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BBA - Biomembranes (v.1788, #9)

Editorial Board (pp. i).

Juvenile hormone binding protein traffic — Interaction with ATP synthase and lipid transfer proteins by Marta Zalewska; Agata Kochman; Jean-Pierre Estève; Frédéric Lopez; Karima Chaoui; Christiane Susini; Andrzej Ożyhar; Marian Kochman ⁎ (pp. 1695-1705).

Juvenile hormone (JH) controls insect development, metamorphosis and reproduction. In insect hemolymph a significant proportion of JH is bound to juvenile hormone binding protein (JHBP), which serves as a carrier supplying the hormone to the target tissues. To shed some light on JHBP passage within insect tissues, the interaction of this carrier with other proteins from Galleria mellonella (Lepidoptera) was investigated. Our studies revealed the presence of JHBP within the tracheal epithelium and fat body cells in both the membrane and cytoplasmic sections. We found that the interaction between JHBP and membrane proteins occurs with saturation kinetics and is specific and reversible. ATP synthase was indicated as a JHBP membrane binding protein based upon SPR-BIA and MS analysis. It was found that in G. mellonella fat body, this enzyme is present in mitochondrial fraction, plasma membranes and cytosol as well. In the model system containing bovine F₁ ATP synthase and JHBP, the interaction between these two components occurs with K_d=0.86 nM. In hemolymph we detected JHBP binding to apolipophorin, arylphorin and hexamerin. These results provide the first demonstration of the physical interaction of JHBP with membrane and hemolymph proteins which can be involved in JHBP molecule traffic.

Keywords: Arylphorin; ATP synthase; Hexamerin; Juvenile hormone binding protein; Lipophorin

Modulation of Kir4.1 and Kir4.1-Kir5.1 channels by extracellular cations by Rikke Søe; Mogens Andreasen; Dan Arne Klaerke (pp. 1706-1713).

This work demonstrates that extracellular Na⁺ modulates the cloned inwardly rectifying K⁺ channels Kir4.1 and Kir4.1-Kir5.1. Whole-cell patch clamp studies on astrocytes have previously indicated that inward potassium currents are regulated by external Na⁺. We expressed Kir4.1 and Kir4.1-Kir5.1 in Xenopus oocytes to disclose if Kir4.1 and/or Kir4.1-Kir5.1 at the molecular level are responsible for the observed effect of [Na⁺]_o and to investigate the regulatory mechanism of external cations further. Our results showed that Na⁺ has a biphasic modulatory effect on both Kir4.1 and Kir4.1-Kir5.1 currents. Depending on the Na⁺-concentration and applied voltage, the inward Kir4.1/Kir4.1-Kir5.1 currents are either enhanced or reduced by extracellular Na⁺. The Na⁺ activation was voltage-independent, whereas the Na⁺-induced reduction of the Kir4.1 and Kir4.1-Kir5.1 currents was both concentration-, time- and voltage-dependent. Our data indicate that the biphasic effect of extracellular Na⁺on the Kir4.1 and Kir4.1-Kir5.1 channels is caused by two separate mechanisms.

Keywords: Kir4.1; Kir4.1-Kir5.1; Sodium; Pore block; K; ⁺; channel; Inward rectifier

Membrane interactions of a self-assembling model peptide that mimics the self-association, structure and toxicity of Aβ(1–40) by Luiz C. Salay; Wei Qi; Ben Keshet; Lukas K. Tamm; Erik J. Fernandez (pp. 1714-1721).

β-amyloid peptide (Aβ) is a primary protein component of senile plaques in Alzheimer's disease (AD) and plays an important, but not fully understood role in neurotoxicity. Model peptides with the demonstrated ability to mimic the structural and toxicity behavior of Aβ could provide a means to evaluate the contributions to toxicity that are common to self-associating peptides from many disease states. In this work, we have studied the peptide–membrane interactions of a model β-sheet peptide, P_11-2 (CH₃CO-Gln-Gln-Arg-Phe-Gln-Trp-Gln-Phe-Glu-Gln-Gln-NH₂), by fluorescence, infrared spectroscopy, and hydrogen–deuterium exchange. Like Aβ(1–40), the peptide is toxic, and conditions which produce intermediate oligomers show higher toxicity against cells than either monomeric forms or higher aggregates of the peptide. Further, P_11-2 also binds to both zwitterionic (POPC) and negatively charged (POPC:POPG) liposomes, acquires a partial β-sheet conformation in presence of lipid, and is protected against deuterium exchange in the presence of lipids. The results show that a simple rationally designed model β-sheet peptide recapitulates many important features of Aβ peptide structure and function, reinforcing the idea that toxicity arises, at least in part, from a common mode of action on membranes that is independent of specific aspects of the amino acid sequence. Further studies of such well-behaved model peptide systems will facilitate the investigation of the general principles that govern the molecular interactions of aggregation-prone disease-associated peptides with cell and/or membrane surfaces.

Keywords: Abbreviations; AD; Alzheimer's disease; Aβ; β-amyloid peptide; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; POPG; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phospho-; rac; -1-glycerol [sodium salt]; DMPC; 1, 2-dimyristoyl-3-; sn; -phosphatidylcholine; LUV; large unilamellar vesicle; SUV; small unilamellar vesicle; AMCA; [6-((7-amino-4-methylcoumarin-3-acetyl)amino) hexanoic acid); TFA; trifluoro acetic acid; PBC; phosphate–borate–citrate; ATR-FTIR; attenuated total reflection-Fourier transform infrared spectroscopy; HX-MS; hydrogen–deuterium exchange mass spectrometry; DMSO; dimethylsulfoxide; DCA; dichloroacetic acidSelf-assembling peptide; Aβ peptide; β-sheet; Lipid membrane; Spectroscopy; HX-MS; Toxicity

Reversible transition between α-helix and β-sheet conformation of a transmembrane domain by Wissam Yassine; Nada Taib; Silvina Federman; Alexandra Milochau; Sabine Castano; Walid Sbi; Claude Manigand; Michel Laguerre; Bernard Desbat; Reiko Oda; Jochen Lang ⁎ (pp. 1722-1730).

Despite the important functions of protein transmembrane domains, their structure and dynamics are often scarcely known. The SNARE proteins VAMP/synaptobrevin and syntaxin 1 are implicated in membrane fusion. Using different spectroscopic approaches we observed a marked sensitivity of their transmembrane domain structure in regard to the lipid/peptide ratio. In the dilute condition, peptides corresponding to the complete transmembrane domain fold into an α-helix inserted at ∼35° to the normal of the membranes, an observation in line with molecular simulations. Upon an increase in the peptide/lipid ratio, the peptides readily exhibited transition to β-sheet structure. Moreover, the insertion angle of these β-sheets increased to 54° and was accompanied by a derangement of lipid acyl chains. For both proteins the transition from α-helix to β-sheet was reversible under certain conditions by increasing the peptide/lipid ratio. This phenomenon was observed in different model systems including multibilayers and small unilamellar vesicles. In addition, differences in peptide structure and transitions were observed when using distinct lipids (DMPC, DPPC or DOPC) thus indicating parameters influencing transmembrane domain structure and conversion from helices to sheets. The putative functional consequences of this unprecedented dynamic behavior of a transmembrane domain are discussed.

Keywords: Vibrational spectroscopy; Molecular dynamics; Vesicle; Membrane protein; SNARE; Synaptobrevin; Syntaxin

Inhibition of cholesterol biosynthesis disrupts lipid raft/caveolae and affects insulin receptor activation in 3T3-L1 preadipocytes by Jana Sánchez-Wandelmer; Alberto Dávalos; Emilio Herrera; Martin Giera; Sonia Cano; Gema de la Peña; Miguel A. Lasunción; Rebeca Busto ⁎ (pp. 1731-1739).

