytometry and confocal microscopy with a fluorescent probe of oxidative potential suggested that oxidation of the plasma membrane occurs during apoptosis stimulated by thapsigargin. When oxidative potential was high, the activity of human group IIa secretory phospholipase A₂ was enhanced 30- to 100-fold compared to that observed with conditions sufficient for maximal hydrolysis by other secretory phospholipase A₂ isoforms. Direct oxidation of cell membranes with either of two oxidizing agents also stimulated hydrolysis by secretory phospholipase A₂. Both oxidizers caused externalization of phosphatidylserine, but a change in lipid order did not always occur. These results demonstrated that membrane oxidation strongly stimulates human group IIa secretory phospholipase A₂ activity toward apoptotic cells. Interestingly, the change in membrane order, previously thought to be imperative for high rates of hydrolysis, was not required when membrane lipids were oxidized. Whether phosphatidylserine exposure is still necessary with oxidation remains unresolved since the two events could not be deconvoluted.Display Omitted► Oxidation of membrane lipids during apoptosis stimulates group IIa sPLA₂ activity. ► Oxidation replaces the need for reduction in membrane order to stimulate hydrolysis. ► For group IIa sPLA₂, oxidation is more effective than other membrane changes. ► Oxidation helps explain sPLA₂ isoform specificity toward different cell death modes.

Keywords: Phospholipase A; ₂; specificity; Merocyanine 540; Hydrolysis kinetics; Patman; Trimethylammonium diphenylhexatriene; C11-BODIPY

A novel antimicrobial peptide derived from modified N-terminal domain of bovine lactoferrin: Design, synthesis, activity against multidrug-resistant bacteria and Candida by Biswajit Mishra; Geeta Devi Leishangthem; Kamaldeep Gill; Abhay K. Singh; Swagata Das; Kusum Singh; Immaculata Xess; Amit Dinda; Arti Kapil; Ishan K. Patro; Sharmistha Dey (pp. 677-686).

Lactoferrin (LF) is believed to contribute to the host's defense against microbial infections. This work focuses on the antibacterial and antifungal activities of a designed peptide, L10 (WFRKQLKW) by modifying the first eight N-terminal residues of bovine LF by selective homologous substitution of amino acids on the basis of hydrophobicity, L10 has shown potent antibacterial and antifungal properties against clinically isolated extended spectrum beta lactamases (ESBL), producing gram-negative bacteria as well as Candida strains with minimal inhibitory concentrations (MIC) ranging from 1 to 8μg/mL and 6.5μg/mL, respectively. The peptide was found to be least hemolytic at a concentration of 800μg/mL. Interaction with lipopolysaccharide (LPS) and lipid A (LA) suggests that the peptide targets the membrane of gram-negative bacteria. The membrane interactive nature of the peptide, both antibacterial and antifungal, was further confirmed by visual observations employing electron microscopy. Further analyses, by means of propidium iodide based flow cytometry, also supported the membrane permeabilization of Candida cells. The peptide was also found to possess anti-inflammatory properties, by virtue of its ability to inhibit cyclooxygenase-2 (COX-2). L10 therefore emerges as a potential therapeutic remedial solution for infections caused by ESBL positive, gram-negative bacteria and multidrug-resistant (MDR) fungal strains, on account of its multifunctional activities. This study may open up new approach to develop and design novel antimicrobials.Antimicrobial activity of a novel peptide by modifying N-terminal bovine lactoferrin against multidrug resistant gram-negative bacteria and fungi.Display Omitted► The peptide, L10 is a modified N-terminal of bovine lactoferrin. ► L10 inhibits the growth of gram-negative bacteria and C. albicans and C. parapsilosis. ► L10 acts via the membrane permeabilization. ► L10 inhibited endotoxin procoagulant activity. ► L10 binds with LA and exhibits anti-inflammatory activity by inhibiting COX-2.

Keywords: Antimicrobial peptide; Lactoferrin; Extended spectrum beta lactamase; Anti-inflammatory; Surface plasmon resonance; Cyclooxygenase

Membrane reconstitution of FtsZ–ZipA complex inside giant spherical vesicles made of E. coli lipids: Large membrane dilation and analysis of membrane plasticity by Lopez-Montero I. López-Montero; Lopez-Navajas P. López-Navajas; J. Mingorance; Velez M. Vélez; M. Vicente; F. Monroy (pp. 687-698).

During the division process of Escherichia coli, the globular protein FtsZ is early recruited at the constriction site. The Z-ring, based on FtsZ filaments associated to the inner cell membrane, has been postulated to exert constriction forces. Membrane anchoring is mediated by ZipA, an essential transmembrane protein able to specifically bind FtsZ. In this work, an artificial complex of FtsZ–ZipA has been reconstituted at the inner side of spherical giant unilamellar vesicles made of E. coli lipids. Under these conditions, FtsZ polymerization, triggered when a caged GTP analogue is UV-irradiated, was followed by up to 40% vesicle inflation. The homogeneous membrane dilation was accompanied by the visualization of discrete FtsZ assemblies at the membrane. Complementary rheological data revealed enhanced elasticity under lateral dilation. This explains why vesicles can undergo large dilations in the regime of mechanical stability. A mechanical role for FtsZ polymers as promoters of membrane softening and plasticization is hypothesized.Display Omitted► Membrane reconstitution of FtsZ–ZipA complexes in giant unilamellar vesicles of E. coli lipids. ► FtsZ polymerization driven by remote UV-controlled ATP-release inside the vesicles. ► Vesicle inflation due to FtsZ polymerization with subsequent membrane dilation. ► Membrane anchoring of FtsZ-polymers through ZipA linker. ► Mechanical plasticization through FtsZ–ZipA membrane complexes.

Keywords: E. coli; Bacterial division; FtsZ; ZipA; Giant vesicles; Membrane plasticity

Identification of two-pore domain potassium channels as potent modulators of osmotic volume regulation in human T lymphocytes by Joseph Andronic; Nicole Bobak; Stefan Bittner; Petra Ehling; Christoph Kleinschnitz; Alexander M. Herrmann; Heiko Zimmermann; Markus Sauer; Heinz Wiendl; Thomas Budde; Sven G. Meuth; Vladimir L. Sukhorukov (pp. 699-707).

Many functions of T lymphocytes are closely related to cell volume homeostasis and regulation, which utilize a complex network of membrane channels for anions and cations. Among the various potassium channels, the voltage-gated K_V1.3 is well known to contribute greatly to the osmoregulation and particularly to the potassium release during the regulatory volume decrease (RVD) of T cells faced with hypotonic environment. Here we address a putative role of the newly identified two-pore domain (K_2P) channels in the RVD of human CD4⁺ T lymphocytes, using a series of potent well known channel blockers. In the present study, the pharmacological profiles of RVD inhibition revealed K_2P5.1 and K_2P18.1 as the most important K_2P channels involved in the RVD of both naïve and stimulated T cells. The impact of chemical inhibition of K_2P5.1 and K_2P18.1 on the RVD was comparable to that of K_V1.3. K_2P9.1 also notably contributed to the RVD of T cells but the extent of this contribution and its dependence on the activation status could not be unambiguously resolved. In summary, our data provide first evidence that the RVD-related potassium efflux from human T lymphocytes relies on K_2P channels.Display Omitted► We analyzed volume regulation properties in human T cells. ► RVD in human T cells is dependent on K_2P channel expression. ► Especially K_2P5.1 and K_2P18.1 have an impact on the process of RVD. ► There is no channel exclusively responsible for the process of volume regulation. ► RVD is an interplay between different channels with partly redundant effects.

