Check out our New Publishers' Select for Free Articles

BBA - Biomembranes (v.1788, #3)

Editorial Board (pp. i).

Expression of sarco/endoplasmic reticulum Ca²⁺ ATPase (SERCA) 3 proteins in two major conformational states in native human cell membranes by Elisabeth Corvazier; Raymonde Bredoux; Tünde Kovács; Jocelyne Enouf ⁎ (pp. 587-599).

The SERCA family includes 3 genes ( SERCA1–3), each of which giving rise to various isoforms. To date, detailed structural data is only available for the SERCA1a isoform. Here, limited trypsinolysis of either human platelet membranes or recombinant SERCA3a in HEK-293 cells followed by Western blotting using antibodies covering different regions of the SERCA3(a) protein revealed two, kinetically distinct, Early ( ETF) and Late ( LTF) Tryptic Fragmentations. The ETF uses many tryptic sites while the LTF uses a unique tryptic site. Using site-directed mutagenesis: i) Arg³³⁴, Arg³⁹⁶ and Arg⁶³⁸ were directly assigned to the ETF and ii) Arg¹⁹⁸ was assigned as the only tryptic site to the LTF. Arg⁶⁷¹, Lys⁷¹²/Lys⁷¹³ and Lys⁷²⁸ were also found to modulate the ETF. SERCA inhibitors Tg and tBHQ induced modest inhibition of the ETF. In contrast, the addition of CaCl₂, EGTA or AlF₄⁻ strikingly modified the ETF without any effect on the LTF. Trypsinolysis of the other recombinant SERCA3b–3f isoforms revealed: i) same ETF and LTF as SERCA3a, with variations of the length of the C-terminal fragments; ii) Arg¹⁰⁰² as an additional tryptic site in SERCA3b–3e isoforms. Taken together, the two distinct SERCA3 fragmentation profiles sign the co-expression of SERCA3 proteins in two conformational states in cell membranes.

Keywords: Abbreviations; SERCA; sarco/endoplasmic reticulum Ca; ²⁺; ATPase; E; 2 and; E; 1; nonphosphorylated forms of Ca; ²⁺; ATPase; Tg; thapsigargin; tBHQ; 2,5-di-; tert; -butyl-1,4-benzohydroquinone; HEK; human embryonic kidney; ETF; Early Tryptic fragmentation; LTF; Late Tryptic fragmentation; aa; amino acidCa; ²⁺; Sarco/endoplasmic reticulum Ca; ²⁺; ATPase; SERCA3; Isoform; Proteolysis trypsin; Site-directed mutagenesis; Platelet; HEK-293 cell; TG; tBHQ

Melting of individual lipid components in binary lipid mixtures studied by FTIR spectroscopy, DSC and Monte Carlo simulations by M. Fidorra; T. Heimburg; H.M. Seeger (pp. 600-607).

Monte Carlo (MC) simulations, Differential Scanning Calorimetry (DSC) and Fourier Transform InfraRed (FTIR) spectroscopy were used to study the melting behavior of individual lipid components in two-component membranes made of DMPC and DSPC. We employed Monte Carlo simulations based on parameters obtained from DSC profiles to simulate the melting of the different lipids as a function of temperature. The simulations show good agreement with the FTIR data recorded for deuterated and non-deuterated lipids, which demonstrates that the information on the differential melting of the individual components is already contained in the calorimetric profiles. In mixtures, both lipids melt over a wide temperature range. As expected, the lipid melting events of the lipid with the lower melting temperature occur on average at lower temperatures. The simulations also yield information on the lateral distribution of the lipids that is neither directly contained in the DSC nor in the FTIR data. In the phase coexistence region, liquid disordered domains are typically richer in the lower-melting-temperature lipid species.

Keywords: Domain formation; Phase separation; FTIR; MC simulation; DSC; Melting of individual lipid components

Role of polyphosphate in regulation of the Streptomyces lividans KcsA channel by Alexander Negoda; Elena Negoda; Mo Xian; Rosetta N. Reusch ⁎ (pp. 608-614).

