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BBA - Biomembranes (v.1808, #7)

Editorial Board (pp. i).

Structure–function relationships in membrane segment 6 of the yeast plasma membrane Pma1 H⁺-ATPase by Manuel Miranda; Juan Pablo Pardo; Valery V. Petrov (pp. 1781-1789).

The crystal structures of the Ca²⁺- and H⁺-ATPases shed light into the membrane embedded domains involved in binding and ion translocation. Consistent with site-directed mutagenesis, these structures provided additional evidence that membrane-spanning segments M4, M5, M6 and M8 are the core through which cations are pumped. In the present study, we have used alanine/serine scanning mutagenesis to study the structure–function relationships within M6 (Leu-721-Pro-742) of the yeast plasma membrane ATPase. Of the 22 mutants expressed and analyzed in secretory vesicles, alanine substitutions at two well conserved residues (Asp-730 and Asp-739) led to a complete block in biogenesis; in the mammalian P-ATPases, residues corresponding to Asp-730 are part of the cation-binding domain. Two other mutants (V723A and I736A) displayed a dramatic 20-fold increase in the IC₅₀ for inorganic orthovanadate compared to the wild-type control, accompanied by a significant reduction in the K_m for Mg-ATP, and an alkaline shift in the pH optimum for ATP hydrolysis. This behavior is apparently due to a shift in equilibrium from the E₂ conformation of the ATPase towards the E₁ conformation. By contrast, the most striking mutants lying toward the extracellular side in a helical structure (L721A, I722A, F724A, I725A, I727A and F728A) were expressed in secretory vesicles but had a severe reduction of ATPase activity. Moreover, all of these mutants but one (F728A) were unable to support yeast growth when the wild-type chromosomal PMA1 gene was replaced by the mutant allele. Surprisingly, in contrast to M8 where mutations S800A and E803Q (Guerra et al., Biochim. Biophys. Acta 1768: 2383–2392, 2007) led to a dramatic increase in the apparent stoichiometry of H⁺ transport, three substitutions (A726S, A732S and T733A) in M6 showed a reduction in the apparent coupling ratio. Taken together, these results suggest that M6 residues play an important role in protein stability and function, and probably are responsible for cation binding and stoichiometry of ion transport as suggested by homology modeling.► Amino acid substitutions of the conserved Asp-730 and D-739 residues led to protein misfolding. ► Mutations on the extracellular side of M6 were expressed in secretory vesicles but had a severe reduction of ATPase activity. ► Mutations of Ala-726, Ala-732 and Thr-733 decreased the coupling ratio of H⁺/ATP.

Keywords: PMA1; H+-ATPase; Plasma membrane ATPase; P2-ATPases; Ion pump; Transmembrane segment 6

Glutamine residues in Q-loops of multidrug resistance protein MRP1 contribute to ATP binding via interaction with metal cofactor by Runying Yang; Yue-xian Hou; Chase A. Campbell; Kanagaraj Palaniyandi; Qing Zhao; Andrew J. Bordner; Xiu-bao Chang (pp. 1790-1796).

Structural analyses of bacterial ATP-binding-cassette transporters revealed that the glutamine residue in Q-loop plays roles in interacting with: 1) a metal cofactor to participate in ATP binding; 2) a putative catalytic water molecule to participate in ATP hydrolysis; 3) other residues to transmit the conformational changes between nucleotide-binding-domains and transmembrane-domains, in ATP-dependent solute transport. We have mutated the glutamines at 713 and 1375 to asparagine, methionine or leucine to determine the functional roles of these residues in Q-loops of MRP1. All these single mutants significantly decreased Mg·ATP binding and increased the K_m (Mg·ATP) and V_max values in Mg·ATP-dependent leukotriene-C4 transport. However, the V_max values of the double mutants Q713N/Q1375N, Q713M/Q1375M and Q713L/Q1375L were lower than that of wtMRP1, implying that the double mutants cannot efficiently bind Mg·ATP. Interestingly, MRP1 has higher affinity for Mn·ATP than for Mg·ATP and the Mn·ATP-dependent leukotriene-C4 transport activities of Q713N/Q1375N and Q713M/Q1375M are significantly higher than that of wtMRP1. All these results suggest that: 1) the glutamine residues in Q-loops contribute to ATP-binding via interaction with a metal cofactor; 2) it is most unlikely that these glutamine residues would play crucial roles in ATP hydrolysis and in transmitting the conformational changes between nucleotide-binding-domains and transmembrane-domains.► The Q residues in Q-loops of MRP1 interact with Mg++ and contribute to Mg·ATP-binding. ► Mutations eliminating amide group significantly decreased Mg·ATP binding. ► Mutations eliminating amide group significantly increased the K_m and V_max values. ► The Q residues in Q-loops of MRP1 may not play a crucial role in ATP hydrolysis. ► May not play crucial roles in transmitting the conformational changes between domains.

