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BBA - Biomembranes (v.1808, #10)

Editorial Board (pp. i).

Structure and membrane orientation of IAPP in its natively amidated form at physiological pH in a membrane environment by Ravi Prakash Reddy Nanga; Jeffrey R. Brender; Subramanian Vivekanandan; Ayyalusamy Ramamoorthy (pp. 2337-2342).

Human islet amyloid polypeptide is a hormone coexpressed with insulin by pancreatic beta-cells. For reasons not clearly understood, hIAPP aggregates in type II diabetics to form oligomers that interfere with beta-cell function, eventually leading to the loss of insulin production. The cellular membrane catalyzes the formation of amyloid deposits and is a target of amyloid toxicity through disruption of the membrane's structural integrity. Therefore, there is considerable current interest in solving the 3D structure of this peptide in a membrane environment. NMR experiments could not be directly utilized in lipid bilayers due to the rapid aggregation of the peptide. To overcome this difficulty, we have solved the structure of the naturally occurring peptide in detergent micelles at a neutral pH. The structure has an overall kinked helix motif, with residues 7–17 and 21–28 in a helical conformation, and with a 3₁₀ helix from Gly 33–Asn 35. In addition, the angle between the N- and C-terminal helices is constrained to 85°. The greater helical content of human IAPP in the amidated versus free acid form is likely to play a role in its aggregation and membrane disruptive activity.Display Omitted► NMR structure of amidated IAPP at pH 7.3 was solved in SDS (PDB2L86). ► C-terminus is more structured compared to the free acid form at low pH in SDS. ► The angle between the N- and C-terminal helices is constrained to 85°.

Keywords: Abbreviations; IAPP; Islet Amyloid Polypeptide; hIAPP; Human Islet Amyloid Polypeptide; SDS; Sodium Dodecyl Sulfate; NMR; Nuclear Magnetic Resonance; CSI; Chemical Shift Index; NOE; Nuclear Overhauser EffectIslet amyloid polypeptide; Amyloid; Aggregation; Peptide; Membrane; Structure

The fusogenic tilted peptide (67–78) of α-synuclein is a cholesterol binding domain by Jacques Fantini; Denis Carlus; Nouara Yahi (pp. 2343-2351).

Parkinson's disease-associated α-synuclein is an amyloidogenic protein not only expressed in the cytoplasm of neurons, but also secreted in the extracellular space and internalized into glial cells through a lipid raft-dependent process. We previously showed that α-synuclein interacts with raft glycosphingolipids through a structural motif common to various viral and amyloidogenic proteins. Here we report that α-synuclein also interacts with cholesterol, as assessed by surface pressure measurements of cholesterol-containing monolayers. Using a panel of recombinant fragments and synthetic peptides, we identified two distinct cholesterol-binding domains in α-synuclein. One of these domains, which corresponds to the tilted peptide of α-synuclein (67–78), bound cholesterol with high affinity and was toxic for cultured astrocytes. Molecular modeling suggested that cholesterol binds to this peptide with a tilt angle of 46°. α-synuclein also contains a cholesterol recognition consensus motif, which had a lower affinity for cholesterol and was devoid of toxicity. This motif is encased in the glycosphingolipid-binding domain (34–45) of α-synuclein. In raft-like model membranes containing both cholesterol and glycosphingolipids, the head groups of glycosphingolipids prevented the accessibility of cholesterol to exogenous ligands. Nevertheless, cholesterol appeared to ‘signal’ its presence by tuning glycosphingolipid conformation, thereby facilitating α-synuclein binding to raft-like membranes. We propose that the association of α-synuclein with lipid rafts involves both the binding of α-synuclein (34–45) to glycosphingolipids, and the interaction of the fusogenic tilted peptide (67–78) with cholesterol. Coincidentally, a similar mechanism is used by viruses (HIV-1, HTLV-I, Ebola) which display a tilted peptide and fuse with host cell membranes through a sphingolipid/cholesterol-dependent process.Display Omitted► α-Synuclein interacts with lipid rafts. ► α-Synuclein displays two cholesterol-binding domains. ► A CRAC domain binds cholesterol with low affinity. ► A tilted peptide binds cholesterol with high affinity. ► α-Synuclein interacts with lipid rafts through a virus-like mechanism.

Keywords: Abbreviations; βMCD; β-methyl-cyclodextrin; CRAC; cholesterol recognition/interaction amino acid consensus sequence; GBM; glycosphingolipid-binding motif; GSL; glycosphingolipid; MTS; 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; Δπ; _max; maximal surface pressure increase; π; _c; critical pressure of insertion; π; _i; initial surface pressure; NAC; sequence 61–95 of α-synuclein, originally termed non β-amyloid component; PD; Parkinson disease; SBD; sphingolipid-binding domain; v; _i; initial velocityLipid raft; Amyloid protein; Glycosphingolipid; Cholesterol; Tilted peptide; Virus fusion

The fusogenic tilted peptide (67–78) of α-synuclein is a cholesterol binding domain by Jacques Fantini; Denis Carlus; Nouara Yahi (pp. 2343-2351).

Multifaceted action of Fuzeon as virus–cell membrane fusion inhibitor by Avraham Ashkenazi; Yael Wexler-Cohen; Yechiel Shai (pp. 2352-2358).

The viral peptide fusion inhibitor Fuzeon (T-20/DP178/enfuvirtide) is an essential part of the drug combination that has significantly increased the quality of life and life span of many acquired immuno-deficiency syndrome (AIDS) patients. Its development as a drug preceded the elucidation of its precise inhibitory mechanism, as well as its molecular targets. The initial model was that Fuzeon inhibits human immunodeficiency virus (HIV) entry by targeting one site within the viral transmembrane envelope protein. Herein, we describe the emerging discoveries that extend this model towards a multifaceted mechanism for the drug in targeting HIV. This significantly advances the understanding of how viruses enter host cells and opens a new window of opportunity for designing future viral fusion inhibitors.► Fuzeon advances the understanding of how viral transmembrane envelope proteins facilitate membrane fusion. ► Fuzeon targets the cell membrane and multiple sites on gp41 and gp120. ► HIV fusion inhibitors are important candidates in AIDS therapy.

Keywords: HIV entry inhibitor; Membrane fusion; Viral envelope protein; Transmembrane protein

Multifaceted action of Fuzeon as virus–cell membrane fusion inhibitor by Avraham Ashkenazi; Yael Wexler-Cohen; Yechiel Shai (pp. 2352-2358).

Keywords: HIV entry inhibitor; Membrane fusion; Viral envelope protein; Transmembrane protein

Multifaceted action of Fuzeon as virus–cell membrane fusion inhibitor by Avraham Ashkenazi; Yael Wexler-Cohen; Yechiel Shai (pp. 2352-2358).

Keywords: HIV entry inhibitor; Membrane fusion; Viral envelope protein; Transmembrane protein

Instability of toxin A subunit of AB₅ toxins in the bacterial periplasm caused by deficiency of their cognate B subunits by Sang-Hyun Kim; Su Hyang Ryu; Sang-Ho Lee; Yong-Hoon Lee; Sang-Rae Lee; Jae-Won Huh; Sun-Uk Kim; Ekyune Kim; Sunghyun Kim; Sangyong Jon; Russell E. Bishop; Kyu-Tae Chang (pp. 2359-2365).

Shiga toxin (STx) belongs to the AB₅ toxin family and is transiently localized in the periplasm before secretion into the extracellular milieu. While producing outer membrane vesicles (OMVs) containing only A subunit of the toxin (STxA), we created specific STx1B- and STx2B-deficient mutants of E. coli O157:H7. Surprisingly, STxA subunit was absent in the OMVs and periplasm of the STxB-deficient mutants. In parallel, the A subunit of heat-labile toxin (LT) of enterotoxigenic E. coli (ETEC) was absent in the periplasm of the LT-B-deficient mutant, suggesting that instability of toxin A subunit in the absence of the B subunit is a common phenomenon in the AB₅ bacterial toxins. Moreover, STx2A was barely detectable in the periplasm of E. coli JM109 when stx2A was overexpressed alone, while it was stably present when stxB was co-expressed. Compared with STx2 holotoxin, purified STx2A was degraded rapidly by periplasmic proteases when assessed for in vitro proteolytic susceptibility, suggesting that the B subunit contributes to stability of the toxin A subunit in the periplasm. We propose a novel role for toxin B subunits of AB₅ toxins in protection of the A subunit from proteolysis during holotoxin assembly in the periplasm.► We expressed the A subunit of Shiga toxin by itself in the bacterial periplasm. ► We found that its stability depended on co-expression of its cognate B subunit. ► We conclude that the B subunit protects the A subunit from periplasmic proteases.

Keywords: Shiga toxin; Heat-labile toxin; Outer membrane; Periplasm; Escherichia coli

Pulmonary surfactant proteins and polymer combinations reduce surfactant inhibition by serum by Karen W. Lu; Perez-Gil Jesús Pérez-Gil; Mercedes Echaide; H. William Taeusch (pp. 2366-2373).

Acute respiratory distress syndrome (ARDS) is an inflammatory condition that can be associated with capillary leak of serum into alveoli causing inactivation of surfactant. Resistance to inactivation is affected by types and concentrations of surfactant proteins, lipids, and polymers. Our aim was to investigate the effects of different combinations of these three components. A simple lipid mixture (DPPC/POPG) or a more complex lipid mixture (DPPC/POPC/POPG/cholesterol) was used. Native surfactant proteins SP-B and SP-C obtained from pig lung lavage were added either singly or combined at two concentrations. Also, non-ionic polymers polyethylene glycol and dextran and the anionic polymer hyaluronan were added either singly or in pairs with hyaluronan included. Non-ionic polymers work by different mechanisms than anionic polymers, thus the purpose of placing them together in the same surfactant mixture was to evaluate if the combination would show enhanced beneficial effects. The resulting surfactant mixtures were studied in the presence or absence of serum. A modified bubble surfactometer was used to evaluate surface activities. Mixtures that included both SP-B and SP-C plus hyaluronan and either dextran or polyethylene glycol were found to be the most resistant to inhibition by serum. These mixtures, as well as some with either SP-B or SP-C with combined polymers were as or more resistant to inactivation than native surfactant. These results suggest that improved formulations of lung surfactants are possible and may be useful in reducing some types of surfactant inactivation in treating lung injuries.► Addition of both SP-B and SP-C to lipids were better than adding either alone. ► Addition of any single polymer improved resistance to inhibition. ► Mixtures with SP-B and SP-C with combined polymers were more resistant to inactivation than native surfactant. ► Simple lipid mixtures work well with non-ionic polymers. ► Complex lipid mixtures work better with HA or HA combined with PEG or dextran.