Keywords: Cell volumetry; Regulatory volume decrease; Osmotic stress; T lymphocytes; Potassium channels; K; _2P; channels

Lipid-like behavior of signal sequence peptides at air–water interface by Ernesto E. Ambroggio; Gerardo D. Fidelio (pp. 708-714).

Several protein transport processes in the cell are mediated by signal sequence peptides located at the N-terminal side of the mature protein sequence. To date, the specific interaction and the stability of these peptides at the amphipathic interface of biological membranes and the relevance of the peptide conformation when they interact with lipids is not clear. We report the surface properties and the peptide–lipid interaction of three signal sequence peptides at the air–NaCl 145mM interface by using the Langmuir monolayer approach. These synthetic peptides have a natural sequence with a non-periodic amphiphilicity, where hydrophobic and hydrophilic residues are located on opposed sides of the peptide primary sequence. We show that signal sequence peptides form insoluble monolayers of high stability against lateral compression. At close packing, peptide molecular area, surface potential and the high stability of the peptide monolayer are indicative that signal sequence peptides are compatible with a β-sheet conformation at the interface. Structure was confirmed with PM-IRRAS and transmission FT-IR studies. The peptides show lateral miscibility with either POPC (a liquid-expanded lipid) or DPPC (a liquid-condensed lipid) in mixed peptide–lipid monolayers. This indicates that signal sequence peptides studied are laterally miscible with phospholipids independent of the phase state of the lipid.Display Omitted► The surface behavior of three hydrophobic signal sequence peptides is reported. ► These highly hydrophobic peptides mimic the surface behavior of lipids. ► A non-periodic amphiphilicity confers to peptide films high lateral stability. ► Surface and structural data show a beta-sheet structure at the air–water interface. ► Signal sequence peptides have lateral empathy with lipids regardless their physical state.

Keywords: Abbreviations; SSP; signal sequence peptide; ASS; human albumin signal sequence peptide; ESS; signal sequence peptide of rat elastase; PM-IRRAS; polarized-modulated infrared reflection-absorption spectroscopy; PSS; dog pretrypsinogen signal sequence peptide; RTP; chloroplast transit peptide of the Ribulose 1,5-bis-phosphate carboxylase-oxygenaseSignal sequence peptide; Peptide monolayer; Peptide–lipid interaction; PM-IRRAS; Lateral surface stability; Interfacial peptide structure

Stability and membrane interactions of an autotransport protein: MD simulations of the Hia translocator domain in a complex membrane environment by Daniel A. Holdbrook; Thomas J. Piggot; Mark S.P. Sansom; Syma Khalid (pp. 715-723).

Hia is a trimeric autotransporter found in the outer membrane of Haemphilus influenzae. The X-ray structure of Hia translocator domain revealed each monomer to consist of an α-helix connected via a loop to a 4-stranded β-sheet, thus the topology of the trimeric translocator domain is a 12-stranded β-barrel containing 3 α-helices that protrude from the mouth of the β-barrel into the extracellular medium. Molecular dynamics simulations of the Hia monomer and trimer have been employed to explore the interactions between the helices, β-barrel and connecting loops that may contribute to the stability of the trimer. In simulations of the Hia monomer we show that the central α-helix may stabilise the fold of the 4-stranded β-sheet. In simulations of the Hia trimer, a H-bond network involving residues in the β-barrel, α-helices and loops has been identified as providing stability for the trimeric arrangement of the monomers. Glutamine residues located in the loops connecting the α-helices to the β-barrel are orientated in a triangular arrangement such that each forms 2 hydrogen bonds to each of the corresponding glutamines in the other loops. In the absence of the loops, the β‐barrel becomes distorted. Simulations show that while the trimeric translocator domain β-barrel is inherently flexible, it is unlikely to accommodate the passenger domain in a folded conformation. Simulations of Hia in an asymmetric model of the outer membrane have revealed membrane–protein interactions that anchor the protein within its native membrane environment.Display Omitted► Hia monomers require the α-helix and α1-β1 loop to remain stable. ► β‐barrel shows substantial flexibility upon removal of the α-helices and α1-β1 loops. ► Specific interactions with the LPS may anchor the protein in the outer membrane.

Keywords: Autotransporter; Membrane protein; Molecular dynamics

Cysteine-rich toxins from Lachesana tarabaevi spider venom with amphiphilic C-terminal segments by Alexey I. Kuzmenkov; Irina M. Fedorova; Alexander A. Vassilevski; Eugene V. Grishin (pp. 724-731).

Venom of Lachesana tarabaevi (Zodariidae, “ant spiders”) exhibits high insect toxicity and serves a rich source of potential insecticides. Five new peptide toxins active against insects were isolated from the venom by means of liquid chromatography and named latartoxins (LtTx). Complete amino acid sequences of LtTx (60-71 residues) were established by a combination of Edman degradation, mass spectrometry and selective proteolysis. Three toxins have eight cysteine residues that form four intramolecular disulfide bridges, and two other molecules contain an additional cystine; three LtTx are C-terminally amidated. Latartoxins can be allocated to two groups with members similar to CSTX and LSTX toxins from Cupiennius salei (Ctenidae) and Lycosa singoriensis (Lycosidae). The interesting feature of the new toxins is their modular organization: they contain an N-terminal cysteine-rich (knottin or ICK) region as in many neurotoxins from spider venoms and a C-terminal linear part alike some cytolytic peptides. The C-terminal fragment of one of the most abundant toxins LtTx-1a was synthesized and shown to possess membrane-binding activity. It was found to assume amphipathic α-helical conformation in membrane-mimicking environment and exert antimicrobial activity at micromolar concentrations. The tails endow latartoxins with the ability to bind and damage membranes; LtTx show cytolytic activity in fly larvae neuromuscular preparations. We suggest a membrane-dependent mode of action for latartoxins with their C-terminal linear modules acting as anchoring devices.Display Omitted► LtTx are modular toxins with a disulfide-rich core and C-terminal linear extensions. ► LtTx comprise 5 new insect toxins belonging to 2 novel families. ► LtTx precursors are conventional prepropeptides. ► The C-terminal amphiphilic fragment of LtTx shows membrane-binding activity. ► Modular design appears widespread in spider toxins.

Keywords: Abbreviations; AMPs; antimicrobial peptides; CITs; cyto-insectotoxins; DOPE; 1,2-dioleoyl-; sn; -glycero-3-phosphoethanolamine; DOPG; 1,2-dioleoyl-; sn; -glycero-3-phospho-; rac; -(1-glycerol); EPSC; excitatory postsynaptic current; EST; expressed sequence tag; GUVs; giant unilamellar vesicles; ICK; inhibitor cystine knot; LD; ₅₀; median lethal dose; LUVs; large unilamellar vesicles; MIC; minimal inhibitory concentration; PQM; processing quadruplet motif; TFA; trifluoroacetic acidNeurotoxin; Membrane-active peptide; Precursor; Amphipathic helix; Inhibitor cystine knot; Membrane anchor

Transmembrane pore formation by the carboxyl terminus of Bax protein by Pranav Garg; Kathleen N. Nemec; Annette R. Khaled; Suren A. Tatulian (pp. 732-742).

