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BBA - Biomembranes (v.1808, #1)

Editorial Board (pp. i).

Reviewer Acknowledgment (pp. iii-v).

Editorial Board (pp. i).

Reviewer Acknowledgment (pp. iii-v).

Editorial Board (pp. i).

Reviewer Acknowledgment (pp. iii-v).

Editorial Board (pp. i).

Reviewer Acknowledgment (pp. iii-v).

A biophysical glance at the outer surface of the membrane transporter SGLT1 by Navneet K. Tyagi; Theeraporn Puntheeranurak; Mobeen Raja; Azad Kumar; Barbara Wimmer; Isabel Neundlinger; Hermann Gruber; Peter Hinterdorfer; Rolf K.H. Kinne (pp. 1-18).

Proteins mediating the transport of solutes across the cell membrane control the intracellular conditions in which life can occur. Because of the particular arrangement of spanning a lipid bilayer and the many conformations required for their function, transport proteins pose significant obstacles for the investigation of their structure–function relation. Crystallographic studies, if available, define the transmembrane segments in a “frozen” state and do not provide information on the dynamics of the extramembranous loops, which are similarly evolutionary conserved and thus as functionally important as the other parts of the protein. The current review presents biophysical methods that can shed light on the dynamics of transporters in the membrane. The techniques that are presented in some detail are single-molecule recognition atomic force microscopy and tryptophan scanning, which can report on the positioning of the loops and on conformational changes at the outer surface. Studies on a variety of symporters are discussed, which use gradients of sodium or protons as energy source to translocate (mainly organic) solutes against their concentration gradients into or out of the cells. Primarily, investigations of the sodium–glucose cotransporter SGLT1 are used as examples for this biophysical approach to understand transporter function.Display Omitted► Biophysical approaches can be used to strengthen functional and structural information of SGLT1. ► Direct evidence of the initial D-glucose-binding site requiring the presence of sodium. ► Large extracellular loops of SGLT1 act as the vestibule for substrate binding. ► Loop 13 of SGLT1 localizes extracellularly. Loop 13 is involved in the binding of phlorizin to SGLT1.

Keywords: Membrane transporter; Sodium/D-glucose cotransporter 1; Atomic force microscopy; Substrate/ inhibitor tagged cantilever; Tryptophan scanning; Membrane topology

Transbilayer organization of membrane cholesterol at low concentrations: Implications in health and disease by Arunima Chaudhuri; Amitabha Chattopadhyay (pp. 19-25).

Cholesterol is an essential and representative lipid in higher eukaryotic cellular membranes and is often found distributed nonrandomly in domains in biological membranes. A large body of literature exists on the organization of cholesterol in plasma membranes or membranes with high cholesterol content. However, very little is known about organization of cholesterol in membranes containing low amounts of cholesterol such as the endoplasmic reticulum or inner mitochondrial membranes. In this review, we have traced the discovery and subsequent development of the concept of transbilayer cholesterol dimers (domains) in membranes at low concentrations. We have further discussed the role of membrane curvature and thickness on the transbilayer organization of cholesterol. Interestingly, this type of cholesterol organization could be relevant in cellular sorting and trafficking, and in pathological conditions.►Transbilayer cholesterol dimers in membranes at low concentrations. ►Role of membrane thickness and curvature on the transbilayer organization of cholesterol. ►Cholesterol organization in cellular trafficking and pathological conditions.

Keywords: Abbreviations; 25-NBD-cholesterol; 25-[; N; -[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-methyl]amino]-27-norcholesterol; DHE; dehydroergosterol (Δ; ^5,7,9(11)22; -ergostatetraen-3β-ol; DAPC; diarachidoyl-; sn; -glycero-3-phosphocholine; DLPC; dilauroyl-; sn; -glycero-3-phosphocholine); DMPC; dimyristoyl-; sn; -glycero-3-phosphocholine; DPPC; dipalmitoyl-; sn; -glycero-3-phosphocholine; DSPC; distearoyl-; sn; -glycero-3-phosphocholine; NBD; 7-nitrobenz-2-oxa-1,3-diazol-4-yl; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine25-NBD-cholesterol; Cholesterol domain; Cholesterol transbilayer dimer; Dehydroergosterol; Membrane curvature; Membrane thickness

Transbilayer organization of membrane cholesterol at low concentrations: Implications in health and disease by Arunima Chaudhuri; Amitabha Chattopadhyay (pp. 19-25).

Effect of trehalose on the interaction of Alzheimer's Aβ-peptide and anionic lipid monolayers by Aslin Izmitli; Carolina Schebor; Michael P. McGovern; Allam S. Reddy; Nicholas L. Abbott; Juan J. de Pablo (pp. 26-33).

The interaction of amyloid β-peptide (Aβ) with cell membranes is believed to play a central role in the pathogenesis of Alzheimer's disease. In particular, recent experimental evidence indicates that bilayer and monolayer membranes accelerate the aggregation and amyloid fibril formation rate of Aβ. Understanding that interaction could help develop therapeutic strategies for treatment of the disease. Trehalose, a disaccharide of glucose, has been shown to be effective in preventing the aggregation of numerous proteins. It has also been shown to delay the onset of certain amyloid-related diseases in a mouse model. Using Langmuir monolayers and molecular simulations of the corresponding system, we study several thermodynamic and kinetic aspects of the insertion of Aβ peptide into DPPG monolayers in water and trehalose subphases. In the water subphase, the insertion of the Aβ peptide into the monolayer exhibits a lag time which decreases with increasing temperature of the subphase. In the presence of trehalose, the lag time is completely eliminated and peptide insertion is completed within a shorter time period compared to that observed in pure water. Molecular simulations show that more peptide is inserted into the monolayer in the water subphase, and that such insertion is deeper. The peptide at the monolayer interface orients itself parallel to the monolayer, while it inserts with an angle of 50° in the trehalose subphase. Simulations also show that trehalose reduces the conformational change that the peptide undergoes when it inserts into the monolayer. This observation helps explain the experimentally observed elimination of the lag time by trehalose and the temperature dependence of the lag time in the water subphase.►Aβ insertion into DPPG monolayers in water exhibits a temperature dependent lag time. ►The presence of trehalose eliminates the lag time before Aβ insertion starts. ►More peptide is inserted into the monolayer in the water subphase. ►Trehalose reduces the conformational change that Aβ undergoes when it inserts. ►Aβ inserts parallel to the monolayer in water and with a 50° angle in the trehalose subphase.

Keywords: Amyloid beta peptide; Trehalose; Monolayer; Alzheimer's disease; Membrane

Probing membrane topology of the antimicrobial peptide distinctin by solid-state NMR spectroscopy in zwitterionic and charged lipid bilayers by Raffaello Verardi; Nathaniel J. Traaseth; Lei Shi; Fernando Porcelli; Luca Monfregola; Stefania De Luca; Pietro Amodeo; Gianluigi Veglia; Andrea Scaloni (pp. 34-40).

Distinctin is a 47-residue antimicrobial peptide, which interacts with negatively charged membranes and is active against Gram-positive and Gram-negative bacteria. Its primary sequence comprises two linear chains of 22 (chain 1) and 25 (chain 2) residues, linked by a disulfide bridge between Cys19 of chain 1 and Cys23 of chain 2. Unlike other antimicrobial peptides, distinctin in the absence of the lipid membrane has a well-defined three-dimensional structure, which protects it from protease degradation. Here, we used static solid-state NMR spectroscopy in mechanically aligned lipid bilayers (charged or zwitterionic) to study the topology of distinctin in lipid bilayers. We found that this heterodimeric peptide adopts an ordered conformation absorbed on the surface of the membrane, with the long helix (chain 2), approximately parallel to the lipid bilayer (~5° from the membrane plane) and the short helix (chain 1) forming a ~24° angle with respect to the bilayer plane. Since the peptide does not disrupt the macroscopic alignment of charged or zwitterionic lipid bilayers at lipid-to-protein molar ratio of 50:1, it is possible that higher peptide concentrations might be needed for pore formation, or alternatively, distinctin elicits its cell disruption action by another mechanism.Display Omitted►In mechanically aligned lipid bilayers, the antimicrobial peptide Distinctin is absorbed on the membrane surface. ►The topology of distinctin does not change upon variation of lipid composition. ►Two different tilt angles were found for the two helical chains of distinctin, with chain 1 tilted by ~24 degrees, while chain 2 by ~5 degrees.

Keywords: Distinctin; Antimicrobial Peptides; Oriented Solid-State NMR; Mechanically Aligned Bilayer; Membrane Protein Topology

Coupled cell-free synthesis and lipid vesicle insertion of a functional oligomeric channel MscL by Catherine Berrier; Ingrid Guilvout; Nicolas Bayan; Kyu-Ho Park; Agnes Mesneau; Mohamed Chami; Anthony P. Pugsley; Alexandre Ghazi (pp. 41-46).

The mechanosensitive channel MscL of the plasma membrane of bacteria is a homopentamer involved in the protection of cells during osmotic downshock. The MscL protein, a polypeptide of 136 residues, was recently shown to require YidC to be inserted in the inner membrane of E. coli. The insertase YidC is a component of an insertion pathway conserved in bacteria, mitochondria and chloroplasts. MscL insertion was independent of the Sec translocon. Here, we report sucrose gradient centrifugation and freeze-etching microscopy experiments showing that MscL produced in a cell-free system complemented with preformed liposomes is able to insert directly in a pure lipid bilayer. Patch-clamp experiments performed with the resulting proteoliposomes showed that the protein was highly active. In vitro cell-free synthesis targeting to liposomes is a new promising expression system for membrane proteins, including those that might require an insertion machinery in vivo. Our results also question the real role of insertases such as YidC for membrane protein insertion in vivo.►The channel MscL can be produced in a cell-free system in the presence of liposomes. ►It inserts directly in the lipid bilayer in the absence of the insertase YidC. ►The inserted channels are highly active, as evidenced by patch-clamp experiments.

Keywords: Membrane protein; Ion channel; Insertase; Cell-free synthesis; Liposome

Monitoring glycolipid transfer protein activity and membrane interaction with the surface plasmon resonance technique by Ohvo-Rekila Henna Ohvo-Rekilä; Peter Mattjus (pp. 47-54).

The glycolipid transfer protein (GLTP) is a protein capable of binding and transferring glycolipids. GLTP is cytosolic and it can interact through its FFAT-like (two phenylalanines in an acidic tract) motif with proteins localized on the surface of the endoplasmic reticulum. Previous in vitro work with GLTP has focused mainly on the complete transfer reaction of the protein, that is, binding and subsequent removal of the glycolipid from the donor membrane, transfer through the aqueous environment, and the final release of the glycolipid to an acceptor membrane. Using bilayer vesicles and surface plasmon resonance spectroscopy, we have now, for the first time, analyzed the binding and lipid removal capacity of GLTP with a completely label-free technique. This technique is focused on the initial steps in GLTP-mediated transfer and the parameters affecting these steps can be more precisely determined. We used the new approach for detailed structure–function studies of GLTP by examining the glycolipid transfer capacity of specific GLTP tryptophan mutants. Tryptophan 96 is crucial for the transfer activity of the protein and tryptophan 142 is an important part of the proteins membrane interacting domain. Further, we varied the composition of the used lipid vesicles and gained information on the effect of membrane properties on GLTP activity. GLTP prefers to interact with more tightly packed membranes, although GLTP-mediated transfer is faster from more fluid membranes. This technique is very useful for the study of membrane–protein interactions and lipid-transfer rates and it can easily be adapted to other membrane-interacting proteins.►Surface plasmon resonance is a useful technique to study lipid transfer proteins. ►Tryptophan 96 is crucial for the transfer activity of glycolipid transfer protein. ►Tryptophan 142 is important for the membrane interaction of GLTP. ►The membrane environment of the substrate affects transfer mediated by GLTP. ►GLTP prefers to interact with more tightly packed membranes.

Keywords: Abbreviations; DPPC; 1,2-dipalmitoyl-; sn; -glycero-3-phosphocholine; GLTP; glycolipid transfer protein; O-SM; N; -oleoyl-sphingomyelin; PGalCer; N; -palmitoyl-galactosylceramide; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; P-SM; N; -palmitoyl-sphingomyelin; SPR; surface plasmon resonance; RU; response unitsGlycolipid transfer protein; GLTP; Surface plasmon resonance; Glycosphingolipid; Lipid transfer; Protein–membrane interaction

Monitoring glycolipid transfer protein activity and membrane interaction with the surface plasmon resonance technique by Ohvo-Rekila Henna Ohvo-Rekilä; Peter Mattjus (pp. 47-54).

Effects of the antimalarial drug primaquine on the dynamic structure of lipid model membranes by Luis G.M. Basso; Renata Z. Rodrigues; Rose M.Z.G. Naal; Antonio J. Costa-Filho (pp. 55-64).

Primaquine (PQ) is a potent therapeutic agent used in the treatment of malaria and its mechanism of action still lacks a more detailed understanding at a molecular level. In this context, we used differential scanning calorimetry (DSC), pressure perturbation calorimetry (PPC), and electron spin resonance (ESR) to investigate the effects of PQ on the lipid phase transition, acyl chain dynamics, and on volumetric properties of lipid model membranes. DSC thermograms revealed that PQ stabilizes the fluid phase of the lipid model membranes and interacts mainly with the lipid headgroups. This result was revealed by the great effect on the pretransition of phosphatidylcholines and the destabilization of the inverted hexagonal phase of a phosphatidylethanolamine bilayer. Spin probes located at different positions along the lipid chain were used to monitor different membrane regions. ESR results indicated that PQ is effective in changing the acyl chain ordering and dynamics of the whole chain of dimyristoylphosphatidylcholine (DMPC) phospholipid in the rippled gel phase. The combined ESR and PPC results revealed that the slight DMPC volume changes at the main phase transition induced by the presence of PQ is probably due to a less dense lipid gel phase. At physiological pH, the cationic amphiphilic PQ strongly interacts with the lipid headgroup region of the bilayers, causing considerable disorganization in the hydrophobic core. These results shed light on the molecular mechanism of primaquine–lipid interaction, which may be useful in the understanding of the complex mechanism of action and/or the adverse effects of this antimalarial drug.► PQ changes the equilibrium constant between DMPC gel-like and fluid-like domains. ► The dynamic structure and volumetric properties of membranes are affected by PQ. ► PQ promotes PC headgroup ordering and PE positive membrane curvature. ► Interactions with membranes may play an important role to the PQ mechanism of action.

Keywords: Primaquine; Model membrane; Electron spin resonance; Calorimetry

Effects of the antimalarial drug primaquine on the dynamic structure of lipid model membranes by Luis G.M. Basso; Renata Z. Rodrigues; Rose M.Z.G. Naal; Antonio J. Costa-Filho (pp. 55-64).

Keywords: Primaquine; Model membrane; Electron spin resonance; Calorimetry

Effects of the antimalarial drug primaquine on the dynamic structure of lipid model membranes by Luis G.M. Basso; Renata Z. Rodrigues; Rose M.Z.G. Naal; Antonio J. Costa-Filho (pp. 55-64).

Keywords: Primaquine; Model membrane; Electron spin resonance; Calorimetry

Effects of the antimalarial drug primaquine on the dynamic structure of lipid model membranes by Luis G.M. Basso; Renata Z. Rodrigues; Rose M.Z.G. Naal; Antonio J. Costa-Filho (pp. 55-64).

Keywords: Primaquine; Model membrane; Electron spin resonance; Calorimetry

Characterisation of the purified human sodium/iodide symporter reveals that the protein is mainly present in a dimeric form and permits the detailed study of a native C-terminal fragment by Sylvaine Huc-Brandt; Didier Marcellin; Fanny Graslin; Olivier Averseng; Laurent Bellanger; Patrick Hivin; Eric Quemeneur; Cécile Basquin; Valérie Navarro; Thierry Pourcher; Elisabeth Darrouzet (pp. 65-77).

The sodium/iodide symporter is an intrinsic membrane protein that actively transports iodide into thyroid follicular cells. It is a key element in thyroid hormone biosynthesis and in the radiotherapy of thyroid tumours and their metastases. Sodium/iodide symporter is a very hydrophobic protein that belongs to the family of sodium/solute symporters. As for many other membrane proteins, particularly mammalian ones, little is known about its biochemistry and structure. It is predicted to contain 13 transmembrane helices, with an N-terminus oriented extracellularly. The C-terminal, cytosolic domain contains approximately one hundred amino acid residues and bears most of the transporter's putative regulatory sites (phosphorylation, sumoylation, di-acide, di-leucine or PDZ-binding motifs). In this study, we report the establishment of eukaryotic cell lines stably expressing various human sodium/iodide symporter recombinant proteins, and the development of a purification protocol which allowed us to purify milligram quantities of the human transporter. The quaternary structure of membrane transporters is considered to be essential for their function and regulation. Here, the oligomeric state of human sodium/iodide symporter was analysed for the first time using purified protein, by size exclusion chromatography and light scattering spectroscopy, revealing that the protein exists mainly as a dimer which is stabilised by a disulfide bridge. In addition, the existence of a sodium/iodide symporter C-terminal fragment interacting with the protein was also highlighted. We have shown that this fragment exists in various species and cell types, and demonstrated that it contains the amino-acids [512–643] from the human sodium/iodide symporter protein and, therefore, the last predicted transmembrane helix. Expression of either the [1–512] truncated domain or the [512–643] domain alone, as well as co-expression of the two fragments, was performed, and revealed that co-expression of [1–512] with [512–643] allowed the reconstitution of a functional protein. These findings constitute an important step towards an understanding of some of the post-translational mechanisms that finely tune iodide accumulation through human sodium/iodide symporter regulation.►The human sodium/iodide symporter (NIS) has been purified for the first time. ►NIS protein exists mainly as a dimer that is stabilised by a disulfide bridge. ►A proteolytic C-terminal fragment of NIS is present in quantity in various species. ►Co-expression of C- and N-terminal fragments reconstitute a functional NIS.