Keywords: Pulmonary surfactant lipid; Surfactant protein; Dextran; Polyethylene glycol; Hyaluronan; Polymer

Pulmonary surfactant proteins and polymer combinations reduce surfactant inhibition by serum by Karen W. Lu; Perez-Gil Jesús Pérez-Gil; Mercedes Echaide; H. William Taeusch (pp. 2366-2373).

Keywords: Pulmonary surfactant lipid; Surfactant protein; Dextran; Polyethylene glycol; Hyaluronan; Polymer

Pulmonary surfactant proteins and polymer combinations reduce surfactant inhibition by serum by Karen W. Lu; Perez-Gil Jesús Pérez-Gil; Mercedes Echaide; H. William Taeusch (pp. 2366-2373).

Keywords: Pulmonary surfactant lipid; Surfactant protein; Dextran; Polyethylene glycol; Hyaluronan; Polymer

Self-directed assembly and clustering of the cytoplasmic domains of inwardly rectifying Kir2.1 potassium channels on association with PSD-95 by Svetlana Fomina; Tina D. Howard; Olivia K. Sleator; Marina Golovanova; Liam O'Ryan; Mark L. Leyland; J. Günter Grossmann; Richard F. Collins; Stephen M. Prince (pp. 2374-2389).

The interaction of the extra-membranous domain of tetrameric inwardly rectifying Kir2.1 ion channels (Kir2.1NC₄) with the membrane associated guanylate kinase protein PSD-95 has been studied using Transmission Electron Microscopy in negative stain. Three types of complexes were observed in electron micrographs corresponding to a 1:1 complex, a large self-enclosed tetrad complex and extended chains of linked channel domains. Using models derived from small angle X-ray scattering experiments in which high resolution structures from X-ray crystallographic and Nuclear Magnetic Resonance studies are positioned, the envelopes from single particle analysis can be resolved as a Kir2.1NC₄:PSD-95 complex and a tetrad of this unit (Kir2.1NC₄:PSD-95)₄. The tetrad complex shows the close association of the Kir2.1 cytoplasmic domains and the influence of PSD-95 mediated self-assembly on the clustering of these channels.► This work provides the first glimpse of ion channel clusters at the molecular level. ► The structures show multivalent interactions between Kir2.1 and PSD-95 that drive the self-assembly of complexes. ► A combination of orthogonal structural techniques allows the positioning of high-resolution structures. ► Consistent models for all of the components involved in the formation of the Kir2.1/PSD-95 complexes are shown.

Keywords: Abbreviations; Kir; inwardly rectifying potassium channel; Kir2.1; inwardly rectifying potassium channel subtype 2.1; Kir2.1NC; (fused amino- and carboxy-terminal) cytoplasmic domains of Kir2.1; Kir2.1NC; ₄; homotetramer of Kir2.1NC; Kir2.1; ₄; homotetramer of Kir2.1; Kv; voltage gated potassium channel; Kv1.2; voltage gated potassium channel subtype 1.2; Kv1.2; ₄; homotetramer of Kv1.2; Kvβ; potassium channel β-subunit; Kvβ; ₄; homotetramer of Kvβ; MAGUK; membrane associated guanylate kinase; PSD; post-synaptic density; PSD-95; 95; kDa protein of the post-synaptic density; PDZ; PSD-95/; Drosophila; discs large tumor suppressor DlgA/Zona occludens ZO-1; SH3; Src homology 3; GK; guanylate kinase; SAXS; small angle X-ray scattering; EM; electron microscopy; nsTEM; Transmission Electron Microscopy in negative stain; LCMS; liquid chromatography mass spectrometry; SEC; size exclusion chromatography; SRS; Synchrotron Radiation Source; CTF; contrast transfer function; FSC; Fourier shell correlation; SDS-PAGE; sodium-dodecylsulphate polyacrylamide gel-electrophoresis; E. coli; Escherichia coli; R; _g; radius of gyrationPotassium channel; MAGUK; Cluster; Electron microscopy; Small angle X-ray scattering

The membrane-active regions of the dengue virus proteins C and E by Nemesio Henrique Nemésio; Francis Palomares-Jerez; Villalain José Villalaín (pp. 2390-2402).

We have identified the membranotropic regions of proteins C and E of DENV virus by performing an exhaustive study of membrane rupture induced by two C and E-derived peptide libraries on model membranes having different phospholipid compositions as well as its ability to modulate the DEPE L_β–L_α and L_α–H_II phospholipid phase transitions. Protein C presents one hydrophobic leakage-prone region coincidental with a proposed membrane interacting domain, whereas protein E presents five membrane-rupture zones coincidental with different significant zones of the protein, i.e., the fusion peptide, a proline-rich sequence, a sequence containing a hydrophobic pocket as well as the stem and transmembrane domains of the protein. The identification of these membrane-active segments supports their role in viral membrane fusion, formation of the replication complex and morphogenesis and therefore attractive targets for development of new anti-viral compounds.► We have carried an exhaustive analysis of the membranotropic regions of DENV C and E proteins. ► C protein displayed one highly positive membrane-interacting region. ► E protein showed five different membranotropic regions. ► These zones should be implicated in the molecular mechanism of membrane fusion, replication and/or budding.

Keywords: Abbreviations; BMP; S,R-bis(monooleoylglycero)phosphate; BPI; Bovine brain L-α-phosphadidylinositol; BPS; Bovine brain L-α-phosphatidylserine; CF; 5-Carboxyfluorescein; CHOL; Cholesterol; CL; 1′,3′-bis[1,2-dimyristoyl-; sn; -glycero-3-phospho]-; sn; -glycerol; DENV; Dengue virus; DEPE; 1,2-Dielaidoyl-sn-glycero-3-phosphatidylethanolamine; DSC; Differential Scanning Calorimetry; EPA; Egg L-α-phosphatidic acid; EPC; Egg L-α-phosphatidylcholine; ER; Endoplasmic reticulum; ESM; Egg sphingomyelin; LUV; Large unilamellar vesicles; MLV; Multilamellar vesicles; NS; Non-structural protein; TFE; Trifluoroethanol; T; _m; Temperature of the gel-to–liquid crystalline phase transition; TM; Transmembrane domain; TPE; Egg trans-esterified L-α-phosphatidylethanolamineC/E dengue proteins; Dengue; Virus–cell entry; Membrane fusion; DENV

The membrane-active regions of the dengue virus proteins C and E by Nemesio Henrique Nemésio; Francis Palomares-Jerez; Villalain José Villalaín (pp. 2390-2402).

Structural characterization of AS1–membrane interactions from a subset of HAMP domains by Unnerstale Sofia Unnerståle; Maler Lena Mäler; Roger R. Draheim (pp. 2403-2412).

HAMP domains convert an extracellular sensory input into an intracellular signaling response in a wide variety of membrane-embedded bacterial proteins. These domains are almost invariably found adjacent to the inner leaflet of the cell membrane. We therefore examined the interaction of peptides corresponding to either AS1 or AS2 of four different, well-characterized HAMP domains with several membrane model systems. The proteins included an Archaeoglobus fulgidus protein (Af1503), the Escherichia coli osmosensor EnvZ_Ec, the E. coli nitrate/nitrite sensor NarX_Ec, and the aspartate chemoreceptor of E. coli (Tar_Ec). Far-UV CD and NMR spectroscopy were used to monitor the induction of secondary structure upon association with neutral or acidic large unilamellar vesicles (LUVs) and bicelles. We observed significant increases in α-helicity within AS1 from NarX_Ec and Tar_Ec but not in AS1 from the other proteins. To characterize these interactions further, we determined the solution structure of AS1 from Tar_Ec associated with acidic bicelles. The bulk of AS1 formed an amphipathic α-helix, whereas the N-terminal control cable, the region between TM2 and AS1, remained unstructured. We observed that the conserved prolyl residue found in AS1 of many membrane-adjacent HAMP domains defined the boundary between the unstructured and helical regions. In addition, two positively charged residues that flank the hydrophobic surface of AS1 are thought to facilitate electrostatic interactions with the membrane. We interpret these results within the context of the helix-interaction model for HAMP signaling and propose roles for AS1–membrane interactions during the membrane assembly and transmembrane communication of HAMP-containing receptors.► AS1-containing peptides from NarX_Ec and Tar_Ec become structured in the presence of membrane mimetics. ► AS1 from Tar_Ec forms an amphipathic α-helix upon association with acidic bicelles. ► Residues comprising the control cable of Tar_Ec remain unstructured. ► A conserved prolyl residue separates the unstructured and helical regions. ► The propensity for AS1–membrane interactions correlates with signaling mechanisms and subcellular localization.

Keywords: Abbreviations; HAMP domain; domain present in; h; istidine kinases,; a; denylyl cyclases,; m; ethyl-accepting proteins and; p; hosphatases; SHK; sensor histidine kinase; MCP; methyl-accepting chemotaxis protein; TM1; first transmembrane helix; TM2; second transmembrane helix; AS1; first amphipathic sequence; AS2; second amphipathic sequence; AS1p; AS1-containing peptide; AS2p; AS2-containing peptide; CTR; connector domain; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; POPG; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phospho-(1′-rac-glycerol); LUV; large unilamellar vesicle; DHPC; 1,2-dihexanoyl-d; ₂₂; -; sn; -glycero-3-phosphocholine; DMPC; 1,2-dimyristoyl-d; ₅₄; -; sn; -glycero-3-phosphocholine; DMPG; 1,2-dimyristoyl-d; ₅₄; -; sn; -glycero-3-phospho-(1′-; rac; -glycerol); Far-UV CD; Far-ultraviolet circular dichroism; PFG-NMR; Pulsed field gradient-nuclear magnetic resonance; TOCSY; Total correlation spectroscopy; NOESY; Nuclear Overhauser effect spectroscopy; PD1; periplasmic domain helix 1; PD4; periplasmic domain helix 4HAMP domain; Signal transduction; Transmembrane communication; AS1–membrane interactions; Helix-interaction model

Structural characterization of AS1–membrane interactions from a subset of HAMP domains by Unnerstale Sofia Unnerståle; Maler Lena Mäler; Roger R. Draheim (pp. 2403-2412).