Bax is a cytosolic protein that responds to various apoptotic signals by binding to the outer mitochondrial membrane, resulting in membrane permeabilization, release of cytochrome c, and caspase-mediated cell death. Currently discussed mechanisms of membrane perforation include formation of hetero-oligomeric complexes of Bax with other pro-apoptotic proteins such as Bak, or membrane insertion of multiple hydrophobic helices of Bax, or formation of lipidic pores physically aided by mitochondrial membrane-inserted proteins. There is compelling evidence provided by our and other groups indicating that the C-terminal “helix 9” of Bax mediates membrane binding and pore formation, yet the mechanism of pore forming capability of Bax C-terminus remains unclear. Here we show that a 20-amino acid peptide corresponding to Bax C-terminus (VTIFVAGVLTASLTIWKKMG) and two mutants where the two lysines are replaced with glutamate or leucine have potent membrane pore forming activities in zwitterionic and anionic phospholipid membranes. Analysis of the kinetics of calcein release from lipid vesicles allows determination of rate constants of pore formation, peptide–peptide affinities within the membrane, the oligomeric state of transmembrane pores, and the importance of the lysine residues. These data provide insight into the molecular details of membrane pore formation by a Bax-derived peptide and open new opportunities for design of peptide-based cytotoxic agents.Display Omitted► The C-terminal peptide of Bax and two mutants form large pores in lipid membranes. ► Calcein release kinetics from vesicles indicate a biphasic process of pore formation. ► Rate constants of pore formation and peptide–peptide affinities are determined. ► Up to eight peptide molecules are involved in a pore structure.

Keywords: Abbreviations; BaxC-KK; C-terminal peptide of wild-type Bax protein: Ac-VTIFVAGVLTASLTIWKKMG-NH; ₂; BaxC-EE; C-terminal peptide of Bax protein with two lysines replaced by glutamates; BaxC-LL; C-terminal peptide of Bax protein with two lysines replaced by leucines; BCL-2; B cell lymphoma 2; DPC; dodecylphosphocholine; LUV; large unilamellar vesicle; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; POPG; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphoglycerolPeptide; Membrane pore; Kinetics; Calcein release; Oligomeric state

Cell-free protein synthesis of membrane (1,3)-β-d-glucan (curdlan) synthase: Co-translational insertion in liposomes and reconstitution in nanodiscs by Agalya Periasamy; Nadim Shadiac; Amritha Amalraj; Garajova Soňa Garajová; Yagnesh Nagarajan; Shane Waters; Haydyn D.T. Mertens; Maria Hrmova (pp. 743-757).

A membrane-embedded curdlan synthase (CrdS) from Agrobacterium is believed to catalyse a repetitive addition of glucosyl residues from UDP-glucose to produce the (1,3)-β-d-glucan (curdlan) polymer. We report wheat germ cell-free protein synthesis (WG-CFPS) of full-length CrdS containing a 6xHis affinity tag and either Factor Xa or Tobacco Etch Virus proteolytic sites, using a variety of hydrophobic membrane-mimicking environments. Full-length CrdS was synthesised with no variations in primary structure, following analysis of tryptic fragments by MALDI-TOF/TOF Mass Spectrometry. Preparative scale WG-CFPS in dialysis mode with Brij-58 yielded CrdS in mg/ml quantities. Analysis of structural and functional properties of CrdS during protein synthesis showed that CrdS was co-translationally inserted in DMPC liposomes during WG-CFPS, and these liposomes could be purified in a single step by density gradient floatation. Incorporated CrdS exhibited a random orientation topology. Following affinity purification of CrdS, the protein was reconstituted in nanodiscs with Escherichia coli lipids or POPC and a membrane scaffold protein MSP1E3D1. CrdS nanodiscs were characterised by small-angle X-ray scattering using synchrotron radiation and the data obtained were consistent with insertion of CrdS into bilayers. We found CrdS synthesised in the presence of the Ac-AAAAAAD surfactant peptide or co-translationally inserted in liposomes made from E. coli lipids to be catalytically competent. Conversely, CrdS synthesised with only Brij-58 was inactive. Our findings pave the way for future structural studies of this industrially important catalytic membrane protein.Display Omitted► Wheat germ cell-free protein synthesis affords a full-length curdlan synthase. ► Up to mg/ml yield of curdlan synthase was obtained in a dialysis mode. ► Active curdlan synthase was made with surfactant peptides or inserted in liposomes. ► Nanodiscs with curdlan synthase were characterised by small-angle X-ray scattering. ► Nanodiscs have a maximum at ~0.1Å that corresponds to a phospholipid bilayer.

Keywords: Abbreviations; β-OG; n; -octyl-β-; d; -glucopyranoside; Brij-58; polyoxyethylene-(20)-cetyl-ether (C16⁄20); CAZy; Carbohydrate-Active enZymes; CF; cell-free; CFPS; cell-free protein synthesis; CHAPS; 3-[(3-cholamidopropyl)dimethylammonio]-1-propansulfonate; CM; catalytic module; CMC; critical micellar concentration; CrdS; curdlan synthase; DDM; n; -dodecyl-β-; d; -maltoside; DM; n; -decyl-β-; d; -maltoside; DMPC; 1,2-dimyristoyl-; sn; -glycero-3-phosphocholine; DOPC; 1,2-dioleoyl-; sn; -glycero-3-phosphocholine; DOPG; 1,2-dioleoyl-; sn; -glycero-3-phospho-(1′-; rac; -glycerol); DOTAP; 1,2-dioleoyl-3-trimethylammonium-propane; DTT; dithiothreitol; EC; Enzyme Commission; EDTA; ethylenediaminetetraacetic acid; GT; glycoside transferase(s); IMAC; Immobilized Metal Affinity Chromatography; IFT; indirect Fourier transformation; MALDI-TOF; Matrix-Assisted Laser Desorption Ionization-Time of Flight; MS; Mass Spectrometry (spectra); M; _r; relative molecular mass; MSP; membrane scaffold protein; NP 40; nonylphenyl-polyethylene-glycol; PDB; Protein Data Bank; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; POPE; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphoethanolamine; PVDF; polyvinylidene fluoride; SAXS; small-angle X-ray scattering; SDS-PAGE; sodium dodecyl sulphate-polyacrylamide gel electrophoresis; SEC; size-exclusion chromatography; SMA; styrene maleic acid; SUMO; small ubiquitin-related modifier; TBS; Tris buffer containing NaCl; Triton X-100; polyethylene-glycol-p-1,1,3,3-tetramethyl-butylphenyl-ether; Tween 80; polyoxyethylene-sorbitan-monolaurate 80; TEM; transmission electron microscopy; TEV; Tobacco Etch Virus; UDP-glucose; uridine-diphosphate glucose; WG-CFPS; wheat germ cell-free protein synthesis; YPD; yeast extract-peptone-dextrose; 3D; three-dimensionalFamily GT2 transferase; Small-angle X-ray scattering; Surfactant; Surfactant peptide; Topology; (1,3)-β-; d; -Glucan

The defensin–lipid interaction: Insights on the binding states of the human antimicrobial peptide HNP-1 to model bacterial membranes by Alessio Bonucci; Enrico Balducci; Sara Pistolesi; Rebecca Pogni (pp. 758-764).