Lipid rafts are plasma membrane microdomains that are highly enriched with cholesterol and sphingolipids and in which various receptors and other proteins involved in signal transduction reside. In the present work, we analyzed the effect of cholesterol biosynthesis inhibition on lipid raft/caveolae composition and functionality and assessed whether sterol precursors of cholesterol could substitute for cholesterol in lipid rafts/caveolae. 3T3-L1 preadipocytes were treated with distal inhibitors of cholesterol biosynthesis or vehicle (control) and then membrane rafts were isolated by sucrose density gradient centrifugation. Inhibition of cholesterol biosynthesis with either SKF 104976, AY 9944, 5,22-cholestadien-3β-ol or triparanol, which inhibit different enzymes on the pathway, led to a marked reduction in cholesterol content and accumulation of different sterol intermediates in both lipid rafts and non-raft domains. These changes in sterol composition were accompanied by disruption of lipid rafts, with redistribution of caveolin-1 and Fyn, impairment of insulin-Akt signaling and the inhibition of insulin-stimulated glucose transport. Cholesterol repletion abrogated the effects of cholesterol biosynthesis inhibitors, reflecting they were specific. Our results show that cholesterol is required for functional raft-dependent insulin signaling.

Keywords: Cholesterol biosynthesis; Sterol; Signal transduction; 3T3-L1

Search for novel neuraminidase inhibitors: Design, synthesis and interaction of oseltamivir derivatives with model membrane using docking, NMR and DSC methods by Charlotte D'Souza; Meena Kanyalkar; Mamata Joshi; Evans Coutinho; Sudha Srivastava ⁎ (pp. 1740-1751).

As a part of our ongoing program of developing novel influenza virus inhibitors, some new derivatives of oseltamivir were prepared by modifying the amino group with glycyl, acetyl, benzyl and prolyl moieties. The interactions of these derivatives with neuraminidase have been probed by molecular modeling techniques. Further, the interaction of these derivatives with model membranes prepared from DPPC and the effect on the thermotropic behavior and polymorphism of the bilayers have been investigated by multinuclear NMR and DSC methods. Results indicate that the glycyl derivative of oseltamivir has the most profound effects on the membrane, compared to other derivatives and seems to be the most promising derivative for further pharmacological evaluation as a neuraminidase inhibitor.

Keywords: Neuraminidase; Oseltamivir; Model membrane; Docking; Nuclear magnetic resonance; Differential scanning calorimeter

The amino acid residues of transmembrane helix 5 of multidrug resistance protein CaCdr1p of Candida albicans are involved in substrate specificity and drug transport by Nidhi Puri; Manisha Gaur; Monika Sharma; Suneet Shukla; Suresh V. Ambudkar; Rajendra Prasad ⁎ (pp. 1752-1761).

In view of the importance of Candida Drug Resistance Protein (Cdr1p) of pathogenic Candida albicans in azole resistance, we have characterized its ability to efflux variety of substrates by subjecting its entire transmembrane segment (TMS) 5 to site directed mutagenesis. All the mutant variants of putative 21 amino acids of TMS 5 and native CaCdr1p were over expressed as a GFP-tagged protein in a heterologous host Saccharomyces cerevisiae. Based on the drug susceptibility pattern, the mutant variants could be grouped into two categories. The variants belonging to first category were susceptible to all the tested drugs, as compared to those belonging to second category which exhibited resistance to selective drugs. The mutant variants of both the categories were analyzed for their ATP catalysis and drug efflux properties. Irrespective of the categories, most of the mutant variants of TMS 5 showed an uncoupling between ATP hydrolysis and drug efflux. The mutant variants such as M667A, F673A, I675A and P678A were an exception since they reflected a sharp reduction in both K_m and V_max values of ATPase activity when compared with WT CaCdr1p-GFP. Based on the competition experiments, we could identify TMS 5 residues which are specific to interact with select drugs. TMS 5 residues of CaCdr1p thus not only impart substrate specificity but also selectively act as a communication link between ATP hydrolysis and drug transport.

Keywords: Candida albicans; Cdr1p; ABC transporter; Multidrug resistance; Fungal transporter; Alanine scanning

Effect of sterol structure on the bending rigidity of lipid membranes: A²H NMR transverse relaxation study by Greger Orädd; Vahid Shahedi; Göran Lindblom (pp. 1762-1771).

The effect of incorporation of 3–43 mol% sterol on the lipid order and bilayer rigidity has been investigated for model membranes of dimyristoylphosphatidylcholine or dipalmitoylphosphatidylcholine.²H NMR spectra and spin-lattice relaxation rates were measured for macroscopically aligned bilayers. The characteristics of spectra obtained at temperatures between 0–60 °C are interpreted in terms of a two-phase coexistence of the liquid disordered and the liquid ordered phases and the data is found to be in agreement with the phase diagram published by Vist and Davis (Biochemistry 29 (1990), pp. 451–464). The bending modulus of the bilayers was calculated from plots of relaxation rate vs. the square of the order parameter at 44 °C. Clear differences were obtained in the efficiency of the sterols to increase the stiffness of the bilayers. These differences are correlated to the ability of the sterols to induce the liquid ordered phase in binary as well as in ternary systems; the only exception being ergosterol, which was found to be unable to induce l_o phases and also had a relatively weak effect on the bilayer stiffness in contrast to earlier reports.

Keywords: Deuterium NMR; Relaxation; Bending rigidity; Sterol; DMPC; DPPC

Lipid reorganization induced by membrane-active peptides probed using differential scanning calorimetry by Pierre Joanne; Cécile Galanth; Nicole Goasdoué; Pierre Nicolas; Sandrine Sagan; Solange Lavielle; Gérard Chassaing; Chahrazade El Amri; Isabel D. Alves ⁎ (pp. 1772-1781).

The overlapping biological behaviors between some cell penetrating peptides (CPPs) and antimicrobial peptides (AMPs) suggest both common and different membrane interaction mechanisms. We thus explore the capacity of selected CPPs and AMPs to reorganize the planar distribution of binary lipid mixtures by means of differential scanning calorimetry (DSC). Additionally, membrane integrity assays and circular dichroism (CD) experiments were performed. Two CPPs (Penetratin and RL16) and AMPs belonging to the dermaseptin superfamily (Drs B2 and C-terminal truncated analog [1–23]-Drs B2 and two plasticins DRP-PBN2 and DRP-PD36KF) were selected. Herein we probed the impact of headgroup charges and acyl chain composition (length and unsaturation) on the peptide/lipid interaction by using binary lipid mixtures. All peptides were shown to be α-helical in all the lipid mixtures investigated, except for the two CPPs and [1–23]-Drs B2 in the presence of zwitterionic lipid mixtures where they were rather unstructured. Depending on the lipid composition and peptide sequence, simple binding to the lipid surface that occur without affecting the lipid distribution is observed in particular in the case of AMPs. Recruitments and segregation of lipids were observed, essentially for CPPs, without a clear relationship between peptide conformation and their effect in the lipid lateral organization. Nonetheless, in most cases after initial electrostatic recognition between the peptide charged amino acids and the lipid headgroups, the lipids with the lowest phase transition temperature were selectively recruited by cationic peptides while those with the highest phase transition were segregated. Membrane activities of CPPs and AMPs could be thus related to their preferential interactions with membrane defects that correspond to areas with marked fluidity. Moreover, due to the distinct membrane composition of prokaryotes and eukaryotes, lateral heterogeneity may be differently affected by cationic peptides leading to either uptake or/and antimicrobial activities.