Keywords: Abbreviations; BCA; bicinchoninic acid; C12E8; octaethylene glycol monododecyl ether; CMV; cytomegalovirus; DAB; 3,3′-diaminobenzidine; DDM; n-dodecyl β-; d; -maltoside; HEK; human embryonic kidney cells; HEPES; 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid; IMAC; immobilized metal affinity chromatography; LDS; Lithium dodecylsulfate; MES; 4-morpholineethanesulfonic acid; MOPS; 4-morpholinepropanesulfonic acid; PAGE; polyacrylamide gel electrophoresis; SEC; size exclusion chromatography; Tris; 2-Amino-2-(hydroxymethyl)-1,3–propanediol; TX–100; Triton X100Thyroid; Sodium/iodide symporter; Membrane protein; Eukaryote; Oligomeric state; Post-translational regulation

Characterization of superoxide overproduction by the D-Loop_Nox4-Nox2 cytochrome b₅₅₈ in phagocytes—Differential sensitivity to calcium and phosphorylation events by Laure Carrichon; Antoine Picciocchi; Franck Debeurme; Federica Defendi; Sylvain Beaumel; Algirdas J. Jesaitis; Marie-Claire Dagher; Marie-José Stasia (pp. 78-90).

NADPH oxidase is a crucial element of phagocytes involved in microbicidal mechanisms. It becomes active when membrane-bound cytochrome b₅₅₈, the redox core, is assembled with cytosolic p47^phox, p67^phox, p40^phox, and rac proteins to produce superoxide, the precursor for generation of toxic reactive oxygen species. In a previous study, we demonstrated that the potential second intracellular loop of Nox2 was essential to maintaining NADPH oxidase activity by controlling electron transfer from FAD to O₂. Moreover, replacement of this loop by the Nox4-D-loop (D-loop_Nox4-Nox2) in PLB-985 cells induced superoxide overproduction. In the present investigation, we demonstrated that both soluble and particulate stimuli were able to induce this superoxide overproduction. Superoxide overproduction was also observed after phosphatidic acid activation in a purified cell-free-system assay. The highest oxidase activity was obtained after ionomycin and fMLF stimulation. In addition, enhanced sensitivity to Ca²⁺ influx was shown by thapsigargin, EDTA, or BTP2 treatment before fMLF activation. Mutated cytochrome b₅₅₈ was less dependent on phosphorylation triggered by ERK1/2 during fMLF or PMA stimulation and by PI3K during OpZ stimulation. The superoxide overproduction of the D-loop_Nox4-Nox2 mutant may come from a change of responsiveness to intracellular Ca²⁺ level and to phosphorylation events during oxidase activation. Finally the D-loop_Nox4-Nox2-PLB-985 cells were more effective against an attenuated strain of Pseudomonas aeruginosa compared to WT-Nox2 cells. The killing mechanism was biphasic, an early step of ROS production that was directly bactericidal, and a second oxidase-independent step related to the amount of ROS produced in the first step.►Superoxide overproduction of the DloopNox4-Nox2 mutant is related to Ca2+ sensitivity.►Superoxide overproduction of the DloopNox4-Nox2 mutant is related to phosphorylation events.►Killing of P. aeruginosa is improved with the DloopNox4-Nox2 mutant.

Keywords: Abbreviations; ROS; reactive oxygen species; CGD; chronic granulomatous disease; PMA; phorbol 12-myristate 13-acetate; fMLF; formyl-methyl-leucyl-phenylalanine; DMF; dimethylformamide; DFP; diisopropylfluorophosphate; SOD; mAb, monoclonal antibody; cyt; b; ₅₅₈; cytochrome; b; ₅₅₈; WT; wildNADPH oxidase; Activation; ROS overproduction; Microbicidy; PLB-985 cells; Neutrophils

Indolicidin action on membrane permeability: Carrier mechanism versus pore formation by Tatyana I. Rokitskaya; Nikolay I. Kolodkin; Elena A. Kotova; Yuri N. Antonenko (pp. 91-97).

Indolicidin, a 13-residue cationic peptide with extremely high tryptophan content, exhibits broad-spectrum antimicrobial as well as hemolytic activity. To gain insight into the mechanism of indolicidin action on membrane permeability, liposome leakage induced by this peptide was studied by using various probes with vesicles of different lipid compositions. In liposomes containing negatively charged lipids, indolicidin induced rather unselective permeabilization. By contrast, the peptide appeared to be selective in provoking leakage of neutral, egg phosphatidylcholine (PC) liposomes: it effectively induced the release of negatively charged fluorescent dyes, carboxyfluorescein (CF), calcein and sulforhodamine B, but was unable to induce the leakage of a neutral compound, glucose, and that of positively charged doxorubicin. Moreover, organic anions, such as fatty acids, were found to suppress the indolicidin-induced CF leakage of egg PC liposomes. Based on these results, we concluded that indolicidin facilitates the dye release from uncharged lipid vesicles not by formation of membrane pores as it is generally accepted for the majority of antimicrobial peptides but rather via translocation of dye molecules across the membrane in the form of dye–peptide complexes, i.e. indolicidin operates as an organic anion carrier. This conclusion was supported by observing the formation of complexes between indolicidin and pyrenebutyrate in solution. The indolicidin analog having only one arginine was ineffective in pyrenebutyrate binding and CF transport. The mode of action proposed here for indolicidin can be related to that previously postulated for oligoarginine derivatives which are able to carry organic anions across liposomal and bulk phase membranes [Sakai N. & Matile S. J. Am. Chem. Soc. 2003, 125:14348–14356]. The newly identified mechanism of peptide ionophoric activity in uncharged lipid membranes may be involved in hemolytic action of indolicidin via induction of plasma membrane permeability for important anionic metabolites which disturbs regulation of osmotic balance ultimately leading to erythrocyte membrane rupture.►Indolicidin induces anion-selective leakage of zwitterionic liposomes. ►This cationic peptide can operate as a carrier of organic anions in lipid membranes. ►A double-arginine motif is necessary for the carrier activity of indolicidin. ►The carrier-type mechanism may be involved in hemolytic action of indolicidin.

Keywords: Abbreviations; BLM; bilayer lipid membrane; CF; carboxyfluorescein; SRB; sulforhodamine B; DPhPC; diphytanoylphosphatidylcholine; DPhPG; diphytanoylphosphatidylglycerol; egg PC; egg yolk phosphatidylcholine; DOPG; 1,2-dioleoyl-; sn; -glycero-3-phospho-(1′-; rac; -glycerol); HK; Hexokinase; G6PDH; Glucose-6-phosphate dehydrogenaseAntimicrobial peptide; Liposome; Leakage; Bilayer lipid membrane; Fluorescence; Arginine

Indolicidin action on membrane permeability: Carrier mechanism versus pore formation by Tatyana I. Rokitskaya; Nikolay I. Kolodkin; Elena A. Kotova; Yuri N. Antonenko (pp. 91-97).

Impact of the biofilm mode of growth on the inner membrane phospholipid composition and lipid domains in Pseudomonas aeruginosa by Hayette Benamara; Christophe Rihouey; Thierry Jouenne; Stéphane Alexandre (pp. 98-105).

Many studies using genetic and proteomic approaches have revealed phenotypic differences between planktonic and sessile bacteria but the mechanisms of biofilm formation and the switch between the two growth modes are not well understood yet. In this study, we focused on inner membrane lipidome modifications when Pseudomonas aeruginosa cells were grown as biofilm. Lipid analyses were performed by Electrospray Ionization Mass Spectrometry. Results showed a drastic decrease of the uneven-numbered chain phospholipids and a slight increase of long chain PEs in sessile organisms as compared with planktonic counterparts, suggesting a better lipid stability in the bilayer and a decrease in membrane fluidity. The impact of sessile growth on lipid domains was then investigated by Brewster Angle Microscopy (BAM) and Atomic Force Microscopy (AFM). Observations showed that inner membrane lipids of P. aeruginosa formed domains when the pressure was close to physiological conditions and that these domains were larger for lipids extracted from biofilm bacteria. This is coherent with the mass spectrometry analyses.► Pseudomonas aeruginosa lipidome is affected by biofilm growth mode. ►Biofilm growth mode leads to a decrease of the uneven-numbered chain phospholipids. ►Accumulation of long chain lipids is also observed. ►Reconstituted lipid monolayers show larger condensed domains with sessile bacteria.

Keywords: Biofilm; Pseudomonas aeruginosa; Lipidome; Inner membrane; Monolayer; Mass spectroscopy

Structure and mechanism of action of a de novo antimicrobial detergent-like peptide by Baptiste Legrand; Mathieu Laurencin; Joe Sarkis; Emilie Duval; Liza Mouret; Jean-François Hubert; Murielle Collen; Vie Véronique Vié; Céline Zatylny-Gaudin; Joël Henry; Michèle Baudy-Floc'h; Arnaud Bondon (pp. 106-116).

The K4 peptide (KKKKPLFGLFFGLF) was recently demonstrated to display good antimicrobial activities against various bacterial strains and thus represents a candidate for the treatment of multiple-drug resistant infections. In this study, we use various techniques to study K4 behaviour in different media: water, solutions of detergent micelles, phospholipid monolayers and suspension of phospholipid vesicles. First, self-assembly of the peptide in water is observed, leading to the formation of spherical objects around 10nm in diameter. The addition of micelles induces partial peptide folding to an extent depending on the charge of the detergent headgroups. The NMR structure of the peptide in the presence of SDS displays a helical character of the hydrophobic moiety, whereas only partial folding is observed in DPC micelles. This peptide is able to destabilize the organization of monolayer membranes or bilayer liposomes composed of anionic lipids. When added on small unilamellar vesicles it generates larger objects attributed to mixed lipid–peptide vesicles and aggregated vesicles. The absence of calcein leakage from liposomes, when adding K4, underlines the original mechanism of this linear amphipathic peptide. Our results emphasize the importance of the electrostatic effect for K4 folding and lipid destabilization leading to the microorganisms' death with a high selectivity for the eukaryotic cells at the MIC. Interestingly, the micrographs obtained by electronic microscopy after addition of peptide on bacteria are also consistent with the formation of mixed lipid–peptide objects. Overall, this work supports a detergent-like mechanism for the antimicrobial activity of this peptide.►Self-assembly properties of de novo antimicrobial peptide. ►Partially helical structure of the peptide in micellar environment. ►Specific interaction with anionic liposomes. ►Formation of mixed lipid/peptide entities and aggregation without membrane disruption.

Keywords: Antimicrobial peptide; Self-assembly; NMR; Circular dichroism; Detergent; Liposome

A novel class of photo-triggerable liposomes containing DPPC:DC_8,9PC as vehicles for delivery of doxorubcin to cells by Amichai Yavlovich; Alok Singh; Robert Blumenthal; Anu Puri (pp. 117-126).

Success of nanoparticle-mediated drug delivery is subject to development of optimal drug release strategies within defined space and time (triggered release). Recently, we reported a novel class of photo-triggerable liposomes prepared from dipalmitoyl phosphatidylcholine (DPPC) and photopolymerizable diacetylene phospholipid (DC₈,₉PC), that efficiently released entrapped calcein (a water soluble fluorescent dye) upon UV (254nm) treatment. To develop these formulations for in vivo applications, we have examined phototriggering of these liposomes by visible light, and the effect of released anticancer drugs on cellular toxicity. Sonicated liposomes containing various ratios of DPPC:DC₈,₉PC and 4mol% DSPE-PEG2000 were loaded with calcein (Ex/Em, 485/517nm) or a chemotherapy drug, Doxorubicin (DOX, Ex/Em 490/590nm). Our initial experiments showed that 514nm laser treatment of liposomes containing 10 or 20mol% DC_8,9PC for 1–3min resulted in significant release of calcein. Based on these results, we performed studies with DOX-loaded liposomes. First, biophysical properties (including liposome size and stability) and DOX encapsulation efficiency of the liposomes were determined. Subsequently, the effect of 514nm laser on DOX release, and cellular toxicity by released DOX were examined. Since liposomes using the 86:10:04 mole ratio of DPPC:DC₈,₉PC:DSPE-PEG2000, showed highest encapsulation of DOX, these formulations were investigated further. We report that (i) liposomes retained about 70% of entrapped DOX at 37°C in the presence of 0–50% serum. (ii) 514nm laser treatment resulted in DOX release from liposomes in a wavelength-specific manner. (iii) Laser treatment of co-cultures containing DOX-loaded liposomes and cells (Raji and MCF-7) resulted in at least 2–3 fold improved cell killing as compared to untreated samples. Taken together, the photo-triggerable liposomes described here may provide a platform for future drug delivery applications. To our knowledge, this is the first report demonstrating improved cell killing following light-triggered release of an encapsulated anticancer agent from photosensitive liposomes.

Keywords: Abbreviations; DPPC; 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; Egg PC; L-α-Phosphatidylcholine (egg, chicken); DC; _8,9; PC; (1,2 bis (tricosa-10 12-diynoyl)-; sn; -glycero-3-phosphocholine); DSPE-PEG2000 (18:0 PEG2 PE); 1,2-Distearoyl-; sn; -Glycero-3 Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-2000] (ammonium salt); HEPES buffer (HBS); 10; mM HEPES, 140; mM NaCl (pH 7.5); PBS; 2.66; mM KCl, 1.47; mM KH; ₂; PO; ₄; , 138; mM NaCl, 8.06; mM Na; ₂; HPO; ₄; -7H; ₂; O (pH 7.1)Polymerizable lipids; Laser; Triggered drug release; Diacetylene phospholipid; Radiation-sensitive liposome; Lipid modification

A novel class of photo-triggerable liposomes containing DPPC:DC_8,9PC as vehicles for delivery of doxorubcin to cells by Amichai Yavlovich; Alok Singh; Robert Blumenthal; Anu Puri (pp. 117-126).

NMR evidence of GM1-induced conformational change of Substance P using isotropic bicelles by Anindita Gayen; Sudipto Kishore Goswami; Chaitali Mukhopadhyay (pp. 127-139).

Substance P (SP) is one of the target neurotransmitters associated with diseases related to chronic inflammation, pain and depression. The selective receptor for SP, NK₁R is located in the heterogeneous microdomains or caveolae in membrane. Gangliosides, specifically GM1, are markers of these heterogeneous sites. Also, gangliosides are considered as important regulatory elements in cell–cell recognition and cell signaling. In the present work, we describe the conformations of Substance P in the presence of ternary membrane systems containing GM1 at the physiological concentration. SP is mostly unstructured in water, but appears as extended 3₁₀ helical or turn III in isotropic bicelles, more pronounced in the presence of GM1. NMR results suggest that, in the GM1 containing bicelles, the peptide is more inserted into the membrane with its C-terminus, while N-terminus lies close to the membrane–water interface. The NMR-derived conformation of SP in GM1 bicelles is docked on homology modeled NK₁R and resulting interactions satisfy reported mutagenesis, fluorescence, photo-affinity labeling and modeling data. The results highlight efficacy of GM1 in membrane in providing structure in an otherwise flexible neurotransmitter Substance P; thus providing indication that it may be useful also for other neurotransmitter peptides/proteins associated with membrane.►Induced structure in Substance P by GM1 containing ternary bicelles. ►Diffusion analysis showing strong interaction of Substance P with GM1 containing ternary bicelles. ►Location of Substance P in bicelles evidenced by paramagnetic quenching and 2D NMR. ►NK₁R receptor-recognized conformation of Substance P in GM1 containing bicelles.

Keywords: Abbreviation; SP; Substance P; NK; ₁; R; Neurokinin-1 receptor; GPCR; G-protein coupled receptor; GM1; Ganglioside Monosialo 1; DMPC; Dimyristoyl-sn-glycero-3-phosphorylcholine; CHAPS; 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate; GT1; Ganglioside Trisialo 1; GD1; Ganglioside Disialo 1; DOPC; Dioleoyl-phosphatidylcholine; DOPG; Dioleoyl-phosphatidylglycerol; POPC; Palmitoyloleoyl phosphatidylcholine; POPG; Palmitoyloleoyl phosphatidylglycerol; DHPC; Dihexanoyl-sn-glycero-3-phosphocholine; DPC; Dodecyl-phosphocholine; SDS; Sodium dodecyl-sulfate; NMR; Nuclear magnetic resonance; TOCSY; Total Correlation Spectroscopy; ROESY; Rotating-frame Overhauser Effect Spectroscopy; DQF-COSY; Double Quantum Filtered-Correlation Spectroscopy; ROE; Rotating-frame NOE; DOSY; Diffusion Ordered Spectroscopy; ADT; AutoDockToolsSubstance P; NK; ₁; R; NMR; GM1; Bicelle

NMR evidence of GM1-induced conformational change of Substance P using isotropic bicelles by Anindita Gayen; Sudipto Kishore Goswami; Chaitali Mukhopadhyay (pp. 127-139).

The vesicle-to-micelle transformation of phospholipid–cholate mixed aggregates: A state of the art analysis including membrane curvature effects by Mustafa M.A. Elsayed; Gregor Cevc (pp. 140-153).