Oleic and linoleic acids are active principles in Nigella sativa and stabilize an E₂P conformation of the Na,K-ATPase. Fatty acids differentially regulate cardiac glycoside interaction with the pump by Yasser A. Mahmmoud; S. Brøgger Christensen (pp. 2413-2420).

Nigella sativa seed oil was found to contain a modulator of Na,K-ATPase. Separation analyses combined with¹H NMR and GCMS identified the inhibitory fraction as a mixture of oleic and linoleic acids. These two fatty acids are specifically concentrated in several medicinal plant oils, and have particularly been implicated in decreasing high blood pressure. The ouabain binding site on Na,K-ATPase has also been implicated in blood pressure regulation. Thus, we aimed to determine how these two molecules modify pig kidney Na,K-ATPase. Oleic and linoleic acids did not modify reactions involving the E₁ (Na⁺) conformations of the Na,K-ATPase. In contrast, K⁺ dependent reactions were strongly modified after treatment. Oleic and linoleic acids were found to stabilize a pump conformation that binds ouabain with high affinity, i.e., an ion free E₂P form. Time-resolved binding assays using anthroylouabain, a fluorescent ouabain analog, revealed that the increased ouabain affinity is unique to oleic and linoleic acids, as compared with γ-linolenic acid, which decreased pump-mediated ATP hydrolysis but did not equally increase ouabain interaction with the pump. Thus, the dynamic changes in plasma levels of oleic and linoleic acids are important in the modulation of the sensitivity of the sodium pump to cardiac glycosides. Given the possible involvement of the cardiac glycoside binding site on Na,K-ATPase in the regulation of hypertension, we suggest oleic acid to be a specific chaperon that modulates interaction of cardiac glycosides with the sodium pump.► Oleic and linoleic acids are active components in Nigella Sativa seeds. ► Oleic and linoleic acids increase the number of static sodium pump units. ► Oleic and linoleic acids enhance ouabain interaction with Na,K-ATPase. ► Oleic and linoleic acids possibly act as Na,K-ATPase chaperons.

Keywords: Abbreviations; EDTA; Ethylenediaminetetraacetic acid; EP; phosphoenzyme; DMSO; Dimethyl sulfoxide; pNPP; para; -nitrophenyl phosphate; TCA; trichloroacetic acidNa,K-ATPase; Nigella Sativa; Oleic acid; Linoleic acid; Ouabain affinity; Anthroyl ouabain

Effects of positively charged arginine residues on membrane pore forming activity of Rev–NIS peptide in bacterial cells by Juneyoung Lee; Hyemin Choi; Jaeyong Cho; Dong Gun Lee (pp. 2421-2427).

Here, we investigated antibacterial effects of Rev–NIS and suggested the role of positively charged amino acids on membrane pore forming activity of the peptide in bacterial cells, by synthesizing two analogs, Anal R and Anal S. Based on the amphipathic property of Rev–NIS, Anal R and Anal S were designed by substituting E¹ and L³ to R and L³ to S, respectively. The circular dichroism (CD) spectroscopy showed that Anal R and Anal S have the same conformation of Rev–NIS, with a significant fraction of helical structure. In succession, the antibacterial susceptibility testing showed that Rev–NIS and its analogs possessed significant activities (Anal R>Rev–NIS>Anal S), without hemolytic effects, against bacterial pathogens including antibiotics-resistant strains. Moreover, the membrane studies, 3,3′-dipropylthiadicarbocyanine iodide (diSC₃5) staining and FITC-dextran (FD) leakage assay demonstrated that the analogs as well as Rev–NIS acted on the bacterial membranes and potently made pores, with the hydrodynamic radius between 1.4nm and 2.3nm. Especially, Anal R made larger pores than other peptides, with the radius between 2.3nm and 3.3nm. These results also corresponded to the result of antibacterial susceptibility testing. In summary, this study indicates that the two arginine residues are more influential than the hydrophobicity or the helicity, regarding the molecular activity of the peptide, and finally suggests that Anal R peptide may be applied to novel antibacterial agents.► We design two analogs of Rev–NIS, substituted with arginine and serine. ► We investigate the antibacterial property of the peptides. ► All the peptides exert the activity on the bacterial membranes. ► Anal R is especially thought to be the most potent antibacterial peptide. ► The net positive charge of arginine residues is one of the most influential factors.

Keywords: Abbreviations; CD; circular dichroism; diSC; ₃; 5; 3,3′-dipropylthiadicarbocyanine iodide; FITC; fluorescein isothiocyanate; FD; FITC-dextran; AMP; antimicrobial peptide; NIS; nuclear entry inhibitory signal; TFE; 2,2,2-trifluoroethanol; SDS; sodium dodecyl sulfate; MIC; minimum inhibitory concentration; ARPA; antibiotics-resistant; Pseudomonas aeruginosa; PE; phosphatidylethanolamine; PG; phosphatidylglycerol; DCM; dichloromethane; DMF; N,N; -dimethylformamide; Fmoc; fluorenylmethyloxycarbonyl; HOBt; 1-hydroxybenzotriazole; DIC; 1,3-diisopropylcarbodiimide; TFA; trifluoroacetic acid; CLSI; clinical and laboratory standards institute; ITO; indium tin oxideHIV-1; Rev protein; Rev–NIS; Analog; Antibacterial peptide

Effects of positively charged arginine residues on membrane pore forming activity of Rev–NIS peptide in bacterial cells by Juneyoung Lee; Hyemin Choi; Jaeyong Cho; Dong Gun Lee (pp. 2421-2427).

Desmosterol replaces cholesterol for ligand binding function of the serotonin_1A receptor in solubilized hippocampal membranes: Support for nonannular binding sites for cholesterol? by Pushpendra Singh; Md. Jafurulla; Yamuna Devi Paila; Amitabha Chattopadhyay (pp. 2428-2434).

The serotonin_1A receptor is an important member of the G-protein coupled receptor family, and is involved in the generation and modulation of a variety of cognitive and behavioral functions. Solubilization of the hippocampal serotonin_1A receptor by CHAPS is accompanied by loss of cholesterol that results in a reduction in specific agonist binding activity. Replenishment of cholesterol to solubilized membranes restores membrane cholesterol content and significantly recovers specific agonist binding. In order to test the stringency of cholesterol requirement, we solubilized native hippocampal membranes followed by replenishment with desmosterol. Desmosterol is the immediate biosynthetic precursor of cholesterol in the Bloch pathway differing only in a double bond at the 24th position. Our results show that replenishment with desmosterol restores ligand binding of serotonin_1A receptors. This is consistent with earlier results showing that desmosterol can replace cholesterol in a large number of cases. However, these results appear to be contradictory to our earlier findings, performed by sterol manipulation utilizing methyl-β-cyclodextrin, in which we observed that replacing cholesterol with desmosterol is unable to restore specific ligand binding of the hippocampal serotonin_1A receptor. We discuss the possible molecular mechanism, in terms of nonannular lipid binding sites around the receptor, giving rise to these differences.Display Omitted► Desmosterol restores ligand binding function of solubilized serotonin_1A receptors. ► Nonannular binding of cholesterol is crucial for the serotonin_1A receptor function. ► Caution should be exercised while interpreting results of cholesterol manipulation.

Keywords: Serotonin; _1A; receptor; Cholesterol; Desmosterol; Ligand binding function; Fluorescence anisotropy; Nonannular binding site

Effects of synaptotagmin 2 on membrane fusion between liposomes that contain SNAREs involved in exocytosis in mast cells by Yumiko Nagai; Satoshi Tadokoro; Hiroki Sakiyama; Naohide Hirashima (pp. 2435-2439).

Mast cells play a pivotal role in allergic responses. Antigen stimulation causes elevation of the intracellular Ca²⁺ concentration, which triggers the exocytotic release of inflammatory mediators such as histamine. Recent research, including our own, has revealed that SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins such as syntaxin-3, -4, SNAP-23, and VAMP-8 are involved in exocytosis. Although exocytosis in mast cells is Ca²⁺ dependent, the target molecule that interacts with Ca²⁺ is not clear. Synaptotagmin is a Ca²⁺ sensor and regulates exocytosis in neuronal cells. However, the role of synaptotagmin 2, a member of the synaptotagmin family, in exocytosis in mast cells remains controversial. In this study, we investigated the role of synaptotagmin 2 by a liposome-based fusion assay. SNARE proteins (SNAP-23, syntaxin-3, VAMP-8) and synaptotagmin 2 were expressed in Escherichia coli and purified as GST-tagged or His-tagged fusion proteins. These SNARE proteins were incorporated by a detergent dialysis method. Membrane fusion between liposomes was monitored by fluorescence resonance energy transfer between fluorescent-labeled phospholipids. In the presence of Ca²⁺, low synaptotagmin 2 concentration inhibited membrane fusion between SNARE-containing liposomes, while high synaptotagmin 2 concentration enhanced membrane fusion. This enhancement required phosphatidylserine as a membrane component. These results suggest that synaptotagmin 2 regulates membrane fusion of SNARE-containing liposomes involved in exocytosis in mast cells, and that this regulation is dependent on synaptotagmin 2 concentration, Ca²⁺, and phosphatidylserine.► Mast cell is a key player in allergic responses. ► Mast cell secretes inflammatory mediators by SNARE-dependent exocytosis. ► We investigated the role of synaptotagmin 2 in exocytosis, using liposomes. ► High concentration of synaptotagmin 2 enhanced the SNARE-mediated liposome fusion. ► Low concentration of synaptotagmin 2 inhibited the liposome fusion.

Keywords: Synaptotagmin; SNARE; Exocytosis; Liposome; Membrane fusion; Mast cell

DODAB:monoolein-based lipoplexes as non-viral vectors for transfection of mammalian cells by J.P. Neves Silva; A.C.N. Oliveira; M.P.P.A. Casal; A.C. Gomes; P.J.G. Coutinho; O.P. Coutinho; M.E.C.D. Real Oliveira (pp. 2440-2449).