Keywords: Abbreviations; AMP; antimicrobial peptide; CD; circular dichroism; CHO; Chinese hamster ovary; CL; cardiolipin; CPP; cell penetrating peptide; DMEM; Dulbecco's modified Eagle's medium; DMPC; 1,2-Dimyristoyl-; sn; -Glycero-3-Phosphocholine; DMPG; 1,2-Dimyristoyl-; sn; -Glycero-3-[Phospho-; rac; -(1-glycerol)]; DPPC; 1,2-Dipalmitoyl-; sn; -Glycero-3-Phosphocholine; DSC; differential scanning calorimetry; DSPC; 1,2-Distearoyl-; sn; -Glycero-3-Phosphocholine; DSPG; 1,2-Distearoyl-; sn; -Glycero-3-[Phospho-; rac; -(1-glycerol)]; EDTA; Ethylenediamine-tetraacetic acid; IPTG; isopropyl-β-; d; -thiogalactopyranoside; LUV; large unilamellar vesicle; MIC; minimal inhibitory concentration; MLV; multilamellar vesicle; ONPG; Ortho-NitroPhenyl-β-Galactoside; ONP; Ortho-NitroPhenol; PBS; phosphate buffer saline; POPG; 1-Palmitoyl-2-Oleoyl-; sn; -Glycero-3-[Phospho-; rac; -(1-glycerol)]; POPC; 1-Palmitoyl-2-Oleoyl-; sn; -Glycero-3-Phosphocholine; RCB; red blood cells; LDH; lactate dehydrogenase; P/L; peptide/lipid ratioCell penetrating peptide; Antimicrobial peptide; Membrane active peptide; Peptide–membrane interaction; Lipid lateral reorganization; Differential scanning calorimetry; Circular dichroism

Membrane-mediated repulsion between gramicidin pores by Doru Constantin (pp. 1782-1789).

We investigated the X-ray scattering signal of highly aligned multilayers of the zwitterionic lipid 1,2-dilauroyl-sn-glycero-3-phosphatidylcholine containing pores formed by the antimicrobial peptide gramicidin as a function of the peptide/lipid ratio. We are able to obtain information on the structure factor of the pore fluid, which then yields the interaction potential between pores in the plane of the bilayers. Aside from a hard core with a radius close to the geometric radius of the pore, we find a repulsive exponential lipid-mediated interaction with a decay length of 2.5 Å and an amplitude that decreases with the pore concentration, in agreement with the hydrophobic matching hypothesis. In dilute systems, the contact value of this interaction is about 30k_B T. Similar results are obtained for gramicidin pores inserted within bilayers formed by the nonionic surfactant pentaethylene glycol monododecyl ether.

Keywords: PACS; 61.05.cf; 87.16.dt; 87.15.ktGramicidin; Small-angle X-ray scattering; In-plane interaction; Hydrophobic mismatch

Fungicidal effect of antimicrobial peptide arenicin-1 by Cana Park; Dong Gun Lee (pp. 1790-1796).

Arenicin-1 is a 21-residue peptide which was derived from Arenicola marina. In this study, we investigated the antifungal effects and its mechanism of action towards human pathogenic fungi. Arenicin-1 exerted remarkable fungicidal activity with both energy-dependent and salt-insensitive manners. To investigate the fungicidal mechanisms of arenicin-1, the membrane interactions of arenicin-1 were examined. Flow cytometric analysis, using propidium iodide (PI) and bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC₄(3)], as well as fluorescence analysis, regarding the membrane probe 1,6-diphenyl-1,3,5-hexatriene (DPH), were conducted against Candida albicans. The results demonstrated that arenicin-1 was associated with lipid bilayers and induced membrane permeabilization. Additionally, the membrane studies in regard to liposomes resembling the phospholipid bilayer of C. albicans confirmed the membrane-disruptive potency of arenicin-1. Therefore, the present study suggests that arenicin-1 exerts its fungicidal effect by disrupting fungal phospholipid membranes.

Keywords: Arenicin-1; Arenicola marina; Antimicrobial peptide; Fungicidal effect; Membrane-active mechanism

The circular bacteriocin, carnocyclin A, forms anion-selective channels in lipid bilayers by Xiandi Gong; Leah A. Martin-Visscher; Drew Nahirney; John C. Vederas; Marek Duszyk ⁎ (pp. 1797-1803).

Bacterial resistance to conventional antibiotics is a major challenge in controlling infectious diseases and has necessitated the development of novel approaches in antimicrobial therapy. One such approach is the use of antimicrobial peptides, such as the bacterially produced bacteriocins. Carnocyclin A (CclA) is a 60-amino acid circular bacteriocin produced by Carnobacterium maltaromaticum UAL307 that exhibits potent activity against many Gram-positive bacteria. Lipid bilayer and single channel recording techniques were applied to study the molecular mechanisms by which CclA interacts with the lipid membrane and exerts its antimicrobial effects. Here we show that CclA can form ion channels with a conductance of 35 pS in 150 mM NaCl solution. This channel displays a linear current–voltage relationship, is anion-selective, and its activation is strongly voltage-dependent. The formation of ion channels by CclA is driven by the presence of a negative membrane potential and may result in dissipation of membrane potential. Carnocyclin A's unique functional activities as well as its circular structure make it a potential candidate for developing novel antimicrobial drugs.

Keywords: Abbreviations; CclA; carnocyclin A; DIDS; 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acidAntimicrobial peptide; Ion channel; Pore formation; Bacterial toxin; Membrane; Lipid bilayer

Molecular dynamics studies of the transmembrane domain of gp41 from HIV-1 by Jong Hwa Kim ⁎; Taryn L. Hartley; A. Rachael Curran; Donald M. Engelman (pp. 1804-1812).

Helix–helix interactions in the putative three-helix bundle formation of the gp41 transmembrane (TM) domain may contribute to the process of virus–cell membrane fusion in HIV-1 infection. In this study, molecular dynamics is used to analyze and compare the conformations of monomeric and trimeric forms of the TM domain in various solvent systems over the course of 4 to 23-ns simulations. The trimeric bundles of the TM domain were stable as helices and remained associated in a hydrated POPE lipid bilayer for the duration of the 23-ns simulation. Several stable inter-chain hydrogen bonds, mostly among the three deprotonated arginine residues located at the center of each of the three TM domains, formed in a right-handed bundle embedded in the lipid bilayer. No such bonds were observed when the bundle was left-handed or when the central arginine residue in each of the three TM helices was replaced with isoleucine (R_I mutant), suggesting that the central arginine residues may play an essential role in maintaining the integrity of the three-helix bundle. These observations suggest that formation of the three-helix bundle of the TM domain may play a role in the trimerization of gp41, thought to occur during the virus–cell membrane fusion process.

Keywords: HIV-1; gp41; Transmembrane (TM); Molecular dynamics

Functional studies of membrane-bound and purified human Hedgehog receptor Patched expressed in yeast by Olivier Joubert; Rony Nehmé; Damien Fleury; Matthieu De Rivoyre; Michel Bidet; Ange Polidori; Martial Ruat; Bernard Pucci; Patrick Mollat; Isabelle Mus-Veteau (pp. 1813-1821).

The Sonic Hedgehog (Shh) signalling pathway plays an important role both in embryonic development and in adult stem cell function. Inappropriate regulation of this pathway is often due to dysfunction between two membrane receptors Patched (Ptc) and Smoothened (Smo), which lead to birth defects, cancer or neurodegenerative diseases. However, little is known about Ptc, the receptor of the Shh protein, and the way Ptc regulates Smo, the receptor responsible for the transduction of the signal. To develop structure–function studies of these receptors, we expressed human Ptc (hPtc) in the yeast Saccharomyces cerevisiae. We demonstrated that hPtc expressed in a yeast membrane fraction is able to interact with its purified ligand Shh, indicating that hPtc is produced in yeast in its native conformational state. Using Surface Plasmon Resonance technology, we showed that fluorinated surfactants preserve the ability of hPtc to interact with its ligand after purification. This is the first report on the heterologous expression and the purification of a native and stable conformation of the human receptor Ptc. This work will allow the scale-up of hPtc production enabling its biochemical characterization, allowing the development of new therapeutic approaches against diseases induced by Shh signalling dysfunction.