We revisited the vesicle-to-micelle transformation in phosphatidylcholine–cholate mixtures paying special attention to the lipid bilayer curvature effects. For this purpose, we prepared unilamellar vesicles with different starting sizes (2 r_v=45–120nm). We then studied mixtures of the unilamellar vesicles (1–8mmol kg^–1) and sodium cholate (0–11.75mmolkg−¹) by static and dynamic light scattering. The transformation generally comprises at least two, largely parallel phenomena; one increases and the other decreases the average mixed aggregate size. In our view, cholate first induces bilayer fluctuations that lead to vesicle asphericity, and then to lipid bilayer poration followed by sealing/reformation (or fusion). The cholate-containing mixed bilayers, whether in vesicular or open form, project thread-like protrusions with surfactant enriched ends even before complete bilayer solubilisation. Increasing cholate concentration promotes detachment of such protrusions (i.e. mixed micelles formation), in parallel to further softening/destabilising of mixed amphipat bilayers over a broad range of concentrations. Vesicles ultimately fragment into mixed thread-like micelles. Higher cholate relative concentrations yield shorter thread-like mixed micelles. Most noteworthy, the cholate-induced bilayer fluctuations, the propensity for large aggregate formation, the transformation kinetics, and the cholate concentration ensuring complete lipid solubilisation all depend on the starting mean vesicle size. The smallest tested vesicles (2 r_v=45nm), with the highest bilayer curvature, require ~30% less cholate for complete solubilisation than the largest tested vesicles (2 r_v=120nm).►The first quantitative study of bilayer curvature effects on bilayer solubilisation. ►Refined and generalized description of vesicle-to-micelle transformation. ►Comparison of the static vs. dynamic light scattering for lecithin/cholate blends. ►Differentiation between vesicle-to-micelle vs. micelle-to-vesicle transformation. ►Discussion of kinetic aspects of morphological changes during lipid solubilisation

Keywords: Cholate; Phosphatidylcholine; Lipid solubilisation; Bilayer curvature; Aggregate size; Static light scattering; Dynamic light scattering

Mitoxantrone is expelled by the ABCG2 multidrug transporter directly from the plasma membrane by Laszlo László Homolya; Orban Tamás I. Orbán; Csanady László Csanády; Balázs Sarkadi (pp. 154-163).

ABC multidrug transporter proteins expel a wide variety of structurally unrelated, mostly hydrophobic compounds from cells. The special role of these transporters both at the physiological barriers and in cancer cells is based on their extremely broad substrate recognition. Since hydrophobic compounds are known to partition into the lipid bilayer and accumulate in membranes, the “classical pump” model for the mechanism of multidrug transporter proteins has been challenged, and alternative models suggesting substrate recognition within the lipid bilayer have been proposed. Although much effort has been made to validate this concept, unambiguous evidence for direct drug extrusion from the plasma membrane has not been provided yet. Here we show a detailed on-line microscopic analysis of cellular extrusion of fluorescent anti-cancer drugs, mitoxantrone and pheophorbide A, by a key human multidrug transporter, ABCG2. Using the fully active GFP-tagged ABCG2 and exploiting the special character of mitoxantrone that gains fluorescence in the lipid environment, we were able to determine transporter-modulated drug concentrations separately in the plasma membrane and the intracellular compartments. Different kinetic models describing the various transport mechanisms were generated and the experimental data were analyzed using these models. On the basis of the kinetic analysis, drug extrusion from the cytoplasm can be excluded, thus, our results indicate that ABCG2 extrudes mitoxantrone directly from the plasma membrane.►Hydrophobic drugs, gaining fluorescence in lipids, are expelled by GFP-tagged ABCG2. ►GFP-tagged ABCG2 allows tracking down the position of the plasma membrane. ►MX concentration can be measured separately in the plasma membrane and cell interior. ►Transport kinetic models disprove drug extrusion from the cytoplasm. ►These models support that MX transported by ABCG2 directly from the plasma membrane.

Keywords: Abbreviations; ABC transporter; ATP-binding cassette transporter; ABCG2; G2 multidrug transporter (breast cancer resistance protein, MXR/BCRP/ABCP); GFP; green fluorescent protein; GFP-G2; ABCG2 protein tagged with GFP; Ko143 or KO; specific inhibitor of ABCG2; MDR1; multidrug resistance transporter 1 (ABCB1, Pgp, P-glycoprotein); MRP1; multidrug resistance-associated protein (ABCC1); MX; Mitoxantrone; Pheo; Pheophorbide A; RMSE; Root Mean Square Error; ROI; Region of InterestABCG2; mitoxantrone; ABC transporter; multidrug resistance; transport kinetic model; hydrophobic vacuum cleaner

Multiple stages of detergent-erythrocyte membrane interaction—A spin label study by Prete Paulo S.C. Preté; Cleyton C. Domingues; Nilce C. Meirelles; Sônia V.P. Malheiros; Goni Félix M. Goñi; Eneida de Paula; Shirley Schreier (pp. 164-170).

The various stages of the interaction between the detergent Triton X-100 (TTX-100) and membranes of whole red blood cells (RBC) were investigated in a broad range of detergent concentrations. The interaction was monitored by RBC hemolysis–assessed by release of intracellular hemoglobin (Hb) and inorganic phosphate–and by analysis of EPR spectra of a fatty acid spin probe intercalated in whole RBC suspensions, as well as pellets and supernatants obtained upon centrifugation of detergent-treated cells. Hemolysis finished at ca. 0.9mM TTX-100. Spectral analysis and calculation of order parameters ( S) indicated that a complex sequence of events takes place, and allowed the characterization of various structures formed in the different stages of detergent–membrane interaction. Upon reaching the end of cell lysis, essentially no pellet was detected, the remaining EPR signal being found almost entirely in the supernatants. Calculated order parameters revealed that whole RBC suspensions, pellets, and supernatants possessed a similar degree of molecular packing, which decreased to a small extent up to 2.5mM detergent. Between 3.2 and 10mM TTX-100, a steep decrease in S was observed for both whole RBC suspensions and supernatants. Above 10mM detergent, S decreased in a less pronounced manner and the EPR spectra approached that of pure TTX-100 micelles. The data were interpreted in terms of the following events: at the lower detergent concentrations, an increase in membrane permeability occurs; the end of hemolysis coincides with the lack of pellet upon centrifugation. Up to 2.5mM TTX-100 the supernatants consist of a (very likely) heterogeneous population of membrane fragments with molecular packing similar to that of whole cells. As the detergent concentration increases, mixed micelles are formed containing lipid and/or protein, approaching the packing found in pure TTX-100 micelles. This analysis is in agreement with the models proposed by Lasch (Biochim. Biophys Acta 1241 (1995) 269-292) and by Le Maire and coworkers (Biochim. Biophys. Acta 1508 (2000) 86-111).► A complex series of events takes place when increasing concentrations of TTX-100 interact with whole red blood cells. ► Detergent binding promoted an increase in permeability, leading to cell lysis. ► The molecular packing accessed by Electron Paramagnetic Resonance, in whole cell suspensions, pellets, and supernatants was very similar until higher detergent concentrations were added. ►Supernatants initially consisted of membrane fragments probably kept in suspension by the formation of edges by detergent molecules surrounding the borders of these fragments, but further detergent addition promoted membrane-to-micelle transition and the aggregates (containing high proportions of detergent) approached the molecular packing of pure TTX-100 micelles.

Keywords: Abbreviations; CMC; critical micelle concentration; EPR; electron paramagnetic resonance; Hb; hemoglobin; Ht; hematocrit; PBS; phosphate buffered saline; RBC; red blood cell; RH; percent relative hemolysis; 5-SASL; 5-doxyl stearic acid spin label; Triton X-100 or TTX-100; t-OctylphenoxypolyethoxyethanolErythrocyte; Membrane; Solubilization; Triton X-100; Hemolysis; EPR

A plausible mode of action of pseudin-2, an antimicrobial peptide from Pseudis paradoxa by Seong-Cheol Park; Jin-Young Kim; Chanyoung Jeong; Suyeon Yoo; Kyung-Soo Hahm; Yoonkyung Park (pp. 171-182).

The search for new antibiotic agents is continuous, reflecting the continuous emergence of antibiotic-resistant pathogens. Among the new agents are the antimicrobial peptides (AMPs), which have the potential to become a leading alternative to conventional antibiotics. Studies for the mechanisms of action of the naturally occurring parent peptides can provide the structural and functional information needed for the development of effective new antibiotic agents. We therefore characterized pseudin-2, an AMP isolated from the skin of the South American paradoxical frog Pseudis paradoxa. We found that pseudin-2 organized to an aggregated state in aqueous solution, but that it dissociated into monomers upon binding to lipopolysaccharide (LPS), even though it did not neutralize LPS in Gram-negative bacteria. In addition, pseudin-2 assumed an α-helical structure in the presence of biological membranes and formed pores in both bacterial and fungal membranes, through which it entered the cytoplasm and tightly bound to RNA. Thus, the potent antimicrobial activity of pseudin-2 likely results from both the formation of pores capable of collapsing the membrane potential and releasing intracellular materials and its inhibition of macromolecule synthesis through its binding to RNA.►We found that pseudin-2 organized to an aggregated state in aqueous solution, but that it dissociated into monomers upon binding to lipopolysaccharide (LPS), even though it did not neutralize LPS in Gram-negative bacteria. ►pseudin-2 assumed an α-helical structure in the presence of biological membranes and formed pores in both bacterial and fungal membranes, through which it entered the cytoplasm and tightly bound to RNA. ►The potent antimicrobial activity of pseudin-2 likely results from both the formation of pores capable of collapsing the membrane potential and releasing intracellular materials and its inhibition of macromolecule synthesis through its binding to RNA.

Keywords: Antimicrobial peptide; pseudin-2; Giant unilamellar vesicle; Toroidal pore; Depolarization; Penetration; RNA-binding

On the association of lipid rafts to the spectrin skeleton in human erythrocytes by Annarita Ciana; Cesare Achilli; Cesare Balduini; Giampaolo Minetti (pp. 183-190).

Lipid rafts are local inhomogeneities in the composition of the plasma membrane of living cells, that are enriched in sphingolipids and cholesterol in a liquid-ordered state, and proteins involved in receptor-mediated signalling. Interactions between lipid rafts and the cytoskeleton have been observed in various cell types. They are isolated as a fraction of the plasma membrane that resists solubilization by nonionic detergents at 4°C (detergent-resistant membranes, DRMs). We have previously described that DRMs are anchored to the spectrin-based membrane skeleton in human erythrocytes and can be released by increasing the pH and ionic strength of the solubilization medium with sodium carbonate. It was unexplained why this carbonate treatment was necessary and why this requirement was not reported by other workers in this area. We show here that when contaminating leukocytes are present in erythrocyte preparations that are subjected to detergent treatment, the isolation of DRMs can occur without the requirement for carbonate treatment. This is due to the uncontrolled breakdown of erythrocyte membrane components by hydrolases that are released from contaminating neutrophils that lead to proteolytic disruption of the supramolecular assembly of the membrane skeleton. Results presented here corroborate the concept that DRMs are anchored to the membrane skeleton through electrostatic interactions that most likely involve the spectrin molecule.►Erythrocyte lipid rafts are tightly anchored to the spectrin membrane skeleton. ►Lipid raft binding to membrane skeleton is sensitive to pH and ionic strength. ►In erythrocyte samples contaminated by neutrophils lipid rafts are easily isolated. ►Proteases from contaminating neutrophils degrade spectrin and release lipid rafts. ►Lipid raft binding is affected by proteolysis of spectrin, not protein 4.1 or ankyrin.

Keywords: Detergent-resistant membranes; Polymorphonuclear granulocytes; Neutrophil elastase; Cathepsin G; Eglin; DFP; p55; Carbonate; Membrane skeleton; Gelatin zymography

On the association of lipid rafts to the spectrin skeleton in human erythrocytes by Annarita Ciana; Cesare Achilli; Cesare Balduini; Giampaolo Minetti (pp. 183-190).

Keywords: Detergent-resistant membranes; Polymorphonuclear granulocytes; Neutrophil elastase; Cathepsin G; Eglin; DFP; p55; Carbonate; Membrane skeleton; Gelatin zymography

On the association of lipid rafts to the spectrin skeleton in human erythrocytes by Annarita Ciana; Cesare Achilli; Cesare Balduini; Giampaolo Minetti (pp. 183-190).

Keywords: Detergent-resistant membranes; Polymorphonuclear granulocytes; Neutrophil elastase; Cathepsin G; Eglin; DFP; p55; Carbonate; Membrane skeleton; Gelatin zymography

On the association of lipid rafts to the spectrin skeleton in human erythrocytes by Annarita Ciana; Cesare Achilli; Cesare Balduini; Giampaolo Minetti (pp. 183-190).

Keywords: Detergent-resistant membranes; Polymorphonuclear granulocytes; Neutrophil elastase; Cathepsin G; Eglin; DFP; p55; Carbonate; Membrane skeleton; Gelatin zymography

Kinetics of degradation of dipalmitoylphosphatidylcholine (DPPC) bilayers as a result of vipoxin phospholipase A₂ activity: An atomic force microscopy (AFM) approach by Konstantin Balashev; Vasil Atanasov; Mariana Mitewa; Svetla Petrova; Bjornholm Thomas Bjørnholm (pp. 191-198).

In this paper we used AFM as an analytical tool to visualize the degradation of a phospholipid bilayer undergoing hydrolysis of the vipoxin's PLA₂. We obtained time series images during the degradation process of supported 1, 2-dipalmitoylphosphatidylcholine (DPPC) bilayers and evaluated the occurrence and the growth rate of the bilayer defects. The special resolution of the AFM images allowed us to measure the area and the perimeter length of these defects and to draw conclusions about the kinetics of the enzyme reaction. Moreover, we also report for some unique characteristics discovered during the vipoxin's PLA₂ action. Experimentally for the first time, we observed the appearance and the growth of three-dimensional (3D), crystal-like structures within the formed defects of the degraded bilayer. In an effort to explain their nature, we applied bearing image analysis to estimate the volume of these crystals and we found that their growth rate follows a similar kinetic pattern as the degradation rate of the supported bilayer.► A degradation of DPPC bilayers as a result of vipoxin’s PLA2 was visualized by AFM. ► Time-series AFM images were obtained during the degradation process. ► The area and the perimeter length of the occurred bilayer defects were measured. ► The appearance and the growth of 3D-crystal-like structures were observed. ► The volume and growth rate of 3D-structures were estimated by applied image analysis.

Keywords: Vipoxin; PLA; ₂; DPPC bilayers; Enzyme hydrolysis; Atomic force microscopy (AFM)

The influence of additives on the nanoscopic dynamics of the phospholipid dimyristoylphosphatidylcholine by Sebastian Busch; Tobias Unruh (pp. 199-208).

The influence of additives on the molecular dynamics of the phospholipid dimyristoylphosphatidylcholine (DMPC) in its fully hydrated liquid crystalline phase was studied. Quasielastic neutron scattering (QENS) was used to detect motions with dimensions of some Ångstroms on two different time scales, namely 60ps and 900ps. The effects of myristic acid, farnesol, cholesterol, and sodium glycocholate could consistently be explained on the basis of collective, flow-like motions of the phospholipid molecules. The influence of the additives on these motions was explained by packing effects, corresponding to the reduction of free volume. Cholesterol was found to decrease the mobility of DMPC seen on the 900ps time scale with increasing cholesterol content. In contrast, all other studied additives have no significant effect on the mobility.►determination of the pico- to nanosecond dynamics of phospholipids by QENS ►mechanistic explanation for the influence of additives on phospholipid dynamics ►effects of farnesol, myristic acid, NaGC, and cholesterol on phospholipid dynamics ►comparison of our QENS results with NMR, MD, FCS, and FRAP.

Keywords: DMPC; Dynamics; QENS; Myristic acid; Farnesol; Cholesterol; Sodium Glycocholate; Dynamic Heterogeneity

The influence of additives on the nanoscopic dynamics of the phospholipid dimyristoylphosphatidylcholine by Sebastian Busch; Tobias Unruh (pp. 199-208).

Keywords: DMPC; Dynamics; QENS; Myristic acid; Farnesol; Cholesterol; Sodium Glycocholate; Dynamic Heterogeneity

The influence of additives on the nanoscopic dynamics of the phospholipid dimyristoylphosphatidylcholine by Sebastian Busch; Tobias Unruh (pp. 199-208).

Keywords: DMPC; Dynamics; QENS; Myristic acid; Farnesol; Cholesterol; Sodium Glycocholate; Dynamic Heterogeneity

The influence of additives on the nanoscopic dynamics of the phospholipid dimyristoylphosphatidylcholine by Sebastian Busch; Tobias Unruh (pp. 199-208).

Keywords: DMPC; Dynamics; QENS; Myristic acid; Farnesol; Cholesterol; Sodium Glycocholate; Dynamic Heterogeneity

Kinetic analysis of the interaction between poly(amidoamine) dendrimers and model lipid membranes by Venkataswarup Tiriveedhi; Kelly M. Kitchens; Kerrick J. Nevels; Hamidreza Ghandehari; Peter Butko (pp. 209-218).