DNA/Cationic liposome complexes (lipoplexes) have been widely used as non-viral vectors for transfection. Neutral lipids in liposomal formulation are determinant for transfection efficiency using these vectors. In this work, we studied the potential of monoolein (MO) as helper lipid for cellular transfection. Lipoplexes composed of pDNA and dioctadecyldimethylammonium bromide (DODAB)/1-monooleoyl-rac-glycerol (MO) at different molar ratios (4:1, 2:1 and 1:1) and at different cationic lipid/DNA ratios were investigated. The physicochemical properties of the lipoplexes (size, charge and structure), were studied by Dynamic Light Scattering (DLS), Zeta Potential (ζ) and cryo-transmission electron microscopy (cryo-TEM). The effect of MO on pDNA condensation and the effect of heparin and heparan sulphate on the percentage of pDNA release from the lipoplexes were also studied by Ethidium Bromide (EtBr) exclusion assays and electrophoresis. Cytotoxicity and transfection efficiency of these lipoplexes were evaluated using 293T cells and compared with the golden standard helper lipids 1,2-dioleoyl-sn-glycero-3-hosphoethanolamine (DOPE) and cholesterol (Chol) as well as with a commercial transfection agent (Lipofectamine™ LTX). The internalization of transfected fluorescently-labeled pDNA was also visualized using the same cell line. The results demonstrate that the presence of MO not only increases pDNA compactation efficiency, but also affects the physicochemical properties of the lipoplexes, which can interfere with lipoplex-cell interactions. The DODAB:MO formulations tested showed little toxicity and successfully mediated in vitro cell transfection. These results were supported by fluorescence microscopy studies, which illustrated that lipoplexes were able to access the cytosol and deliver pDNA to the nucleus. DODAB:MO-based lipoplexes were thus validated as non-toxic, efficient lipofection vectors for genetic modification of mammalian cells. Understanding the relation between structure and activity of MO-based lipoplexes will further strengthen the development of these novel delivery systems.Display Omitted► We studied the potential of monoolein as new helper lipid for non-viral transfection. ► We examined the physicochemical characteristics of DODAB:MO lipoplexes. ► Cytotoxicity and transfection efficiency were evaluated in 293T cells. ► MO favors DNA condensation and affects the structural properties of lipoplexes. ► MO-based lipoplexes were validated as non-toxic and efficient lipofection vectors.

Keywords: Abbreviations; DODAB; dioctadecyldimethylammonium bromide; MO; 1-monooleoyl-rac-glycerol; DOPE; 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DOTMA; 2,3-diokeyloxypropyl-1-trimethyl ammonium chloride; Ch; cholesterol; DLS; Dynamic Light Scattering; ζ-potential; Zeta potential; cryo-TEM; cryo-transmission electron microscopyMonoolein-based lipoplexes; pDNA complexation; Transfection; Cytotoxicity

Molecular dynamics simulation of phase transitions in model lung surfactant monolayers by Susan L. Duncan; Indranil S. Dalal; Ronald G. Larson (pp. 2450-2465).

To explore the role of individual lung surfactant components in liquid-condensed (LC)/liquid-expanded (LE) phase transitions the MARTINI coarse-grained (CG) model is used to simulate monolayers containing DPPC and additional lipid or peptide components. Our analysis suggests that the LC phase forms from the LE phase via a nucleation and growth mechanism, while the LC–LE transition occurs by melting that originates from defects in the monolayer. On the time scale of our simulations, DPPC monolayers display a substantial hysteresis between the ordering and disordering transitions, which is decreased by the addition of a second component. In binary mixtures of DPPC with lung surfactant peptide fragment SP-B_1–25, the ordered side of the hysteresis loop is abolished altogether, suggesting that SP-B_1–25 effectively nucleates disorder in the monolayer on heating. SP-B_1–25 is observed to perturb the packing of the surrounding lipids leading to local fluidization of the monolayer and to aggregate within the LE phase. In 1:1 DPPC:POPC monolayers, a high concentration of unsaturated phospholipid leads to a substantial decrease in the LC–LE and LE–LC transition temperatures. Adding cholesterol to pure DPPC increases the LC–LE and LE–LC transition temperatures and increases the order on the disordered side of the hysteresis loop leading to a phase of intermediate order, which could be the liquid-disordered (Ld) phase. Cholesterol is also observed to show a preference for LC–LE domain boundaries. The results of our molecular dynamics simulations coincide with many experimental observations and can help provide insight into the physiological roles of individual surfactant components.► Molecular dynamics simulation of liquid-condensed–liquid-expanded phase transitions. ► Liquid-condensed phase forms via nucleation and growth. ► Disordering transition occurs by melting originating at defects. ► SP-B_1–25 aggregates in the liquid-expanded phase and nucleates disorder in the monolayer. ► Cholesterol displays a preference for the interface between the ordered and disordered phases.

Keywords: Pulmonary surfactant; Liquid condensed; Liquid expanded; Phase coexistence; Nucleation; Melting

Molecular dynamics simulation of phase transitions in model lung surfactant monolayers by Susan L. Duncan; Indranil S. Dalal; Ronald G. Larson (pp. 2450-2465).

Keywords: Pulmonary surfactant; Liquid condensed; Liquid expanded; Phase coexistence; Nucleation; Melting

Molecular dynamics simulation of phase transitions in model lung surfactant monolayers by Susan L. Duncan; Indranil S. Dalal; Ronald G. Larson (pp. 2450-2465).

Keywords: Pulmonary surfactant; Liquid condensed; Liquid expanded; Phase coexistence; Nucleation; Melting

Glycolipid–cholesterol monolayers: Towards a better understanding of the interaction between the membrane components by Beata Korchowiec; Jacek Korchowiec; Masakatsu Hato; Ewa Rogalska (pp. 2466-2476).

In this work, the interaction between a synthetic analog of archaeal lipids and cholesterol was studied using Langmuir technique. The lipid, β-Mal₃O(C16+₄)₂, contained phytanyl chains attached via two ether bonds to the sn-2 carbon of the glycerol backbone. The preliminary studies showed that monolayers formed with the pure lipid have a liquid-like character; here, a hypothesis that admixing cholesterol to β-Mal₃O(C16+₄)₂ could confer a higher rigidity on the films was tested.To check this proposal, two-dimensional miscibility of cholesterol and β-Mal₃O(C16+₄)₂ in monomolecular films was studied using surface pressure and surface potential measurements, as well as Brewster angle microscopy and polarization-modulation infrared reflection absorption spectroscopy. The stability of the monomolecular films was evaluated based on thermodynamics of mixing of cholesterol and β-Mal₃O(C16+₄)₂. Atomic level information concerning the orientation of molecules and the degree of hydration of polar headgroups was obtained from molecular dynamics simulations.► The presence of cholesterol in glycolipid films yields condensed phase domains with an organized molecular arrangement. ► The formation of condensed phase domains is driven by chain ordering of both cholesterol and β-Mal₃O(C₁₆₊₄)₂. ► The maltooligosaccharide headgroup has a strong propensity for hydrogen bonding.

Keywords: Model biological membranes; Binary mixture; Langmuir monolayers; Lipid miscibility; Molecular dynamics simulation

The hERG K⁺ channel S4 domain L532P mutation: Characterization at 37°C by Yi H. Zhang; Charlotte K. Colenso; Richard B. Sessions; Christopher E. Dempsey; Jules C. Hancox (pp. 2477-2487).

hERG ( human Ether-à-go-go Related Gene) is responsible for ion channels mediating rapid delayed rectifier potassium current, I_Kr, which is key to cardiac action potential repolarization. Gain-of-function hERG mutations give rise to the SQT1 variant of the Short QT Syndrome (SQTS). Reggae mutant zebrafish, with a S4 zERG mutation (Leucine499Proline; L499P), display arrhythmic features analogous to those seen in the SQTS. The affected S4 domain ERG residue is highly conserved. This study was executed to determine how the homologous hERG mutation (L532P) influences channel function at 37°C. Whole-cell measurements of current (I_hERG) were made from HEK 293 cells expressing WT or L532P hERG. The half maximal activation voltage (V_0.5) of L532P I_hERG was positively shifted by ~+36mV compared to WT I_hERG; however at negative voltages a pronounced L532P I_hERG was observed. Both activation and deactivation time-courses were accelerated for L532P I_hERG. The inactivation V_0.5 for L532P I_hERG was shifted by ~+32mV. Under action potential (AP) voltage-clamp, L532P I_hERG exhibited a dome-shaped current peaking at ~+16mV, compared to ~−31mV for WT-I_hERG. The L532P mutation produced an ~5-fold increase in the IC₅₀ for dronedarone inhibition of I_hERG. Homology modeling indicated that the L532 residue within the S4 helix lies closely apposed to the S5 region of an adjacent hERG subunit. Alterations to the S4 domain structure and, potentially, to interactions between adjacent hERG subunits are likely to account for the functional effects of this mutation.► The L532P hERG channel mutation was studied at 37°C. ► The L532P mutation significantly modified activation and inactivation kinetics. ► hERG current elicited by AP voltage clamp was also significantly altered. ► The mutation also decreased channel sensitivity to inhibition by dronedarone. ► Homology modeling provides a structural context for the effects of this mutation.

Keywords: Arrhythmia; Dronedarone; hERG; I; _Kr; Short QT syndrome; Potassium channel

The hERG K⁺ channel S4 domain L532P mutation: Characterization at 37°C by Yi H. Zhang; Charlotte K. Colenso; Richard B. Sessions; Christopher E. Dempsey; Jules C. Hancox (pp. 2477-2487).

Keywords: Arrhythmia; Dronedarone; hERG; I; _Kr; Short QT syndrome; Potassium channel

The hERG K⁺ channel S4 domain L532P mutation: Characterization at 37°C by Yi H. Zhang; Charlotte K. Colenso; Richard B. Sessions; Christopher E. Dempsey; Jules C. Hancox (pp. 2477-2487).

Keywords: Arrhythmia; Dronedarone; hERG; I; _Kr; Short QT syndrome; Potassium channel

Laminar shear stress modulates the activity of heterologously expressed P2X₄ receptors by Sarah Kessler; Wolfgang G. Clauss; Martin Fronius (pp. 2488-2495).