Keywords: Abbreviations; Sf21; Spodoptera frugiperda; 21; ABC; ATP binding cassette; P-gp; P-glycoprotein; BCRP; breast cancer resistance protein; MRP1; multidrug resistance protein 1; NBD; nucleotide binding domain; TMD; transmembrane domain; LTC4; leukotriene C4; 8-N; ₃; ATP; 8-azidoadenosine 5′-triphosphate; PBS; phosphate-buffered saline; EGTA; ethylene glycol-bis(β-aminoethyl ether); N; ,; N; ,; N; ,; N; -tetraacetic acid; SDS; sodium dodecyl sulfateMRP1; Glutamine residue in Q-loop; Metal cofactor; ATP binding/hydrolysis; ATP dependent; LTC4 transport; K; _m; and; V; _max; values

Destabilization exerted by peptides derived from the membrane-proximal external region of HIV-1 gp41 in lipid vesicles supporting fluid phase coexistence by Apellaniz Beatriz Apellániz; Garcia-Saez Ana García-Sáez; Shlomo Nir; José L. Nieva (pp. 1797-1805).

The human immonodeficiency virus (HIV) envelope is enriched in cholesterol and sphingomyelin, two lipids that sustain the formation of laterally segregated liquid-ordered fluid domains in model systems. Several evidences indicate that the high lipid order existing at the envelope may play a role in HIV pathogenesis. A putative mechanism might involve the modulation of the membrane-perturbing function of the gp41 membrane-proximal external region (MPER). To test such hypothesis, we investigate here the effect of lipid phase coexistence on the membrane-restructuring properties of NpreTM and CpreTM, two peptides based on the amino- and carboxy-terminal MPER sequences, respectively. Fluid phase coexistence elicited the fusogenic activity of NpreTM at high membrane doses and stimulated “graded” leakage at low doses. By comparison, the effect on CpreTM was restricted to an enhancement of “all-or-none” leakage that was consistent with the promotion of its surface aggregation. Confocal microscopy of single vesicles revealed the preference of both peptides for liquid-disordered domains. Accordingly, we speculate that confinement into envelope fluid nanodomains might boost the distinct capacities of HIV MPER hydrophobic modules for inducing membrane defects during fusion.► We investigate fluid phase coexistence effects on the membrane-restructuring properties of two HIV MPER-derived peptides. ► For NpreTM, fluid phase coexistence elicited vesicle fusion and “graded” membrane permeabilization. ► For CpreTM, fluid phase coexistence enhanced “all-or-none” leakage but did not promote fusion. ► Confocal microscopy of single vesicles revealed the preference of both peptides for liquid-disordered domains. ► We infer that MPER crowding into fluid nanodomains may boost its membrane-restructuring activity during HIV fusion.

Keywords: MPER; HIV gp41; Membrane fusion; Lytic pore; Lipid rafts; Lo–Ld coexistence

Thermodynamic invariants of gel to the liquid-crystal 1,2-diacylphosphatidylcholines transition by Sergey A. Potekhin; Alexander A. Senin; Nickolay N. Abdurakhmanov; Railya S. Khusainova (pp. 1806-1810).

The bilayer phase transitions from the ripple gel phase ( P′_β) to the liquid-crystal phase ( L_α) of a series of 1,2-diacylphosphatidylcholines containing a linear saturated acyl chain ( C=14–19) have been studied by high-pressure scanning microcalorimetry. It has been shown that at ambient pressure, the transition temperature increases non-linearly depending on the acyl chain length. Pressure stabilizes the gel phase of lipids in a similar way; the pressure derivatives of the logarithm transition temperature as function of pressure are identical for all lipids. Based on the results obtained it has been concluded that the ratio γ of volume to enthalpy increments upon transitions in 1,2-diacylphosphatidylcholines is not dependent on the acyl chain length. When pressure grows, this ratio decreases drastically remaining identical for all lipids studied. Besides it has been demonstrated that increments of coefficients of thermal expansibility and isothermal compressibility are also rigidly bound to each other. Semi-empirical equations permitting to estimate volume parameters of the gel-to-liquid transition for 1,2-diacylphosphatidylcholines are given. The reasons for invariance of γ are discussed.► Pressure shifts of the transition temperature according to the classical relation. ► We studied effect of pressure on 6 phospholipids with different acyl chain lengths. ► The ratio of volume-to-enthalpy increments is the same for all lipids. ► Increments of expansibility and compressibility are also rigidly bound to each other.

Keywords: High pressure; Belayer membrane; Phase transition; Diacylphosphatidylcholine; Volume and enthalpy increment; Isothermal compressibility; Isobaric expansibility

A mechanistic investigation of cell-penetrating Tat peptides with supported lipid membranes by Stefania Piantavigna; George A. McCubbin; Solveig Boehnke; Bim Graham; Leone Spiccia; Lisandra L. Martin (pp. 1811-1817).