Keywords: Abbreviations; Hh; Hedgehog; Shh; Sonic Hedgehog; hSmo; human Smoothened; hPtc; human Patched; AOX1; alcohol oxidase 1; PMSF; phenylmethylsulfonyl fluoride; CBD; calmodulin binding domain; HA; hemagglutinin A; PCR; polymerase chain reaction; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; DTT; dithiothreitol; DDM; n; -dodecyl-β-; d; -maltoside; Ni-NTA; Ni; ²⁺; -nitrilotriacetate; FSs; fluorinated surfactants; F; ₆; -TAC; C; ₆; F; ₁₃; C; ₂; H; ₄; -S-poly-; Tris; -(hydroxymethyl)aminomethane; F; ₈; -TAC; C; ₈; F; ₁₇; C; ₂; H; ₄; -S-poly-; Tris; -(hydroxymethyl)aminomethane; SPR; surface plasmon resonanceHeterologous expression; Membrane receptor; Patched; Sonic Hedgehog; Fluorinated surfactants

Structural and spectropotentiometric analysis of Blastochloris viridis heterodimer mutant reaction center by Nina S. Ponomarenko; Liang Li; Antony R. Marino; Valentina Tereshko; Agnes Ostafin; Julia A. Popova; Edward J. Bylina; Rustem F. Ismagilov; James R. Norris Jr. (pp. 1822-1831).

Heterodimer mutant reaction centers (RCs) of Blastochloris viridis were crystallized using microfluidic technology. In this mutant, a leucine residue replaced the histidine residue which had acted as a fifth ligand to the bacteriochlorophyll (BChl) of the primary electron donor dimer M site (HisM200). With the loss of the histidine-coordinated Mg, one bacteriochlorophyll of the special pair was converted into a bacteriopheophytin (BPhe), and the primary donor became a heterodimer supermolecule. The crystals had dimensions 400×100×100 μm, belonged to space group P4₃2₁2, and were isomorphous to the ones reported earlier for the wild type (WT) strain. The structure was solved to a 2.5 Å resolution limit. Electron-density maps confirmed the replacement of the histidine residue and the absence of Mg. Structural changes in the heterodimer mutant RC relative to the WT included the absence of the water molecule that is typically positioned between the M side of the primary donor and the accessory BChl, a slight shift in the position of amino acids surrounding the site of the mutation, and the rotation of the M194 phenylalanine. The cytochrome subunit was anchored similarly as in the WT and had no detectable changes in its overall position. The highly conserved tyrosine L162, located between the primary donor and the highest potential heme C₃₈₀, revealed only a minor deviation of its hydroxyl group. Concomitantly to modification of the BChl molecule, the redox potential of the heterodimer primary donor increased relative to that of the WT organism (772 mV vs. 517 mV). The availability of this heterodimer mutant and its crystal structure provides opportunities for investigating changes in light-induced electron transfer that reflect differences in redox cascades.

Keywords: Blastochloris viridis; Heterodimer mutant; Reaction center structure; Primary donor redox potential; Photosynthetic reaction center; Microfluidic

Interactions of oritavancin, a new lipoglycopeptide derived from vancomycin, with phospholipid bilayers: Effect on membrane permeability and nanoscale lipid membrane organization by Oscar Domenech; Grégory Francius; Paul M. Tulkens; Françoise Van Bambeke; Yves Dufrêne; Marie-Paule Mingeot-Leclercq ⁎ (pp. 1832-1840).

Antibiotics acting on bacterial membranes are receiving increasing attention because of widespread resistance to agents acting on other targets and of potentially improved bactericidal effects. Oritavancin is a amphiphilic derivative of vancomycin showing fast and extensive killing activities against multi-resistant (including vancomycin insusceptible) Gram-positive organisms with no marked toxicity towards eukaryotic cells. We have undertaken to characterize the interactions of oritavancin with phospholipid bilayers, using liposomes (LUV) and supported bilayers made of cardiolipin (CL) or phosphatidylglycerol (POPG) and phosphatidylethanolamine (POPE), all abundant in Gram-positive organisms. Changes in membrane permeability were followed by the release of calcein entrapped in liposomes at self-quenching concentrations, and changes in nanoscale lipid organization examined by Atomic Force Microscopy (AFM). Oritavancin caused a fast (<5 min) and complete (>95%) release of calcein from CL:POPE liposomes, and a slower but still substantial (50% in 60 min) release from POPG:POPE liposomes, which was (i) concentration-dependent (0–600 nM; [microbiologically meaningful concentrations]); (ii) enhanced by an increase in POPG:POPE ratio, and decreased when replacing POPG by DPPG. AFM of CL:POPE supported bilayers showed that oritavancin (84 nM) caused a remodeling of the lipid domains combined with a redisposition of the drug and degradation of the borders. In all the above studies, vancomycin was without a significant effect at 5.5 μM. Electrostatic interactions, together with lipid curvature, lipid polymorphism as well of fluidity play a critical role for the permeabilization of lipid bilayer and changes in lipid organization induced by oritavancin.

Keywords: Vancomycin; Oritavancin; POPE; POPG; POPC; Cardiolipin; Calcein release; AFM; Membrane permeability

Pore size of swelling-activated channels for organic osmolytes in Jurkat lymphocytes, probed by differential polymer exclusion by Vladimir L. Sukhorukov; Dennis Imes; Michael W. Woellhaf; Joseph Andronic; Martin Kiesel; Ryo Shirakashi; Ulrich Zimmermann; Heiko Zimmermann (pp. 1841-1850).

The present study explores the impact of the molecular size on the permeation of low-molecular-weight polyethylene glycols (PEG200–1500) through the plasma membrane of Jurkat cells under iso- and hypotonic conditions. To this end, we analyzed the cell volume responses to PEG-substituted solutions of different osmolalities (100–300 mOsm) using video microscopy. In parallel experiments, the osmotically induced changes in the membrane capacitance and cytosolic conductivity were measured by electrorotation (ROT). Upon moderate swelling in slightly hypotonic solutions (200 mOsm), the lymphocyte membrane remained impermeable to PEG300–1500, which allowed the cells to accomplish regulatory volume decrease (RVD). During RVD, lymphocytes released intracellular electrolytes through the swelling-activated pathways, as proved by a decrease of the cytosolic conductivity measured by electrorotation. RVD also occurred in strongly hypotonic solutions (100 mOsm) of PEG600–1500, whereas 100 mOsm solutions of PEG300–400 inhibited RVD in Jurkat cells. These findings suggest that extensive hypotonic swelling rendered the cell membrane highly permeable to PEG300–400, but not to PEG600–1500. The swelling-activated channels conducting PEG300–400 were inserted into the plasma membrane from cytosolic vesicles via swelling-mediated exocytosis, as suggested by an increase of the whole cell capacitance. Using the hydrodynamic radii R_h of PEGs (determined by viscosimetry), the observed size-selectivity of membrane permeation yielded an estimate of ∼0.74 nm for the cut-off radius of the swelling-activated channel for organic osmolytes. Unlike PEG300–1500, the smallest PEG (PEG200, R_h=0.5 nm) permeated the lymphocyte membrane under isotonic conditions thus leading to a continuous isotonic swelling. The results are of interest for biotechnology and biomedicine, where PEGs are widely used for cryopreservation of cells and tissues.

Keywords: Cell volumetry; Regulatory volume decrease; Polyethylene glycol; Osmotic stress; T-lymphocyte; Hydrodynamic radius; Volume-sensitive channel; Electrorotation

Interaction of the chemopreventive agent resveratrol and its metabolite, piceatannol, with model membranes by Olga Wesołowska; Michał Kużdżał; Janez Štrancar; Krystyna Michalak (pp. 1851-1860).