We used fluorescence spectroscopy and surface tensiometry to study the interaction between low-generation (G1 and G4) poly(amidoamine) (PAMAM) dendrimers, potential vehicles for intracellular drug delivery, and model lipid bilayers. Membrane association of fluorescently labeled dendrimers, measured by fluorescence anisotropy, increased with increasing size of the dendrimer and with increasing negative charge density in the membrane, indicating the electrostatic nature of the interaction. When the membrane was doped with pyrene-labeled phosphatidyl glycerol (pyrene-PG), pyrene excimer fluorescence demonstrated a dendrimer-induced selective aggregation of negatively charged lipids when the membrane was in the liquid crystalline state. A nonlinear Stern–Volmer quenching of dendrimer fluorescence with cobalt bromide suggested a dendrimer-induced aggregation of lipid vesicles, which increased with the dendrimer's generation number. Surface tensiometry measurements showed that dendrimers penetrated into the lipid monolayer only at subphysiologic surface pressures (<30mN/m). We conclude that the low-generation PAMAM dendrimers associate with lipid membranes predominantly electrostatically, without significantly compromising the bilayer integrity. They bind stronger to membranes with higher fluidity and lower surface pressure, which are characteristic of rapidly dividing cells.► PAMAM dendrimers insert into lipid monolayers at subphysiologic surface pressures. ► PAMAM dendrimers bind stronger to fluid membranes than to the rigid ones. ► PAMAM dendrimers induce aggregation of lipid vesicles.

Keywords: Cell-penetrating molecules; Fluorescence spectroscopy; Membrane fluidity; PAMAM dendrimers; Quenching; Resonance energy transfer

Tryptophan orientations in membrane-bound gramicidin and melittin—a comparative linear dichroism study on transmembrane and surface-bound peptides by Frida R. Svensson; Per Lincoln; Norden Bengt Nordén; Esbjorner Elin K. Esbjörner (pp. 219-228).

In the search for methods to study structure and function of membrane-associated proteins and peptides flow linear dichroism, LD, spectroscopy has emerged as a promising technique. Using shear-aligned lipid vesicles, conformations and binding geometries of membrane-bound bio-macromolecules can be assessed. Here we investigate anchoring properties and specific orientations of tryptophan relative to the peptide backbone and to the membrane normal for the model peptides gramicidin and melittin. We have monitored the conformational change associated with the refolding of non-channel gramicidin into its channel form, and quantitatively determined the average orientations of its tryptophan transition moments, suggesting that these residues adopt a well-defined orientation at the membrane interface. An important conclusion regards the structural variation of gramicidin between these two distinct transmembrane forms. Whilst circular dichroism (CD) spectra, as has been reported before, vary strongly between the two forms suggesting their structures might be quite different, the LD results clearly evidence both the peptide backbone orientation and tryptophan side-chain positioning to be very similar. The latter are oriented in accord with what is expected from their role to anchor peptide termini to the membrane surface. The variations in CD could be due to, the in LD observed, minor shifts in mutual orientation and distance between neighbouring tryptophans sensitively determining their exciton interactions. Our data dispute that the non-channel form of membrane-bound gramicidin would be any of the intertwined forms often observed in crystal as the positioning of tryptophans along the peptide axis would not be compatible with the strong interfacial positioning observed here. The general role of tryptophans as interfacial anchors is further assessed for melittin whose conformation shows considerable angular spread, consistent with a carpet model of its mechanism for induced membrane leakage, and a predominantly surface-aligned membrane orientation governed by amphipathic interactions.►LD probes structural details of gramicidin non-channel/channel conversion. ►Membrane positioning of tryptophans in gramicidin is driven by interfacial forces. ►Melittin orients predominantly along the surface of neutral and acidic membranes.

Keywords: Abbreviations; CD; circular dichroism; DOPC; 1,2-dioleoyl-; sn; -glycero-3-phosphocholine; DOPG; 1,2-dioleoyl-; sn; -glycero-3-phosphoglycerol; LD; linear dichroism; LD; ^r; reduced linear dichroism; LUV; Large unilamellar lipid vesicle; NMR; nuclear magnetic resonance; TEM; Thulstrup–Eggers–Michl's methodFlow linear dichroism spectroscopy; Transition moment orientation; Ion-channel; Membrane-active peptide; Membrane interface

The intermembrane ceramide transport catalyzed by CERT is sensitive to the lipid environment by Jessica Tuuf; Matti A. Kjellberg; Julian G. Molotkovsky; Kentaro Hanada; Peter Mattjus (pp. 229-235).

The in vitro activity of the ceramide transporter, CERT has been studied using a fluorescence assay. CERT is responsible for the in vivo non-vesicular trafficking of ceramide between the endoplasmic reticulum and Golgi. In this study we have examined how the membrane environment surrounding the ceramide substrate, the membrane packing density and the membrane charge, are affecting the ceramide transfer activity. To examine this we have used an anthrylvinyl-labeled ceramide analogue. We found that if ceramide is in a tightly packed environment such as in sphingomyelin or dipalmitoylphosphatidylcholine containing membranes, the CERT transfer activity is markedly reduced. Ceramide in fluid membranes on the other hand are available for CERT mediated transfer. CERT also favors membranes that contain phosphatidylinositol 4-monophospate, due to its binding capacity of the pleckstrin homology domain towards phosphatidylinositol 4-monophospate. From this study we conclude that the membrane matrix surrounding ceramide, that is ceramide miscibility, is largely affecting the transfer activity of CERT.►Ceramide in fluid membranes is available for CERT mediated transfer. ►Ceramide transfer from tightly packed environment such as in sphingomyelin is markedly reduced. ►CERT favors membranes that contain phosphatidylinositol 4-monophospate, PI4P. ►The membrane matrix surrounding ceramide is largely affecting the transfer activity of CERT.

Keywords: Abbreviations; AV-Cer; N; -[12-(9-anthryl)-11; E; -dodecenoyl]sphingosine; CERT; ceramide transfer protein; DiO-C; ₁₆; 3,3′-dihexadecyloxacarbocyanine perchlorate; DOPC; 1,2-dioleoyl-; sn; -glycero-3-phosphocholine; DPPC; 1,2-dipalmitoyl-; sn-; glycero-3-phosphocholine; ER; endoplasmic reticulum; FFAT; two phenylalanines in an acidic tract; GLTP; glycolipid transfer protein; GlcCer; glucosylceramide; GPBP; Goodpasture antigen-binding protein; PH; pleckstrin homology; PI; phosphatidylinositol; PI4P; phosphatidylinositol 4-monophosphate; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; P-SM; palmitoyl-sphingomyelin; RET; resonance energy transfer; SM; sphingomyelin; START; steroidogenic acute regulatory protein-related lipid transfer; VAP; VAMP-associated proteinCERT; Ceramide; Resonance energy transfer; Substrate specificity; Phosphatidylinositol 4-monophosphate; Lipid transfer

The intermembrane ceramide transport catalyzed by CERT is sensitive to the lipid environment by Jessica Tuuf; Matti A. Kjellberg; Julian G. Molotkovsky; Kentaro Hanada; Peter Mattjus (pp. 229-235).

Liposome-incorporated DHA increases neuronal survival by enhancing non-amyloidogenic APP processing by Gunter P. Eckert; Steffi Chang; Janett Eckmann; Ekaterini Copanaki; Stephanie Hagl; Uwe Hener; Muller Walter E. Müller; Kogel Donat Kögel (pp. 236-243).

The fluidity of neuronal membranes plays a pivotal role in brain aging and neurodegeneration. In this study, we investigated the role of the omega-3 fatty acid docosahexaenoic acid (DHA) in modulation of membrane fluidity, APP processing, and protection from cytotoxic stress. To this end, we applied unilamellar transfer liposomes, which provided protection from oxidation and effective incorporation of DHA into cell membranes. Liposomes transferring docosanoic acid (DA), the completely saturated form of DHA, to the cell cultures served as controls. In HEK-APP cells, DHA significantly increased membrane fluidity and non-amyloidogenic processing of APP, leading to enhanced secretion of sAPPα. This enhanced secretion of sAPPα was associated with substantial protection against apoptosis induced by ER Ca²⁺ store depletion. sAPPα-containing supernatants obtained from HEK-APP cells exerted similar protective effects as DHA in neuronal PC12 cells and HEK293 control cells. Correlating to further increased sAPPα levels, supernatants obtained from DHA-treated HEK-APP cells enhanced protection, whereas supernatants obtained from DHA-treated HEK293 control cells did not inhibit apoptosis, likely due to the low expression of endogenous APP and negligible sAPPα secretion in these cells. Further experiments with the small molecule inhibitors LY294002 and SP600125 indicated that sAPPα-induced cytoprotection relied on activation of the anti-apoptotic PI3K/Akt pathway and inhibition of the stress-triggered JNK signaling pathway in PC12 cells. Our data suggest that liposomal DHA is able to restore or maintain physiological membrane properties, which are required for neuroprotective sAPPα secretion and autocrine modulation of neuronal survival.► Liposomal DHA increases the fluidity of cell membranes. ► DHA enhances non-amyloidogenic processing of APP. ► Enhanced secretion of sAPPα is associated with autocrine protection against apoptosis induced by ER Ca²⁺ store depletion. ► sAPPα-dependent cytoprotection relies on activation of the anti-apoptotic PI3K/Akt pathway and inhibition of the stress-triggered JNK signaling pathway.

Keywords: Abbreviations; ADAM; a disintegrin and metalloprotease; AD; Alzheimer's disease; APP; amyloid precursor protein; ATP; adenosine-triphosphate; BSA; bovine serum albumin; CTF; C-terminal fragment; DA; docosanoic acid; DHA; docosahexaenoic acid; DMEM; Dulbecco's modified Eagle's medium; JNK; c-Jun N-terminal kinase; LS; liposomes; MMP; mitochondrial membrane potential; MLV; multilamellar vesicles; NPD1; neuroprotectin D1; PC; phosphaditylcholine; PUFA; polyunsaturated fatty acid; SERCA; sarco/endoplasmic reticulum Ca; ²⁺; -ATPase; TBS; Tris-buffered salineMembrane fluidity; Omega-3 fatty acids; Stress signaling; Alpha secretase; Apoptosis

Effects of peptide hydrophobicity on its incorporation in phospholipid membranes — an NMR and ellipsometry study by Oradd Greger Orädd; Artur Schmidtchen; Martin Malmsten (pp. 244-252).

Effects of peptide hydrophobicity on lipid membrane binding, incorporation, and defect formation was investigated for variants of the complement-derived antimicrobial peptide CNY21 (CNYITELRRQHARASHLGLAR), in anionic 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE)/1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) and zwitterionic 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) membranes. Using a method combination of, e.g., ellipsometry, CD, and fluorescence spectroscopy, it was shown that peptide adsorption, as well as peptide-induced liposome leakage and bactericidal potency against Escherichia coli and Pseudomonas aeruginosa, was promoted by increasing the hydrophobicity of CNY21 through either substituting the two histidines (H) in CNY21 with more hydrophobic leucine (L) residues, or end-tagging with tritryptophan (WWW). Fluorescence spectroscopy revealed that both CNY21WWW and the WWW tripeptide localized to the polar headgroup region of these phospholipid membranes. Deuterium NMR experiments on macroscopically oriented membranes containing fully (palmitoyl) deuterated POPC (POPC-d₃₁) demonstrated that both CNY21L and CNY21WWW induced disordering of the lipid membrane. In contrast, for cholesterol-supplemented POPC-d₃₁ bilayers, peptide-induced disordering was less pronounced in the case of CNY21L, indicating that the peptide is unable to partition to the interior of the lipid membrane in the presence of cholesterol. CNY21WWW, on the other hand, retained its membrane-disordering effect also for cholesterol-supplemented POPC-d₃₁. These findings were supported by pulsed field gradient NMR experiments where the lateral lipid diffusion was determined in the absence and presence of peptides. Overall, the results provide some mechanistic understanding to previously observed effects of peptide hydrophobization through point mutations and end-tagging, particularly so for complement-based antimicrobial peptides.►Effects of different hydrophobic modifications in CNY21 antimicrobial peptide were investigated by NMR and ellipsometry. ►Both hydrophobic modifications promote peptide binding to membranes, membrane disruption, and bacterial killing. ►CNY21L penetrates into the membrane interior in the absence of cholesterol, but is unable to do so in the presence of cholesterol. ►CNY21WWW displays relatively shallow localization in the membrane irrespective of cholesterol, but is not excluded from the membrane by cholesterol.

Keywords: AMP; Antimicrobial peptide; Ellipsometry; Liposome; Membrane; NMR

Effects of peptide hydrophobicity on its incorporation in phospholipid membranes — an NMR and ellipsometry study by Oradd Greger Orädd; Artur Schmidtchen; Martin Malmsten (pp. 244-252).

Keywords: AMP; Antimicrobial peptide; Ellipsometry; Liposome; Membrane; NMR

Effects of peptide hydrophobicity on its incorporation in phospholipid membranes — an NMR and ellipsometry study by Oradd Greger Orädd; Artur Schmidtchen; Martin Malmsten (pp. 244-252).

Keywords: AMP; Antimicrobial peptide; Ellipsometry; Liposome; Membrane; NMR

Effects of peptide hydrophobicity on its incorporation in phospholipid membranes — an NMR and ellipsometry study by Oradd Greger Orädd; Artur Schmidtchen; Martin Malmsten (pp. 244-252).

Keywords: AMP; Antimicrobial peptide; Ellipsometry; Liposome; Membrane; NMR

Specific inhibition of a pathogenic receptor tyrosine kinase by its transmembrane domain by Lijuan He; Nadia Shobnam; Kalina Hristova (pp. 253-259).

The transmembrane (TM) domains of receptor tyrosine kinases (RTKs) are believed to be important players in RTK signal transduction. However, the degree of specificity and promiscuity of RTK TM domain lateral interactions in mammalian membranes has not been assessed in detail in the literature. A technique to probe the occurrence of interactions between TM domains and their biological significance is to evaluate the propensity for formation of heterodimers of a full-length RTK and its TM domain. Here we examine if the inhibition of two RTK pathogenic mutants, Neu/V664E and FGFR3/A391E, can be achieved by the TM domains of Neu, Neu/V664E, FGFR3 and FGFR3/A391E. We show that the TM domain of Neu/V664E specifically inhibits the phosphorylation of full-length Neu/V664E, while the wild-type Neu TM domain does not. In addition, Neu/V664E TM domain does not affect the phosphorylation levels of full-length FGFR3/A391E. The results suggest that TM domain peptides could be exploited in the future for the development of specific inhibitors of mutant RTKs.►The TM domain of Neu/V664E specifically inhibits the phosphorylation of full-length Neu/V664E. ►The TM domain of wild-type Neu does not inhibit the phosphorylation of full-length Neu/V664E. ►Neu/V664E TM domain does not affect the phosphorylation of full-length FGFR3/A391E.

Keywords: Receptor tyrosine kinase; Signaling; Transmembrane domain

Specific inhibition of a pathogenic receptor tyrosine kinase by its transmembrane domain by Lijuan He; Nadia Shobnam; Kalina Hristova (pp. 253-259).

Keywords: Receptor tyrosine kinase; Signaling; Transmembrane domain

Specific inhibition of a pathogenic receptor tyrosine kinase by its transmembrane domain by Lijuan He; Nadia Shobnam; Kalina Hristova (pp. 253-259).

Keywords: Receptor tyrosine kinase; Signaling; Transmembrane domain

Specific inhibition of a pathogenic receptor tyrosine kinase by its transmembrane domain by Lijuan He; Nadia Shobnam; Kalina Hristova (pp. 253-259).

Keywords: Receptor tyrosine kinase; Signaling; Transmembrane domain

Amino acid derivatives are substrates or non-transported inhibitors of the amino acid transporter PAT2 (slc36a2) by Noel Edwards; Catriona M.H. Anderson; Kelly M. Gatfield; Mark P. Jevons; Vadivel Ganapathy; David T. Thwaites (pp. 260-270).

The H⁺-coupled amino acid transporter PAT2 (SLC36A2) transports the amino acids proline, glycine, alanine and hydroxyproline. A physiological role played by PAT2 in amino acid reabsorption in the renal proximal tubule is demonstrated by mutations in SLC36A2 that lead to an iminoglycinuric phenotype (imino acid and glycine uria) in humans. A number of proline, GABA and tryptophan derivatives were examined to determine if they function either as transported substrates or non-transported inhibitors of PAT2. The compounds were investigated following heterologous expression of rat PAT2 in Xenopus laevis oocytes. PAT2 function was characterised by: radiotracer uptake and competition (cis-inhibition) studies; radiotracer efflux and trans-stimulation; and measurement of substrate-induced positive inward current by two-electrode voltage-clamp. In general, the proline derivatives appeared to be transported substrates and the relative ability to induce current flow was closely related to the inhibitory effects on PAT2-mediatedl-[³H]proline uptake. In contrast, certain heterocyclic GABA derivatives (e.g.l-pipecolic acid) were translocated only slowly. Finally, the tryptophan derivatives inhibited PAT2 function but did not undergo transport.l-Proline uptake was inhibited by 5-hydroxy-l-tryptophan (IC₅₀ 1.6±0.4mM), α-methyl-d,l-tryptophan (3.5±1.5mM),l-tryptophan, 1-methyl-l-tryptophan and indole-3-propionic acid. Although neither 5-hydroxy-l-tryptophan nor α-methyl-d,l-tryptophan were able to elicit inward current in PAT2-expressing oocytes both reduced the current evoked byl-proline. 5-Hydroxy-l-tryptophan and α-methyl-d,l-tryptophan were unable to trans-stimulatel-proline efflux from PAT2-expressing oocytes, confirming that the two compounds act as non-transported blockers of PAT2. These two tryptophan derivatives should prove valuable experimental tools in future investigations of the physiological roles of PAT2.►PAT2 (SLC36A2) transports proline, glycine, alanine and hydroxyproline. ►Heterocyclic proline derivatives are transported substrates. ►Heterocyclic GABA derivatives are translocated slowly. ►Tryptophan derivatives (e.g. α-methyl-d,l-tryptophan) are non-transported inhibitors.