The multifarious Tat peptide derived from the HIV-1 virus exhibits antimicrobial activity. In this article, we use Quartz Crystal Microbalance with Dissipation monitoring (QCM-D) to investigate the mechanisms of action of Tat (44–57) and Tat (49–57) on bacterial-mimetic 1,2-dimyristoyl- sn-glycero-3-phosphocholine (DMPC)/1,2-dimyristoyl- sn-glycero-3-phospho- rac-(1-glycerol) (sodium salt) (DMPG) membranes. The results reveal that both peptides disrupt DMPC/DMPG membranes via a surface-active (carpet-like) mechanism. The magnitude of this disruption is dependent on both membrane and peptide properties. Firstly, less disruption was observed on the more negatively charged membranes. Secondly, less disruption was observed for the longer and slightly more hydrophobic Tat (44–57) peptide. As a comparison, the behaviour of the two Tat peptides on mammalian-mimetic DMPC/cholesterol membranes was investigated. Consistent with the literature no membrane disruption was observed. These results suggest that both electrostatic and hydrophobic interactions, as well as peptide geometry, determine the antimicrobial activity of Tat. This should guide the development of more potent Tat antibiotics.Display Omitted► Tat peptides disrupt bacterial-mimetic membranes via surface-active mechanisms. ► Peptide binding increases with greater negatively charged lipid composition. ► Membrane disruption decreases with increasing negative membrane charge. ► Membrane disruption less for Tat (44–57) compared with Tat (49–57). ► Both Tat peptides bind to mammalian-mimetic membranes without disruption.

Keywords: Tat peptide; Quartz crystal microbalance; Disruption; Antimicrobial peptide; Peptide antibiotic

Cpt-cAMP activates human epithelial sodium channels via relieving self-inhibition by Raul Molina; Dong-Yun Han; Xue-Feng Su; Run-Zhen Zhao; Meimi Zhao; Gretta M. Sharp; Yongchang Chang; Hong-Long Ji (pp. 1818-1826).

External Na⁺ self-inhibition is an intrinsic feature of epithelial sodium channels (ENaC). Cpt-cAMP regulates heterologous guinea pig but not rat αβγ ENaC in a ligand-gated manner. We hypothesized that cpt-cAMP may eliminate the self-inhibition of human ENaC thereby open channels. Regulation of self-inhibition by this compound in oocytes was analyzed using the two-electrode voltage clamp and Ussing chamber setups. External cpt-cAMP stimulated human but not rat and murine αβγ ENaC in a dose- and external Na⁺ concentration-dependent fashion. Intriguingly, cpt-cAMP activated human δβγ more potently than αβγ channels, suggesting that structural diversity in ectoloop between human α, δ, and those ENaC of other species determines the stimulating effects of cpt-cAMP. Cpt-cAMP increased the ratio of stationary and maximal currents. Mutants having abolished self-inhibition (β_ΔV348 and γ_H233R) almost completely eliminated cpt-cAMP mediated activation of ENaC. On the other hand, mutants both enhancing self-inhibition and elevating cpt-cAMP sensitivity increased the stimulating effects of the compound. This compound, however, could not activate already fully opened channels, e.g., degenerin mutation (αβ_S520Cγ) and the proteolytically cleaved ENaC by plasmin. Cpt-cAMP activated native ENaC to the same extent as that for heterologous ENaC in human lung epithelial cells. Our data demonstrate that cpt-cAMP, a broadly used PKA activator, stimulates human αβγ and δβγ ENaC channels by relieving self-inhibition.► Cpt-cAMP activates human αβγ and δβγ ENaC channels as a ligand. ► Cpt-cAMP is a self-inhibition releaser. ► Cpt-cAMP eventually modulates ENaC gating.

Keywords: Epithelial sodium channels; Cpt-cAMP; Self inhibition; Mutagenesis; Electrophysiology

On-line identification of P-glycoprotein substrates by monitoring of extracellular acidification and respiration rates in living cells by Swen Seeland; Alexander Treiber; Mathias Hafner; Jörg Huwyler (pp. 1827-1831).

The influence of P-glycoprotein (ABCB1) in drug resistance as well as drug absorption and disposition is an important factor to be considered during the development of new drugs. Thus, the early identification and exclusion of compounds showing a high affinity towards P-glycoprotein can help to select drug candidates. The aim of our study was to implement a label-free assay for the identification of P-glycoprotein substrates in living cells. For this approach, a multiparametric, chip-based sensor system was used to determine extracellular acidification, cell respiration and adhesion upon stimulation with P-glycoprotein substrates. Using L-MDR1 cells, a human P-glycoprotein overexpressing cell line, the influence of P-glycoprotein activity was determined for seven different compounds, demonstrating the applicability of the system for P-glycoprotein substrate identification. Effects were concentration dependent, as shown for the P-glycoprotein substrate verapamil, and were associated with cellular acidification and respiration. P-glycoprotein ATPase activation by verapamil could be described by a Michaelis–Menten type kinetic profile showing saturation at high substrate concentrations. The Michaelis–Menten constants K_M were determined to be 0.92μM (calculated based on extracellular acidification) and 4.9μM (calculated based on cellular respiration). Control experiments using 100nM of the P-glycoprotein inhibitor elacridar indicated that the observed effects were related to P-glycoprotein ATPase activity. In contrast, wild-type LLC-PK1 cells not expressing P-glycoprotein were not responsive towards stimulation with different P-glycoprotein substrates. Summarizing these findings, the used microsensor system is a generic system suitable for the identification of P-glycoprotein substrates. In contrast to biochemical P-glycoprotein assays, activation of the drug efflux pump can be monitored on-line in living cells to identify P-glycoprotein substrates and to study the molecular mechanisms of adenosintriphosphate-dependent active transport.► Identification of P-gp substrates by a label-free cytosensor assay. ► P-gp substrates induce cellular acidification and respiration. ► Effects were concentration dependent (Michaelis–Menten type kinetics). ► Inhibitors of P-gp demonstrate specificity of the assay. ► P-gp deficient control cells showed no stimulation effects.