Resveratrol and piceatannol are plant-derived polyphenols possessing extremely wide range of biological activities such as cancer chemopreventive, cardio- and neuroprotective, antioxidant, anti-inflammatory, anticancer and lifespan extending properties. Despite great interest in these stilbenes, their interactions with lipid bilayers have not been extensively studied. In the present work, the interaction of both resveratrol and piceatannol with model membranes composed of phosphatidylcholine (DMPC and DPPC) was investigated by means of fluorescence spectroscopy, differential scanning calorimetry (DSC) and electron spin resonance spectroscopy (ESR). Generalized polarization of two fluorescent probes Laurdan and Prodan measured in pure lipid and lipid:stilbene mixtures revealed that resveratrol and piceatannol changed bilayer properties in both gel-like and liquid crystalline phase and interacted with lipid headgroup region of the membrane. These findings were corroborated by DSC experiments in which the stilbene-induced decrease of lipid melting temperature and transition cooperativity were recorded. Resveratrol and piceatannol restricted also the ESR-measured mobility of spin probes GluSIN18, 5DSA and 16DSA with nitroxide group localized at different depths. Since the most pronounced effect was exerted on the spin probe located near membrane surface, we concluded that also ESR results pointed to the preferential interaction of resveratrol and piceatannol with headgroup region of lipid bilayer.

Keywords: Abbreviations; DSC; differential scanning calorimetry; ESR; electron spin resonance; DPPC; 1,2-dipalmitoyl-; sn; -glycero-3-phosphocholine; DMPC; 1,2-dimyristoyl-; sn; -glycero-3-phosphocholine; GP; generalized polarization; T; _M; temperature of the main phospholipid phase transition; Δ; H; transition enthalpy; DSPC; 1,2-distearoyl-; sn; -glycero-3-phosphocholine; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; POPS; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphoserine; DEPE; 1,2-dielaidoyl-; sn; -glycero-3-phosphoethanolamineResveratrol; Piceatannol; Phospholipid bilayer; Fluorescence spectroscopy; Microcalorimetry; Electron spin resonance

The ATRA-dependent overexpression of the glutamate transporter EAAC1 requires RARβ induction by Massimiliano G. Bianchi; Gian C. Gazzola; Silvia Cagnin; Hiroyuki Kagechika; Ovidio Bussolati ⁎ (pp. 1861-1868).

The mechanisms underlying trafficking and membrane targeting of EAAC1, the rodent counterpart of the human EAAT3 carrier for anionic amino acids, are well characterized. In contrast, much less is known on the regulation of Slc1a1, the gene that encodes for the transporter. We have recently found that all- trans retinoic acid (ATRA) stimulates EAAC1 expression and anionic amino acid transport in C6 rat glioma cells. We report here that the ATRA effect on EAAC1 activity was inhibited by the specific RAR antagonist LE540 and mimicked by Am80, a RAR agonist, but not by the RXR agonist HX630. Moreover, the ATRA-dependent induction of Slc1a1 mRNA required the synthesis of a protein intermediate and was not associated with changes in the messenger half-life. ATRA treatment induced the expression of both Rarb mRNA and RARβ protein several hours before the induction of Slc1a1, while the mRNA for RFX1, a transcription factor recently involved in Slc1a1 transcription, was unchanged. In addition, Rarb silencing markedly inhibited the ATRA-dependent increase of both Rarb and Slc1a1 mRNAs. We conclude that in C6 glioma cells the induction of Slc1a1 by ATRA requires the synthesis of RARβ, suggesting that the receptor is involved in the regulation of the transporter gene.

Keywords: Abbreviations; ATRA; all-; trans; retinoic acid; CNS; Central Nervous System; DMEM; Dulbecco's modified Eagle Medium; EAAC1; Excitatory Amino Acid Carrier 1; EAAT; Excitatory Amino Acid Transporter; EBSS; Earle's Balanced Salt Solution; FBS; Fetal Bovine Serum; Rarb; retinoic acid receptor, beta, gene; RAR; Retinoic Acid Receptors; RXR; Retinoid X ReceptorsEAAT3; C6 cells; Glioma; Retinoids; RXR; Slc1a1; Transport

The effect of salt and pH on block liposomes studied by cryogenic transmission electron microscopy by Alexandra Zidovska; Kai K. Ewert; Joel Quispe; Bridget Carragher; Clinton S. Potter; Cyrus R. Safinya (pp. 1869-1876).

Recently, we have reported the discovery of block liposomes (BLs), a new class of liquid (chain-melted) vesicles, formed in mixtures of the curvature-stabilizing hexadecavalent cationic lipid MVLBG2, the neutral lipid 1,2-dioleoyl- sn-glycero-3-phosphatidylcholine (DOPC), and water with no added salt. BLs consist of connected spheres, pears, tubes, or rods. Unlike in typical liposome systems, where spherical vesicles, tubular vesicles, and cylindrical micelles are separated on the macroscopic scale, shapes remain connected and are separated only on the nanometer scale within a single BL. Here, we report structural studies of the effect of salt and pH on the BL phase, carried out using differential interference contrast microscopy (DIC) and cryogenic transmission electron microscopy (cryo-TEM). Addition of salt screens the electrostatic interactions; in low-salt conditions, partial screening of electrostatic interactions leads to a shape transition from BLs to bilamellar vesicles, while in the high-salt regime, a shape transition from BLs to liposomes with spherical morphologies occurs. This demonstrates that strong electrostatic interactions are essential for BL formation. Understanding the control of liposome shape evolution is of high interest because such shape changes play an important role in many intracellular processes such as endocytosis, endoplasmatic reticulum-associated vesiculation, vesicle recycling and signaling.

Keywords: Abbreviations; BLs; block liposomes; CL; cationic lipid; cryo-TEM; cryo transmission electron microscopy; DIC; differential interference contrast microscopy; DOPC; 1,2-dioleoyl-; sn; -glycero-3-phosphatidylcholineBlock liposome; Charged membrane; Curvature stabilization; Cryo-TEM

Thermotropic and structural evaluation of the interaction of natural sphingomyelins with cholesterol by Peter J. Quinn ⁎; Claude Wolf (pp. 1877-1889).

The structural transitions in aqueous dispersions of egg-sphingomyelin and bovine brain-sphingomyelin and sphingomyelin co-dispersed with different proportions of cholesterol were compared during temperature scans between 20° and 50 °C using small-angle and wide-angle X-ray scattering techniques. The Bragg reflections observed in the small-angle scattering region from pure phospholipids and codispersions of sphingomyelin:cholesterol in molar ratios 80:20 and 50:50 could all be deconvolved using peak fitting methods into two coexisting lamellar structures. Electron density profiles through the unit cell normal to the bilayer plane were calculated to derive bilayer and water layer thicknesses of coexisting structures at 20° and 50 °C. Codispersions of sphingomyelin:cholesterol in a molar ratio 60:40 consisted of an apparently homogeneous bilayer structure designated as liquid-ordered phase. Curve fitting analysis of the wide-angle scattering bands were applied to correlate changes in packing arrangements of hydrocarbon in the hydrophobic domain of the bilayer with changes in enthalpy recorded by differential scanning calorimetry. At 20 °C the wide-angle scattering bands of both pure sphingomyelins and codispersions of sphingomyelin and cholesterol could be deconvolved into two symmetric components. A sharp component located at a d-spacing of 0.42 nm was assigned to a gel phase in which the hydrocarbon chains are oriented perpendicular to the bilayer plane. A broader symmetric band centered at d-spacings in the region of 0.44 nm was assigned as disordered hydrocarbon in dispersions of pure sphingomyelin and as liquid-ordered phase in codispersions of sphingomyelin and cholesterol. It is concluded from the peak fitting analysis that cholesterol is excluded from gel phases of egg and brain sphingomyelins at 20 °C. The gel phases coexist with liquid-ordered phase comprised of egg-sphingomyelin and 27 mol% cholesterol and brain-sphingomyelin and 33 mol% cholesterol, respectively. Correlation of the disappearance of gel phase during heating scans and the enthalpy change recorded by calorimetry in codispersions of sphingomyelin and cholesterol leads to the conclusion that a major contribution to the broadened phase transition endotherm originates from dilution of the cholesterol-rich liquid-ordered phase by mobilization of sphingomyelin from the melting gel phase.