Keywords: Abbreviations; 1-ACHC; 1-aminocyclohexanecarboxylic acid; C2-ACHC; cis-2-aminocyclohexanecarboxylic acid; C4-ACHC; cis-4-aminocyclohexanecarboxylic acid; CHDP; cis-4-hydroxy-; d; -proline; CHLP; cis-4-hydroxy-; l; -proline; DHP; 3,4-dehydro-; d; ,; l; -proline; Guv; guvacine; 3-IPA; indole-3-propionic acid; Isoguv; isoguvacine; Isonip; isonipecotic acid; Me-Trp; α-methyl-; d; ,; l; -tryptophan; 1-Me-; d; -Trp; 1-methyl-; d; -tryptophan; 1-Me-; l; -Trp; 1-methyl-; l; -tryptophan; Nip; nipecotic acid; OH-Trp; 5-hydroxy-; l; -tryptophan; d; -Pip; d; -pipecolic acid; l; -Pip; l; -pipecolic acid; Sar; sarcosine; SLC; solute carrier family; T2-ACHC; trans-2-aminocyclohexanecarboxylic acid; TCA; l; -thiaproline (; l; -thiazolidine-4-carboxylic acid); T4HP; trans-4-hydroxy-; l; -proline; T3HP; trans-3-hydroxy-; l; -proline; Trypt; tryptaminePAT2; Amino acid transport; SLC36; PAT1; Tryptophan; Inhibitor

α-Hemolysin pore formation into a supported phospholipid bilayer using cell-free expression by Jerome Chalmeau; Nadezda Monina; Jonghyeon Shin; Christophe Vieu; Vincent Noireaux (pp. 271-278).

Cell-free protein synthesis is becoming a serious alternative to cell-based protein expression. Cell-free systems can deliver large amounts of cytoplasmic recombinant proteins after a few hours of incubation. Recent studies have shown that membrane proteins can be also expressed in cell-free reactions and directly inserted into phospholipid membranes. In this work, we present a quantitative method to study in real time the concurrent cell-free expression and insertion of membrane proteins into phospholipid bilayers. The pore-forming protein α-hemolysin, fused to the reporter protein eGFP, was used as a model of membrane protein. Cell-free expression of the toxin in solution and inside large synthetic phospholipid vesicles was measured by fluorometry and fluorescence microscopy respectively. A quartz crystal microbalance with dissipation was used to characterize the interaction of the protein with a supported phospholipid bilayer. The cell-free reaction was directly incubated onto the bilayer inside the microbalance chamber while the frequency and the dissipation signals were monitored. The presence of pores in the phospholipid bilayer was confirmed by atomic force microscopy. A model is presented which describes the kinetics of adsorption of the expressed protein on the phospholipid bilayer. The combination of cell-free expression, fluorescence microscopy and quartz crystal microbalance-dissipation is a new quantitative approach to study the interaction of membrane proteins with phospholipid bilayers.►The toxin α-hemolysin-eGFP is synthesized in a cell-free system. ►The toxin is expressed in vesicles and on a supported phospholipid bilayer. ►α-hemolysin-eGFP pore formation is studied with a quartz crystal microbalance. ►Adsorption kinetics of the toxin onto the membrane is described. ►Pores are visualized by atomic force microscopy.

Keywords: Abbreviations; QCM-D; quartz crystal microbalance-dissipation; SPB; supported phospholipid bilayer; AFM; atomic force microscopy; eGFP; enhanced Green Fluorescent Protein; αHL-eGFP; α-hemolysin-eGFP fusion protein; PBS; phosphate buffered saline; EggPC; l; -α-lysophosphatidylcholine; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphatidylcholine; Δ; f; frequency shift; Δ; D; dissipation shiftCell-free expression; α-hemolysin; Quartz crystal microbalance-dissipation; Fluorescence microscopy

α-Hemolysin pore formation into a supported phospholipid bilayer using cell-free expression by Jerome Chalmeau; Nadezda Monina; Jonghyeon Shin; Christophe Vieu; Vincent Noireaux (pp. 271-278).

Effects of bilayer composition and physical properties on the phospholipase C and sphingomyelinase activities of Clostridium perfringens α-toxin by Patricia Urbina; Flores-Diaz Marietta Flores-Díaz; Alape-Giron Alberto Alape-Girón; Alicia Alonso; Goni Félix M. Goñi (pp. 279-286).

α-Toxin, a major determinant of Clostridium perfringens toxicity, exhibits both phospholipase C and sphingomyelinase activities. Our studies with large unilamellar vesicles containing a variety of lipid mixtures reveal that both lipase activities are enhanced by cholesterol and by lipids with an intrinsic negative curvature, e.g. phosphatidylethanolamine. Conversely lysophospholipids, that possess a positive intrinsic curvature, inhibit the α-toxin lipase activities. Phospholipids with a net negative charge do not exert any major effect on the lipase activities, and the same lack of effect is seen with the lysosomal lipid bis (monoacylglycero) phosphate. Ganglioside GT1b has a clear inhibitory effect, while the monosialic ganglioside GM3 is virtually ineffectual even when incorporated at 6mol % in the vesicles. The length of the lag periods appears to be inversely related to the maximum (post-lag) enzyme activities. Moreover, and particularly in the presence of cholesterol, lag times increase with pH. Both lipase activities are sensitive to vesicle size, but in opposite ways: while phospholipase C is higher with larger vesicles, sphingomyelinase activity is lower. The combination of our results with previous structural studies suggests that α-toxin lipase activities have distinct, but partially overlapping and interacting active sites.► α-Toxin, a major determinant of Clostridium perfringens toxicity, exhibits both phospholipase C and sphingomyelinase activities. ► Both activities are enhanced by cholesterol and by phospholipids with intrinsic negative curvatures, and inhibited by lipids with positive intrinsic curvatures. ► Phospholipids with a net negative charge in the headgroup do not exert any marked effects on the lipase activities. ► Sphingomyelinase activity decreases with increasing vesicle diameter, while phospholipase C activity increases with increasing vesicle size. ► The two lipase activities in the toxin appear to have distinct, but partially overlapping and interacting active sites.

Keywords: Abbreviations; BMP; bis (monoacylglycero)phosphate; BSA; bovine serum albumin; Chol; cholesterol; DOPA; dioleoyl phosphatidic acid; DOPC; dioleoyl phosphatidylcholine; DOPE; dioleoyl phosphatidylethanolamine; DOPG; dioleoyl phosphatidylglycerol; DOPS; dioleoyl phosphatidylserine; LBPA; lysobisphosphatidic acid; PI; phosphatidylinositol; PLC; phospholipase C; SMase; sphingomyelinasePhospholipase C; Sphingomyelinase; Cholesterol; Lipid curvature; Lipid negative charge; Lysosomal lipids; Clostridium perfringens

The prediction and characterization of YshA, an unknown outer-membrane protein from Salmonella typhimurium by Thomas C. Freeman Jr.; Samuel J. Landry; William C. Wimley (pp. 287-297).

We have developed an effective pathway for the prediction and characterization of novel transmembrane β-barrel proteins. The Freeman–Wimley algorithm, which is a highly accurate prediction method based on the physicochemical properties of experimentally characterized transmembrane β barrel (TMBB) structures, was used to predict TMBBs in the genome of Salmonella typhimurium LT2. The previously uncharacterized product of gene yshA was tested as a model for validating the algorithm. YshA is a highly conserved 230-residue protein that is predicted to have 10 transmembrane β-strands and an N-terminal signal sequence. All of the physicochemical and spectroscopic properties exhibited by YshA are consistent with the prediction that it is a TMBB. Specifically, recombinant YshA localizes to the outer membrane when expressed in Escherichia coli; YshA has a β-sheet-rich secondary structure with stable tertiary contacts in the presence of detergent micelles or when reconstituted into a lipid bilayer. When in a lipid bilayer, YshA forms a membrane-spanning pore with an effective radius of ~0.7nm. Taken together, these data substantiate the predictions made by the Freeman–Wimley algorithm by showing that YshA is a TMBB protein.► Outer-membrane protein genomics and proteomics of Salmonella typhimurium. ► Ysha/OmpL a conserved, putative outer-membrane protein of unknown function. ► Biophysical characterization demonstrates refolding in detergents and bilayers. ► Outer-membrane protein identification algorithm is validated.

Keywords: Salmonella; Beta barrel; Outer-membrane protein; Structure prediction

The prediction and characterization of YshA, an unknown outer-membrane protein from Salmonella typhimurium by Thomas C. Freeman Jr.; Samuel J. Landry; William C. Wimley (pp. 287-297).

Keywords: Salmonella; Beta barrel; Outer-membrane protein; Structure prediction

The prediction and characterization of YshA, an unknown outer-membrane protein from Salmonella typhimurium by Thomas C. Freeman Jr.; Samuel J. Landry; William C. Wimley (pp. 287-297).

Keywords: Salmonella; Beta barrel; Outer-membrane protein; Structure prediction

The prediction and characterization of YshA, an unknown outer-membrane protein from Salmonella typhimurium by Thomas C. Freeman Jr.; Samuel J. Landry; William C. Wimley (pp. 287-297).

Keywords: Salmonella; Beta barrel; Outer-membrane protein; Structure prediction

Laurdan and di-4-ANEPPDHQ do not respond to membrane-inserted peptides and are good probes for lipid packing by Jelena Dinic; Biverstahl Henrik Biverståhl; Maler Lena Mäler; Ingela Parmryd (pp. 298-306).

Laurdan and di-4-ANEPPDHQ are used as probes for membrane order, with a blue shift in emission for membranes in liquid-ordered (lo) phase relative to membranes in liquid-disordered (ld) phase. Their use as membrane order probes requires that their spectral shifts are unaffected by membrane proteins, which we have examined by using membrane inserting peptides and large unilamellar vesicles (LUVs). The transmembrane polypeptides, mastoparan and bovine prion protein-derived peptide (bPrPp), were added to LUVs of either lo or ld phase, up to 1:10 peptide/total lipid ratio. The excitation and emission spectra of laurdan and di-4-ANEPPDHQ in both lipid phases were unaltered by peptide addition. The integrity and size distribution of the LUVs upon addition of the polypeptides were determined by dynamic light scattering. The insertion efficiency of the polypeptides into LUVs was determined by measuring their secondary structure by circular dichroism. Mastoparan had an α-helical and bPrPp a β-strand conformation compatible with insertion into the lipid bilayer. Our results suggest that the presence of proteins in biological membranes does not influence the spectra of laurdan and di-4-ANEPPDHQ, supporting that the dyes are appropriate probes for assessing lipid order in cells.►Membrane-inserted peptides do not affect the lo to ld spectral shift of laurdan. ►Membrane-inserted peptides do not affect the lo to ld spectral shift of di-4-ANEPPDHQ. ►Neither α-helical nor β-structure peptides affected the spectra of laurdan. Neither α-helical nor β-structure peptides affected the spectra of di-4-ANEPPDHQ. ►LUVs retained their integrity at high concentrations of membrane inserted peptides. ►Laurdan and di-4-ANEPPDHQ are appropriate for assessing lipid order in cells.

Keywords: Abbreviations; bPrPp; bovine prion protein-derived peptide; DLS; dynamic light scattering; DOPC; 1,2-dioleoyl-; sn; -glycero-3-phosphocholine; DPPG; 1,2-dihexadecanoyl-; sn; -glycero-3-phospho-(1'-rac-glycerol); GP; generalized polarization; GUVs; giant unilamellar vesicles; LUVs; large unilamellar vesicles; laurdan; 6-dodecanoyl-2-dimethyl-aminonaphthalene; ld; liquid disordered; lo; liquid ordered; PBS; phosphate-buffered salinedi-4-ANEPPDHQ; Laurdan; ld phase; Lipid rafts; lo phase; Membrane order

Correlated AFM and NanoSIMS imaging to probe cholesterol-induced changes in phase behavior and non-ideal mixing in ternary lipid membranes by Christopher R. Anderton; Kaiyan Lou; Peter K. Weber; Ian D. Hutcheon; Mary L. Kraft (pp. 307-315).

Cholesterol is believed to be an important component in compositionally distinct lipid domains in the cellular plasma membrane, which are referred to as lipid rafts. Insight into how cholesterol influences the interactions that contribute to plasma membrane organization can be acquired from model lipid membranes. Here we characterize the lipid mixing and phase behavior exhibited by¹⁵N-dilaurolyphosphatidycholine (¹⁵N-DLPC)/deuterated distearoylphosphatiylcholine (D₇₀-DSPC) membranes with various amounts of cholesterol (0, 3, 7, 15 or 19mol%) at room temperature. The microstructures and compositions of individual membrane domains were determined by imaging the same membrane locations with both atomic force microscopy (AFM) and high-resolution secondary ion mass spectrometry (SIMS) performed with a Cameca NanoSIMS 50. As the cholesterol composition increased from 0 to 19mol%, the circular ordered domains became more elongated, and the amount of¹⁵N-DLPC in the gel-phase domains remained constant at 6–7mol%. Individual and micron-sized clusters of nanoscopic domains enriched in D₇₀-DSPC were abundant in the 19mol% cholesterol membrane. AFM imaging showed that these lipid domains had irregular borders, indicating that they were gel-phase domains, and not non-ideally mixed lipid clusters or nanoscopic liquid-ordered domains.►Lo-Ld phase separation does not occur in DLPC/DSPC membranes containing cholesterol. ►Correlated AFM and NanoSIMS imaging identifies lipid domain structure and composition. ►Cholesterol addition causes elongation of gel-phase domains in DLPC/DSPC membranes. ►Ordered domains in DLPC/DSPC membranes with ≤19mol% cholesterol contain <7mol% DLPC.

Keywords: Abbreviations; FRET; fluorescence resonance energy transfer; AFM; atomic force microscopy; SIMS; secondary ion mass spectrometry; ¹⁵; N-DLPC; ¹⁵; N-1,2-dilauryl-; sn; -glycero-3-phosphocholine; D; ₇₀; -DSPC; 1,2-distearoyl-D; ₇₀; -; sn; -glycero-3-phosphocholine; NBD-PC; 1-palmitoyl-2-{12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]lauroyl}-; sn; -glycero-3-phosphocholine; PC; phosphatidylcholine; GUVs; unilamellar vesicles, ROIs, regions of interest; T; _m; melting transition temperatureSecondary ion mass spectrometry; Atomic force microscopy; Lipid phase behavior; Cholesterol; Lipid composition; Phase separation

Rb⁺ occlusion stabilized by vanadate in gastric H⁺/K⁺-ATPase at 25°C by Mónica R. Montes; Alejandro J. Spiaggi; José L.E. Monti; Flemming Cornelius; Claus Olesen; Patricio J. Garrahan; Rolando C. Rossi (pp. 316-322).

Despite its similarity with the Na⁺/K⁺-ATPase, it has not been possible so far to isolate a K⁺-occluded state in the H⁺/K⁺-ATPase at room temperature. We report here results on the time course of formation of a state containing occluded Rb⁺ (as surrogate for K⁺) in H⁺/K⁺-ATPase from gastric vesicles at 25°C. Alamethicin (a pore-forming peptide) showed to be a suitable agent to open vesicles, allowing a more efficient removal of Rb⁺ ions from the intravesicular medium than C₁₂E₈ (a non-ionic detergent). In the presence of vanadate and Mg²⁺, the time course of [⁸⁶Rb]Rb⁺ uptake displayed a fast phase due to Rb⁺ occlusion. The specific inhibitor of the H⁺/K⁺-ATPase SCH28080 significantly reduces the amount of Rb⁺ occluded in the vanadate–H⁺/K⁺-ATPase complex. Occluded Rb⁺ varies with [Rb⁺] according to a hyperbolic function with K_0.5=0.29±0.06mM. The complex between the Rb⁺-occluded state and vanadate proved to be very stable even after removal of free Mg²⁺ with EDTA. Our results yield a stoichiometry lower than one occluded Rb⁺ per phosphorylation site, which might be explained assuming that, unlike for the Na⁺/K⁺-ATPase, Mg²⁺-vanadate is unable to recruit all the Rb⁺-bound to the Rb⁺-occluded form of the Rb⁺–vanadate–H⁺/K⁺-ATPase complex.► The time course of Rb+ occlusion in gastric H+/K+-ATPase was measured at 25°C. ► Rb+ occlusion was detectable only in the presence of vanadate. ► The enzyme-vanadate complex holding occluded Rb+ is very stable. ► A fraction of the bound Rb+ might not be occluded even in media with vanadate. ► Alamethicin showed to be a suitable permeabilizing agent for gastric vesicles.

Keywords: H; ⁺; /K; ⁺; -ATPase; Rb; ⁺; -occlusion; Gastric vesicle; Alamethicin; Vanadate

Lipid chain branching at the iso- and anteiso-positions in complex chlamydia membranes: A molecular dynamics study by Joseph B. Lim; Jeffery B. Klauda (pp. 323-331).