We examine the hypotheses that the Streptomyces lividans potassium channel KcsA is gated at neutral pH by the electrochemical potential, and that its selectivity and conductance are governed at the cytoplasmic face by interactions between the KcsA polypeptides and a core molecule of inorganic polyphosphate (polyP). The four polypeptides of KcsA are postulated to surround the end unit of the polyP molecule with a collar of eight arginines, thereby modulating the negative charge of the polyP end unit and increasing its preference for binding monovalent cations. Here we show that KcsA channels can be activated in planar lipid bilayers at pH 7.4 by the chemical potential alone. Moreover, one or both of the C-terminal arginines are replaced with residues of progressively lower basicity–lysine, histidine, valine, asparagine–and the effects of these mutations on conductance and selectivity for K⁺ over Mg²⁺ is tested in planar bilayers as a function of Mg²⁺ concentration and pH. As the basicity of the C-terminal residues decreases, Mg²⁺ block increases, and Mg²⁺ becomes permeant when medium pH is greater than the pI of the C-terminal residues. The results uphold the premise that polyP and the C-terminal arginines are decisive elements in KcsA channel regulation.

Keywords: Streptomyces lividans; KcsA; Inorganic polyphosphate; Ion channel; Selectivity

The YheI/YheH heterodimer from Bacillus subtilis is a multidrug ABC transporter by Cristina Torres; Carmen Galián; Christoph Freiberg; Jean-Raphaël Fantino; Jean-Michel Jault ⁎ (pp. 615-622).

ABC (ATP-binding cassette) transporters form the largest family of membrane proteins in micro-organisms where they are able to transport a wide variety of substrates against a concentration gradient, in an ATP-dependent process. Two genes from the same putative Bacillus subtilis operon, yheI and yheH, encoding possibly two different ABC transporters, were overexpressed in Escherichia coli in high yield, either separately or jointly. Using membrane vesicles, it is shown here that both subunits were required to detect, (i) the transport of four structurally unrelated drugs, and (ii) a vanadate-sensitive ATPase activity. Mutation of the invariant Walker-A lysine to an alanine residue in both subunits led to an inactive transporter. Moreover, after membrane solubilization by detergent, both wild-type subunits co-purified on a Ni-Agarose affinity column while only the YheH subunit contained a hexa-histidine tag. This shows that YheI and YheH are indeed able to interact together to form a heterodimer. Importantly, expression of both yheI and yheH genes in B. subtilis could be strongly stimulated by addition of sub-inhibitory concentrations of various unrelated antibiotics. Therefore, B. subtilis YheI/YheH forms a new heterodimeric multidrug ABC transporter possibly involved in multiple antibiotic resistance in vivo.

Keywords: ABC transporter; Multidrug; Overexpression; Drug transport; ATP-binding cassette

How does the Bax-α1 targeting sequence interact with mitochondrial membranes? The role of cardiolipin by Marc-Antoine Sani; Erick J. Dufourc ⁎; Gerhard Gröbner ⁎ (pp. 623-631).

A key event in programmed cell death is the translocation of the apoptotic Bax protein from the cytosol towards mitochondria. The first helix localized at the N-terminus of Bax (Bax-α1) can act here as an addressing sequence, which directs activated Bax towards the mitochondrial surface. Solid state NMR (nuclear magnetic resonance), CD (circular dichroism) and ATR (attenuated total reflection) spectroscopy were used to elucidate this recognition process of a mitochondrial membrane system by Bax-α1. Two potential target membranes were studied, with the outer mitochondrial membrane (OM) mimicked by neutral phospholipids, while mitochondrial contact sites (CS) contained additional anionic cardiolipin.¹H and³¹P magic angle spinning (MAS) NMR revealed Bax-α1 induced pronounced perturbations in the lipid headgroup region only in presence of cardiolipin. Bax-α1 could not insert into CS membranes but at elevated concentrations it inserted into the hydrophobic core of cardiolipin-free OM vesicles, thereby adopting β-sheet-like features, as confirmed by ATR. CD studies revealed, that the cardiolipin mediated electrostatic locking of Bax-α1 at the CS membrane surface promotes conformational changes into an α-helical state; a process which seems to be necessary to induce further conformational transition events in activated Bax which finally causes irreversible membrane permeabilization during the mitochondrial apoptosis.