Antimicrobial peptides are an important component of innate immunity and have generated considerable interest as a new potential class of natural antibiotics. The biological activity of antimicrobial peptides is strongly influenced by peptide–membrane interactions. Human Neutrophil Peptide 1 (HNP-1) is a 30 aminoacid peptide, belonging to the class of α-defensins. Many biophysical studies have been performed on this peptide to define its mechanism of action. Combining spectroscopic and thermodynamic analysis, insights on the interaction of the α-defensin with POPE:POPG:CL negative charged bilayers are given. The binding states of the peptide below and above the threshold concentration have been analyzed showing that the interaction with lipid bilayers is dependent by peptide concentration. These novel results that indicate how affinity and biological activities of natural antibiotics are depending by their concentration, might open new way of investigation of the antimicrobial mode of action.Display Omitted► HNP-1 antimicrobial peptide interacts with lipid bilayer. ► Model membranes mimicking composition of Gram-negative bacteria inner membrane ► The mode of peptide–membrane interaction is peptide concentration dependent. ► Peptide insertion at threshold concentration alters lipid packing and dynamics. ► Different binding modes of peptide below and above the threshold concentration have been highlighted.

Keywords: Abbreviations; AMP; antimicrobial peptide; HNP-1; human neutrophil peptide 1; EPR; Electron Paramagnetic Resonance; CD; Circular Dichroism; POPG; 1-palmitoyl-2-oleoyl-; sn; -glycerol-3-phosphatidylglycerol; POPE; 1-palmitoyl-2-oleoyl-; sn; -glycerol-3-phosphatidylethanolamine, CL, 1,1′,2,2′-tetramyristoyl cardiolipin ammonium salt; MOPS; 3-(N-morpholino) propanesulfonic acid; LUV; Large Unilamellar Vesicle; PCSL; phosphatidylcholine spin-labels; DMPC:DMPG; 1,2-dimyristoyl-; sn; -glycero-3-phosphocholine:1,2-dimyristoyl-; sn; -glycero-3-phosphatidylglycerolHNP-1; Antimicrobial peptide; Bacterial model membrane; Bilayer interaction; Spectroscopic analysis; Partition coefficient

Voltage-dependent inhibition of outward Kir2.1 currents by extracellular spermine by Hsueh-Kai Chang; Ru-Chi Shieh (pp. 765-775).

Outward currents through inward rectifier Kir2.1 channels play crucial roles in controlling the electrical properties of excitable cells. Extracellular monovalent and divalent cations have been shown to reduce outward K⁺ conductance. In the present study, we examined whether spermine, with four positive charges, also inhibits outward Kir2.1 currents. We found that extracellular spermine inhibits steady-state outward Kir2.1 currents, an effect that increases as the voltage becomes more depolarizing, similar to that observed for intracellular spermine. However, several lines of evidence suggest that extracellular spermine does not inhibit outward currents by entering the cytoplasmic pore. Site-directed mutagenesis studies support that extracellular spermine directly interacts with the extracellular domain. In addition, we found that the voltage-dependent decay of outward Kir2.1 currents was necessary for inhibition by extracellular spermine. Further, a region at or near the selectivity filter and the cytoplasmic pore are involved in the voltage-dependent decay and thus in the inhibition of outward currents by extracellular spermine. Taken together, the data suggest that extracellular spermine bound to the mouth of the extracellular pore may induce an allosteric effect on voltage-dependent decay of outward currents, a process in which a region in the vicinity of the selectivity filter and cytoplasmic pore are involved. This study reveals that the extracellular pore domain, the selectivity filter and the cytoplasmic pore are in communication and this coupling is involved in modulating K⁺ conduction in the Kir2.1 channel.Display Omitted► Extracellular spermine inhibits outward Kir2.1 currents. ► The effect is stronger at more depolarized voltages. ► The inhibition involves extracellular pore, selectivity filter and cytoplasmic pore. ► Coupling of these domains is important for Kir2.1 conduction.

Keywords: Kir2.1 channel; Voltage-dependence; Extracellular spermine; Cytoplasmic pore; Selectivity filter

Lipid–protein nanodiscs promote in vitro folding of transmembrane domains of multi-helical and multimeric membrane proteins by Zakhar O. Shenkarev; Ekaterina N. Lyukmanova; Ivan O. Butenko; Lada E. Petrovskaya; Alexander S. Paramonov; Mikhail A. Shulepko; Oksana V. Nekrasova; Mikhail P. Kirpichnikov; Alexander S. Arseniev (pp. 776-784).

Production of helical integral membrane proteins (IMPs) in a folded state is a necessary prerequisite for their functional and structural studies. In many cases large-scale expression of IMPs in cell-based and cell-free systems results in misfolded proteins, which should be refolded in vitro. Here using examples of the bacteriorhodopsin ESR from Exiguobacterium sibiricum and full-length homotetrameric K⁺ channel KcsA from Streptomyces lividans we found that the efficient in vitro folding of the transmembrane domains of the polytopic and multimeric IMPs could be achieved during the protein encapsulation into the reconstructed high-density lipoprotein particles, also known as lipid–protein nanodiscs. In this case the self-assembly of the IMP/nanodisc complexes from a mixture containing apolipoprotein, lipids and the partially denatured protein solubilized in a harsh detergent induces the folding of the transmembrane domains. The obtained folding yields showed significant dependence on the properties of lipids used for nanodisc formation. The largest recovery of the spectroscopically active ESR (~60%) from the sodium dodecyl sulfate (SDS) was achieved in the nanodiscs containing anionic saturated lipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPG) and was approximately twice lower in the zwitterionic DMPC lipid. The reassembly of tetrameric KcsA from the acid-dissociated monomer solubilized in SDS was the most efficient (~80%) in the nanodiscs containing zwitterionic unsaturated lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The charged and saturated lipids provided lower tetramer quantities, and the lowest yield (<20%) was observed in DMPC. The overall yield of the ESR and KcsA folding was mainly restricted by the efficiency of the protein encapsulation into the nanodiscs.Display Omitted► Protein/nanodisc complexes can be assembled using SDS denatured membrane proteins. ► Obtained complexes enclose the folded polytopic and multimeric membrane domains. ► Nanodiscs promote in vitro folding of the 7-helical bacteriorhodopsin ESR. ► Nanodiscs promote in vitro folding of the homotetrameric K⁺ channel KcsA. ► Optimization of lipid composition is required to achieve efficient folding

Keywords: Abbreviations; CF; cell-free; DDM; n-dodecyl-β-; d; -maltopyranoside; DMPC; 1,2-dimyristoyl-sn-glycero-3-phosphocholine; DMPG; 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol; DOPG; 1,2-dioleoyl-sn-glycero-3-phosphoglycerol; DOPE; 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; ESR; bacteriorhodopsin from; Exiguobacterium sibiricum; IMP; integral membrane protein; KcsA; K; ⁺; -channel from; Streptomyces lividans; LPN; lipid–protein nanodisc; mKcsA; monomeric form of KcsA; MSP; membrane scaffold protein; MSP; ⁺; MSP with the; N; -terminal His; ₆; -tag sequence; MSP; ⁻; MSP without His; ₆; -tag; POPC; 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; R; _St; hydrodynamic radius of a particle, Stokes radius; SEC; size exclusion chromatography; SDS; sodium dodecyl sulfate; tKcsA; tetrameric form of KcsA; TM; transmembraneBacteriorhodopsin ESR; Potassium channel KcsA; Reconstructed high-density lipoprotein; Membrane protein folding; Membrane mimetic; Cell-free expression

Cholesterol's interactions with serine phospholipids — A comparison of N-palmitoyl ceramide phosphoserine with dipalmitoyl phosphatidylserine by Christian Sergelius; Shou Yamaguchi; Tetsuya Yamamoto; Oskar Engberg; Shigeo Katsumura; J. Peter Slotte (pp. 785-791).