Keywords: Liquid-ordered phase; X-ray diffraction; Differential scanning calorimetry; Bilayer phase separation; Phospholipid–cholesterol interaction

Detection of ligand-induced CNTF receptor dimers in living cells by fluorescence cross correlation spectroscopy by Felix Neugart; Andrea Zappe; Deborah M. Buk; Inna Ziegler; Steffen Steinert; Monika Schumacher; Eva Schopf; Ralph Bessey; Kathrin Wurster; Carsten Tietz; Michael Börsch; Jörg Wrachtrup; Lutz Graeve ⁎ (pp. 1890-1900).

Ciliary neurotrophic factor (CNTF) signals via a receptor complex consisting of the specific CNTF receptor (CNTFR) and two promiscuous signal transducers, gp130 and leukemia inhibitory factor receptor (LIFR). Whereas earlier studies suggested that the signaling complex is a hexamer, more recent analyses strongly support a tetrameric structure. However, all studies so far analyzed the stoichiometry of the CNTF receptor complex in vitro and not in the context of living cells. We generated and expressed in mammalian cells acyl carrier protein-tagged versions of both CNTF and CNTFR. After labeling CNTF and CNTFR with different dyes we analyzed their diffusion behavior at the cell surface. Fluorescence (cross) correlation spectroscopy (FCS/FCCS) measurements reveal that CNTFR diffuses with a diffusion constant of about 2×10−⁹ cm² s−¹ independent of whether CNTF is bound or not. FCS and FCCS measurements detect the formation of receptor complexes containing at least two CNTFs and CNTFRs. In addition, we measured Förster-type fluorescence resonance energy transfer between two differently labeled CNTFs within a receptor complex indicating a distance of 5–7 nm between the two. These findings are not consistent with a tetrameric structure of the CNTFR complex suggesting that either hexamers and or even higher-order structures (e.g. an octamer containing two tetramers) are formed.

Keywords: CNTFR; Fluorescence cross correlation spectroscopy; IL-6-type cytokine; Receptor complex; Stoichiometry; Dimer

Apolipophorin III lysine modification: Effect on structure and lipid binding by Lesley J. Vasquez; Gezman E. Abdullahi; Chung-Ping Leon Wan; Paul M.M. Weers ⁎ (pp. 1901-1906).

Apolipophorin III (apoLp-III) from Locusta migratoria was used as a model to investigate apolipoprotein lipid binding interactions. ApoLp-III contains eight lysine residues, of which seven are located on one side of the protein. To investigate the role of positive charges on lipid binding, lysine residues were acetylated by acetic anhydride. The degree of acetylation was analyzed by SDS-PAGE and MALDI-TOF, indicating a maximum of eight acetyl additions. Modified apoLp-III remained α-helical, but displayed a decreased α-helical content (from 78 to 54%). Acetylation resulted in a slight increase in protein stability, as indicated by a change in the midpoint of guanidine–HCl induced denaturation from 0.55 (unmodified) to 0.65 M (acetylated apoLp-III). Lipid bound apoLp-III, either acetylated or unmodified, displayed similar increases in helical content and midpoint of guanidine–HCl-induced denaturation of ∼4 M. The ability to solubilize vesicles of dimyristoylphosphatidylcholine remained unchanged. However, the rate to solubilize dimyristoylphosphatidylglycerol vesicles was reduced two-fold. In addition, a decreased ability to stabilize diacylglycerol-enriched low density lipoproteins was observed. This indicated that lysine residues are not critical for the protein's ability to bind to zwitterionic phospholipids. Since binding interactions with ionic phospholipids and lipoproteins were affected by acetylation, lysine side-chains may play a modulating role in the interaction with more complex lipid surfaces encountered in vivo.

Keywords: Apolipoprotein; Apolipophorin; Lipoprotein; Lipid; DMPC; DMPG

Cholesterol modulates the exposure and orientation of pulmonary surfactant protein SP-C in model surfactant membranes by L. Gómez-Gil; J. Pérez-Gil; E. Goormaghtigh (pp. 1907-1915).

Cholesterol is the major neutral lipid in lung surfactant, accounting for up to 8–10% of surfactant mass, while surfactant protein SP-C (∼4.2 kDa) accounts for no more than 1–1.5% of total surfactant weight but plays critical roles in formation and stabilization of pulmonary surfactant films. It has been reported that surfactant protein SP-C interacts with cholesterol in lipid/protein interfacial films and this interaction could have a potential role on modulating surfactant function. In the present study, we have analyzed the effect of cholesterol on the structure, orientation and dynamic properties of SP-C embedded in physiologically relevant model membranes. The presence of cholesterol does not induce substantial changes in the secondary structure of SP-C, as analyzed by Attenuated Reflection Fourier Transformed Infrared spectroscopy (ATR-FTIR). However, the presence of cholesterol modifies the orientation of the transmembrane helix and the dynamic properties of the protein, as demonstrated by hydrogen/deuterium exchange kinetics. The effect of cholesterol on SP-C reconstituted in zwitterionic, entirely fluid, membranes made of POPC (palmitoyloleoylphospatidylcholine) or in anionic membranes with coexistence of ordered and disordered phases, such as those made of dipalmitoylphosphatidylcholine (DPPC):POPC:Palmitoyloleoylphosphatidylglycerol (POPG) (50:25:15) is dual. Cholesterol decreases the exposure of the protein to the aqueous environment and the tilt of its transmembrane helical segment up to a ratio Cholesterol:SP-C of 4.8 and 2.4 (mol/mol) in the two lipid systems tested, respectively, and it increases the exposure and tilt at higher cholesterol proportions. The results presented here suggest the existence of an interaction between SP-C and cholesterol-enriched phases, with consequences on the behavior of the protein, which could be of relevance for cholesterol-dependent structure–function relationships in pulmonary surfactant membranes and films.

Keywords: Transmembrane protein; Cholesterol; ATR-FTIR; Structure; Membrane protein dynamics; Lipid–protein interaction

Oligotryptophan-tagged antimicrobial peptides and the role of the cationic sequence by Adam A. Strömstedt ⁎; Mukesh Pasupuleti; Artur Schmidtchen; Martin Malmsten (pp. 1916-1923).

The effects of varying the cationic sequence of oligotryptophan-tagged antimicrobial peptides were investigated in terms of peptide adsorption to model lipid membranes, liposome leakage induction, and antibacterial potency. Heptamers of lysine (K7) and arginine (R7) were lytic against Escherichia coli bacteria at low ionic strength. In parallel, both peptides adsorbed on to bilayers formed by E. coli phospholipids, and caused leakage in the corresponding liposomes. K7 was the more potent of the two peptides in causing liposome leakage, although the adsorption of this peptide on E. coli membranes was lower than that of R7. The bactericidal effect, liposome lysis, and membrane adsorption were all substantially reduced at physiological ionic strength. When a tryptophan pentamer tag was linked to the C-terminal end of these peptides, substantial peptide adsorption, membrane lysis, and bacterial killing were observed also at high ionic strength, and also for a peptide of lower cationic charge density (KNKGKKN-W5). Strikingly, the order of membrane lytic potential of the cationic peptides investigated was reversed when tagged. This and other aspects of peptide behavior and adsorption, in conjunction with effects on liposomes and bacteria, suggest that tagged and untagged peptides act by different lytic mechanisms, which to some extent counterbalance each other. Thus, while the untagged peptides act by generating negative curvature strain in the phospholipid membrane, the tagged peptides cause positive curvature strain. The tagged heptamer of arginine, R7W5, was the best candidate for E. coli membrane lysis at physiological salt conditions and proved to be an efficient antibacterial agent.