Keywords: Apoptosis; Bax N-terminal; Cardiolipin; Solid-State NMR

Kinematic viscosity of therapeutic pulmonary surfactants with added polymers by Karen W. Lu ⁎; Jesús Pérez-Gil; H. William Taeusch (pp. 632-637).

The addition of various polymers to pulmonary surfactants improves surface activity in experiments both in vitro and in vivo. Although the viscosity of surfactants has been investigated, the viscosity of surfactant polymer mixtures has not. In this study, we have measured the viscosities of Survanta and Infasurf with and without the addition of polyethylene glycol, dextran or hyaluronan. The measurements were carried out over a range of surfactant concentrations using two concentrations of polymers at two temperatures. Our results indicate that at lower surfactant concentrations, the addition of any polymers increased the viscosity. However, the addition of polyethylene glycol and dextran to surfactants at clinically used concentrations can substantially lower viscosity. Addition of hyaluronan at clinical surfactant concentrations slightly increased Infasurf viscosity and produced little change in Survanta viscosity. Effects of polymers on viscosity correlate with changes in size and distribution of surfactant aggregates and the apparent free volume of liquid as estimated by light microscopy. Aggregation of surfactant vesicles caused by polymers may therefore not only improve surface activity as previously shown, but may also affect viscosity in ways that could improve surfactant distribution in vivo.

Keywords: Pulmonary surfactant; Viscosity; Dextran; Polyethylene glycol; Hyaluronan

Validation of all-atom phosphatidylcholine lipid force fields in the tensionless NPT ensemble by Justine Taylor; Nava E. Whiteford; Geoff Bradley; Graeme W. Watson ⁎ (pp. 638-649).

A recently defined charge set, to be used in conjunction with the all-atom CHARMM27r force field, has been validated for a series of phosphatidylcholine lipids. The work of Sonne et al. successfully replicated experimental bulk membrane behaviour for dipalmitoylphosphatidylcholine (DPPC) under the isothermal-isobaric (NPT) ensemble. Previous studies using the defined CHARMM27r charge set have resulted in lateral membrane contraction when used in the tensionless NPT ensemble, forcing the lipids to adopt a more ordered conformation than predicted experimentally. The current study has extended the newly defined charge set to 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphatidylcholine (POPC) and 1-palmitoyl-2-docosahexaenoyl- sn-glycero-3-phosphatidylcholine (PDPC). Molecular dynamics simulations were run for each of the lipids (including DPPC) using both the CHARMM27r charge set and the newly defined modified charge set. In all three cases a significant improvement was seen in both bulk membrane properties and individual atomistic effects. Membrane width, area per lipid and the depth of water penetration were all seen to converge to experimental values. Deuterium order parameters generated with the new charge set showed increased disorder across the width of the bilayer and reflected both results from experiment and similar simulations run with united atom models. These newly validated models can now find use in mixed biological simulations under the tensionless ensemble without concern for lateral contraction.

Keywords: Phospholipid bilayer; Molecular dynamics; Simulation

Peptide induced demixing in PG/PE lipid mixtures: A mechanism for the specificity of antimicrobial peptides towards bacterial membranes? by Ahmad Arouri; Margitta Dathe; Alfred Blume (pp. 650-659).

Antimicrobial peptides attract a lot of interest as potential candidates to overcome bacterial resistance. So far, nearly all the proposed scenarios for their mechanism of action are associated with perforating and breaking down bacterial membranes after a binding process. In this study we obtained additional information on peptide induced demixing of bacterial membranes as a possible mechanism of specificity of antimicrobial peptides. We used DSC and FT-IR to study the influence of a linear and cyclic arginine- and tryptophan-rich antimicrobial peptide having the same sequence (RRWWRF) on the thermotropic phase transitions of lipid membranes. The cyclization of the peptide was found to enhance its antimicrobial activity and selectivity ( Dathe, M. Nikolenko, H. Klose, J. Bienert, M. Biochemistry 43 (2004) 9140–9150). A particular preference of the binding of the peptides to DPPG headgroups compared to other headgroups of negatively charged phospholipids, namely DMPA, DPPS and cardiolipin was observed. The main transition temperature of DPPG bilayers was considerably decreased by the bound peptides. The peptides caused a substantial down-shift of the transition of DPPG/DMPC. In contrast, they induced a demixing in DPPG/DPPE bilayers and led to the appearance of two peaks in the DSC curves indicating a DPPG-peptide-enriched domain and a DPPE-enriched domain. These results could be confirmed by FT-IR-spectroscopic measurements. We therefore propose that the observed peptide-induced lipid demixing in PG/PE-membranes could be a further specific effect of the antimicrobial peptides operating only on bacterial membranes, which contain appreciable amounts of PE and PG, and which could in principle also occur in liquid–crystalline membranes.