Keywords: Abbreviations; MDR1; multi-drug resistance gene 1 or ABCB1; P-gp; P-glycoprotein; ATP; adenosintriphosphate; ECAR; extracellular acidification rate; OCR; oxygen consumption rate; SEM; standard error of the mean; IDES; interdigitated electrode structure; ISFET; ion-sensitive field effect transistorP-glycoprotein; ABCB1; Drug transport; Multi-drug resistance; Microsensor

Comparative study of clinical pulmonary surfactants using atomic force microscopy by Hong Zhang; Qihui Fan; Yi E. Wang; Charles R. Neal; Yi Y. Zuo (pp. 1832-1842).

Clinical pulmonary surfactant is routinely used to treat premature newborns with respiratory distress syndrome, and has shown great potential in alleviating a number of neonatal and adult respiratory diseases. Despite extensive study of chemical composition, surface activity, and clinical performance of various surfactant preparations, a direct comparison of surfactant films is still lacking. In this study, we use atomic force microscopy to characterize and compare four animal-derived clinical surfactants currently used throughout the world, i.e., Survanta, Curosurf, Infasurf and BLES. These modified-natural surfactants are further compared to dipalmitoyl phosphatidylcholine (DPPC), a synthetic model surfactant of DPPC:palmitoyl-oleoyl phosphatidylglycerol (POPG) (7:3), and endogenous bovine natural surfactant. Atomic force microscopy reveals significant differences in the lateral structure and molecular organization of these surfactant preparations. These differences are discussed in terms of DPPC and cholesterol contents. We conclude that all animal-derived clinical surfactants assume a similar structure of multilayers of fluid phospholipids closely attached to an interfacial monolayer enriched in DPPC, at physiologically relevant surface pressures. This study provides the first comprehensive survey of the lateral structure of clinical surfactants at various surface pressures. It may have clinical implications on future application and development of surfactant preparations.Display Omitted► We conduct a comprehensive comparison of lateral structure of clinical surfactants. ► Clinical surfactants are compared to synthetic and bovine natural surfactants. ► All surfactant films form multilayer structures regardless of the DPPC content. ► Cholesterol regulates phospholipid phase separation and surfactant film collapse.

Keywords: Surfactant replacement therapy; Dipalmitoyl phosphatidylcholine; Cholesterol; Domain formation; Surface tension; Monolayer

Linker and/or transmembrane regions of influenza A/Group-1, A/Group-2, and type B virus hemagglutinins are packed differently within trimers by Larisa V. Kordyukova; Marina V. Serebryakova; Anton A. Polyansky; Ekaterina A. Kropotkina; Andrei V. Alexeevski; Michael Veit; Roman G. Efremov; Irina Yu. Filippova; Lyudmila A. Baratova (pp. 1843-1854).

Influenza virus hemagglutinin is a homotrimeric spike glycoprotein crucial for virions' attachment, membrane fusion, and assembly reactions. X-ray crystallography data are available for hemagglutinin ectodomains of various types/subtypes but not for anchoring segments. To get structural information for the linker and transmembrane regions of hemagglutinin, influenza A (H1–H16 subtypes except H8 and H15) and B viruses were digested with bromelain or subtilisin Carlsberg, either within virions or in non-ionic detergent micelles. Proteolytical fragments were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Within virions, hemagglutinins of most influenza A/Group-1 and type B virus strains were more susceptible to digestion with bromelain and/or subtilisin compared to A/Group-2 hemagglutinins. The cleavage sites were always located in the hemagglutinin linker sequence. In detergent, 1) bromelain cleaved hemagglutinin of every influenza A subtype in the linker region; 2) subtilisin cleaved Group-2 hemagglutinins in the linker region; 3) subtilisin cleaved Group-1 hemagglutinins in the transmembrane region; 4) both enzymes cleaved influenza B virus hemagglutinin in the transmembrane region. We propose that the A/Group-2 hemagglutinin linker and/or transmembrane regions are more tightly associated within trimers than type A/Group-1 and particularly type B ones. This hypothesis is underpinned by spatial trimeric structure modeling performed for transmembrane regions of both Group-1 and Group-2 hemagglutinin representatives. Differential S-acylation of the hemagglutinin C-terminal anchoring segment with palmitate/stearate residues possibly contributes to fine tuning of transmembrane trimer packing and stabilization since decreased stearate amount correlated with deeper digestion of influenza B and some A/Group-1 hemagglutinins.► Enzymatic digestion of influenza hemagglutinin within virions or micelles was done. ► MALDI–TOF MS analysis of extracted anchoring segments revealed digestion sites. ► HA linkers/TM domains possess a quaternary structure in the absence of ectodomains. ► Differences between HAs belonging to different phylogenetic groups exist. ► First spatial models for A/Group-1 and Group-2 HA TM homotrimers were created. ► S-acylation might contribute to fine tuning of TM packing and stabilization.