Keywords: AMP; Antimicrobial; Peptide; End-tagged; Phospholipid; Bilayer; Membrane; Liposome; Bacteria; Ellipsometry; Oligotryptophan; Oligolysine; Oligoarginine

The interaction of the Bax C-terminal domain with membranes is influenced by the presence of negatively charged phospholipids by Alessio Ausili; Ana de Godos; Alejandro Torrecillas; Senena Corbalán-García; Juan C. Gómez-Fernández (pp. 1924-1932).

The C-terminal domain of the pro-apoptotic protein Bax (Bax-C) is supposed to act as a membrane anchor motif when Bax is activated leading to programmed cell death. A synthetic peptide which imitates this domain has been used to study the mechanism of peptide–phospholipid interaction. We have used static and MAS-NMR techniques to show that the interaction of Bax-C with membranes is modulated by the presence of a negatively charged phospholipid like phosphatidylglycerol. Bax-C slightly shifted upfield the³¹P resonances coming from phosphatidylglycerol and phosphatidylcholine. However the width of the resonance peaks was considerably higher when phosphatidylglycerol was present. Bax-C substantially decreased the T₁ relaxation times of phosphatidylglycerol and those of phosphatidylcholine when mixtured with phosphatidylglycerol, but T₁ values were not decreased when phosphatidylcholine was the only phospholipid present in the membrane.¹³C-MAS-NMR showed that T₁ values were decreased when Bax-C was incorporated into the lipid vesicles and this reduction affected similarly to carbons located in different regions of the membrane when the only phospholipid present was phosphatidylcholine. However, when phosphatidylglycerol was also present, the decrease in T₁ affected considerably more to some carbons in the polar region. These results indicate that Bax-C interacts differently with the polar part of the membrane depending on whether phosphatidylglycerol is present or not, suggesting that an electrostatic interaction of Bax-C with the membrane determines the location of this domain. Fluorescence spectroscopy showed that the Trp residues of Bax-C were placed in a microenvironment more hydrophobic and less accessible to quenching by acrylamide when phosphatidylglycerol was present.

Keywords: Abbreviations; CS; chemical shift; CSA; chemical shift anisotropy; FWHH; full width at half height; MLV; lipid multilamellar vesicles; MAS; magic angle spinning; POPC; 1-palmitoyl-2-oleoyl-; sn; -3-phosphocholine; POPG; 1-palmitoyl-2-oleoyl-; sn; -3-phospho-rac-glycerol; TFE; 2,2,2-trifluoroethanolBax; MAS-NMR; ³¹; P-NMR; ¹³; C-NMR; Protein–lipid interaction; Apoptosis

Calcium enhances the proteolytic activity of BACE1: An in vitro biophysical and biochemical characterization of the BACE1–calcium interaction by Michael Hayley; Samantha Perspicace; Therese Schulthess; Joachim Seelig (pp. 1933-1938).

BACE1 is a novel type I transmembrane aspartyl protease that has been implicated in the pathogenesis of Alzheimer's disease. Cleavage of the amyloid precursor protein by the β-secretase, BACE1, is the first step in the production of the Aβ peptide and is a prime target for therapeutic intervention. Using circular dichroism, we reveal that the secondary structure of BACE1 in a membrane environment is significantly different from what was determined from the previously resolved crystal structure, and, we provide the first evidence that show differences in stability between the active (pH 4.8) and inactive (pH 7.4) forms of BACE1. In this study we have also examined Ca²⁺ binding to BACE1, the effect of this binding on the secondary and tertiary structural characteristics of BACE1, and the influence of this binding on the specific activity of the purified protein. Circular dichroism and endogenous tryptophan fluorescence measurements demonstrated that the secondary and tertiary structures, respectively, are sensitive to increasing concentrations of Ca²⁺. Isothermal titration calorimetry was then used to characterize the Ca²⁺–BACE1 interaction in more detail. Our results suggest that there is a high affinity of binding ( k_d=2.0×μM) between Ca²⁺ and BACE1 and that the binding process was exothermic (Δ H=−3.5 kcal/mol). We also could demonstrate that low concentrations of Ca²⁺ (μM range) significantly increased the proteolytic activity of BACE1. Collectively, these results identify a direct interaction between BACE1 and Ca²⁺ and suggest that under physiological conditions, the function(s) of BACE1 must also be influenced by Ca²⁺.

Keywords: BACE1; Calcium; Alzheimer's disease; Calcium dysregulation

Voltage-dependent opening of HCN channels: Facilitation or inhibition by the phytoestrogen, genistein, is determined by the activation status of the cyclic nucleotide gating ring by Anjali O. Rozario; Harma K. Turbendian; Keri J. Fogle; Nelson B. Olivier; Gareth R. Tibbs ⁎ (pp. 1939-1949).

Investigation of the mechanistic bases and physiological importance of cAMP regulation of HCN channels has exploited an arginine to glutamate mutation in the nucleotide-binding fold, an approach critically dependent on the mutation selectively lowering the channel's nucleotide affinity. In apparent conflict with this, in intact Xenopus oocytes, HCN and HCN-RE channels exhibit qualitatively and quantitatively distinct responses to the tyrosine kinase inhibitor, genistein — the estrogenic isoflavonoid strongly depolarizes the activation mid-point of HCN1-R538E, but not HCN1 channels (+9.8 mV±0.9 versus +2.2 mV±0.6) and hyperpolarizes gating of HCN2 (−4.8 mV±1.0) but depolarizes gating of HCN2-R591E (+13.2 mV±2.1). However, excised patch recording, X-ray crystallography and modeling reveal that this is not due to either a fundamental effect of the mutation on channel gating per se or of genistein acting as a mutation-sensitive partial agonist at the cAMP site. Rather, we find that genistein equivalently moves both HCN and HCN-RE channels closer to the open state (rendering the channels inherently easier to open but at a cost of decreasing the coupling energy of cAMP) and that the anomaly reflects a balance of these energetic effects with the isoform-specific inhibition of activation by the nucleotide gating ring and relief of this by endogenous cAMP. These findings have specific implications with regard to findings based on HCN-RE channels and kinase antagonists and general implications with respect to interpretation of drug effects in mutant channel backgrounds.

Keywords: cAMP; HCN channel; Genistein; I; _H; Allosteric modulation; Voltage-dependent

Organization and synergistic binding of copine I and annexin A1 on supported lipid bilayers observed by atomic force microscopy by Carl E. Creutz ⁎; J. Michael Edwardson (pp. 1950-1961).

The transduction of signals across the plasma membrane of cells after receptor activation frequently involves the assembly of interacting protein molecules on the cytoplasmic face of the membrane. However, the structural organization and dynamics of the formation of such complexes has not been well defined. In this study atomic force microscopy was used to monitor the assemblies formed in vitro by two classes of calcium-dependent, membrane-binding proteins that participate in the formation of signaling complexes on membranes – the annexins and the copines. When applied to supported lipid bilayers composed of 25% brain phosphatidylserine and 75% dioleyl phosphatidylcholine in the presence of 1 mM Ca²⁺ both human annexin A1 and human copine I bound only to specialized domains that appeared to be 0.5 to 1.0 nm lower than the rest of the bilayer. These domains may be enriched in phosphatidylserine and have a more disordered structure allowing probe penetration. Confinement of the binding of the proteins to these domains may be important in the process of concentrating other signaling proteins bound to the copine or annexin. The binding of the annexin promoted the growth of the domains and created additional binding space for the copine. This may reflect a general ability of annexins to alter membrane structure in such a way that C2 domain-containing proteins like copine can bind. Copine I formed a reticular lattice composed of linear elements approximately 45 nm long on the specialized domains. This lattice might provide a scaffold for the assembly and interaction of copine target proteins in signaling complexes.