In this study we have prepared ceramide phosphoserine (CerPS) and examined its sterol-interacting properties. CerPS is a hydrogen-bonding sphingolipid, but its head group differs from that found in sphingomyelin (SM). Based on diphenylhexatriene steady-state anisotropy measurements, we observed that fully hydrated N-palmitoyl CerPS had a gel-to-liquid crystalline phase transition temperature of about 51°C in 50mM sodium phosphate buffer (pH 7.4). This was close to the T_m measured for 1,2-dipalmitoyl- sn-glycero-3-phosphoserine (DPPS) bilayers (T_m 50.5°C). Based on cholestatrienol (CTL) quenching experiments in liquid disordered ternary bilayers (containing 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphcholine; POPC), cholesterol/CTL formed sterol-enriched ordered domains with CerPS. These had similar thermostability as the sterol domains formed with N-palmitoyl SM. Cholesterol failed to form sterol-enriched ordered domains with DPPS under comparable conditions. Based on the equilibrium partitioning of CTL, we observed that the affinity of sterol for bilayers containing POPC/CerPS/cholesterol (6:3:1 by mol) was much higher than the affinity measured for control fluid POPC/cholesterol (9:1 by mol) bilayers, but slightly less than seen for comparable PSM-containing bilayers. We conclude that the phosphoserine head group was less efficient than the phosphocholine head group in stabilizing sterol/sphingolipid interaction. However, hydrogen bonding apparently can overcome some of the negative effects of the phosphoserine head group, since CerPS interacted more favorably with cholesterol compared to DPPS.Display Omitted► A ceramide with a phosphoserine (CerPS) headgroup was synthesized. ► The biophysical properties of CerPS were assessed in bilayers. ► The CerPS gel phase was more stable than that of sphingomyelin. ► Cholesterol interacted with CerPS but not with dipalmitoyl PS. ► We discuss the importance of hydrogen bonding for interactions.

Keywords: Abbreviations; 7SLPC; 1-palmitoyl-2-stearoyl-(7-doxyl)-; sn; -glycero-3-phosphocholine; CerPS; N; -palmitoyl ceramide phosphoserine; CTL; cholesta-5,7 (11)-trien-3-beta-ol; DPH; 1,6-diphenyl-1,3,5-hexatriene; DPPS; dipalmitoyl-; sn; -glycero-3-phosphoserine; mβCD; methylβcyclodextrin; PC; phosphatidylcholine; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; PSM; N; -palmitoyl-; d; -; erythro; -sphingomyelin; SM; sphingomyelin; tPA; trans; -parinaric acidSterol domain; Lateral structure; Lipid interaction; Laurdan; Cholestatrienol; trans; -Parinaric acid

Reduced steric hindrance and optimized spatial arrangement of carbohydrate ligands in imprinted monolayers for enhanced protein binding by Haifu Zheng; Xuezhong Du (pp. 792-800).

Imprinted monolayers provide several advantages over bulk imprinting methods. This is especially important for large templates such as proteins. Concanavalin A (Con A)-imprinted binary monolayers consisting of glycolipids with oligo(ethylene glycol) (OEG) spacers and zwitterionic phospholipids (DPPC) were constructed and investigated. The shorter phosphorylcholine (PC) headgroups with an almost flat-on orientation in the binary monolayers gave rise to reduced steric hindrance favorable to the accommodation of Con A with greater ease and facilitated the access of the OEG-linked mannose moieties for enhanced protein binding. Further enhanced binding resulted from optimized spatial rearrangement of the glycolipids at the air–water interface directed by Con A in the subphase to create bivalent binding sites and to minimize steric crowding of neighboring mannose ligands. The combination of the exposed carbohydrate ligands from biologically inert surfaces and the optimized ligand arrangement is the most reasonable solution to enhancement of protein affinity. The bivalent carbohydrate binding sites protruding from the imprinted monolayers were created to be complementary to the Con A binding pockets. This strategy generates tailor-made surfaces with enhanced protein binding and opens the possibility of controlled assembly of intellectual biomaterials and preparation of biosensors.Display Omitted► protein-imprinted monolayers favorable to mass transfer and signal transduction ► Con A binding to glycolipid-containing binary monolayers at the air–water interface ► protrusion of carbohydrate ligands from the interface for protein binding ► protein-directed optimization of spatial arrangement and reduce of steric hindrance ► preservation of multivalent protein interactions for enhanced binding

Keywords: Lipid monolayer; Multivalent protein binding; Protein imprinting; Infrared spectroscopy; Surface plasmon resonance

Effects of surfactin on membrane models displaying lipid phase separation by Magali Deleu; Joseph Lorent; Laurence Lins; Robert Brasseur; Nathalie Braun; Karim El Kirat; Tommy Nylander; Dufrene Yves F. Dufrêne; Marie- Paule Mingeot-Leclercq (pp. 801-815).

Surfactin, a bacterial amphiphilic lipopeptide is attracting more and more attention in view of its bioactive properties which are in relation with its ability to interact with lipids of biological membranes. In this work, we investigated the effect of surfactin on membrane structure using model of membranes, vesicles as well as supported bilayers, presenting coexistence of fluid-disordered (DOPC) and gel (DPPC) phases. A range of complementary methods was used including AFM, ellipsometry, dynamic light scattering, fluorescence measurements of Laurdan, DPH, calcein release, and octadecylrhodamine B dequenching. Our findings demonstrated that surfactin concentration is critical for its effect on the membrane. The results suggest that the presence of rigid domains can play an essential role in the first step of surfactin insertion and that surfactin interacts both with the membrane polar heads and the acyl chain region. A mechanism for the surfactin lipid membrane interaction, consisting of three sequential structural and morphological changes, is proposed. At concentrations below the CMC, surfactin inserted at the boundary between gel and fluid lipid domains, inhibited phase separation and stiffened the bilayer without global morphological change of liposomes. At concentrations close to CMC, surfactin solubilized the fluid phospholipid phase and increased order in the remainder of the lipid bilayer. At higher surfactin concentrations, both the fluid and the rigid bilayer structures were dissolved into mixed micelles and other structures presenting a wide size distribution.Display Omitted► At low conc., surfactin (SF) binds to the boundary between gel and fluid domains. ► At conc. close to CMC, SF induces a high ordering effect of the bilayer. ► At high conc., SF destabilizes totally the bilayer with formation of large particles. ► SF interacts both with the membrane polar heads and the acyl chain region.