Membranes in the intracellular eubacterial parasite Chlamydia trachomatis consist of the elementary body (EB) and reticular body (RB), and contain methyl branches at the iso- and anteiso-positions for some phospholipid chains. Acyl chain branching is the focus of this study. Molecular dynamics simulations were used to study bilayers of 1-13-methylpentadecanoyl-2-palmitoyl-phosphatidylcholine (13-MpPPC), 1-14-methylpentadecanoyl-2-palmitoyl-phosphatidylcholine (14-MpPPC), and diphytanoylphosphatidylcholine (DPhPC). These three membranes were simulated at 323K and simulations of DPhPC at 298K were also performed for better comparison to existing experimental data. Two simulations of representative EB and RB membranes of C. trachomatis composed of nine different lipid components were performed at 310.15K, to accurately reflect compositions determined by experiment and physiological conditions. Based on nearly 0.5μs of simulation data, we report that branching increases average lipid surface area, area elastic moduli, and lipid axial relaxation times, while decreasing lipid chain order. Branching also has a distinct effect on electron density profiles. Due to their high cholesterol concentrations, the EB and RB membranes were found to have relatively high area elastic moduli, which may have important biological implications.Display Omitted► Single-branched bilayers have properties similar to those of non-branched bilayers except with regards to lipid chain order, since methyl branches induce sharp decreases in order. ► Increased lipid chain branching dramatically affects bilayer properties by increasing surface area per lipid, lateral elastic moduli, and lipid axial relaxation times; decreasing lipid chain order; and producing unique electron density profiles. ► The elementary and reticular bodies of Chlamydia trachomatis have high lateral elastic moduli due to their cholesterol concentrations, which may aid in their biological roles.

Keywords: Chlamydia trachomatis; Lipid chain branching; Cholesterol; Complex membranes; Molecular dynamics

Lipid chain branching at the iso- and anteiso-positions in complex chlamydia membranes: A molecular dynamics study by Joseph B. Lim; Jeffery B. Klauda (pp. 323-331).

Keywords: Chlamydia trachomatis; Lipid chain branching; Cholesterol; Complex membranes; Molecular dynamics

Lipid chain branching at the iso- and anteiso-positions in complex chlamydia membranes: A molecular dynamics study by Joseph B. Lim; Jeffery B. Klauda (pp. 323-331).

Keywords: Chlamydia trachomatis; Lipid chain branching; Cholesterol; Complex membranes; Molecular dynamics

Lipid chain branching at the iso- and anteiso-positions in complex chlamydia membranes: A molecular dynamics study by Joseph B. Lim; Jeffery B. Klauda (pp. 323-331).

Keywords: Chlamydia trachomatis; Lipid chain branching; Cholesterol; Complex membranes; Molecular dynamics

Interaction of chromium(III) complexes with model lipid bilayers: Implications on cellular uptake by Natesan Sella Raja; Kamatchi Sankaranarayanan; Aruna Dhathathreyan; Balachandran Unni Nair (pp. 332-340).

To understand molecular cytotoxicity of chromium(III) and how it affects the stability of biological membranes, studies on the interaction of chromium(III) complexes aquapentaminechromium complex (complex I) and trans- [Cr(5-methoxysalcyclohex) (H₂O)₂] ClO₄ (complex II) with model biomembranes have been carried out. Langmuir films of dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidic acid (DPPA), dioctadecyldimethylammoniumbromide (DOMA) at air/water interface interacting with the chromium(III) complexes have been characterized using the surface pressure-molecular area (π-A) isotherms. Initial surface pressures changes for the two complexes show that the chromium(III) complexes inserted in the Langmuir films and complex I interacted strongly compared to complex II. Supported bilayers (SB) of the lipids on solid substrates formed by hydrating their Langmuir-Blodgett films (LB films) have been characterized using linear dichroic spectra, low angle X-ray diffraction and steady state fluorescence anisotropy. Depending on the geometry of the ligands and concentration, the complexes either insert in the alkyl or in the head group region of the SB and sometimes in both regions. The Supported lipid bilayers are well-layered and at low concentration, the metal complexes are incorporated near the head group region. Order and increase in lamellar spacing show stronger interaction of complex I with the lipids compared with complex II. This study provides some insights into the mechanism of chromium(III) toxicity and uptake of chromium(III) by the cells.Chromium(III) complexes interact with supported lipid bilayers leading to changes in their structural organization. These changes are determined by both ligand structure and the charge of the chromium(III) complex.Display Omitted► Geometry and charge of ligand in Cr(III) complexes influence the interaction with model lipid films. ► Cellular uptake of chromium(III) ions can be regulated by the ligand structures. ► This study is helpful in understanding of chromium(III) toxicity and development of chromium(III) supplements.

Keywords: Langmuir films; Phospholipids; Cr(III) complexes; Supported multibilayers; Fluorescence anisotropy; Toxicity of Cr(III)

Gemini surfactant dimethylene-1,2-bis(tetradecyldimethylammonium bromide)-based gene vectors: A biophysical approach to transfection efficiency by Ana M.S. Cardoso; Henrique Faneca; João A.S. Almeida; Alberto A.C.C. Pais; Eduardo F. Marques; Maria C. Pedroso de Lima; Amália S. Jurado (pp. 341-351).

Cationic liposomes have been proposed as biocompatible gene delivery vectors, able to overcome the barriers imposed by cell membranes. Besides lipids, other surfactant molecules have been successfully used in the composition of gene carriers. In the present work, we used a Gemini surfactant, represented by the general structure [C₁₄H₂₉(CH₃)₂N⁺(CH₂)₂N⁺(CH₃)₂C₁₄H₂₉]2Br⁻ and herein designated 14–2–14, to prepare cationic gene carriers, both as the sole component and in combination with neutral helper lipids, cholesterol and DOPE. The effectiveness of three Gemini-based formulations, namely neat 14–2–14, 14–2–14:Chol (1:1 molar ratio) and 14–2–14:Chol:DOPE (2:1:1 molar ratio), to mediate gene delivery was evaluated in DNA mixtures of +/− charge ratios ranging from 1/1 to 12/1. After ruling out cytotoxicity as responsible for the differences observed in the transfection competence, structural and physical properties of the vector were investigated, using several techniques. The size and surface charge density (zeta potential) of surfactant-based structures were determined by conventional techniques and the thermotropic behaviour of aqueous dispersions of surfactant/lipid/DNA formulations was monitored by fluorescence polarization of DPH and DPH-PA probes. The capacity of lipoplexes to interact with membrane-mimicking lipid bilayers was evaluated, using the PicoGreen assay and a FRET technique. Our data indicate inefficiency of the neat 14–2–14 formulation for gene delivery, which could result from the large dimensions of the particles and/or from its relative incompetence to release DNA upon interaction with anionic lipids. The addition of cholesterol or cholesterol and DOPE conferred to Gemini-based gene carrier transfection activity at specific ranges of +/− charge ratios. Fluorescence polarization data suggest that an order parameter within a specific range was apparently needed for complexes to display maximal transfection efficiency. The transfection-competent formulations showed to be efficiently destabilized by interaction with different anionic and zwitterionic bilayers, including those containing PS and cardiolipin. These data are discussed in terms of the potential of these formulations to address different intracellular targets.►The Gemini surfactant 14–2–14 offers a high level of DNA protection. ►14–2–14 based gene carriers assisted by helper lipids are transfection competent. ►Anionic lipids promote DNA exposure by Chol or Chol+DOPE assisted Gemini systems.

Keywords: Abbreviations; DMPC; dimyristoylphosphatidylcholine; DOPC; dioleoylphosphatidylcholine; DOPE; dioleoylphosphatidylethanolamine; DPH; 1,6-diphenyl-1,3,5-hexatriene; DPH-PA; 3-(p-(6-phenyl)-1,3,5-hexatrienyl)phenylpropionic acid; FRET; Förster Resonance Energy Transfer; MLV; multillamelar vesicles; NBD-PE; N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)-phosphatidylethanolamine; PA; phosphatidic acid; PS; phosphatidylserine; Rho-PE; rhodamine-phosphatidylethanolamine; SUV; small unillamelar vesicles; CL; cardiolipinGene delivery; Gemini surfactant; Lipoplex; Helper lipid; Fluorescence polarization; FRET

Apolipoprotein E LDL receptor-binding domain-containing high-density lipoprotein: A nanovehicle to transport curcumin, an antioxidant and anti-amyloid bioflavonoid by Panupon Khumsupan; Ricardo Ramirez; Darin Khumsupan; Vasanthy Narayanaswami (pp. 352-359).

Curcumin is an antioxidant and anti-inflammatory bioflavonoid that has been recently identified as an anti-amyloid agent as well. To make it more available in its potent form as a potential amyloid disaggregation agent, we employed high-density lipoproteins (HDL), which are lipid–protein complexes that transport plasma cholesterol, to transport curcumin. The objective of this study was to employ reconstituted HDL containing human apoE3 N-terminal (NT) domain, as a vehicle to transport curcumin. The NT domain serves as a ligand to mediate binding and uptake of lipoprotein complexes via the low-density lipoprotein receptor (LDLr) family of proteins located at the cell surface. Reconstituted HDL was prepared with phospholipids and recombinant apoE3-NT domain in the absence or presence of curcumin. Non-denaturing polyacrylamide gel electrophoresis indicated that the molecular mass and Stokes' diameter of HDL bearing curcumin were ~670kDa and ~17nm, respectively, while electron microscopy revealed the presence of discoidal particles. Fluorescence emission spectra of HDL bearing (the intrinsically fluorescent) curcumin indicated that the wavelength of maximal fluorescence emission (λ_max) of curcumin was ~495nm, which is highly blue-shifted compared to λ_max of curcumin in solvents of varying polarity (λ_max ranging from 515–575nm) or in aqueous buffers. In addition, an enormous enhancement in fluorescence emission intensity was noted in curcumin-containing HDL compared to curcumin in aqueous buffers. Curcumin fluorescence emission was quenched to a significant extent by lipid-based quenchers but not by aqueous quenchers. These observations indicate that curcumin has partitioned efficiently into the hydrophobic milieu of the phospholipid bilayer of HDL. Functional assays indicated that the LDLr-binding ability of curcumin-containing HDL with apoE3-NT is similar to that of HDL without curcumin. Taken together, we report that apoE-containing HDL has a tremendous potential as a ‘nanovehicle’ with a homing device to transport curcumin to target sites.Apolipoprotein E LDL receptor-binding domain-containing high-density lipoprotein: A nanovehicle to transport curcumin, an antioxidant and anti-amyloid bioflavonoid.Display Omitted►Incorporation of curcumin, an anti-amyloid bioflavonoid into HDL ►Curcumin embedded in hydrophobic milieu of HDL ►HDL with apoE as a ‘nanovehicle’ to target curcumin to cell surface LDL receptor

Keywords: Apolipoprotein E; High density lipoprotein; Curcumin; Nanovehicle; Alzheimer's disease; Anti-amyloid; Nanodisc; Fluorescence

Solution structure of subunit F (Vma7p) of the eukaryotic V₁V_O ATPase from Saccharomyces cerevisiae derived from SAXS and NMR spectroscopy by Sandip Basak; Shovanlal Gayen; Youg R. Thaker; Malathy S.S. Manimekalai; Manfred Roessle; Cornelia Hunke; Gruber Gerhard Grüber (pp. 360-368).

Vacuolar ATPases use the energy derived from ATP hydrolysis, catalyzed in the A₃B₃ sector of the V₁ ATPase to pump protons via the membrane-embedded V_O sector. The energy coupling between the two sectors occurs via the so-called central stalk, to which subunit F does belong. Here we present the first low resolution structure of recombinant subunit F (Vma7p) of a eukaryotic V-ATPase from Saccharomyces cerevisiae, analyzed by small angle X-ray scattering (SAXS). The protein is divided into a 5.5nm long egg-like shaped region, connected via a 1.5nm linker to a hook-like segment at one end. Circular dichroism spectroscopy revealed that subunit F comprises of 43% α-helix, 32% β-sheet and a 25% random coil arrangement. To determine the localization of the N- and C-termini in the protein, the C-terminal truncated form of F, F_1–94 was produced and analyzed by SAXS. Comparison of the F_1–94 shape with the one of subunit F showed the missing hook-like region in F_1–94, supported by the decreased D_max value of F_1–94 (7.0nm), and indicating that the hook-like region consists of the C-terminal residues. The NMR solution structure of the C-terminal peptide, F_90–116, was solved, displaying an α-helical region between residues 103 and 113. The F_90–116 solution structure fitted well in the hook-like region of subunit F. Finally, the arrangement of subunit F within the V₁ ATPase is discussed.Display Omitted►First low resolution structure of subunit F of a eukaryotic V-ATPase. ►Shape of the truncated form of F, F_1–94, allows the localization of the N- and C-termini in the entire protein. ►NMR solution structure of C-terminal peptide, F_90–116. ►New insight into the static and mechanistic properties of subunit F inside the V₁V_O ATPase.

Keywords: Abbreviations; DSS; 2, 2-dimethyl-2-silapentane-5-sulphonate; NMR; nuclear magnetic resonance; NOE; nuclear Overhauser effect; NOESY; NOE spectroscopy; NMRSD; normalized root mean square deviation; RMSD; root mean square distanceVacuolar ATPase; V; ₁; V; _O; ATPase; V; ₁; ATPase; Vma7p; Subunit F; Small angle X-ray scattering; NMR spectroscopy

Oligomeric structure of a cathelicidin antimicrobial peptide in dodecylphosphocholine micelle determined by NMR spectroscopy by Rathi Saravanan; Surajit Bhattacharjya (pp. 369-381).

The broad spectrum of antibacterial activities of host defense cationic antimicrobial peptides (AMPs) arises from their ability to perturb membrane integrity of the microbes. The mechanisms are often thought to require assembly of AMPs on the membrane surface to form pores. However, three dimensional structures in the oligomeric form of AMPs in the context of lipid membranes are largely limited. Here, we demonstrate that a 22-residue antimicrobial peptide, termed VK22, derived from fowlicidin-1, a cathelicidin family of AMP from chicken oligomerizes into a predominantly tetrameric state in zwitterionic dodecylphosphocholine (DPC) micelles. An ensemble of NMR structures of VK22 determined in 200mM perdeuterated DPC, from 755 NOE constrains including 19 inter-helical NOEs, had revealed an assembly of four helices arranged in anti-parallel fashion. Hydrogen bonds, C^αH―O=C types, and van der Waals interactions among the helical sub-units appear to be involved in the stabilization of the quaternary structures. The central region of the barrel shaped tetrameric bundle is non-polar with clusters of aromatic residues, whereas all the cationic residues are positioned at the termini. Paramagnetic spin labeled NMR experiments indicated that the tetrameric structure is embedded into micelles such that the non-polar region located inside the lipid acyl chains. Structure and micelle localization of a monomeric version, obtained from substitution of two Tyr residues with Ala, of the peptide is also compared. The mutated peptide VK22AA has been found be localized at the surface of the micelles. The tetrameric structure of VK22 delineates a small water pore that can be larger in the higher order oligomers. As these results provide structural insights, at atomic resolution, into the oligomeric states of a helical AMP in lipid environment, the structural details may be further utilized for the design of novel self-assembled membrane protein mimics.Display Omitted► Oliogomeric structure of antimicrobial peptide promotes membrane pore formation. ► Tetrameric structure of an antimicrobial peptide in lipid micelle. ► View of pore made by the oligomer.

Keywords: Antimicrobial peptide (AMP); NMR; Oligomeric AMP; Antibiotic resistance

Oligomeric structure of a cathelicidin antimicrobial peptide in dodecylphosphocholine micelle determined by NMR spectroscopy by Rathi Saravanan; Surajit Bhattacharjya (pp. 369-381).

Keywords: Antimicrobial peptide (AMP); NMR; Oligomeric AMP; Antibiotic resistance

Oligomeric structure of a cathelicidin antimicrobial peptide in dodecylphosphocholine micelle determined by NMR spectroscopy by Rathi Saravanan; Surajit Bhattacharjya (pp. 369-381).

Keywords: Antimicrobial peptide (AMP); NMR; Oligomeric AMP; Antibiotic resistance

Oligomeric structure of a cathelicidin antimicrobial peptide in dodecylphosphocholine micelle determined by NMR spectroscopy by Rathi Saravanan; Surajit Bhattacharjya (pp. 369-381).

Keywords: Antimicrobial peptide (AMP); NMR; Oligomeric AMP; Antibiotic resistance

Different membrane behaviour and cellular uptake of three basic arginine-rich peptides by Astrid Walrant; Isabelle Correia; Chen-Yu Jiao; Olivier Lequin; Eric H. Bent; Goasdoue Nicole Goasdoué; Claire Lacombe; Gérard Chassaing; Sandrine Sagan; Isabel D. Alves (pp. 382-393).

Cell penetrating peptides (CPPs) are peptides displaying the ability to cross cell membranes and transport cargo molecules inside cells. Several uptake mechanisms (endocytic or direct translocation through the membrane) are being considered, but the interaction between the CPP and the cell membrane is certainly a preliminary key point to the entry of the peptide into the cell. In this study, we used three basic peptides: RL9 (RRLLRRLRR-NH₂), RW9 (RRWWRRWRR-NH₂) and R9 (RRRRRRRRR-NH₂). While RW9 and R9 were internalised into wild type Chinese Hamster Ovary cells (CHO) and glycosaminoglycan-deficient CHO cells, at 4°C and 37°C, RL9 was not internalised into CHO cells. To better understand the differences between RW9, R9 and RL9 in terms of uptake, we studied the interaction of these peptides with model lipid membranes. The effect of the three peptides on the thermotropic phase behaviour of a zwitterionic lipid (DMPC) and an anionic lipid (DMPG) was investigated with differential scanning calorimetry (DSC). The presence of negative charges on the lipid headgroups appeared to be essential to trigger the peptide/lipid interaction. RW9 and R9 disturbed the main phase transition of DMPG, whereas RL9 did not induce significant effects. Isothermal titration calorimetry (ITC) allowed us to study the binding of these peptides to large unilamellar vesicles (LUVs). RW9 and R9 proved to have about ten fold more affinity for DSPG LUVs than RL9. With circular dichroism (CD) and NMR spectroscopy, the secondary structure of RL9, RW9 and R9 in aqueous buffer or lipid/detergent conditions was investigated. Additionally, we tested the antimicrobial activity of these peptides against Escherichia coli and Staphylococcus aureus, as CPPs and antimicrobial peptides are known to share several common characteristics. Only RW9 was found to be mildly bacteriostatic against E. coli. These studies helped us to get a better understanding as to why R9 and RW9 are able to cross the cell membrane while RL9 remains bound to the surface without entering the cell. ►The RW9 and R9 peptides are cell penetrating peptides but RL9 is not internalised. ►The three peptides bind to the cell membrane. ►RW9 and R9 increase the fluidity of negatively charged lipid bilayers, RL9 does not. ►RW9 and R9 have ten fold more affinity for negatively charged liposomes than RL9. ►RL9 and RW9 become helical in presence of negatively charged liposomes.