Keywords: Abbreviations; MALDI–TOF MS; matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; HA; hemagglutinin; NA; neuraminidase; NP; nucleoprotein; TM; transmembraneHemagglutinin; Viral membrane; Linker and transmembrane region; MALDI–TOF MS; Phylogenetic groups of influenza virus; Molecular modeling

Acr3p is a plasma membrane antiporter that catalyzes As(III)/H⁺ and Sb(III)/H⁺ exchange in Saccharomyces cerevisiae by Ewa Maciaszczyk-Dziubinska; Magdalena Migocka; Robert Wysocki (pp. 1855-1859).

Resistance to arsenical compounds in Saccharomyces cerevisiae as well as in a growing number of prokaryotes and eukaryotes is mediated by members of the Acr3 family of transporters. In yeast cells, it has been clearly shown that Acr3p is localized to the plasma membrane and facilitates efflux of trivalent arsenic and antimony. However, until now, the energy dependence and kinetic properties of Acr3 proteins remained uncharacterized. In this work, we show that arsenite and antimonite uptake into everted membrane vesicles via the yeast Acr3 transporter is coupled to the electrochemical potential gradient of protons generated by the plasma membrane H⁺-translocating P-type ATPase. These results strongly indicate that Acr3p acts as a metalloid/H⁺ antiporter. Two differential kinetic assays revealed that Acr3p-mediated arsenite/H⁺ and antimonite/H⁺ exchange demonstrates Michaelis–Menten-type saturation kinetics characterized by a maximum flux for permeating metalloids. The approximate K_m values for arsenite and antimonite transport were the same, suggesting that Acr3p exhibits similar low affinity for both metalloids. Nevertheless, the maximal velocity of the transport at saturation concentrations of metalloids was approximately 3 times higher for arsenite than for antimonite. These findings may explain a predominant role of Acr3p in conferring arsenite tolerance in S. cerevisiae.► Acr3 mediates As(III)/H⁺ and Sb(III)/H⁺ antiport. ► Acr3 transports As(III) three times faster than Sb(III). ► Acr3 plays a predominant role in As(III) resistance in budding yeast.

Keywords: Abbreviations; As(III); arsenite; As(V); arsenate; Sb(III); antimonite; GFP; green fluorescent proteinAntimonite; Arsenite; Antiport; Acr3; Saccharomyces cerevisiae; Yeast

Binding of cell-penetrating penetratin peptides to plasma membrane vesicles correlates directly with cellular uptake by Amand Helene L. Åmand; Bostrom Carolina L. Boström; Per Lincoln; Norden Bengt Nordén; Esbjorner Elin K. Esbjörner (pp. 1860-1867).

Cell-penetrating peptides (CPPs) gain access to intracellular compartments mainly via endocytosis and have capacity to deliver macromolecular cargo into cells. Although the involvement of various endocytic routes has been described it is still unclear which interactions are involved in eliciting an uptake response and to what extent affinity for particular cell surface components may determine the efficiency of a particular CPP. Previous biophysical studies of the interaction between CPPs and either lipid vesicles or soluble sugar-mimics of cell surface proteoglycans, the two most commonly suggested CPP binding targets, have not allowed quantitative correlations to be established. We here explore the use of plasma membrane vesicles (PMVs) derived from cultured mammalian cells as cell surface models in biophysical experiments. Further, we examine the relationship between affinity for PMVs and uptake into live cells using the CPP penetratin and two analogs enriched in arginines and lysines respectively. We show, using centrifugation to sediment PMVs, that the amount of peptide in the pellet fraction correlates linearly with the degree of cell internalization and that the relative efficiency of all-arginine and all-lysine variants of penetratin can be ascribed to their respective cell surface affinities. Our data show differences between arginine- and lysine-rich variants of penetratin that has not been previously accounted for in studies using lipid vesicles. Our data also indicate greater differences in binding affinity to PMVs than to heparin, a commonly used cell surface proteoglycan mimic. Taken together, this suggests that the cell surface interactions of CPPs are dependent on several cell surface moieties and their molecular organization on the plasma membrane.Display Omitted► Affinity of penetratin variants for plasma membrane vesicles (PMVs) was examined. ► Binding to PMVs is arginine-dependent and correlates with uptake in live cells. ► PMVs have a membrane composition representative of the cells they were derived from. ► The results suggest that membrane affinity is a key property for an efficient CPP.