Keywords: Surfactin; Membrane interaction; Phase coexistence; Laurdan and DPH fluorescence; Ellipsometry; AFM

HIV-1 p6 — a structured to flexible multifunctional membrane-interacting protein by Sara Marie Øie Solbak; Tove Ragna Reksten; Friedrich Hahn; Victor Wray; Petra Henklein; Peter Henklein; Øyvind Halskau; Ulrich Schubert; Torgils Fossen (pp. 816-823).

The human immunodeficiency virus type 1 (HIV-1) p6 protein has recently been recognized as a docking site for several cellular and viral binding partners and is important for the formation of infectious viruses. Most of its known functions are suggested to occur under hydrophobic conditions near the cytoplasmic membrane, where the protein is presumed to exist in its most structured state. Although p6 is involved in manifold specific interactions, the protein has previously been considered to possess a random structure in aqueous solution. We show that p6 exhibits a defined structure with N- and C-terminal helical domains, connected by a flexible hinge region in 100mM dodecylphosphocholine micelle solution at pH 7 devoid of any organic co-solvents, indicating that this is a genuine limiting structural feature of the molecule in a hydrophobic environment. Furthermore, we show that p6 directly interacts with a cytoplasmic model membrane through both N-terminal and C-terminal regions by use of surface plasmon resonance (SPR) spectroscopy. Phosphorylation of Ser-40 located in the center of the C-terminal α-helix does not alter the secondary structure of the protein but amplifies the interaction with membranes significantly, indicating that p6 binds to the polar head groups at the surface of the cytoplasmic membrane. The increased hydrophobic membrane interaction of p6^23-52 S40F correlated with the observed increased amount of the polyprotein Gag in the RIPA insoluble fraction when Ser40 of p6 was mutated with Phe indicating that p6 modulates the membrane interactions of HIV-1 Gag.Display Omitted► HIV-1 p6 exhibits N- and C-terminal helical domains in micelle solution at pH 7. ► p6 interacts with a cytoplasmic model membrane through N- and C-terminal regions. ► Phosphorylation of Ser-40 does not alter the secondary structure of the protein. ► Phosphorylation of Ser-40 amplifies the interaction of p6 with membranes. ► Effects of p6 S40F mutation indicate p6 modulates membrane interactions of HIV Gag.

Keywords: Abbreviations; ALIX; ALG-2 interacting protein 1/X; DPC; Dodecylphosphatidylcholine; HIV-1; human immunodeficiency virus type 1; L-domains; late assembly domains; NMR; nuclear magnetic resonance; NOESY; Nuclear Overhauser Effect Spectroscopy; SPR; surface plasmon resonance; TOCSY; Total Correlation Spectroscopy; Tsg101; tumor susceptibility gene 101; Vpr; viral protein RHIV-1 p6; NMR; Biacore; SPR; Phosphorylation; Protein–membrane interaction

Structure and dynamics of the two amphipathic arginine-rich peptides RW9 and RL9 in a lipid environment investigated by solid-state NMR and MD simulations by Kristina Witte; Bjoern E.S. Olausson; Astrid Walrant; Isabel D. Alves; Alexander Vogel (pp. 824-833).

Cell penetrating peptides (CPPs) are able to cross membranes without using receptors but only little information about the underlying mechanism is available. In this work, we investigate the interaction of the two arginine-rich CPPs RW9 and RL9 with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG), and POPC/POPG membranes with varying POPG content using isothermal titration calorimetry (ITC), solid-state nuclear magnetic resonance (NMR) spectroscopy, and molecular dynamics (MD) simulations. Both peptides were derived from the known CPP penetratin and it was shown previously that RW9 is able to penetrate membranes better than RL9. Overall, the results show that both RW9 and RL9 have a relatively small influence on the membrane. They increase the order of the lipids in the headgroup region and reduce order in the acyl chains indicating that they are located in the lipid/water interface. In addition, the flexibility of the membrane is slightly increased by both peptides but RW9 has a larger influence than RL9. The differences observed in the influences on POPC and POPG as well as MD simulations on the mixed POPC/POPG bilayers of 850ns length each show that both peptides preferentially associate with and enrich the charged PG lipids almost 2fold in an area of 12Å around the peptides. As expected, we could not observe any membrane crossing on the simulation time scale of 850ns but observed that some peptides flipped their orientation during binding to the membrane. Interestingly, all observed flips coincided with structural changes in the peptides indicating that structural changes or flexibility might play a role during the binding of arginine-rich CPPs to membranes.Display Omitted► RW9 and RL9 are located in the lipid/water interface. ► Both peptides have small influence on membrane slightly increasing its flexibility. ► Both peptides enrich POPG almost 2fold in an area of 12Å around them. ► Both peptides reverse their orientation during binding to the membrane. ► Structural changes were observed during peptide reorientation.

Keywords: Cell penetrating peptides; Peptide/lipid interactions; Calorimetry; Molecular dynamics; ²; H and; ³¹; P solid-state NMR; Order parameter

Rapid bactericidal action of alpha-mangostin against MRSA as an outcome of membrane targeting by Jun-Jie Koh; Shengxiang Qiu; Hanxun Zou; Rajamani Lakshminarayanan; Jianguo Li; Xiaojun Zhou; Charles Tang; Padmanabhan Saraswathi; Chandra Verma; Donald T.H. Tan; Ai Ling Tan; Shouping Liu; Roger W. Beuerman (pp. 834-844).

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) has created the need for better therapeutic options. In this study, five natural xanthones were extracted and purified from the fruit hull of Garcinia mangostana and their antimicrobial properties were investigated. α-Mangostin was identified as the most potent among them against Gram-positive pathogens (MIC=0.78–1.56μg/mL) which included two MRSA isolates. α‐Mangostin also exhibited rapid in vitro bactericidal activity (3-log reduction within 5min). In a multistep (20 passage) resistance selection study using a MRSA isolated from the eye, no resistance against α-mangostin in the strains tested was observed. Biophysical studies using fluorescence probes for membrane potential and permeability, calcein encapsulated large unilamellar vesicles and scanning electron microscopy showed that α‐mangostin rapidly disrupted the integrity of the cytoplasmic membrane leading to loss of intracellular components in a concentration-dependent manner. Molecular dynamic simulations revealed that isoprenyl groups were important to reduce the free energy for the burial of the hydrophobic phenyl ring of α-mangostin into the lipid bilayer of the membrane resulting in membrane breakdown and increased permeability. Thus, we suggest that direct interactions of α-mangostin with the bacterial membrane are responsible for the rapid concentration-dependent membrane disruption and bactericidal action.Display Omitted► α-Mangostin showed potent antimicrobial properties against Gram-positive bacteria. ► α-Mangostin could achieve 3-log reduction in 5min. ► No observable pathogen resistance against α-mangostin up to 20 passages ► Biophysical studies showed that α-mangostin is membrane targeting. ► Isoprenyl groups were important for α-mangostin to penetrate into lipid bilayers.