Keywords: Abbreviations; APA; Amino pentanoic acid; CD; Circular dichroism; CHO; Chinese hamster ovary; CPP; Cell-penetrating peptide; CS; Chondroitin sulfate; CSD; Chemical shift deviation; D8PG; Dioctanoyl phosphatidylglycerol; DiPoPE; Dipalmitoleoyl phosphatidylethanolamine; DLS; Dynamic light scattering; DMPC; Dimyristoyl phosphatidylcholine; DMPG; Dimyristoyl phosphatidylglycerol; DPC; Dodecyl phosphocholine; DSC; Differential scanning calorimetry; DSPG; Distearoyl phosphatidylglycerol; HS; Heparan sulfate; HSQC; Heteronuclear single quantum correlation; ITC; Isothermal titration calorimetry; LDH; Lactate dehydrogenase; LUV; Large unilamellar vesicle; MIC; Minimal inhibitory concentration; MLV; Multi lamellar vesicle; NOE; Nuclear Overhauser effect; NOESY; NOE spectroscopy; PA; Phosphatidic acid; PC; Phosphatidylcholine; PE; Phosphatidylethanolamine; PG; Phosphatidylglycerol; PI; Phosphatidylinositol; PS; Phosphatidylserine; SDS; Sodium dodecyl sulfate; TOCSY; Total correlation spectroscopyCell penetrating peptide; Polyarginine; Cellular uptake; Peptide lipid interactions; Calorimetry; NMR

Quantitative analysis of influenza M2 channel blockers by Peleg Astrahan; Ravenna Flitman-Tene; Estelle R. Bennett; Miriam Krugliak; Chaim Gilon; Isaiah T. Arkin (pp. 394-398).

The influenza M2 H⁺ channel enables the concomitant acidification of the viral lumen upon endosomic internalization. This process is critical to the viral infectivity cycle, demonstrated by the fact that M2 is one of only two targets for anti-flu agents. However, aminoadamantyls that block the M2 channel are of limited therapeutic use due to the emergence of resistance mutations in the protein. Herein, using an assay that involves expression of the protein in Escherichia coli with resultant growth retardation, we present quantitative measurements of channel blocker interactions. Comparison of detailed K_s measurements of different drugs for several influenza channels, shows that the swine flu M2 exhibits the highest resistance to aminoadamantyls of any channel known to date. From the perspective of the blocker, we show that rimantadine is consistently a better blocker of M2 than amantadine. Taken together, such detailed and quantitative analyses provide insight into the mechanism of this important and pharmaceutically relevant channel blocker system.

Keywords: Influenza; Channel; Channel blocker

Quantitative analysis of influenza M2 channel blockers by Peleg Astrahan; Ravenna Flitman-Tene; Estelle R. Bennett; Miriam Krugliak; Chaim Gilon; Isaiah T. Arkin (pp. 394-398).

Keywords: Influenza; Channel; Channel blocker

Quantitative analysis of influenza M2 channel blockers by Peleg Astrahan; Ravenna Flitman-Tene; Estelle R. Bennett; Miriam Krugliak; Chaim Gilon; Isaiah T. Arkin (pp. 394-398).

Keywords: Influenza; Channel; Channel blocker

Quantitative analysis of influenza M2 channel blockers by Peleg Astrahan; Ravenna Flitman-Tene; Estelle R. Bennett; Miriam Krugliak; Chaim Gilon; Isaiah T. Arkin (pp. 394-398).

Keywords: Influenza; Channel; Channel blocker

Do main location within the cystic fibrosis transmembrane conductance regulator protein investigated by electron microscopy and gold labelling by Liang Zhang; Luba A. Aleksandrov; John R. Riordan; Robert C. Ford (pp. 399-404).

The domain organisation of the cystic fibrosis transmembrane conductance regulator (CFTR) protein was studied using electron microscopy of detergent-solubilised dimeric complexes. Ni-NTA nanogold labelling data suggest that in the nonphosphorylated, nucleotide-free state, the C-terminus is intimately associated with the cytoplasmic ATP-binding regions, whilst part of the regulatory domain occupies a position close to the cytoplasmic surface of the lipid membrane. Removal of the entire second nucleotide binding domain (NBD2) results in a deficit in the CFTR structure that is consistent with the size and shape of a single NBD. The data suggest that NBD2 lies closer to the C2 symmetry axis than the first nucleotide binding domain (NBD1) and that NBD2 from one CFTR monomer also contacts NBD1 from the opposing one. These data suggest that current homology models for CFTR based on other ATP-binding cassette proteins appear to be reasonable, at least to low resolution. We also find that Ni-NTA nanogold labelling of an internal hexa-Histidine sequence is a valuable approach to locate individual protein domains.► Structure of the cystic fibrosis transmembrane conductance regulator protein at low resolution. ► Labelling of the R region by a 1.8-nm-diameter nanogold shows a location close to the cytoplasmic face of the membrane for residues around 696. ► Extracellular loop 4 is resistant to labelling by nanogold. ► Nucleotide-binding domain 2 is localized in the full-length CFTR and forms a compact 3- to 4-nm-diameter domain. ► Current homology models for CFTR based on the Sav1866 ABC transporter appear to be valid.

Keywords: Abbreviations; 2D; two-dimensional; 3D; three-dimensional; CFTR; cystic fibrosis transmembrane conductance regulator; DDM; dodecyl maltoside; EM; electron microscopy; NBD; nucleotide binding domain; NTA; nitrilotriacetic acid; PAGE; polyacrylamide gel electrophoresis; SEC; size exclusion chromatography; SDS; sodium dodecylsulphate; TMD; transmembrane domainCystic fibrosis; Chloride channel; Structure; Electron microscopy; CFTR; Ni-NTA nanogold

Ethanol effects on binary and ternary supported lipid bilayers with gel/fluid domains and lipid rafts by Marques Joaquim T. Marquês; Ana S. Viana; Rodrigo F.M. De Almeida (pp. 405-414).

Ethanol–lipid bilayer interactions have been a recurrent theme in membrane biophysics, due to their contribution to the understanding of membrane structure and dynamics. The main purpose of this study was to assess the interplay between membrane lateral heterogeneity and ethanol effects. This was achieved by in situ atomic force microscopy, following the changes induced by sequential ethanol additions on supported lipid bilayers formed in the absence of alcohol. Binary phospholipid mixtures with a single gel phase, dipalmitoylphosphatidylcholine (DPPC)/cholesterol, gel/fluid phase coexistence DPPC/dioleoylphosphatidylcholine (DOPC), and ternary lipid mixtures containing cholesterol, mimicking lipid rafts (DOPC/DPPC/cholesterol and DOPC/sphingomyelin/cholesterol), i.e., with liquid ordered/liquid disordered (ld/lo) phase separation, were investigated. For all compositions studied, and in two different solid supports, mica and silicon, domain formation or rearrangement accompanied by lipid bilayer thinning and expansion was observed. In the case of gel/fluid coexistence, low ethanol concentrations lead to a marked thinning of the fluid but not of the gel domains. In the case of ld/lo all the bilayer thins simultaneously by a similar extent. In both cases, only the more disordered phase expanded significantly, indicating that ethanol increases the proportion of disordered domains. Water/bilayer interfacial tension variation and freezing point depression, inducing acyl chain disordering (including opening and looping), tilting, and interdigitation, are probably the main cause for the observed changes. The results presented herein demonstrate that ethanol influences the bilayer properties according to membrane lateral organization.► Gel/fluid and raft-like domains affect distinctly ethanol-membrane interactions. ► Liquid ordered and disordered undergo large thickness reduction at low ethanol levels. ► Moderate ethanol levels induce only a small thickness reduction (tilting) of the gel. ► The liquid disordered phase always goes through a marked lateral expansion. ► Lipid rafts and ethanol effects are clearly observed on silicon surface by liquid AFM.

Keywords: Ethanol–membrane interactions; Liquid AFM; Liquid ordered liquid disordered; Bilayer expansion; Silicon; Sphingomyelin

Ethanol effects on binary and ternary supported lipid bilayers with gel/fluid domains and lipid rafts by Marques Joaquim T. Marquês; Ana S. Viana; Rodrigo F.M. De Almeida (pp. 405-414).

Keywords: Ethanol–membrane interactions; Liquid AFM; Liquid ordered liquid disordered; Bilayer expansion; Silicon; Sphingomyelin

Ethanol effects on binary and ternary supported lipid bilayers with gel/fluid domains and lipid rafts by Marques Joaquim T. Marquês; Ana S. Viana; Rodrigo F.M. De Almeida (pp. 405-414).

Keywords: Ethanol–membrane interactions; Liquid AFM; Liquid ordered liquid disordered; Bilayer expansion; Silicon; Sphingomyelin

Ethanol effects on binary and ternary supported lipid bilayers with gel/fluid domains and lipid rafts by Marques Joaquim T. Marquês; Ana S. Viana; Rodrigo F.M. De Almeida (pp. 405-414).

Keywords: Ethanol–membrane interactions; Liquid AFM; Liquid ordered liquid disordered; Bilayer expansion; Silicon; Sphingomyelin

Conformational plasticity of the influenza A M2 transmembrane helix in lipid bilayers under varying pH, drug binding, and membrane thickness by Fanghao Hu; Wenbin Luo; Sarah D. Cady; Mei Hong (pp. 415-423).

Membrane proteins change their conformations to respond to environmental cues, thus conformational plasticity is important for function. The influenza A M2 protein forms an acid-activated proton channel important for the virus lifecycle. Here we have used solid-state NMR spectroscopy to examine the conformational plasticity of membrane-bound transmembrane domain of M2 (M2TM).¹³C and¹⁵N chemical shifts indicate coupled conformational changes of several pore-facing residues due to changes in bilayer thickness, drug binding, and pH. The structural changes are attributed to the formation of a well-defined helical kink at G34 in the drug-bound state and in thick lipid bilayers, nonideal backbone conformation of the secondary-gate residue V27 in the presence of drug, and nonideal conformation of the proton-sensing residue H37 at high pH. The chemical shifts constrained the ( ϕ, ψ) torsion angles for three “basis” states, the equilibrium among which explains the multiple resonances per site in the NMR spectra under different combinations of bilayer thickness, drug binding, and pH conditions. Thus, conformational plasticity is important for the proton conduction and inhibition of M2TM. The study illustrates the utility of NMR chemical shifts for probing the structural plasticity and folding of membrane proteins.►Influenza M2 proton channel shows conformational plasticity due to the environment. ►Thicker lipid membranes and drug binding promote kinked M2TM helices. ►Channel opening promotes a more ideal helical conformation at histidine-37. ►Equilibrium between different conformations is shifted by the environment.

Keywords: Conformational change; Influenza virus; M2 proton channel; Amantadine; Membrane thickness; Solid-state NMR

Monitoring membrane binding and insertion of peptides by two-color fluorescent label by V.Y. Postupalenko; V.V. Shvadchak; G. Duportail; V.G. Pivovarenko; A.S. Klymchenko; Mely Y. Mély (pp. 424-432).

Herein, we developed an approach for monitoring membrane binding and insertion of peptides using a fluorescent environment-sensitive label of the 3-hydroxyflavone family. For this purpose, we labeled the N-terminus of three synthetic peptides, melittin, magainin 2 and poly-l-lysine capable to interact with lipid membranes. Binding of these peptides to lipid vesicles induced a strong fluorescence increase, which enabled to quantify the peptide–membrane interaction. Moreover, the dual emission of the label in these peptides correlated well with the depth of its insertion measured by the parallax quenching method. Thus, in melittin and magainin 2, which show deep insertion of their N-terminus, the label presented a dual emission corresponding to a low polar environment, while the environment of the poly-l-lysine N-terminus was rather polar, consistent with its location close to the bilayer surface. Using spectral deconvolution to distinguish the non-hydrated label species from the hydrated ones and two photon fluorescence microscopy to determine the probe orientation in giant vesicles, we found that the non-hydrated species were vertically oriented in the bilayer and constituted the best indicators for evaluating the depth of the peptide N-terminus in membranes. Thus, this label constitutes an interesting new tool for monitoring membrane binding and insertion of peptides.Display Omitted►We developed an approach for monitoring membrane binding and insertion of peptides using a fluorescent environment-sensitive label of the 3-hydroxyflavone family. ►The dual emission of the label covalently bound to the N-terminus of three peptides correlates well with the depth of its insertion measured by the parallax quenching method. ►Orientation of the label bound to the peptides can also be determined by two photon microscopy on giant vesicles.

Keywords: Fluorescence; 3-hydroxyflavone; Lipid membrane; Melittin; Peptide–membrane interaction

Ketoconazole and miconazole alter potassium homeostasis in Saccharomyces cerevisiae by Martha Calahorra; Carlos Lozano; Sanchez Norma Silvia Sánchez; Pena Antonio Peña (pp. 433-445).

The effects of ketoconazole and miconazole uptake on K⁺ transport and the internal pH of Saccharomyces cerevisiae were studied. The uptake of both drugs was very fast, linear with concentration and not dependent on glucose, indicating entrance by diffusion and concentrating inside. Low (5.0μM) to intermediate concentrations (40μM) of both drugs produced a glucose-dependent K⁺ efflux; higher ones also produced a small influx of protons, probably through a K⁺/H⁺ exchanger, resulting in a decrease of the internal pH of the cells and the efflux of material absorbing at 260nm and phosphate. The cell membrane was not permeabilized. The K⁺ efflux with miconazole was dependent directly on the medium pH. This efflux results in an increased membrane potential, responsible for an increased Ca²⁺ uptake and other effects. These effects were not observed with two triazolic antifungals. A decrease of the Zeta (ζ) potential was observed at low concentrations of miconazole. Although the main effect of these antifungals is the inhibition of ergosterol synthesis, K⁺ efflux is an important additional effect to be considered in their therapeutic use. Under certain conditions, the use of single mutants of several transporters involved in the movements of K⁺ allowed to identify the participation of several antiporters in the efflux of the cation.► Imidazolic antifungals produce K⁺ efflux and hyperpolarize the plasma membrane. ► Hyperpolarization increases Ca²⁺ uptake resulting in cell death. ► Cation/proton exchangers appear to be involved in the effects.

Keywords: Abbreviations; Bicine-TEA; N,N; -Bis(2-hydroxyethyl)glycine, adjusted to pH 8.0 with triethanolamine; CCCP; carbonyl cyanide 3-chlorophenylhydrazone; CFU; colony forming units; CH; ₂; Cl; ₂; dichloromethane; DiSC; ₃; (3); 3,3′-dipropylthiacarbocyanine or cyanine; FCCP; carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; HOMOPIPES-TEA; Homopiperazine-N,N′-bis-2-(ethane sulfonic acid) adjusted to pH 4.0 with triethanolamine; K; ketoconazole; M; miconazole; MES-TEA; morpholino ethanesulfonic acid adjusted to pH 6.0 with triethanolamine; PMP; plasma membrane potential; TEA; triethanolamineAntifungal; Yeast ion transport; Yeast transport; Potassium transport; Ketoconazole; Miconazole

Ketoconazole and miconazole alter potassium homeostasis in Saccharomyces cerevisiae by Martha Calahorra; Carlos Lozano; Sanchez Norma Silvia Sánchez; Pena Antonio Peña (pp. 433-445).

The intrinsically disordered late embryogenesis abundant protein LEA18 from Arabidopsis thaliana modulates membrane stability through binding and folding by Michaela Hundertmark; Rumiana Dimova; Jan Lengefeld; Robert Seckler; Dirk K. Hincha (pp. 446-453).

Intrinsically disordered proteins (IDPs) constitute a substantial part of cellular proteomes. Late embryogenesis abundant (LEA) proteins are mostly predicted to be IDPs associated with dehydration tolerance in many plant, animal and bacterial species. Their functions, however, are largely unexplored and also their structure and interactions with potential target molecules have only recently been experimentally investigated in a small number of proteins. Here, we report on the structure and interactions with membranes of the Pfam LEA_1 protein LEA18 from the higher plant Arabidopsis thaliana. This functionally uncharacterized positively charged protein specifically aggregated and destabilized negatively charged liposomes. Isothermal titration calorimetry showed binding of the protein to both charged and uncharged membranes. LEA18 alone was largely unstructured in solution. While uncharged membranes had no influence on the secondary structure of LEA18, the protein partially folded into β-sheet structure in the presence of negatively charged liposomes. These data suggest that LEA18 does not function as a membrane stabilizing protein, as suggested for other LEA proteins. Instead, a possible function of LEA18 could be the composition-dependent modulation of membrane stability, e.g., during signaling or vesicle-mediated transport.►LEA18 is a plant IDP that induces leakage from liposomes during freezing. ►In solution only negatively charged membranes are destabilized and aggregated. ►Binding to negatively charged membranes induces partial folding in LEA18. ►ITC data show strong binding to negatively charged and weak binding to neutral lipids.