Keywords: Abbreviations; AFM; atomic force microscopy; DOPC; 1,2-Dioleoyl-; sn; -glycero-3-phosphatidylcholine; HBS; HEPES-buffered saline [100 mM NaCl and 20 mM HEPES (pH 7.5)]; PS; l-α-phosphatidylserineAnnexin; Copine; Supported bilayer; Atomic force microscopy; Lipid domain

Histidine 440 controls the opening of colicin E1 channels in a lipid-dependent manner by Alexander A. Sobko; Tatyana I. Rokitskaya; Elena A. Kotova (pp. 1962-1966).

The in vitro activity of many pore-forming toxins, in particular, the rate of increase in the membrane conductance induced by the channel-forming domain (P178) of colicin E1 is maximum at an acidic pH. However, after P178 binding at acidic conditions, a subsequent pH shift from 4 to 6 on both sides of the planar bilayer lipid membrane caused a large increase in the trans-membrane current which was solely due to an increase in the number of open channels. This effect required the presence of anionic lipid. Replacing the His440 residue of P178 by alanine eliminated the pH-shift effect thereby showing that it is associated with deprotonation of this histidine residue. It was concluded that alkalinization-induced weakening of the electrostatic interactions between colicin and the membrane surface facilitates conformational changes required for the transition of membrane-bound colicin molecules to an active channel state.

Keywords: Abbreviations; BLM; bilayer lipid membrane; P178; 178-residue C-terminal colicin E1 polypeptide prepared by thermolysin proteolysis; DPhPC; 1,2-diphytanoyl-sn-glycero-3-phosphocholine; DPhPG; 1,2-diphytanoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt); DOPC; 1,2-dioleoyl-sn-glycero-3-phosphocholine; DOPG; 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt); SDS; sodium dodecyl sulfate; cmc; critical micelle concentrationColicin E1; Ion channel; Bilayer lipid membrane; Histidine; pH effect

Anti-inflammatory peptides grab on to the whiskers of atherogenic oxidized lipids by Raquel F. Epand; Vinod K. Mishra; Mayakonda N. Palgunachari; G.M. Anantharamaiah; Richard M. Epand ⁎ (pp. 1967-1975).

The peptide 4F is known to have potent anti-atherogenic activity. 4F is an 18 residue peptide that has a sequence capable of forming a class A amphipathic helix. Several other class A amphipathic helical, 18 residue peptides with the same polar face but with increasing Phe residues on the nonpolar face have been synthesized with varying degrees of biological activity. In this work we compared the properties of the original 2F peptide, modeled on the consensus sequence of the amphipathic helical segments of the apolipoprotein A-I with the peptide 4F that has two Leu residues replaced with Phe. We demonstrate that the more biologically active 4F peptide has the greatest affinity for binding to several molecular species of oxidized lipids. Lipoprotein particles can be formed by solubilizing 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) with peptides. These solubilized lipoprotein particles extract oxidized lipid from liposomes of POPC containing 5 mol% of oxidized lipid. The peptides with the strongest anti-atherogenic activity interact most strongly with the oxidized lipid. The results show that there is a correlation between the biological potency of these peptides and their ability to interact with certain specific cytotoxic lipids, suggesting that this interaction may contribute favourably to their biological properties.

Keywords: Abbreviations; POPC; 1-palmitoyl-2-oleoyl phosphatidylcholine; SOPC; 1-stearoyl-2-oleoyl phosphatidylcholine; DEPE; dielaidoyl phophatidylethanolamine; PGPC; 1-palmitoyl-2-glutaryl-; sn; -glycero-3-phosphocholine; Azelaoyl PAF; 1-hexadecyl-2-azelaoyl-; sn; -glycero-3-phosphocholine; KDdiA-PC; 1-palmitoyl-2-(4-keto-dodec-3-ene-dioyl)phosphatidylcholine; KOdiA-PC; 1-palmitoyl-2-(5-keto-6-octene-dioyl)phosphatidylcholine; LPC; lysophosphatidylcholine; LUV; large unilamellar vesiclesAtherosclerosis; Amphipathic helix; Lipid oxidation product; Peptide-lipid interaction; Fluorescence

Biophysical studies of the membrane location of the voltage-gated sensors in the HsapBK and KvAP K⁺ channels by Henrik Biverståhl; Jesper Lind; Andrea Bodor; Lena Mäler ⁎ (pp. 1976-1986).

The membrane location of two fragments in two different K⁺-channels, the KvAP (from Aeropyrum pernix) and the HsapBK (human) corresponding to the putative “paddle” domains, has been investigated by CD, fluorescence and NMR spectroscopy. Both domains interact with q=0.5 phospholipid bicelles, DHPC micelles and with POPC vesicles. CD spectra demonstrate that both peptides become largely helical in the presence of phospholipid bicelles. Fluorescence quenching studies using soluble acrylamide or lipid-attached doxyl-groups show that the arginine-rich domains are located within the bilayered region in phospholipid bicelles. Nuclear magnetic relaxation parameters, T₁ and¹³C–¹H NOE, for DMPC in DMPC/DHPC bicelles and for DHPC in micelles showed that the lipid acyl chains in the bicelles become less flexible in the presence of either of the fragments. An even more pronounced effect is seen on the glycerol carbons.²H NMR spectra of magnetically aligned bicelles showed that the peptide derived from KvAP had no or little effect on bilayer order, while the peptide derived from HsapBK had the effect of lowering the order of the bilayer. The present study demonstrates that the fragments derived from the full-length proteins interact with the bilayered interior of model membranes, and that they affect both the local mobility and lipid order of model membrane systems.

Keywords: Abbreviations; NMR; nuclear magnetic resonance; CD; circular dichroism; NOE; nuclear Overhauser enhancement; DHPC; 1,2-dihexanoyl-; sn; -glycero-3-phosphatidylcholine; DMPC; 1,2-dimyristoyl-; sn; -glycero-3-phosphatidylcholine; POPC; 1-palmitoyl-2-oleoyl-phosphatidylcholineK; ⁺; -channel; Voltage-sensor; Membrane; Bicelle; Micelle; NMR; Fluorescence

Photoaffinity labeling the agonist binding domain of α4β4 and α4β2 neuronal nicotinic acetylcholine receptors with [¹²⁵I]epibatidine and 5[¹²⁵I]A-85380 by Ayman K. Hamouda; Xiaochun Jin; Mitesh Sanghvi; Shouryadeep Srivastava; Akash Pandhare; Phaneendra K. Duddempudi; Joe Henry Steinbach; Michael P. Blanton (pp. 1987-1995).

The development of nicotinic acetylcholine receptor (nAChR) agonists, particularly those that discriminate between neuronal nAChR subtypes, holds promise as potential therapeutic agents for many neurological diseases and disorders. To this end, we photoaffinity labeled human α4β2 and rat α4β4 nAChRs affinity-purified from stably transfected HEK-293 cells, with the agonists [¹²⁵I]epibatidine and 5[¹²⁵I]A-85380. Our results show that both agonists photoincorporated into the β4 subunit with little or no labeling of the β2 and α4 subunits respectively. [¹²⁵I]epibatidine labeling in the β4 subunit was mapped to two overlapping proteolytic fragments that begin at β4V102 and contain Loop E (β4I109–P120) of the agonist binding site. We were unable to identify labeled amino acid(s) in Loop E by protein sequencing, but we were able to demonstrate that β4Q117 in Loop E is the principal site of [¹²⁵I]epibatidine labeling. This was accomplished by substituting residues in the β2 subunit with the β4 homologs and finding [¹²⁵I]epibatidine labeling in β4 and β2F119Q subunits with little, if any, labeling in α4, β2, or β2S113R subunits. Finally, functional studies established that the β2F119/β4Q117 position is an important determinant of the receptor subtype-selectivity of the agonist 5I-A-85380, affecting both binding affinity and channel activation.

Keywords: Cys-loop receptor; Neuronal nicotinic receptor; HEK-293 cell; Affinity-purification; Photoaffinity labeling; Protein sequencing

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BBA - Biomembranes (v.1788, #9)

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BBA - Biomembranes (v.1788, #9)