Keywords: Abbreviations; MRSA; methicillin-resistant; Staphylococcus aureus; EtOAc; ethyl acetate; DOPE; 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine; DOPG; 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodium salt); CFU; Colony Forming Units; LUV; large unilamellar vesicle; EtBr; ethidium bromide; DiSC3-5; 3,3′-dipropylthiadicarbocyanide iodide; MHB; Mueller Hinton Broth; MIC; minimum inhibitory concentration; DMF; N,N-dimethylformamide; TSA; Tryptic Soy Agar; MD; molecular dynamics; SPC; simple point charge model; HEPES; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; NaCl; sodium chloride; EDTA; ethylenediaminetetraacetic acid; RBC; red blood cell; ATB; Automated force field Topology Builder; SEM; scanning electron microscopyMangosteen; Garcinia mangostana; α-Mangostin; Antimicrobial; MRSA; Membrane targeting

Role of terminal dipole charges in aggregation of α-helix pair in the voltage gated K⁺ channel by Lipika Adhya; Tarunendu Mapder; Samit Adhya (pp. 845-850).

The voltage sensor domain (VSD) of the potassium ion channel KvAP is comprised of four (S1–S4) α-helix proteins, which are encompassed by several charged residues. Apart from these charges, each peptide α-helix having two inherent equal and opposite terminal dipolar charges behave like a macrodipole. The activity of voltage gated ion channel is electrostatic, where all the charges (charged residues and dipolar terminal charges) interact with each other and with the transmembrane potential. There are evidences that the role of the charged residues dominate the stabilization of the conformation and the gating process of the ion channel, but the role of the terminal dipolar charges are never considered in such analysis. Here, using electrostatic theory, we have studied the role of the dipolar terminal charges in aggregation of the S3b–S4 helix pair of KvAP in the absence of any external field (V=0). A system attains stability, when its potential energy reaches minimum values. We have shown that the presence of terminal dipole charges (1) change the total potential energy of the charges on S3b–S4, affecting the stabilization of the α-helix pair within the bilayer lipid membrane and (2) the C- and the N-termini of the α-helices favor a different dielectric medium for enhanced stability. Thus, the dipolar terminal charges play a significant role in the aggregation of the two neighboring α-helices.Display Omitted► Inherent existence of the dipolar charges in α-helices of VSD ► Dipolar charges affect the potential energy of a pair of α-helices. ► Dipolar charges exposed to lipid membrane enhances stability.

Keywords: Macrodipole; Dielectric constant; Potential energy; Lipid membrane; Electrostatic theory

The determinants of hydrophobic mismatch response for transmembrane helices by Armando J. de Jesus; Toby W. Allen (pp. 851-863).

Hydrophobic mismatch arises from a difference in the hydrophobic thickness of a lipid membrane and a transmembrane protein segment, and is thought to play an important role in the folding, stability and function of membrane proteins. We have investigated the possible adaptations that lipid bilayers and transmembrane α-helices undergo in response to mismatch, using fully-atomistic molecular dynamics simulations totaling 1.4μs. We have created 25 different tryptophan-alanine-leucine transmembrane α-helical peptide systems, each composed of a hydrophobic alanine–leucine stretch, flanked by 1–4 tryptophan side chains, as well as the β-helical peptide dimer, gramicidin A. Membrane responses to mismatch include changes in local bilayer thickness and lipid order, varying systematically with peptide length. Adding more flanking tryptophan side chains led to an increase in bilayer thinning for negatively mismatched peptides, though it was also associated with a spreading of the bilayer interface. Peptide tilting, bending and stretching were systematic, with tilting dominating the responses, with values of up to ~45° for the most positively mismatched peptides. Peptide responses were modulated by the number of tryptophan side chains due to their anchoring roles and distributions around the helices. Potential of mean force calculations for local membrane thickness changes, helix tilting, bending and stretching revealed that membrane deformation is the least energetically costly of all mismatch responses, except for positively mismatched peptides where helix tilting also contributes substantially. This comparison of energetic driving forces of mismatch responses allows for deeper insight into protein stability and conformational changes in lipid membranes.Display Omitted► Hydrophobic mismatch perturbations are explored with a family of WALP variants and gramicidin A. ► Mismatch responses depend on hydrophobic length and number and placement of tryptophan side chains. ► Free energy calculations explain the dominant responses to positive and negative mismatches. ► These studies reveal the fundamental driving forces for protein folding and function in membranes.

Keywords: Protein–lipid interactions; Membrane protein; Hydrophobic mismatch; Molecular dynamics; Free energy

The role of tryptophan side chains in membrane protein anchoring and hydrophobic mismatch by Armando J. de Jesus; Toby W. Allen (pp. 864-876).

Tryptophan (Trp) is abundant in membrane proteins, preferentially residing near the lipid–water interface where it is thought to play a significant anchoring role. Using a total of 3μs of molecular dynamics simulations for a library of hydrophobic WALP-like peptides, a long poly-Leu α-helix, and the methyl-indole analog, we explore the thermodynamics of the Trp movement in membranes that governs the stability and orientation of transmembrane protein segments. We examine the dominant hydrogen-bonding interactions between the Trp and lipid carbonyl and phosphate moieties, cation–π interactions to lipid choline moieties, and elucidate the contributions to the thermodynamics that serve to localize the Trp, by ~4kcal/mol, near the membrane glycerol backbone region. We show a striking similarity between the free energy to move an isolated Trp side chain to that found from a wide range of WALP peptides, suggesting that the location of this side chain is nearly independent of the host transmembrane segment. Our calculations provide quantitative measures that explain Trp's role as a modulator of responses to hydrophobic mismatch, providing a deeper understanding of how lipid composition may control a range of membrane active peptides and proteins.Display Omitted► The interactions of tryptophan side chains with lipid components lead to interfacial anchoring. ► Free energies for tryptophan movement are reported for a family of transmembrane helices and analog molecule. ► Comparisons reveal the origins of membrane anchoring and perturbations. ► Calculations explain the role of tryptophan in modulating responses to hydrophobic mismatch. ► These studies help to understand the activities of all membrane active peptides and proteins.

Keywords: Protein–lipid interactions; Tryptophan; Membrane protein; Hydrophobic mismatch; Molecular Dynamics; Free energy

Wavelength dependence of patman equilibration dynamics in phosphatidylcholine bilayers by Hannabeth Franchino; Evan Stevens; Jennifer Nelson; Thomas A. Bell; John D. Bell (pp. 877-886).

Assessment of the equilibration kinetics of Patman at the edges of its emission spectra provided additional insights about membrane properties beyond those obtained from end-point fluorescence measurements. Upon introduction of the probe to aqueous suspensions of liposomes, the emission intensity slowly increased about 10-fold ( t_½=~100s). The rate of equilibration depended on emission wavelength, and was usually faster at 500 than at 435nm. However, this trend was reversed for equilibration with lipids at their phase transition temperature. The apparent rotational motion of the dye also differed between the long and short emission wavelengths but did not display the slow equilibration time dependence observed with intensity measurements. These results suggested that slow equilibration reflects relaxation of the immediate membrane microenvironment around the probe rather than slow insertion into the membrane. The data were rationalized with a model that allows two membrane/probe configurations with distinct microenvironments. The analysis suggests that by monitoring the equilibration pattern of Patman, inferences can be made regarding the polarity of two microenvironments occupied by the probe, the distribution of the probe among those microenvironments, and the kinetics with which they relax to equilibrium.Display Omitted► Patman equilibration kinetics are complex. ► The rate of Patman equilibration depends on emission wavelength. ► Equilibration reflects microenvironment relaxation around the probe. ► The complexity identifies at least two microenvironment configurations. ► Analysis of kinetics provides refined observations of membrane microenvironments.