Keywords: Abbreviations; DSC; Differential scanning calorimetry; FT-IR; Fourier transform infrared; DMPC; 1,2-dimyristoyl-; sn; -glycero-3-phosphocholine; DPPE; 1,2-dipalmitoyl-; sn; -glycero-3-phosphoethanolamine; DPPG; 1,2-dipalmitoyl-; sn; -glycero-3-phospho-; rac; -(1-glycerol); DMPA; 1,2-dimyristoyl-; sn; -glycero-3-phosphatidic acid; DPPS; 1,2-dipalmitoyl-; sn; -glycero-3-phospho-; l; -serine; TMCL; 1,1′,2,2′-tetramyristoyl cardiolipin; PC; Phosphatidylcholine; PE; Phosphatidylethanolamine; PG; Phosphatidylglycerol; PA; Phosphatidic acid; PS; Phosphatidylserine; CL; Cardiolipin; CAMP; Cationic antimicrobial peptide; T; _m; Lipid phase transition temperatureAntimicrobial peptide; Model lipid membrane; Lipid demixing; Lipid–peptide interaction; DSC; FT-IR

Lipid binding properties of 4E10, 2F5, and WR304 monoclonal antibodies that neutralize HIV-1 by Gary R. Matyas; Zoltan Beck; Nicos Karasavvas; Carl R. Alving (pp. 660-665).

Two human mAbs (2F5 and 4E10), originally derived from HIV-1-infected patients, are important, but rare, mAbs that exhibit broad cross-clade neutralizing activities against HIV-1. In addition to peptide sequences on the gp41 envelope protein, both antibodies reportedly also bound specifically to several phospholipid antigens. However, the phospholipid binding property of 2F5 has been disputed and, because of uncertainly regarding phospholipid binding, the modeling of neutralizing mechanisms has been difficult. To explore this issue, we examined the binding of 4E10 and 2F5 to a broad range of lipid antigens by ELISA. 4E10 and 2F5 both bound to a variety of purified phospholipids, and 4E10 bound, but 2F5 did not bind, to cardiolipin. Both mAbs also bound to a sulfated glycolipid, sulfogalactosyl ceramide (sulfatide), and to two neutral glycolipids, galactosyl ceramide and glucosyl ceramide, but not to other galactosyl glycolipids. 4E10, but not 2F5, also bound to cholesterol, although both mAbs bound to squalene. Interestingly, 4E10, but not 2F5, exhibited striking binding to lipid A, the lipid moiety of Gram-negative bacterial lipopolysaccharide. The binding properties of 4E10 to phospholipids, sulfatide, cholesterol, squalene, and lipid A were similar to those of a neutralizing murine mAb (WR304) induced by liposomes containing phosphatidylinositol phosphate and lipid A, although WR304 did not bind to neutral glycolipids. The discovery of a binding specificity of 4E10 for lipid A, a widely used vaccine adjuvant, suggests that innate immunity stimulated by lipid A could have played a role for induction of multispecific antibodies that simultaneously recognize both HIV-1 protein and lipid antigens.

Keywords: HIV-1; Antibody; Phospholipid; Cholesterol; Glycolipid; Lipid A

Membrane microdomains: Role of ceramides in the maintenance of their structure and functions by Galya Staneva; Albena Momchilova; Claude Wolf; Peter J. Quinn; Kamen Koumanov (pp. 666-675).