Keywords: Abbreviations; CF; Carboxyfluorescein; CHO-K1; Chinese hamster ovarian cell; CPP; Cell-penetrating peptide; DTT; Dithiothreitol; FA; Formaldehyde; FITC; Fluorescein isothiocyanate; FM4-64; N; -(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridiniumdibromide; GABA; Gamma-amino-butyric acid; HBS; Hepes-buffered saline; HEPES; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; PMV; Plasma membrane vesicleCell-penetrating peptide; Penetratin; Plasma membrane vesicles; Membrane affinity; Arginine; Lysine

Recent progress in the study of G protein-coupled receptors with molecular dynamics computer simulations by Alan Grossfield (pp. 1868-1878).

G protein-coupled receptors (GPCRs) are a large, biomedically important family of proteins, and the recent explosion of new high-resolution structural information about them has provided an enormous opportunity for computational modeling to make major contributions. In particular, molecular dynamics simulations have become a driving factor in many areas of GPCR biophysics, improving our understanding of lipid–protein interaction, activation mechanisms, and internal hydration. Given that computers will continue to get faster and more structures will be solved, the importance of computational methods will only continue to grow, particularly as simulation research is more closely coupled to experiment.► G protein-coupled receptors (GPCRs) are biomedically important. ► Molecular dynamics simulations provide crucial insight into structure and dynamics. ► Recent crystal structures have increased the importance of GPCR simulations.

Keywords: G protein-coupled receptor; Molecular dynamics; Hydration; Lipid–protein interactions; Oligomerization; Convergence

Structural transitions in the intrinsically disordered plant dehydration stress protein LEA7 upon drying are modulated by the presence of membranes by Antoaneta V. Popova; Michaela Hundertmark; Robert Seckler; Dirk K. Hincha (pp. 1879-1887).

Dehydration stress-related late embryogenesis abundant (LEA) proteins have been found in plants, invertebrates and bacteria. Most LEA proteins are unstructured in solution, but some fold into amphipathic α-helices during drying. The Pfam LEA_4 (Group 3) protein LEA7 from the higher plant Arabidopsis thaliana was predicted to be 87% α-helical, while CD spectroscopy showed it to be largely unstructured in solution and only 35% α-helical in the dry state. However, the dry protein contained 15% β-sheets. FTIR spectroscopy revealed the β-sheets to be largely due to aggregation. β-Sheet content was reduced and α-helix content increased when LEA7 was dried in the presence of liposomes with secondary structure apparently influenced by lipid composition. Secondary structure was also affected by the presence of membranes in the fully hydrated state. A temperature-induced increase in the flexibility of the dry protein was also only observed in the presence of membranes. Functional interactions of LEA7 with membranes in the dry state were indicated by its influence on the thermotropic phase transitions of the lipids and interactions with the lipid headgroup phosphates.►LEA7 is a plant IDP that is abundant in seeds and in vegetative tissues under stress conditions. ►It is largely unstructured in solution, but folds and partially aggregates upon dehydration. ►The presence of membranes induces folding into an α-helix during drying and suppresses aggregation. ►LEA7 interacts with lipid membranes in the dry state, partially through the lipid phosphate groups, and depresses the gel to liquid-crystalline phase transition temperature of the membranes.

Keywords: Abbreviation; CD; circular dichroism; EPE; egg phosphatidylethanolamine; EPG; egg phosphatidylglycerol; FTIR; Fourier-transform infrared; GRAVY; grand average of hydropathy; IDP; intrinsically disordered protein; LEA; late embryogenesis abundant; LG; β-lactoglobulin; POPC; 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine; T; _m; gel to liquid-crystalline phase transition temperatureDesiccation; CD spectroscopy; FTIR spectroscopy; LEA protein; Protein–membrane interactions; Protein secondary structure

A CRAC-like motif in BAX sequence: Relationship with protein insertion and pore activity in liposomes by Martinez-Abundis Eduardo Martínez-Abundis; Francisco Correa; Rodriguez Emma Rodríguez; Elizabeth Soria-Castro; Rodriguez-Zavala José S. Rodríguez-Zavala; Diana Pacheco-Alvarez; Cecilia Zazueta (pp. 1888-1895).