Keywords: Phase transition; Solvatochromatic; Membrane water; Fluorescence spectroscopy; Fluorescence anisotropy; Generalized polarization

Molecular details of membrane fluidity changes during apoptosis and relationship to phospholipase A₂ activity by Elizabeth Gibbons; Katalyn R. Pickett; Michael C. Streeter; Ashley O. Warcup; Jennifer Nelson; Allan M. Judd; John D. Bell (pp. 887-895).

Secretory phospholipase A₂ exhibits much greater activity toward apoptotic versus healthy cells. Various plasma membrane changes responsible for this phenomenon have been proposed, including biophysical alterations described as “membrane fluidity” and “order.” Understanding of these membrane perturbations was refined by applying studies with model membranes to fluorescence measurements during thapsigargin-induced apoptosis of S49 cells using probes specific for the plasma membrane: Patman and trimethylammonium-diphenylhexatriene. Alterations in emission properties of these probes corresponded with enhanced susceptibility of the cells to hydrolysis by secretory phospholipase A₂. By applying a quantitative model, additional information was extracted from the kinetics of Patman equilibration with the membrane. Taken together, these data suggested that the phospholipids of apoptotic membranes display greater spacing between adjacent headgroups, reduced interactions between neighboring lipid tails, and increased penetration of water among the heads. The phase transition of artificial bilayers was used to calibrate quantitatively the relationship between probe fluorescence and the energy of interlipid interactions. This analysis was applied to results from apoptotic cells to estimate the frequency with which phospholipids protrude sufficiently at the membrane surface to enter the enzyme's active site. The data suggested that this frequency increases 50–100-fold as membranes become susceptible to hydrolysis during apoptosis.Display Omitted► Reduced membrane order during apoptosis enhances secretory phospholipase A2 activity. ► Patman equilibration kinetics differ between healthy cells and apopotic cells. ► The probability of a lipid protruding from the membrane increases 100-fold during apoptosis.

Keywords: Two-photon microscopy; Merocyanine 540; Laurdan; Hydrolysis kinetic; Patman; Diphenylhexatriene

Interplay of mycolic acids, antimycobacterial compounds and pulmonary surfactant membrane: A biophysical approach to disease by Marina Pinheiro; Juan J. Giner-Casares; Lucio Marlene Lúcio; João M. Caio; Cristina Moiteiro; José L.F.C. Lima; Salette Reis; Luis Camacho (pp. 896-905).

This work focuses on the interaction of mycolic acids (MAs) and two antimycobacterial compounds (Rifabutin and N′-acetyl-Rifabutin) at the pulmonary membrane level to convey a biophysical perspective of their role in disease. For this purpose, accurate biophysical techniques (Langmuir isotherms, Brewster angle microscopy, and polarization-modulation infrared reflection spectroscopy) and lipid model systems were used to mimic biomembranes: MAs mimic bacterial lipids of the Mycobacterium tuberculosis (MTb) membrane, whereas Curosurf® was used as the human pulmonary surfactant (PS) membrane model. The results obtained show that high quantities of MAs are responsible for significant changes on PS biophysical properties. At the dynamic inspiratory surface tension, high amounts of MAs decrease the order of the lipid monolayer, which appears to be a concentration dependent effect. These results suggest that the amount of MAs might play a critical role in the initial access of the bacteria to their targets. Both molecules also interact with the PS monolayer at the dynamic inspiratory surface. However, in the presence of higher amounts of MAs, both compounds improve the phospholipid packing and, therefore, the order of the lipid surfactant monolayer. In summary, this work discloses the putative protective effects of antimycobacterial compounds against the MAs induced biophysical impairment of PS lipid monolayers. These protective effects are most of the times overlooked, but can constitute an additional therapeutic value in the treatment of pulmonary tuberculosis (Tb) and may provide significant insights for the design of new and more efficient anti-Tb drugs based on their behavior as membrane ordering agents.Display Omitted► A biophysical approach to TB was established by Langmuir isotherms, BAM and PM-IRRAS. ► High quantities of MAs enhanced the permeability of the PS model. ► The antimycobacterial compounds (RFB and RFB2) seem to be membrane ordering agents. ► In the MAs' presence, RFB and RFB2 improve the order of the PS model.

Keywords: Abbreviations; BAM; Brewster angle microscopy; DPPC; Dipalmitoylphosphatidylcholine; DPPG; Dipalmitoylphosphatidylglycerol; LC; Liquid-condensed; LE; Liquid-expanded; LUVs; Large unilamellar vesicles; LPC; Lysophosphatidylcholine; MAs; Mycolic acids; MTb; Mycobacterium tuberculosis; RFB; Rifabutin; RFB2; N′; -acetyl-rifabutin; PE; Phosphatildylethanolamine; PG; Phosphatidylglycerol; PhS; Phosphatildylserine; PI; Phosphatildylinositol; PM-IRRAS; Polarization-modulation infrared reflection spectroscopy; PS; Pulmonary surfactant; SM; Sphingomyelin; SP; Surfactant protein; Tb; TuberculosisAnti-tuberculosis drug; Brewster angle microscopy; Langmuir monolayer; Mycolic acid; Polarization-modulation infrared reflection spectroscopy; Pulmonary surfactant

New insights into lipid-Nucleoside Diphosphate Kinase-D interaction mechanism: Protein structural changes and membrane reorganisation by L. Francois-Moutal; O. Maniti; O. Marcillat; T. Granjon (pp. 906-915).

Nucleoside Diphosphate Kinases (NDPKs) have long been considered merely as housekeeping enzymes. The discovery of the NME1 gene, an anti-metastatic gene coding for NDPK-A, led the scientific community to re-evaluate their role in the cell. It is now well established that the NDPK family is more complex than what was first thought, and despite the increasing amount of evidence suggesting the multifunctional role of nm23/NDPKs, the specific functions of each family member are still elusive. Among these isoforms, NDPK-D is the only one to present a mitochondria-targeting sequence. It has recently been shown that this protein is able to bind and cross-link with mitochondrial membranes, suggesting that NDPK-D can mediate contact sites and contributes to the mitochondrial intermembrane space structuring. To better understand the influence of NDPK-D on mitochondrial lipid organisation, we analysed its behaviour in different lipid environments. We found that NDPK-D not only interacts with CL or anionic lipids, but is also able to bind in a non negligible manner to zwitterionic PC. NDPK-D alters membrane organisation in terms of fluidity, hydration and lipid clustering, effects which depend on lipid structure. Changes in the protein structure after lipid binding were evidenced, both by fluorescence and infrared spectroscopy, regardless of membrane composition. Taking into account all these elements, a putative mechanism of interaction between NDPK-D and zwitterionic or anionic lipids was proposed.Display Omitted► NDPKD binds not only to CL or anionic lipids, but also to zwitterionic PC membranes. ► NDPKD decreases phosphate and ester moiety hydration whatever the lipid head group. ► NDPKD increases anionic acyl chain hydration and induces lipid clustering. ► Changes in NDPKD structure after lipid binding were evidenced.

Keywords: NDPK-D; Cardiolipin; Mitochondrion; Fluorescence; Infrared; Brewster angle microscopy

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BBA - Biomembranes (v.1828, #2)