Free-standing giant unilamellar vesicles were used to visualize the complex lateral heterogeneity, induced by ceramide in the membrane bilayer at micron scale using C₁₂-NBD-PC probe partitioning under the fluorescence microscope. Ceramide gel domains exist as leaf-like structures in glycerophospholipid/ceramide mixtures. Cholesterol readily increases ceramide miscibility with glycerophospholipids but cholesterol–ceramide interactions are not involved in the organization of the liquid-ordered phase as exemplified by sphingomyelin/cholesterol mixtures. Sphingomyelin stabilizes the gel phase and thus decreases ceramide miscibility in the presence of cholesterol. Gel/liquid-ordered/liquid-disordered phase coexistence was visualized in quaternary phosphatidylcholine/sphingomyelin/ceramide/cholesterol mixtures as occurrence of dark leaf-like and circular domains within a bright liquid phase. Sphingomyelin initiates specific ceramide–sphingomyelin interactions to form a highly ordered gel phase appearing at temperatures higher than pure ceramide gel phase in phosphatidylcholine/ceramide mixtures. Less sphingomyelin is engaged in formation of liquid-ordered phase leading to a shift in its formation to lower temperatures. Sphingomyelinase activity on substrate vesicles destroys micron L_o domains but induces the formation of a gel-like phase. The activation of phospholipase A₂ by ceramide on heterogeneous membranes was visualized. Changes in the phase state of the membrane bilayer initiates such morphological processes as membrane fragmentation, budding in and budding out was demonstrated.

Keywords: Ceramide; Cholesterol; Sphingomyelin; Raft; Sphingomyelinase; Phospholipase A; ₂; GUV; Lipid signaling pathway

Transport of glutathione transferase-fold structured proteins into living cells by Melanie J. Morris; Scott J. Craig; Theresa M. Sutherland; Philip G. Board; Marco G. Casarotto (pp. 676-685).

Glutathione transferases are a family of enzymes that are traditionally known to contribute to the phase II class of detoxification reactions. However, a novel property of the Schistosoma japonicum glutathione transferase (Sj.GST26) involves its translocation from the external medium into a variety of different cell types. Here we explore the efficiency and mechanism of cell entry for this class of protein. Using flow cytometry and confocal microscopy, we have examined the internalisation of Sj.GST26 into live cells under a variety of conditions designed to shed light on the mode of cellular uptake. Our results show that Sj.GST26 can effectively enter cells through an energy-dependent event involving endocytosis. More specifically, Sj.GST26 was found to colocalise with transferrin within the cell indicating that the endocytosis process involves clathrin-coated pits. A comprehensive study into the cellular internalisation of proteins from other classes within the GST structural superfamily has also been conducted. These experiments suggest that the ‘GST-fold’ structural motif influences cellular uptake, which presents a novel glimpse into an unknown aspect of GST function.

Keywords: Abbreviations; PTD; protein transduction domain; GST; glutathione transferase; GSH; glutathione; GSH-S; glutathione synthetase; CLIC2; chloride intracellular channel 2; GDAP1; ganglioside-induced differentiation-associated protein 1; Grx2; glutaredoxin-2; BSA; bovine serum albumin; GFP; green fluorescent protein; PBS; phosphate-buffered saline; FACS; fluorescence-activated cell sorting; OG; Oregon Green; TRITC; tetramethylrhodamine isothiocyanate; 7-AAD; 7-amino-actinomycin D; ER; endoplasmic reticulum; Sj.GST26; Schistosoma japonicum; GST; TCEP; tris-(2-carboxyethyl)phosphine; PNRC; perinuclear recycling compartment; CME; clathrin-mediated endocytosis; Tfn; transferrinGlutathione transferase; Endocytosis; Protein structure; Protein transduction domain

Solid-state NMR and molecular dynamics simulations reveal the oligomeric ion-channels of TM2-GABA_A stabilized by intermolecular hydrogen bonding by Senthil K. Kandasamy; Dong-Kuk Lee; Ravi P.R. Nanga; Jiadi Xu; Jose S. Santos; Ronald G. Larson; Ayyalusamy Ramamoorthy ⁎ (pp. 686-695).