Several proteins that interact with cholesterol have a highly conserved sequence, corresponding to the cholesterol recognition/interaction amino acid consensus. Since cholesterol has been proposed to modulate both oligomerization and insertion of the pro-apoptotic protein BAX, we investigated the existence of such a motif in the BAX sequence. Residues 113 to 119 of the recombinant BAX α5-helix,LFYFASK, correspond with the sequence motif described for the consensus pattern, –L/V-(X)(1–5)-Y-(X)(1–5)-R/K. Functional characterization of the point mutations, K119A, Y115F, and L113A in BAX, was performed in liposomes supplemented with cholesterol, comparing binding, integration, and pore forming activities. Our results show that the mutations Y115F and L113A changed the cholesterol-dependent insertion observed in the wild type protein. In addition, substitutions in the BAX sequence modified the concentration dependency of carboxyfluorescein release in liposomes, although neither pore activity of the wild type or of any of the mutants significantly increased in cholesterol-enriched liposomes. Thus, while it is likely that the putative CRAC motif in BAX accounts for its enhanced insertion in cholesterol-enriched liposomes; the pore forming properties of BAX did not depend on cholesterol content in the membranes, albeit those mutations changed the pore channeling activity of the protein.► BAX α5-helix point mutations changed cholesterol-dependent insertion observed in the wild type protein. ► Substitutions in the BAX α5-helix sequence modified the protein channel properties. ► BAX pore activity was not dependent on cholesterol content in the membrane.

Keywords: Abbreviations; mPTP; mitochondrial permeability transition pore; CRAC; cholesterol recognition/interaction amino acid consensusBax; Cholesterol; CRAC motif

An atomic force microscopy study of Galleria mellonella apolipophorin III effect on bacteria by Agnieszka Zdybicka-Barabas; Barbara Januszanis; Pawel Mak; Cytrynska Małgorzata Cytryńska (pp. 1896-1906).

Apolipophorin III (apoLp-III) is an abundant hemolymph protein involved in lipid transport and immune response in insects. As revealed by LIVE/DEAD staining, incubation of Gram-negative and Gram-positive bacteria in the presence of Galleria mellonella apoLp-III led to growth inhibition of selected bacteria. An atomic force microscopy (AFM) study of bacterial cells after apoLp-III treatment showed considerable alterations in the cell surface of Bacillus circulans, Klebsiella pneumoniae and Salmonella typhimurium. Our results clearly demonstrate that apoLp-III disturbed the proper structure of the bacterial cell surface. The alterations were dissimilar to those caused by cationic antimicrobial peptide, cecropin B, suggesting a different mode of action against bacteria. The present results indicate that AFM provides a powerful tool for studying the interactions of apoLp-III with microbial cells.► G. mellonella apolipophorin III causes perturbations in the structure of bacterial cell envelope. ► ApoLp-III and cecropin B cause dissimilar alterations of bacterial cell surface. ► Cell surface of M. luteus, non-sensitive to apoLp-III bacteria, is almost unaffected by apoLp-III.

Keywords: Apolipophorin III; Antimicrobial peptides; Cecropin; Atomic force microscopy; Galleria mellonella

The lipid composition of a cell membrane modulates the interaction of an antiparasitic peptide at the air–water interface by Rondinelli D. Herculano; Felippe J. Pavinatto; Luciano Caseli; Claudius D'Silva; Osvaldo N. Oliveira Jr. (pp. 1907-1912).

The antiparasitic property of peptides is believed to be associated with their interactions with the protozoan membrane, which calls for research on the identification of membrane sites capable of peptide binding. In this study we investigated the interaction of a lipophilic glutathioine peptide known to be effective against the African Sleeping Sickness ( ASS — African Trypanosomiasis) and cell membrane models represented by Langmuir monolayers. It is shown that even small amounts of the peptide affect the monolayers of some phospholipids and other lipids, which points to a significant interaction. The latter did not depend on the electrical charge of the monolayer-forming molecules but the peptide action was particularly distinctive for cholesterol + sphingomyelin monolayers that roughly resemble rafts on a cell membrane. Using in situ polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS), we found that the orientation of the peptide is affected by the phospholipids and dioctadecyldimethylammonium bromide (DODAB), but not in monolayers comprising cholesterol + sphingomyelin. In this mixed monolayer resembling rafts, the peptide still interacts and has some induced order, probably because the peptide molecules are fitted together into a compact monolayer. Therefore, the lipid composition of the monolayer modulates the interaction with the lipophilic glutathioine peptide, and this may have important implications in understanding how the peptide acts on specific sites of the protozoan membrane.► Antiparasitic peptides were incorporated in membrane models. ► Small amounts of peptide affect the monolayers of lipids. ► Surface pressure isotherms point to interactions between lipids and peptides. ► Interactions do not depend on the electrical charge. ► The lipid composition modulates the interaction with the lipophilic peptide.

Keywords: Cell membrane model; Langmuir monolayer; Raft; Lipophilic peptide; African Sleeping Sickness

Relationship between membrane permeability and specificity of human secretory phospholipase A₂ isoforms during cell death by Jennifer Nelson; Elizabeth Gibbons; Katalyn R. Pickett; Michael Streeter; Ashley O. Warcup; Celestine H.-Y. Yeung; Allan M. Judd; John D. Bell (pp. 1913-1920).