P2X₄ receptors are involved in mechanotransduction processes, but it is unknown whether or not P2X₄ receptors form mechanosensitive ion channels. This study questioned, whether laminar shear stress (LSS) can modulate P2X₄ receptor activity. Mouse P2X₄ receptor was cloned and heterologously expressed in Xenopus laevis oocytes. In two-electrode-voltage-clamp experiments the application of ATP (100μM) produced a transient inward current that was decreased by about 50% upon a second ATP application, corresponding to the desensitization behavior of P2X₄ receptors. In P2X₄ expressing oocytes LSS (shear forces of ~5.1dynes/cm²) did not produce any effect. However, LSS modulated the response of P2X₄ to ATP. With LSS (~5.1dynes/cm²) the desensitization of the current due to the second ATP application was diminished. Ivermectin (IVM), a compound which stabilizes the open state of P2X₄ receptors, mimicked the effect of LSS (~5.1dynes/cm²), since there was no additional effect of LSS after pre-incubation with IVM detected. This indicates that LSS like IVM stabilizes the open state of the receptor, although the particular mechanism remains unknown. These data demonstrate that LSS modulates the activity of P2X₄ receptors by eliminating the desensitization of the receptors in response to ATP probably by stabilizing the open state of the channel.► Laminar shear stress (LSS) modulates the ATP-mediated response of P2X4 receptors by preventing desensitization. ► Ivermectin (IVM), which stabilzes the open state of P2X4 receptors, mimicked the effect of LSS. ► P2X4 receptors are capable to sense physical forces and shear forces in particular. ► This feature is important since P2X4 receptors are expressed in endothelial cells, which are permanently exposed to LSS.

Keywords: Abbreviations; LSS; laminar shear stress; ATP; adenosine-5′-triphosphate; IVM; ivermectin; DEG/ENaC; Degenerin/epithelial Na; ⁺; channel; TRP; transient receptor potential; ENaC; epithelial Na; ⁺; channel; HEPES; 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid; TEVC; two-electrode-voltage-clamp; ORi; oocyte Ringer's solution; DMSO; dimethyl sulfoxide; PPADS; pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid); TM; transmembrane domainP2X; ₄; receptor; Laminar shear stress; ATP; Mechanosensitive ion channels; Ivermectin

Laminar shear stress modulates the activity of heterologously expressed P2X₄ receptors by Sarah Kessler; Wolfgang G. Clauss; Martin Fronius (pp. 2488-2495).

Oxidative stress increases angiotensin receptor type I responsiveness by increasing receptor degree of aggregation using image correlation spectroscopy by Titiwat Sungkaworn; Yongwimon Lenbury; Varanuj Chatsudthipong (pp. 2496-2500).

Oxidative stress and hyper-functioning of angiotensin II receptor type I (AT₁R) are commonly observed in hypertensive patients but the relationship between oxidative stress and AT₁R function is still unclear. We investigated the effects of H₂O₂ treatment on AT₁R-mediated intracellular calcium [Ca²⁺]_i signaling and its cell surface distribution pattern in HEK cells stably expressing EGFP-tagged rat AT₁R using image correlation spectroscopy (ICS). Following H₂O₂ treatment (50–800μM), [Ca²⁺]_i was significantly increased upon angiotensin II stimulation. Similarly ICS revealed a significant increase in degree of AT₁R aggregation in H₂O₂ treated group during Ang II activation but no difference in cluster density compared with untreated control cells or those with N-acetyl cysteine pretreatment. Thus, oxidative stress-induced AT₁R hyper-responsiveness can be attributed by an increase in cell surface receptor aggregation state, possibly stemming in part from oxidant-related increase receptor–receptor interactions.►We use ICS to characterize cell surface AT₁R distribution pattern in living cells. ►Oxidative stress induces aggregation and hyper-function of AT₁R. ►AT₁R distribution pattern markedly affects its signaling i.e. increased [Ca²⁺]_i. ►This is possibly caused by oxidant-induced receptor dimerization.

Keywords: Abbreviations; AT; ₁; R; angiotensin II receptor type I; AT; ₂; R; angiotensin II receptor type II; Ang II; angiotensin II; BMP; bone morphogenetic protein; CD; cluster density; DA; degree of aggregation; EGFP; enhanced green fluorescent protein; GPCR; G - protein coupled receptor; HBSS; Hanks' balanced salt solution; HEK cell; human embryonic kidney cell; ICS; image correlation spectroscopy; IP3; inositol triphosphate; [Ca; ²⁺; ]; _i; intracellular calcium concentration; MTT; thiazolyl blue tetrazolium bromide; NAC; N; -acetyl cysteine; NOX1; NADPH oxidase 1; RAS; renin angiotensin system; ROS; reactive oxygen speciesOxidative stress; Angiotensin receptor; Image correlation spectroscopy; Aggregation

Osmotin from Calotropis procera latex: New insights into structure and antifungal properties by Cleverson Diniz Teixeira de Freitas; José Luiz de Souza Lopes; Leila Maria Beltramini; Raquel Sombra Basílio de Oliveira; José Tadeu Abreu Oliveira; Márcio Viana Ramos (pp. 2501-2507).

This study aimed at investigating the structural properties and mechanisms of the antifungal action of CpOsm, a purified osmotin from Calotropis procera latex. Fluorescence and CD assays revealed that the CpOsm structure is highly stable, regardless of pH levels. Accordingly, CpOsm inhibited the spore germination of Fusarium solani in all pH ranges tested. The content of the secondary structure of CpOsm was estimated as follows: α-helix (20%), β-sheet (33%), turned (19%) and unordered (28%), RMSD 1%. CpOsm was stable at up to 75°C, and thermal denaturation (T_m) was calculated to be 77.8°C. This osmotin interacted with the negatively charged large unilamellar vesicles (LUVs) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-1-glycerol (POPG), inducing vesicle permeabilization by the leakage of calcein. CpOsm induced the membrane permeabilization of spores and hyphae from Fusarium solani, allowing for propidium iodide uptake. These results show that CpOsm is a stable protein, and its antifungal activity involves membrane permeabilization, as property reported earlier for other osmotins and thaumatin-like proteins.Display Omitted►In this study we reported new insights into structure and antifungal properties of a latex protein. ►The protein was highly stable and active regardless pH conditions. ►It interacted and induced permeabilization of negatively charged large unilamellar vesicles. ►It also induced the membrane permeabilization of spores and hyphae from Fusarium solani, allowing for propidium iodide uptake.

Keywords: Calcein; Fusarium solani; Laticifer protein; Propidium iodide; Thaumatin-like protein

G117C MelB, a mutant melibiose permease with a changed conformational equilibrium by C. Ganea; K. Meyer-Lipp; R. Lemonnier; A. Krah; G. Leblanc; K. Fendler (pp. 2508-2516).

Replacement of the glycine at position 117 by a cysteine in the melibiose permease creates an interesting phenotype: while the mutant transporter shows still transport activity comparable to the wild type its pre steady-state kinetic properties are drastically altered. The transient charge displacements after substrate concentration jumps are strongly reduced and the fluorescence changes disappear. Together with its maintained transport activity this indicates that substrate translocation in G117C melibiose permease is not impaired but that the initial conformation of the mutant transporter differs from that of the wild type permease. A kinetic model for the G117C melibiose permease based on a rapid dynamic equilibrium of the substrate free transporter is proposed. Implications of the kinetic model for the transport mechanism of the wild type permease are discussed.► Steady-state kinetics of G117C MelB like wild type. ► Pre steady-state kinetics altered in G117C MelB. ► Initial configuration of G117C MelB differs from wild type. ► Rapid dynamic equilibrium of substrate free transporter.

Keywords: Abbreviations; MelB; melibiose permease; E. coli; Escherichia coli; SSM; solid supported membrane; c-less; cysteine-less; KPi; potassium phosphate; Lapao; 3-(laurylamido)-N,N′-dimethylaminopropylamine oxide; PC; phosphatidylcholine; Dns; ²; -S-Gal; 2′-(; N; -dansyl)-aminoethyl-1-thio-β-; d; -galactopyranoside; [; ³; H]αNPG; p; -Nitrophenyl α-d-(6-; ³; H)galactopyranoside; NEM; N-ethylmaleimide; PCMBS; p-chloromercuribenzenesulphonate; MTS; methanethiosulfonate; KPi; potassium phosphate; RSO; right-side-out; ISO; inside-out; IMV; inverted membrane vesicles; FRET; fluorescence resonance energy transfer; Trp-fluorescence; tryptophan-fluorescence; FTIR; Fourier transform infraredSolid supported membrane; Electrophysiology; Fluorescence spectroscopy; Kinetic model; Transport mechanism; Sugar transport

G117C MelB, a mutant melibiose permease with a changed conformational equilibrium by C. Ganea; K. Meyer-Lipp; R. Lemonnier; A. Krah; G. Leblanc; K. Fendler (pp. 2508-2516).

Lipid composition modulates the interaction of peptides deriving from herpes simplex virus type I glycoproteins B and H with biomembranes by Giuseppe Vitiello; Annarita Falanga; Massimiliano Galdiero; Derek Marsh; Stefania Galdiero; Gerardino D'Errico (pp. 2517-2526).

Lipid membranes play a key role in the viral life cycle. Enveloped viruses particularly require a sequence of fusion and fission events between the viral envelope and the target membranes for entry into the cell and egress from it. These processes are controlled by one or more viral glycoproteins that undergo conformational changes favoring the necessary micro- and mesoscopic lipid re-arrangements. Multiple regions from these glycoproteins are thought to interact with the membranes, according to a concerted mechanism, in order to generate the distortion necessary for fusion. In this work, we perform an EPR study on the role played by the membrane composition in tuning the interaction between lipid bilayers and two peptides, gH626–644 and gB632–650, that are highly fusogenic fragments of the gH and gB glycoproteins of herpes simplex virus. Our results show that both peptides interact with lipid bilayers, perturbing the local lipid packing. gH626–644 localizes close to the hydrophilic bilayer surface, while gB632–650 penetrates deeply into the membrane. Chain perturbation by the peptides increases in the presence of charged phospholipids. Finally, cholesterol does not alter the ability of gB632–650 to penetrate deeply in the membrane, whereas it limits penetration of the gH626–644 peptide to the more external layer. The different modes of interaction result in a higher fusogenic ability of gB632–650 towards cholesterol-enriched membranes, as demonstrated by lipid mixing assays. These results suggest that the mechanism of action of the gH and gB glycoproteins is modulated by the properties and composition of the phospholipid bilayer.► Two peptides deriving from HSV glycoproteins B and H interact with lipid bilayers. ► Peptide insertion perturbs lipid packing and dynamics. ► Bilayer composition modulates the peptide–membrane interaction. ► Anionic phospholipids enhance the peptide–bilayer interaction. ► Cholesterol reduces the gH peptide–bilayer interaction.