The second transmembrane (TM2) domain of GABA_A receptor forms the inner-lining surface of chloride ion-channel and plays important roles in the function of the receptor protein. In this study, we report the first structure of TM2 in lipid bilayers determined using solid-state NMR and MD simulations. The interatomic¹³C–¹⁵N distances measured from REDOR magic angle spinning experiments on multilamellar vesicles, containing a TM2 peptide site specifically labeled with¹³C′ and¹⁵N isotopes, were used to determine the secondary structure of the peptide. The¹⁵N chemical shift and¹H–¹⁵N dipolar coupling parameters measured from PISEMA experiments on mechanically aligned phospholipid bilayers, containing a TM2 peptide site specifically labeled with¹⁵N isotopes, under static conditions were used to determine the membrane orientation of the peptide. Our results reveal that the TM2 peptide forms an alpha helical conformation with a tilted transmembrane orientation, which is unstable as a monomer but stable as pentameric oligomers as indicated by MD simulations. Even though the peptide consists of a number of hydrophilic residues, the transmembrane folding of the peptide is stabilized by intermolecular hydrogen bondings between the side chains of Ser and Thr residues as revealed by MD simulations. The results also suggest that peptide–peptide interactions in the tilted transmembrane orientation overcome the hydrophobic mismatch between the peptide and bilayer thickness.

Keywords: Abbreviations; CD; circular dichroism; CP; cross polarization; DMPC; 1,2-dimyristoyl-; sn; -glycero-3-phosphatidylcholine; GABA; γ-amino butyric acid; MAS; magic angle spinning; MD; molecular dynamics; MLVs; multilamellar vesicles; NMR; nuclear magnetic resonance; PISA; polarity index slant angle; PISEMA; polarization inversion spin exchange at the magic angle; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphatidylcholine; POPG; 1-palmitoyl-2-oleoyl-phosphatidylglycerol; REDOR; rotational echo double resonance; RF; radio frequency; SEMA; spin exchange at the magic angle; SUV; small unilamellar vesicle; TFE; trifluoroethanol; TM; tansmembrane; TPPM; two-phase pulse-modulationGABA receptor; Membrane protein; Ion channel; Solid state NMR; Lipid bilayer; Structure

Prediction of the most favorable configuration in the ACBP–membrane interaction based on electrostatic calculations by Diego F. Vallejo; Fernando Zamarreño; Diego M.A. Guérin; J. Raul Grigera; Marcelo D. Costabel ⁎ (pp. 696-700).

Acyl-CoA binding proteins (ACBPs) are highly conserved 10 kDa cytosolic proteins that bind medium- and long-chain acyl-CoA esters. They act as intracellular carriers of acyl-CoA and play a role in acyl-CoA metabolism, gene regulation, acyl-CoA-mediated cell signaling, transport-mediated lipid synthesis, membrane trafficking and also, ACBPs were indicated as a possible inhibitor of diazepam binding to the GABA-A receptor. To estimate the importance of the non-specific electrostatic energy in the ACBP–membrane interaction, we computationally modeled the interaction of HgACBP with both anionic and neutral membranes. To compute the Free Electrostatic Energy of Binding ( dE), we used the Finite Difference Poisson Boltzmann Equation (FDPB) method as implemented in APBS. In the most energetically favorable orientation, ACBP brings charged residues Lys18 and Lys50 and hydrophobic residues Met46 and Leu47 into membrane surface proximity. This conformation suggests that these four ACBP amino acids are most likely to play a leading role in the ACBP–membrane interaction and ligand intake. Thus, we propose that long range electrostatic forces are the first step in the interaction mechanism between ACBP and membranes.

Keywords: ACBP; HgACBP; Protein–membrane interaction

Calcium inhibits diacylglycerol uptake by serum albumin by Hasna Ahyayauch; Gorka Arana; Jesús Sot; Alicia Alonso; Félix M. Goñi ⁎ (pp. 701-707).

Serum albumin is an abundant protein in blood plasma, that is well-known for its ability to transport hydrophobic biomolecules and drugs. Recent hypotheses propose that serum albumin plays a role in the regulation of lipid metabolism in addition to its lipid transport properties. The present work explores the capacity of bovine serum albumin (BSA) to extract diacylglycerols (DAG) from phospholipid bilayers, and the inhibition of such interaction by divalent cations. Quantitative measurements using radioactive DAG and morphological evidence derived from giant unilamellar vesicles examined by confocal microscopy provide concurrent results. BSA extracts DAG from vesicles consisting of phosphatidylinositol/DAG. Long, saturated DAG species are incorporated more readily than the shorter-chain or unsaturated ones. Divalent cations hinder DAG uptake by BSA. For Ca²⁺, the concentration causing half-maximal inhibition is ≈10 μM; 90% inhibition is caused by 100 μM Ca²⁺. Sr²⁺ requires concentrations one order of magnitude higher, while Mg²⁺ has virtually no effect. As an example on how DAG uptake by BSA, and its inhibition by Ca²⁺, could play a regulating role in lipid metabolism, a PI-specific phospholipase C has been assayed in the presence of BSA and/or Ca²⁺. BSA activates the enzyme by removing the end-product DAG, but the activation is reverted by Ca²⁺ that inhibits DAG uptake.