Keywords: Abbreviations; CD; circular dichroism; CF; carboxyfluorescein; EPE; egg phosphatidylethanolamine; EPG; egg phosphatidylglycerol; FRET; fluorescence resonance energy transfer; IDP; intrinsically disordered protein; ITC; isothermal titration calorimetry; LEA; late embryogenesis abundant; MBP; myelin basic protein; NBD-PE; N-(7-nitro-2,1,3-benzoxadiazol-4-yl) dipalmitoyl-phosphatidylethanolamine; POPC; 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine; Rh-PE; N-(lissamine Rhodamine B sulfonyl) dipalmitoyl-phosphatidylethanolamineIntrinsically disordered protein; Late embryogenesis abundant protein; Membrane stability; Protein–membrane interaction; Protein folding

Electro-pharmacological profile of a mitochondrial inner membrane big-potassium channel from rat brain by J. Fahanik-babaei; A. Eliassi; A. Jafari; R. Sauve; S. Salari; R. Saghiri (pp. 454-460).

Recent studies have indicated a calcium-activated large conductance potassium channel in rat brain mitochondrial inner membrane (mitoBK channel). Accordingly, we have characterized the functional and pharmacological profile of a BK channel from rat brain mitochondria in the present study. Brain mitochondrial inner membrane preparations were subjected to SDS-PAGE analysis and channel protein reconstitution into planar lipid bilayers. Western blotting and antibodies directed against various cellular proteins revealed that mitochondrial inner membrane fractions did not contain specific proteins of the other subcellular compartments except a very small fraction of endoplasmic reticulum. Channel incorporation into planar lipid bilayers revealed a voltage dependent 211 pS potassium channel with a voltage for half activation ( V_1/2) of 11.4±1.1mV and an effective gating charge z_d of 4.7±0.9. Gating and conducting behaviors of this channel were unaffected by the addition of 2.5mM ATP, and 500 nM charybdotoxin (ChTx), but the channel appeared sensitive to 100 nM iberiotoxin (IbTx). Adding 10mM TEA at positive potentials and 10mM 4-AP at negative or positive voltages inhibited the channel activities. These results demonstrate that the mitoBK channel, present in brain mitochondrial inner membrane, displays different pharmacological properties than those classically described for plasma membrane, especially in regard to its sensitivity to iberiotoxin and charybdotoxin sensitivity.►Brain mitoBK channel has different pharmacological properties than those classically described for plasma membrane. ►Brain mitoBK channel is sensitive to iberiotoxin, but not to charybdotoxin.

Keywords: Abbreviation; EGTA; ethylene glycol-bis (2 amino-ethylether)-; N,N,N',N'; -tetraacetic acid; HEPES; 4-(2-Hydroxyethyl) piperazine-1-ethanesulfonic acid, N-(2-Hydroxyethyl) piperazine-N'-(2-ethanesulfonic acid) potassium salt; BSA; bovine serum albumin; mitoBK; mitochondrial big Ca; ²⁺; -activated potassium channel; mitoK; _ATP; mitochondrial ATP-sensitive K; ⁺; channel; Trizma base; Tris [hydroxymethyl] aminomethane; cox; cytochorom oxydase antibody; IbTx; iberiotoxin; ChTx; charybdotoxin; TEA; tetra-ethyl ammonium; 4-AP; 4-aminopyridine; SDS-PAGE; sodium dodecyl sulfate-polyacryl-amide gel electrophoresis; 58KGP; 58K Golgi proteinMitochondria; Potassium channels; MitoBK channel; Charybdotoxin; Iberiotoxin

Electro-pharmacological profile of a mitochondrial inner membrane big-potassium channel from rat brain by J. Fahanik-babaei; A. Eliassi; A. Jafari; R. Sauve; S. Salari; R. Saghiri (pp. 454-460).

Cholesterol depletion enhances adrenergic signaling in cardiac myocytes by Yamuna Devi Paila; Ekta Jindal; Shyamal K. Goswami; Amitabha Chattopadhyay (pp. 461-465).

Cardiac myocytes endogenously express α and β adrenergic receptors, prototypes of the G-protein coupled receptor superfamily. Depending upon the dose of norepinephrine (agonist) exposure, hypertrophy and apoptosis are initiated by differential induction of two discrete constituents of the transcription factor AP-1, i.e., FosB and Fra-1. We explored differential adrenergic signaling as a paradigm for understanding how cholesterol dictates cells to choose hypertrophy or apoptosis. For this, we used fosB and fra-1 promoter–reporter constructs for monitoring adrenergic signaling. We show that cholesterol depletion enhances norepinephrine-mediated signaling in cardiac myocytes. Importantly, this increased signaling is reduced to original level upon cholesterol replenishment. We used specific ligands for α and β adrenergic receptors and show that the enhanced signaling upon cholesterol depletion is a combined effect of both α and β adrenergic receptors. These results constitute the first report demonstrating the effect of cholesterol on adrenergic signaling using a direct end-point gene expression.► Cellular cholesterol and adrenergic signaling in cardiac myocytes. ► Differential adrenergic signaling results in hypertrophy or apotosis. ► Adrenergic signaling monitored using direct end-point gene expression assay. ► Restoration of adrenergic signaling upon cholesterol replenishment.

Keywords: Abbreviations; GPCR; G-protein coupled receptor; MβCD; methyl-β-cyclodextrin; NE; norepinephrineAdrenergic receptors; Adrenergic signaling; Cholesterol depletion; Cholesterol replenishment; H9c2 cardiac myocytes; MβCD

Cholesterol depletion enhances adrenergic signaling in cardiac myocytes by Yamuna Devi Paila; Ekta Jindal; Shyamal K. Goswami; Amitabha Chattopadhyay (pp. 461-465).

Curcumin is a lipid dependent inhibitor of the Na,K-ATPase that likely interacts at the protein-lipid interface by Yasser A. Mahmmoud (pp. 466-473).

Curcumin is an important nutraceutical widely used in disease treatment and prevention. We have previously suggested that curcumin interferes with K⁺ binding to pig kidney Na,K-ATPase by interaction with its extracellular domains. The aim of this study was to further characterize the site of curcumin interaction with the ATPase. We have performed pair inhibitor studies and investigated the sided action of curcumin on pig kidney Na,K-ATPase reconstituted into lipid vesicles of defined composition. An addition of curcumin to either the intracellular or extracellular domains of the Na,K-ATPase produced similar inhibition. The lipid environment and temperature strongly influenced the potency of the drug. Curcumin inhibition decreased following insertion of the ATPase in sphingomyelin-cholesterol ‘raft’ domains and fully abolished following treatment with non-ionic detergents. The drug induced cross-linking of membrane embedded domains of the Na,K-ATPase. We conclude that curcumin interacts with Na,K-ATPase at the protein-lipid interface. Non-annulus lipids likely participate in this interaction. These results provide new information on the molecular mechanism of curcumin action and explain (at least partly) the ambiguous effectiveness of this polyphenol in the different systems.►Curcumin interacts with the Na,K-ATPase from within the membrane area. ►Curcumin potency is strongly modulated by the physical properties of the membrane. ►Recruitment of Na,K-ATPase into different membrane domains affects curcumin potency. ►Membrane lipids participate in the interaction of curcumin with the Na,K-ATPase.

Keywords: Abbreviations; BS; ³; Bis-(sulfosuccinimidyl)suberate; CFTR; cystic fibrosis transmembrane conductance regulator; DMSO; dimethyl sulfoxide; ECL; enhanced chemiluminescence; EC; ₅₀; ligand concentration producing half-maximum effect; Na,K-ATPase; sodium and potassium activated adenosine triphosphatase; PBS; phosphate-buffered saline; PVDF; Polyvinylidene fluoride; Tricine; N-[2-hydroxy-1,1 bis(hydroxymethyl)ethyl]glycineCurcumin; Diferuloylmethane; Na,K-ATPase; Membrane composition; P-type ATPases; Lipid raft

Curcumin is a lipid dependent inhibitor of the Na,K-ATPase that likely interacts at the protein-lipid interface by Yasser A. Mahmmoud (pp. 466-473).

Liposomes alter thermal phase behavior and composition of red blood cell membranes by Christoph Stoll; Hart Stadnick; Oliver Kollas; Jelena L. Holovati; Birgit Glasmacher; Jason P. Acker; Willem F. Wolkers (pp. 474-481).

Unilamellar liposomes composed of natural phospholipids provide a new promising class of protective agents for hypothermic storage, cryopreservation, or freeze-drying of red blood cells (RBCs). In this study, FTIR spectroscopy, MALDI-TOF MS, and colorimetric assays were used to investigate the effects of liposomes composed of a homologous series of linear saturated phosphatidylcholine phospholipids (18:0; 16:0; 14:0; 12:0) on RBC membranes. RBCs were incubated with liposomes at 37°C and both the liposomal and the RBC fraction were analyzed after incubation. FTIR studies showed that liposomes composed of short acyl chain length lipids cause an increase in RBC membrane conformational disorder at suprazero temperatures, whereas long acyl chain length lipids were found to have little effects. The increased lipid conformational disorder in the RBC membranes coincided with a decrease in the cholesterol-to-phospholipid ratio. The opposite effects were found in the liposomes after incubation with RBCs. MALDI-TOF MS analysis showed the presence of short acyl chain length lipids (14:0 and 12:0) in RBC membranes after incubation, which was not observed after incubation with liposomes containing long acyl chain length lipids (18:0 and 16:0). Liposomes alter RBC membrane properties by cholesterol depletion and lipid addition.►Liposomes alter red blood cell (RBC) membrane properties by cholesterol depletion and lipid addition. ►Cholesterol transfers from RBCs to liposomes. ►Acyl chain length determines liposome RBC interactions. ►Short acyl chain length liposomes increase RBC membrane conformational disorder.

Keywords: Abbreviations; ACN; acetonitrile; DHB; 2,5-dihydroxybenzoic acid; DLPC; 1,2-dilauroyl-; sn; -glycero-3-phosphocholine; DMPC; 1,2-dimyristoyl phosphatidylcholine; DPPC; 1,2-dipalmitoyl-; sn; -glycero-3-phosphocholine; DSPC; 1,2-distearoyl-; sn; -glycero-3-phosphocholine; FA; formic acid; FTIR; Fourier transform infrared spectroscopy; LUV; large unilamellar vesicles; MALDI-TOF MS; matrix-assisted laser desorption/ionization–time of flight mass spectrometry; T; _m; fluid-to-gel membrane phase transition temperature; PEG; poly(ethylene glycol)Fourier transform infrared spectroscopy; Lipid phase behavior; Liposome; Erythrocyte; MALDI-TOF mass spectrometry; Cholesterol transfer

Expression, refolding, and initial structural characterization of the Y. pestis Ail outer membrane protein in lipids by Leigh A. Plesniak; Radhakrishnan Mahalakshmi; Candace Rypien; Yuan Yang; Jasmina Racic; Francesca M. Marassi (pp. 482-489).

Ail is an outer membrane protein and virulence factor of Yersinia pestis, an extremely pathogenic, category A biothreat agent, responsible for precipitating massive human plague pandemics throughout history. Due to its key role in bacterial adhesion to host cells and bacterial resistance to host defense, Ail is a key target for anti-plague therapy. However, little information is available about the molecular aspects of its function and interactions with the human host, and the structure of Ail is not known. Here we describe the recombinant expression, purification, refolding, and sample preparation of Ail for solution and solid-state NMR structural studies in lipid micelles and lipid bilayers. The initial NMR and CD spectra show that Ail adopts a well-defined transmembrane β-sheet conformation in lipids. ►Y. pestis Ail outer membrane protein is expressed and purified in high yields. ►Y. pestis Ail is refolded quantitatively in lipid micelles, bicelles, and bilayers. ►Y. pestis Ail yields high-quality solution and solid-state NMR spectra in these environments. ►NMR studies show that Y. pestis Ail adopts a well-defined transmembrane β-sheet structure.

Keywords: Ail; Yersinia pestis; Outer membrane protein; Bilayer; Bicelle; Micelle; NMR

Comparative study of the membrane-permeabilizing activities of mastoparans and related histamine-releasing agents in bacteria, erythrocytes, and mast cells by Satoshi Nakao; Keiko Komagoe; Tsuyoshi Inoue; Takashi Katsu (pp. 490-497).

The membrane-permeabilizing activities of mastoparans and related histamine-releasing agents were compared through measurements of K⁺ efflux from bacteria, erythrocytes, and mast cells. Changes in bacterial cell viability, hemolysis, and histamine release, as well as in the shape of erythrocytes were also investigated. The compounds tested were mastoparans (HR1, a mastoparan from Polistes jadwagae, and a mastoparan from Vespula lewisii), granuliberin R, mast cell-degranulating peptide, and compound 48/80, as well as antimicrobial peptides, such as magainin I, magainin II, gramicidin S, and melittin. We used a K⁺-selective electrode to determine changes in the permeability to K⁺ of the cytoplasmic membranes of cells. Consistent with the surface of mast cells becoming negatively charged during histamine release, due to the translocation of phosphatidylserine to the outer leaflet of the cytoplasmic membrane, histamine-releasing agents induced K⁺ efflux from mast cells, dependent on their ability to increase the permeability of bacterial cytoplasmic membranes rich in negatively charged phospholipids. The present results demonstrated that amphiphilic peptides, possessing both histamine-releasing and antimicrobial capabilities, induced the permeabilization of the cytoplasmic membranes of not only bacteria but mast cells. Mastoparans increased the permeability of membranes in human erythrocytes at higher concentrations, and changed the normal discoid shape to a crenated form. The structural requirement for making the crenated form was determined using compound 48/80 and its constituents (monomer, dimer, and trimer), changing systematically the number of cationic charges of the molecules.►Mastoparans and related histamine-releasing agents induce K⁺ efflux from mast cells, dependent on their ability to cause K⁺ efflux from bacteria. ►Mastoparans change the normal discoid shape of human erythrocytes to a crenated form at the concentration causing hemolysis. ►The structural requirement for making the crenated form is determined using compound 48/80 and its constituents, changing systematically the number of cationic charges of the molecules.

Keywords: Mastoparan; Compound 48/80; Antimicrobial peptide; Histamine-releasing agent; Membrane permeability; Cell shape

Structure and lipid interactions of an anti-inflammatory and anti-atherogenic 10-residue class G^⁎ apolipoprotein J peptide using solution NMR by Vinod K. Mishra; Mayakonda N. Palgunachari; Jason S. Hudson; Ronald Shin; Tamara D. Keenum; N. Rama Krishna; G.M. Anantharamaiah (pp. 498-507).

The surprising observation that a 10-residue class G^⁎ peptide from apolipoprotein J, [113–122]apoJ, possesses anti-inflammatory and anti-atherogenic properties prompted us to delineate its structural characteristics in the presence of normal and oxidized lipid. Towards this, we have determined high-resolution structure of [113–122]apoJ in solution using nuclear magnetic resonance (NMR) spectroscopy and studied its interaction with lipids, including oxidized lipids, using a number of biophysical methods. Circular dichroism and NMR studies established that in the presence of dodecylphosphocholine (DPC) micelle, this peptide adopts amphipathic α-helical structure. The observed Nuclear Overhauser effects indicate that the amphipathic helical structure of the peptide is stabilized by the N-terminal acetyl and C-terminal amide blocking groups. We used isothermal titration calorimetry to measure binding enthalpy of the peptide with DPC micelle, an oxidized lipid, 1-(palmitoyl)-2-(5-keto-6-octene-dioyl) phosphatidylcholine (KOdiA-PC), and the mixture of these two lipids (5mol% KOdiA-PC in DPC micelle). We find that the peptide binding with DPC micelle is associated with an enthalpy change (−16.75±0.16 Kcal/mol) much larger than that resulting from the binding with KodiA-PC (−3.67±0.13 Kcal/mol). Incorporation of a small amount of KOdiA-PC (5mol%) in DPC micelle also results in the lowering of peptide binding enthalpy (−13.43±0.18 Kcal/mol). These results are consistent with overall negative charge and altered conformational properties of oxidized sn-2 chain of KOdiA-PC. Our results have unambiguously established the amphipathic α-helical structure of [113–122]apoJ peptide in the presence of DPC micelle as well as its ability to bind oxidized lipid. These in vitro results help explain the previously observed anti-inflammatory and anti-atherosclerotic properties of this peptide.►A 10-residue peptide from apolipopotein J adopts an amphipathic α-helical structure in the presence of dodecylphosphocholine micelle. ►Solution NMR structure of the 10-residue peptide in the presence of dodecylphosphocholine micelle has been determined. ►Ability of this peptide to associate with oxidized lipid helps explain its anti-inflammatory and anti-atherogenic properties.

Keywords: Abbreviations; ApoA-I; apolipoprotein A-I; CSI; chemical shift index; DPC; dodecyphosphocholine; DSS; sodium 2,2-dimethyl-2-silapentane-5-sulfonate; KOdiA-PC; 1-palmitoyl-2-(5-keto-6-octene dioyl)phosphatidylcholine; NMR; nuclear magnetic resonance; NOE; nuclear Overhauser effect; NOESY; NOE spectroscopy; TOCSY; total correlation spectroscopy; rmsd; root–mean–square deviation; TPPI; time proportional phase incrementationApolipoprotein J; Class G* helix; Peptide; Dodecylphosphocholine micelle; Oxidized lipid; 1-Palmitoyl-2-(5-keto-6-octene dioyl)phosphatidylcholine; Nuclear magnetic resonance spectroscopy; Isothermal titration calorimetry; Circular dichroism

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BBA - Biomembranes (v.1808, #1)