Keywords: Phospholipid; Cholesterol; Fusion peptide; Viral fusion; Herpes simplex virus; Electron paramagnetic resonance

Cholesterol regulates prokaryotic Kir channel by direct binding to channel protein by Dev K. Singh; Tzu-Pin Shentu; Decha Enkvetchakul; Irena Levitan (pp. 2527-2533).

Cholesterol is a major regulator of a variety of ion channels but the mechanisms underlying cholesterol sensitivity of ion channels are still poorly understood. The key question is whether cholesterol regulates ion channels by direct binding to the channel protein or by altering the physical environment of lipid bilayer. In this study, we provide the first direct evidence that cholesterol binds to prokaryotic Kir channels, KirBac1.1, and that cholesterol binding is essential for its regulatory effect. Specifically, we show that cholesterol is eluted together with the KirBac1.1 protein when separated on an affinity column and that the amount of bound cholesterol is proportional to the amount of the protein. We also show that cholesterol binding to KirBac1.1 is saturable with a K_D of 390μM. Moreover, there is clear competition between radioactive and non-radioactive cholesterol for the binding site. There is no competition, however, between cholesterol and 5-Androsten 3β-17 β-diol, a sterol that we showed previously to have no effect on KirBac1.1 function. Finally, we show that cholesterol–KirBac1.1 binding is significantly inhibited by trifluoperazine, known to inhibit cholesterol binding to other proteins, and that inhibition of cholesterol–KirBac1.1 binding results in full recovery of the channel activity. Collectively, results from this study indicate that cholesterol-induced suppression of KirBac1.1 activity is mediated by direct interaction between cholesterol and the channel protein.► Channel activity of Kir2 sub-family of inward rectifiers is strongly suppressed by the elevation of cellular cholesterol. ► The goal of this study is to find the mechanism by which cholesterol regulate the function of Kir channels. ► Provides first direct evidence for specific binding between cholesterol and ion channel. ► Cholesterol–Kir binding is required for cholesterol suppression of the channel function. ► There is competition between radioactive and non-radioactive cholesterol for the binding site.

Keywords: Cholesterol; KirBac1.1; Lipid–protein interaction; Lipid-bilayer; Ion channel

Cholesterol regulates prokaryotic Kir channel by direct binding to channel protein by Dev K. Singh; Tzu-Pin Shentu; Decha Enkvetchakul; Irena Levitan (pp. 2527-2533).

Keywords: Cholesterol; KirBac1.1; Lipid–protein interaction; Lipid-bilayer; Ion channel

Cholesterol regulates prokaryotic Kir channel by direct binding to channel protein by Dev K. Singh; Tzu-Pin Shentu; Decha Enkvetchakul; Irena Levitan (pp. 2527-2533).

Keywords: Cholesterol; KirBac1.1; Lipid–protein interaction; Lipid-bilayer; Ion channel

Influence of the lipid composition of biomimetic monolayers on the structure and orientation of the gp41 tryptophan-rich peptide from HIV-1 by Gladys Matar; Françoise Besson (pp. 2534-2543).

The tryptophan-rich peptide of gp41 (so-called gp41W), one of the two envelope glycoproteins of HIV-1, is known to play a crucial role in the fusion between this virus and the host cell membranes. The influence of lipids on this role was investigated using different lipid monolayers at the air–water interface. Gp41W affinity for the lipid monolayer was measured by following the peptide-induced variation in the lateral surface pressure and we demonstrated that gp41W binds to monolayers containing the saturated zwitterionic dipalmitoylphosphatidylcholine (DPPC) as well as to the anionic dipalmitoylphosphatidylglycerol (DPPG) and to mixed monolayers containing DPPC and cholesterol (Chol). The secondary structure of gp41W in the presence of these lipid monolayers was determined by polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS). The data showed that gp41W was an oriented α-helix in the presence of DPPG. However this spectroscopic method was unable to detect the gp41W structure in the presence of DPPC and DPPC/Chol monolayer. The peptide-induced modifications of the DPPC/Chol, DPPC and DPPG monolayer morphology were analyzed by Brewster angle microscopy (BAM). The peptide-induced changes in the DPPG monolayer morphology suggest that gp41W disturbed the lipid intermolecular interactions. Furthermore the peptide delayed the condensed state of DPPC and DPPC/Chol, indicating that, although gp41W was not detected by PM-IRRAS, it was present in these lipid monolayers.► Gp41W is the Trp-rich peptide of the HIV-1 glycoprotein 41. ► Its interaction with DPPC, DPPC/Chol and DPPG monolayers is surface pressure-dependent. ► Gp41W keeps its α-helix conformation in the presence of DPPG monolayers.

Keywords: Abbreviations; BAM; Brewster angle microscopy; Chol; cholesterol; DPPC; dipalmitoylphosphatidylcholine; DPPG; dipalmitoylphosphatidylglycerol; ET; exposure time; GL; grey level; П/A; surface pressure/area; Π; _i; initial surface pressure; PM-IRRAS; polarization modulation infrared reflection absorption spectroscopyBrewster angle microscopy; Langmuir monolayer; Membrane proximal external region of gp41; Peptide orientation; PM-IRRAS

Influence of the lipid composition of biomimetic monolayers on the structure and orientation of the gp41 tryptophan-rich peptide from HIV-1 by Gladys Matar; Françoise Besson (pp. 2534-2543).

Directed evolution of efficient secretion in the SRP-dependent export of TolB by Yaramah M. Zalucki; William M. Shafer; Michael P. Jennings (pp. 2544-2550).

Signal sequence non-optimal codons have been shown to be important for the folding and efficient export of maltose binding protein (MBP), a SecB dependent protein. In this study, we analysed the importance of signal sequence non-optimal codons of TolB, a signal recognition particle (SRP) dependent exported protein. The protein production levels of wild type TolB (TolB-wt) and a mutant allele of TolB in which all signal sequence non-optimal codons were changed to a synonymous optimal codon (TolB-opt), revealed that TolB-opt production was 12-fold lower than TolB-wt. This difference could not be explained by changes in mRNA levels, or plasmid copy number, which was the same in both strains. A directed evolution genetic screen was used to select for mutants in the TolB-opt signal sequence that resulted in higher levels of TolB production. Analysis of the 46 independent TolB mutants that reverted to wild type levels of expression revealed that at least four signal sequence non-optimal codons were required. These results suggest that non-optimal codons may be required for the folding and efficient export of all proteins exported via the Sec system, regardless of whether they are dependent on SecB or SRP for delivery to the inner membrane.► Signal sequence rare codons are required for export of SRP-dependent proteins. ► Selection experiment demonstrates that >3 rare-codons required for efficient export. ► Study confirms that rare codons are required for both SRP and SecB dependent export.

Keywords: Protein export; Codon usage; Evolution; Signal recognition particle

Directed evolution of efficient secretion in the SRP-dependent export of TolB by Yaramah M. Zalucki; William M. Shafer; Michael P. Jennings (pp. 2544-2550).

Keywords: Protein export; Codon usage; Evolution; Signal recognition particle

Directed evolution of efficient secretion in the SRP-dependent export of TolB by Yaramah M. Zalucki; William M. Shafer; Michael P. Jennings (pp. 2544-2550).

Keywords: Protein export; Codon usage; Evolution; Signal recognition particle

The B°AT1 amino acid transporter from rat kidney reconstituted in liposomes: Kinetics and inactivation by methylmercury by Francesca Oppedisano; Lorena Pochini; Broer Stefan Bröer; Cesare Indiveri (pp. 2551-2558).

The neutral amino acid transporter B°-like from rat kidney, previously reconstituted in liposomes, was identified as B°AT1 by a specific antibody. Collectrin was present in the brush-border extract but not in functionally active proteoliposomes, indicating that it was not required for the transport function. Neutral amino acids behaved as competitive inhibitors of the glutamine transport mediated by B°AT1 with half saturation constants ranging from 0.13 to 4.74mM. The intraliposomal half saturation constant for glutamine was 2.0mM. By a bisubstrate kinetic analysis of the glutamine-Na⁺ cotransport, a random simultaneous mechanism was found. Methylmercury and HgCl₂ inhibited the transporter; the inhibition was reversed by dithioerythritol, Cys and, at a lower extent, N-acetylcysteine but not by S-carboxymethylcysteine. The IC₅₀ of the transporter for methylmercury and HgCl₂ was 1.88 and 1.75μM, respectively. The reagents behaved as non-competitive inhibitors toward both glutamine and Na⁺ and no protection by glutamine or Na⁺ was found for the two inhibitors.B°AT1 transporter reconstituted in liposomes does not require collectrin for function. ► The transporter catalyses Gln-Na⁺ cotransport by random simultaneous mechanism. ► Methylmercury inactivates the transporter; N-acetylcysteine reverses this effect. ► A CXXC motif of B°AT1 close to the membrane is a possible binding site of mercurials.

Keywords: Abbreviations; C; ₁₂; E; ₈; octaethylene glycol monododecyl ether; DTE; 1,4-dithioerythritol; Cys; cysteine; NAC; N-acetylcysteine; CMC; S-carboxymethylcysteinePlasma membrane; Transport; Liposome; Methylmercury; Amino acids; SLC6A19

The effect of variable liposome brightness on quantifying lipid–protein interactions using fluorescence correlation spectroscopy by Ana M. Melo; Manuel Prieto; Ana Coutinho (pp. 2559-2568).