Keywords: Serum albumin; Diacylglycerol; Calcium; Divalent cation; Phospholipase C

Study of bradykinin conformation in the presence of model membrane by Nuclear Magnetic Resonance and molecular modelling by Claudia Bonechi ⁎; Sandra Ristori; Giacomo Martini; Silvia Martini; Claudio Rossi (pp. 708-716).

The conformation of bradykinin (BK), Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9, was investigated by Nuclear Magnetic Resonance (NMR) spectroscopy and Monte Carlo simulation in two different media, i.e. in pure aqueous solution and in the presence of phospholipid vesicles. Monolamellar liposomes are a good model for biological membranes and mimic the environment experienced by bradykinin when interacting with G-protein coupled receptors (GPCRs). The NMR spectra showed that lipid bilayers induced a secondary structure in the otherwise inherently flexible peptide. The results of ensemble calculations revealed conformational changes occurring rapidly on the NMR time scale and allowed for the identification of different families of conformations that were averaged to reproduce the NMR observables. These structural results supported the hypothesis of the central role played by the peptide C-terminal domain in biological environments, and provided an explanation for the different biological behaviours observed for bradykinin.

Keywords: Bradykinin; Monolamellar liposome; NMR; DOPC/DOPE

Characterization of the kinetics and mechanisms of inhibition of drugs interacting with the S. cerevisiae multidrug resistance pumps Pdr5p and Snq2p by Tomáš Hendrych; Marie Kodedová; Karel Sigler; Dana Gášková (pp. 717-723).

We have developed a novel screening method that measures the kinetics and potencies of inhibitors of the yeast multidrug resistance pumps Pdr5p and Snq2p. The assay uses the potentiometric fluorescent probe diS-C₃(3) (as a benchmark substrate of both pumps) to distinguish drugs with minimal effects on plasma membrane potential as a marker of side-effects on membrane function and integrity. Using FK506, its structural analog rapamycin and enniatin B, we showed that our assay can also be used to determine the minimum drug concentration causing an immediate inhibitory effect and to compare the inhibitory potencies of the drug on the two pumps. We found that the protonophore CCCP effectively inhibits the transport of diS-C₃(3) by both pumps and confirmed the activation of membrane H⁺-ATPase by CCCP.

Keywords: Yeast MDR pump; Pump inhibitor; Fluorescence probe diS-C; ₃; (3); Membrane potential

Atomic force microscopy differentiates discrete size distributions between membrane protein containing and empty nanolipoprotein particles by Craig D. Blanchette; Jenny A. Cappuccio; Edward A. Kuhn; Brent W. Segelke; W. Henry Benner; Brett A. Chromy; Matthew A. Coleman; Graham Bench; Paul D. Hoeprich; Todd A. Sulchek (pp. 724-731).

To better understand the incorporation of membrane proteins into discoidal nanolipoprotein particles (NLPs) we have used atomic force microscopy (AFM) to image and analyze NLPs assembled in the presence of bacteriorhodopsin (bR), lipoprotein E4 n-terminal 22k fragment scaffold and DMPC lipid. The self-assembly process produced two distinct NLP populations: those containing inserted bR (bR-NLPs) and those that did not (empty-NLPs). The bR-NLPs were distinguishable from empty-NLPs by an average increase in height of 1.0 nm as measured by AFM. Streptavidin binding to biotinylated bR confirmed that the original 1.0 nm height increase corresponds to br-NLP incorporation. AFM and ion mobility spectrometry (IMS) measurements suggest that NLP size did not vary around a single mean but instead there were several subpopulations, which were separated by discrete diameters. Interestingly, when bR was present during assembly the diameter distribution was shifted to larger particles and the larger particles had a greater likelihood of containing bR than smaller particles, suggesting that membrane proteins alter the mechanism of NLP assembly.