Fluorescence correlation spectroscopy (FCS) has been increasingly used to study the binding of fluorescently-labeled peptides and proteins to phospholipid vesicles. In this work, we present a new method to analyze partition data obtained by this technique based on the assumption that the number of fluorescently-labeled protein molecules bound per liposome follows a Poisson distribution. To not overestimate the recovered partition coefficients, we first show that the variation in liposome brightness caused by this statistical distribution must be considered explicitly in data analysis when the parameter used to establish the partition curves is the fractional instead of the absolute amplitudes associated with the slowest diffusing particles in the system (lipid vesicles), a choice frequently made in FCS partition studies. We further extend the theoretical model describing the membrane partition of a fluorescently-labeled protein by considering the presence of a trace amount of free fluorescent dye (non-binding component) in the system. We show that this situation can account for an apparent maximal binding level lower than 100% in the experimental partitioning curves obtained for Alexa 488 fluorescently-labeled lysozyme and liposomes prepared with variable anionic phospholipid content. The extreme sensitivity of the FCS technique allowed uncoupling lysozyme partition from the protein-induced liposome aggregation, confirming that lysozyme binding to negatively charged liposomes is dominantly driven by electrostatic interactions.Display Omitted► A new formalism to measure membrane partition coefficients using FCS is presented. ► Proteins are assumed to distribute among the liposomes according to a Poisson law. ► Presence of vestigial free dye causes an apparent maximal protein bound fraction lower than 100%. ► Lysozyme binding to POPS-containing liposomes is dominantly driven by electrostatic interactions. ► FCS measurements are easily uncoupled from potential interfering protein-mediated liposome aggregation.

Keywords: Abbreviations; AC; autocorrelation; Alexa 488; Alexa Fluor 488 (carboxylic acid, succinimidyl ester, mixed isomers, dilithium salt); Alexa488-lysozyme; lysozyme conjugated with the fluorescent dye Alexa Fluor 488; BODIPY-PC; 2-(4,4-difluoro-5methyl-4-bora-3a,4a-diaza-; s; -indacene-3-dodecanoyl)-1-hexadecanoyl-; sn; -glycero-3-phosphocholine; BSA; bovine serum albumin; FCS; fluorescence correlation spectroscopy; LUV; large unilamellar vesicles; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; POPS; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphoserineMembrane partition; Molecular brightness; Poisson distribution; Lysozyme; Anionic phospholipid vesicles; Electrostatic interaction

The effect of variable liposome brightness on quantifying lipid–protein interactions using fluorescence correlation spectroscopy by Ana M. Melo; Manuel Prieto; Ana Coutinho (pp. 2559-2568).

Activation of phospholipase A2 by Hsp70 in vitro by Ajay K. Mahalka; Christian Code; Behnam Rezaijahromi; Thomas Kirkegaard; Jaattela Marja Jäättelä; Paavo K.J. Kinnunen (pp. 2569-2572).

We recently suggested a novel mechanism for the activation of phospholipase A2 (PLA2), with a (catalytically) highly active oligomeric state, which subsequently becomes inactivated by conversion into amyloid. This process can be activated by lysophosphatidylcholine which promotes both oligomerization and amyloid activation/inactivation. The heat shock protein 70 (Hsp70), has been demonstrated to be able to revert the conversion of α-synuclein and Alzheimer β-peptide to amyloid fibrils in vitro. Accordingly, we would expect Hsp70 to sustain the lifetime of the active state of the enzyme oligomer by attenuating the conversion of the enzyme oligomers into inactive amyloid. Here we show that Hsp70 activates PLA2 in vitro, in a manner requiring ATP and Mg²⁺.► Here we demonstrate the activation of a secretory phospholipase A2 by Hsp70 in vitro. ► This supports the mechanism of phospholipase A2 regulation by oligomerization–amyloid formation acting as an on–off switch for enzyme activity. ► The activation reported here requires ATP and Mg2+, thus demonstrating that catalytic activity of HSp70 is involved. ► This mechanism may well be involved in the activation of other membrane associated enzymes, such as sphingomyelinase.

Keywords: Abbreviations; C28-O-PHPM; 1-octosanyl-2-(pyren-1-yl)hexanoyl-sn-glycero-3-phosphatidylmonomethyl ester; CMC; critical micelle concentration; DPPC; 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; EM; electron microscopy; FFA; free fatty acid; Hsp70; human heat shock protein 70; LUV; large unilamellar vesicles; lysoPC; lysophosphatidylcholine; PAGE; polyacrylamide gel electrophoresis; PLA2; phospholipase A2; SDS; sodium dodecyl sulfate; T; _m; main phase transition temperature; ThT; thioflavin T; X; mole fractionPhospholipase A2; Heat shock protein 70; Oligomerization; Amyloid formation

Activation of phospholipase A2 by Hsp70 in vitro by Ajay K. Mahalka; Christian Code; Behnam Rezaijahromi; Thomas Kirkegaard; Jaattela Marja Jäättelä; Paavo K.J. Kinnunen (pp. 2569-2572).

Correlation of structural and functional thermal stability of the integral membrane protein Na,K-ATPase by Andrew J. Miles; B.A. Wallace; Mikael Esmann (pp. 2573-2580).

The membrane-bound cation-transporting P-type Na,K-ATPase isolated from pig kidney membranes is much more resistant towards thermal inactivation than the almost identical membrane-bound Na,K-ATPase isolated from shark rectal gland membranes. The loss of enzymatic activity is correlated well with changes in protein structure as determined using synchrotron radiation circular dichroism (SRCD) spectroscopy. The enzymatic activity is lost at a 12°C higher temperature for pig enzyme than for shark enzyme, and the major changes in protein secondary structure also occur at T_m's that are ~10–15°C higher for the pig than for the shark enzyme. The temperature optimum for the rate of hydrolysis of ATP is about 42°C for shark and about 57°C for pig, both of which are close to the temperatures for onset of thermal unfolding. These results suggest that the active site region may be amongst the earliest parts of the structure to unfold. Detergent-solubilized Na,K-ATPases from the two sources show the similar differences in thermal stability as the membrane-bound species, but inactivation occurs at a lower temperature for both, and may reflect the stabilizing effect of a bilayer versus a micellar environment.► Na,K-ATPase from pig kidney is thermally more stable than that of shark. ► Na,K-ATPase activity has optimum at 42°C for shark and 57°C for pig. ► Synchrotron radiation circular dichroism is used for structure determination. ► Secondary structure is lost at a ~12°C lower temperature for shark than pig. ► Loss of structure correlates well with loss of enzyme activity.

Keywords: Abbreviations; C; ₁₂; E; ₈; octaethyleneglycoldodecylmonoether; DSC; differential scanning calorimetry; DSSP; decentralized software services protocol; FXYD; the family of regulatory small peptides interacting with Na,K-ATPase; HT; high tension; SRCD; synchrotron radiation circular dichroism; SVD; single value deconvolution; T; _m; midpoint temperatureSynchrotron radiation circular dichroism (SRCD) spectroscopy; Na,K-ATPase; Membrane protein; Thermal melt curves; Enzyme activity measurements

Correlation of structural and functional thermal stability of the integral membrane protein Na,K-ATPase by Andrew J. Miles; B.A. Wallace; Mikael Esmann (pp. 2573-2580).

Cationic amphipathic peptides accumulate sialylated proteins and lipids in the plasma membrane of eukaryotic host cells by Julian Weghuber; Michael C. Aichinger; Mario Brameshuber; Stefan Wieser; Verena Ruprecht; Birgit Plochberger; Josef Madl; Andreas Horner; Siegfried Reipert; Karl Lohner; Tamás Henics; Schutz Gerhard J. Schütz (pp. 2581-2590).

Cationic antimicrobial peptides (CAMPs) selectively target bacterial membranes by electrostatic interactions with negatively charged lipids. It turned out that for inhibition of microbial growth a high CAMP membrane concentration is required, which can be realized by the incorporation of hydrophobic groups within the peptide. Increasing hydrophobicity, however, reduces the CAMP selectivity for bacterial over eukaryotic host membranes, thereby causing the risk of detrimental side-effects. In this study we addressed how cationic amphipathic peptides—in particular a CAMP with Lysine–Leucine–Lysine repeats (termed KLK)—affect the localization and dynamics of molecules in eukaryotic membranes. We found KLK to selectively inhibit the endocytosis of a subgroup of membrane proteins and lipids by electrostatically interacting with negatively charged sialic acid moieties. Ultrastructural characterization revealed the formation of membrane invaginations representing fission or fusion intermediates, in which the sialylated proteins and lipids were immobilized. Experiments on structurally different cationic amphipathic peptides (KLK, 6-MO-LF11-322 and NK14-2) indicated a cooperation of electrostatic and hydrophobic forces that selectively arrest sialylated membrane constituents.► Cationic antimicrobial peptide KLK affects eukaryotic host cells ► KLK induces accumulation of plasma mebrane proteins/lipids ► Dependent on sialic acid residues ► Application of KLK leads to a recycling inhibition of sialyted molecules

Keywords: CAMPs; KLK; Sialic acids; Plasma membrane accumulation

Biochemical and biophysical characterisation yields insights into the mechanism of a Cd/Zn transporting ATPase purified from the hyperaccumulator plant Thlaspi caerulescens by Barbara Leitenmaier; Annelie Witt; Annabell Witzke; Anastasia Stemke; Wolfram Meyer-Klaucke; Peter M.H. Kroneck; Kupper Hendrik Küpper (pp. 2591-2599).

TcHMA4 (GenBank no.AJ567384), a Cd/Zn transporting ATPase of the P_1B-type (=CPx-type) was isolated and purified from roots of the Cd/Zn hyperaccumulator Thlaspi caerulescens. Optimisation of the purification protocol, based on binding of the natural C-terminal His-tag of the protein to a Ni-IDA metal affinity column, yielded pure, active TcHMA4 in quantities sufficient for its biochemical and biophysical characterisation with various techniques. TcHMA4 showed activity with Cu(2+), Zn(2+) and Cd(2+) under various concentrations (tested from 30nM to 10μM), and all three metal ions activated the ATPase at a concentration of 0.3μM. Notably, the enzyme worked best at rather high temperatures, with an activity optimum at 42°C. Arrhenius plots yielded interesting differences in activation energy. In the presence of zinc it remained constant ( E_A=38kJ⋅mol⁻¹) over the whole concentration range while it increased from 17 to 42kJ⋅mol⁻¹ with rising copper concentration and decreased from 39 to 23kJ⋅mol⁻¹ with rising cadmium concentration. According to EXAFS the TcHMA4 appeared to bind Cd(2+) mainly by thiolate sulphur from cysteine, and not by imidazole nitrogen from histidine.► TcHMA4, a Cd/Zn-ATPase has been purified from roots in its native state. ► TcHMA4 displayed highest activity with submicromolar concentrations of Cd²⁺, Cu²⁺ and Zn²⁺. ► Concentration-dependent changes in activation energy were different for the three metals tested. ► The temperature optimum for ATPase activity was reached at approximately 40°C for all tested metals. ► EXAFS measurements showed that Cd in TcHMA4 is bound by S from cysteines and not by N from histidines.

Keywords: Abbreviations; DDM; n-dodecyl-β-; d; -maltoside; E; _A; activation energy; EXAFS; extended X-ray absorption fine structure; TCEP; tris(2-carboxyethyl)phosphine; XAS; X-ray absorption spectroscopyMetal specificity; Hyperaccumulator; Natural overexpression; Cd/Zn ATPase; Activation energy; Temperature optimum

Interaction between sodium dodecyl sulfate and membrane reconstituted aquaporins: A comparative study of spinach SoPIP2;1 and E. coli AqpZ by Jesper S. Hansen; Ardcharaporn Vararattanavech; Inés Plasencia; Per Jr Greisen; Julie Bomholt; Jaume Torres; Emneus Jenny Emnéus; Helix-Nielsen Claus Hélix-Nielsen (pp. 2600-2607).

This study describes the interaction between sodium dodecyl sulfate (SDS) and membrane proteins reconstituted into large unilamellar lipid vesicles and detergent micelles studied by circular dichroism (CD) and polarity sensitive probe labeling. Specifically, we carried out a comparative study of two aquaporins with high structural homology SoPIP2;1 and AqpZ using identical reconstitution conditions. Our CD results indicate that SDS, when added to membrane-reconstituted aquaporins in concentrations below the SDS critical micelle concentration (CMC, ~8mM), causes helical rearrangements of both aquaporins. However, we do not find compelling evidence for unfolding. In contrast when SDS is added to detergent stabilized aquaporins, SoPIP2;1 partly unfolds, while AqpZ secondary structure is unaffected. Using a fluorescent polarity sensitive probe (Badan) we show that SDS action on membrane reconstituted SoPIP2;1 as well as AqpZ is associated with initial increased hydrophobic interactions in protein transmembrane (TM) spanning regions up to a concentration of 0.1× CMC. At higher SDS concentrations TM hydrophobic interactions, as reported by Badan, decrease and reach a plateau from SDS CMC up to 12.5× CMC. Combined, our results show that SDS does not unfold neither SoPIP2;1 nor AqpZ during transition from a membrane reconstituted form to a detergent stabilized state albeit the native folds are changed.►The interaction between SDS and membrane-reconstituted and detergent micelle stabilized aquaporins is addressed. ► SDS imposes helical rearrangements of membrane-reconstituted aquaporins without compelling evidence for unfolding. ► SDS causes partial unfolding of SoPIP2;1 but not of AqpZ, when added to detergent stabilized aquaporins. ► We address interactions in membrane protein TM regions by creating fluorescent polarity sensitive aquaporins using Badan. ► We show that SDS promotes increased hydrophobic TM interactions up to a threshold of 0.1× CMC.

Keywords: Abbreviations; SDS; sodium dodecyl sulfate; CD; circular dichroism; CMC; critical micelle concentration; TM; transmembrane; DGK; diacylglycerol kinase; BR; Bacteriorhodopsin; DM; decylmaltoside; DDM; dodecylmaltoside; DMPC; L-α-1,2-dimyristoylphosphatidylcholine; CHAPS; 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; DOPC; 1,2-dioleoyl-; sn; -glycero-3-phosphocholine; DPhPC; 1,2-diphytanoyl-; sn; -glycero-3-phosphocholine; OG; Octyl-β-; d; -glucopyranoside; PBS; phosphate buffered salineMembrane protein; Aquaporin; Sodium dodecyl sulfate; Folding; Reconstitution; Lipid membrane

Effect of membrane tension on the electric field and dipole potential of lipid bilayer membrane by Dora Toledo Warshaviak; Michael J. Muellner; Mirianas Chachisvilis (pp. 2608-2617).

The dipole potential of lipid bilayer membrane controls the difference in permeability of the membrane to oppositely charged ions. We have combined molecular dynamics (MD) simulations and experimental studies to determine changes in electric field and electrostatic potential of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid bilayer in response to applied membrane tension. MD simulations based on CHARMM36 force field showed that electrostatic potential of DOPC bilayer decreases by ~45mV in the physiologically relevant range of membrane tension values (0 to 15dyn/cm). The electrostatic field exhibits a peak (~0.8×10⁹V/m) near the water/lipid interface which shifts by 0.9Å towards the bilayer center at 15dyn/cm. Maximum membrane tension of 15dyn/cm caused 6.4% increase in area per lipid, 4.7% decrease in bilayer thickness and 1.4% increase in the volume of the bilayer. Dipole-potential sensitive fluorescent probes were used to detect membrane tension induced changes in DOPC vesicles exposed to osmotic stress. Experiments confirmed that dipole potential of DOPC bilayer decreases at higher membrane tensions. These results are suggestive of a potentially new mechanosensing mechanism by which mechanically induced structural changes in the lipid bilayer membrane could modulate the function of membrane proteins by altering electrostatic interactions and energetics of protein conformational states.►Electrostatic potential of lipid bilayer was calculated using molecular dynamics simulations. ►Simulated dipole potential decreases with applied membrane tension. ►Experimental dipole potential decreases with applied membrane tension. ►Electric field profile within bilayer is modified by applied membrane tension. ►Membrane tension leads to changes in the lipid bilayer structure.

Keywords: Abbreviations; FSS; fluid shear stress; MD; molecular dynamics; LUV; large unilamelar vesicles; DOPC; 1,2-dioleoyl-sn-glycero-3-phosphocholine; COM; center of mass; PMF; potential of mean force; PME; particle mesh Ewald method; LJ; Lennard–Jones potentialDipole potential; Lipid bilayer; Mechanosensing; Fluid shear stress; GPCR; Membrane tension

Effect of membrane tension on the electric field and dipole potential of lipid bilayer membrane by Dora Toledo Warshaviak; Michael J. Muellner; Mirianas Chachisvilis (pp. 2608-2617).

Unexpected wide substrate specificity of C. perfringens α-toxin phospholipase C by Patricia Urbina; M. Isabel Collado; Alicia Alonso; Goni Félix M. Goñi; Flores-Diaz Marietta Flores-Díaz; Alape-Giron Alberto Alape-Girón; Jean-Marie Ruysschaert; Marc F. Lensink (pp. 2618-2627).

Clostridium perfringens phospholipase C (CpPLC), also called α-toxin, is the main virulence factor for gas gangrene in humans. The lipase activity serves the bacterium to generate lipid signals in the host eukaryotic cell, and ultimately to degrade the host cell membranes. Several previous reports indicated that CpPLC was specific for phosphatidylcholine and sphingomyelin. Molecular docking studies described in this paper predict favorable interactions of the CpPLC active site with other phospholipids, e.g. phosphatidylethanolamine, phosphatidylinositol and, to a lesser extent, phosphatidylglycerol. On the basis of these predictions, we have performed experimental studies showing α-toxin to degrade all the phospholipids mentioned above. The molecular docking data also provide an explanation for the observed lower activity of CpPCL on sphingomyelin as compared to the glycerophospholipids.► Lipase activity of bacterial phospholipases. ► Combination of in-silico and in-vitro studies. ► α-toxin aspecifically hydrolyses a wide variety of phospholipids. ► This helps the bacterium to infect a greater variety of host tissues.

Keywords: Abbreviations; Ala and A; alanine; Asp and D; aspartic acid; BcPLC; Bacillus cereus; phospholipase C; BSA; bovine serum albumin; CbPLC; Clostridium bifermentans; phospholipase C; CDCl; ₃; -d; deuterated chloroform; Chol; cholesterol; CpPLC; Clostridium perfringens; phospholipase C; C-terminal; carboxyl-terminal, DAG, diacylglycerol; DHPC; (3)-3,4-di-n-hexanoyloxybutyl-1-phosphocholine; DOPC; dioleoylphosphatidylcholine; DOPG; dioleoylphosphatidylglycerol; D5PC; dipentanoylphosphatidylcholine; D5PE; dipentanoylethanolamine; D5PG; dipentanoylglycerol; D5PI; dipentanoylinositol; D5PS; dipentanoylserine; EDTA-Cs; cesium ethylenediamine-tetra-acetate; EF hand; helix-loop-helix structural domain; Glu and E; glutamic acid; G; glycine; GTP; guanosine triphosphate; His and H; histidine; I; isoleucine; K; lysine; LGA; Lamarckian genetic algorithm; LUV; large unilamellar vesicle; MLV; multilamellar vesicle; N; asparagine; N-domain; amino-terminal domain; PC; phosphatidylcholine; PE; phosphatidylethanolamine; PG; phosphatidylglycerol; P-group; phosphate-group; Phe and F; phenilalanine; PI; phosphatidylinositol; PLC-B; Listeria monocytogenes; phospholipase C; PlcH; Pseudomonas aeruginosa; phospholipase C; PlcHR; ₂; Pseudomonas aeruginosa; hetrodimeric complex composed of a phospholipase C and a chaperone protein; P; proline; PS; phosphatidylserine; Rac1; Ras-related C3 botulinum toxin substrate 1; RMSD; root mean square deviation; S; serine; SM; sphingomyelin; TIM barrel; triosephosphateisomerase barrel; Trp and W; tryptophane; Tyr and Y; tyrosine; Zn; zinc; 5SM; pentanoyl sphingomyelin; 5C; five carbon; ³¹; P-NMR; 31-phosphorus nuclear magnetic resonanceBacterial phospholipase; Docking; Cell membrane; Pathogenicity; Sphingomyelinase; Phospholipid

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BBA - Biomembranes (v.1808, #10)