Keywords: Apolipoprotein; Nanolipoprotein particle; Nanodisc; Atomic force microscopy; Membrane protein; Bacteriorhodopsin; NLP

Infrared spectroscopy studies of mixtures prepared with synthetic ceramides varying in head group architecture: Coexistence of liquid and crystalline phases by M. Janssens; G.S. Gooris; J.A. Bouwstra ⁎ (pp. 732-742).

The barrier function of the skin is provided by the stratum corneum (SC), the outermost layer of the skin. Ceramides (CERs), cholesterol (CHOL) and free fatty acids (FFAs) are present in SC and form highly ordered crystalline lipid lamellae. These lamellae are crucial for a proper skin barrier function. In the present study, Fourier transform infrared spectroscopy was used to examine the lipid organization of mixtures prepared from synthetic CERs with CHOL and FFAs. The conformational ordering and lateral packing of these mixtures showed great similarities to the lipid organization in SC and lipid mixtures prepared with native CERs. Therefore, mixtures with synthetic CERs serve as an excellent tool for studying the effect of molecular architecture of CER subclasses on the lipid phase behavior. In SC the number of OH-groups in the head groups of CER subclasses varies. Furthermore, acylCERs with a linoleic acid chemically bound to a long acyl chain are also identified. The present study revealed that CER head group architecture affects the lateral packing and conformational ordering of the CER:CHOL:FFA mixtures. Furthermore, while the majority of the lipids form a crystalline packing, the linoleate moiety of the acylCERs participates in a “pseudo fluid” phase.

Keywords: Ceramides; Skin; Lipid organization; Infrared spectroscopy; Stratum corneum

Mechanistic role of ergosterol in membrane rigidity and cycloheximide resistance in Saccharomyces cerevisiae by Fumiyoshi Abe ⁎; Toshiki Hiraki (pp. 743-752).

Mutants of Saccharomyces cerevisiae defective in the late steps of ergosterol biosynthesis are viable but accumulate structurally altered sterols within the plasma membrane. Despite the significance of pleiotropic abnormalities in the erg mutants, little is known about how sterol alterations mechanically affect the membrane structure and correlate with individual mutant phenotypes. Here we demonstrate that the membrane order and occurrence of voids are determinants of membrane rigidity and hypersensitivity to a drug. Among five ergΔ mutants, the erg2Δ mutant exhibited the most marked sensitivity to cycloheximide. Notably, measurement of time-resolved anisotropy indicated that the erg2Δ mutation decreased the membrane order parameter ( S), and dramatically increased the rotational diffusion coefficient ( D_w) of 1-[4-(trimethylamino)pheny]-6-phenyl-1,3,5-hexatriene (TMA-DPH) in the plasma membrane by 8-fold, providing evidence for the requirement of ergosterol for membrane integrity. The IC₅₀ of cycloheximide was closely correlated with S/ D_w in these strains, suggesting that the membrane disorder and increasing occurrence of voids within the plasma membrane synergistically enhance passive diffusion of cycloheximide across the membrane. Exogenous ergosterol partially restored the membrane properties in the upc2-1erg2Δ strain. In this study, we describe the ability of ergosterol to adjust the dynamic properties of the plasma membrane, and consider the relevance of drug permeability.

Keywords: Abbreviations; DPH; 1,6-diphenyl-1,3,5-hexatriene; TMA-DPH; 1-[4-(trimethylamino)pheny]-6-phenyl-1,3,5-hexatriene; TCSPC; time-correlated single-photon counting; DMPC; 1,2-dimyristoyl-; sn; -glycero-3-phosphocholine; r; _s; steady-state anisotropy; r; ₀; maximum anisotropy; r; _∞; limiting anisotropy; θ; rotational correlation time; S; order parameter; D; _w; rotational diffusion coefficientErgosterol; TMA-DPH; Fluorescence anisotropy; Membrane rigidity and fluidity; Drug permeability

Sections

Personal tools

BBA - Biomembranes (v.1788, #3)

Sections

Personal tools

Local resources

BBA - Biomembranes (v.1788, #3)