During apoptosis, a number of physical changes occur in the cell membrane including a gradual increase in permeability to vital stains such as propidium iodide. This study explored the possibility that one consequence of membrane changes concurrent with early modest permeability is vulnerability to degradation by secretory phospholipase A₂. The activity of this hydrolytic enzyme toward mammalian cells depends on the health of the cell; healthy cells are resistant, but they become susceptible early during programmed death. Populations of S49 lymphoma cells during programmed death were classified by flow cytometry based on permeability to propidium iodide and susceptibility to secretory phospholipase A₂. The apoptotic inducers thapsigargin and dexamethasone caused modest permeability to propidium iodide and increased staining by merocyanine 540, a dye sensitive to membrane perturbations. Various secretory phospholipase A₂ isozymes (human groups IIa, V, X, and snake venom) preferentially hydrolyzed the membranes of cells that displayed enhanced permeability. In contrast, cells exposed briefly to a calcium ionophore showed the increase in cell staining intensity by merocyanine 540 without accompanying uptake of propidium iodide. Under that condition, only the snake venom and human group X enzymes hydrolyzed cells that were dying. These results suggested that cells showing modest permeability to propidium iodide during the early phase of apoptosis are substrates for secretory phospholipase A₂ and that specificity among isoforms of the enzyme depends on the degree to which the membrane has been perturbed during the death process. This susceptibility to hydrolysis may be important as part of the signal to attract macrophages toward apoptotic cells.► Early apoptotic cells display modest permeability to propidium iodide ► These cells are susceptible to hydrolysis by secretory phospholipase A₂ (sPLA₂) ► Cells dying by acute calcium loading do not show the modest permeability ► The differences in permeability corresponds to specificity of human sPLA₂ isoforms

Keywords: Apoptosis; Merocyanine 540; Propidium iodide; Fluorescence spectroscopy; Membrane biophysics

Reorganization of the actin cytoskeleton upon G-protein coupled receptor signaling by Sourav Ganguly; Roopali Saxena; Amitabha Chattopadhyay (pp. 1921-1929).

The actin cytoskeleton is involved in a multitude of cellular responses besides providing structural support. While the role of the actin cytoskeleton in cellular processes such as trafficking and motility has been extensively studied, reorganization of the actin cytoskeleton upon signaling by G-protein coupled receptors (GPCRs) represents a relatively unexplored area. The G-protein coupled receptor superfamily is an important protein family in mammals, involved in signal transduction across membranes. G-protein coupled receptors act as major signaling hubs and drug targets. The serotonin_1A receptor is a representative member of the G-protein coupled receptor superfamily and plays a crucial role in the generation and modulation of various cognitive, developmental and behavioral functions. In order to monitor the changes in the actin cytoskeleton upon serotonin_1A receptor signaling in a quantitative manner, we developed an approach based on high magnification imaging of F-actin in cells, followed by image reconstruction. Our results suggest that the actin cytoskeleton is reorganized in response to serotonin_1A receptor signaling. In addition, we show that reorganization of the actin cytoskeleton is strongly dependent on adenosine 3′,5′-cyclic monophosphate level, and is mediated by the activation of protein kinase A. Our results are consistent with the possibility of a feedback mechanism involving the actin cytoskeleton, adenosine 3′,5′-cyclic monophosphate level and the serotonin_1A receptor.► Serotonin_1A receptor signaling leads to reorganization of the actin cytoskeleton. ► Cytoskeletal destabilization is mediated by changes in cAMP level. ► We propose the possibility of a feedback mechanism under these conditions.

Keywords: Abbreviations; 5-HT; 5-hydroxytryptamine (serotonin); AC; adenylyl cyclase; cAMP; adenosine 3′,5′-cyclic monophosphate; CD; cytochalasin D; DMSO; dimethyl sulphoxide; ERM; ezrin/radixin/moesin; Fsk; forskolin; GPCR; G-protein coupled receptor; H-89; N-[2-(; p; -bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide; IBMX; 3-isobutyl-1-methylxanthine; Jas; jasplakinolide; LatA; latrunculin A; PDZ; PSD95/DlgA/ZO-1; PKA; protein kinase AActin; cAMP; Cytoskeleton; GPCR; Serotonin; _1A; receptor

Lipid raft detecting in membranes of live erythrocytes by Ilya Mikhalyov; Andrey Samsonov (pp. 1930-1939).

The fluorescent probe N-(BODIPY^®-FL-propionyl)-neuraminosyl-GM₁ (BODIPY-GM₁) was used to detect lipid rafts in living red blood cells (RBCs) membranes. The probe was detected with fluorescence video microscopy and was found to be uniformly distributed along plasma membrane at room temperature (23°C). At 4°C some probe clearly phase-separated to yield detectable bright spots that were smaller than spatial resolution. As measured by spectrofluorometry, in addition to a major fluorescence peak caused by emissions from monomers, the probe exhibited a red-shifted peak that is characteristic of a BODIPY fluorophore at high local concentrations, indicating that some probe had clustered. Red-shifted fluorescence was the greatest at 4°C, intermediate at 23°C, and the smallest at 37°C. Treating the RBCs with methyl-β-cyclodextrin to remove cholesterol eliminated the red-shifted peak. This strongly indicates that the presence of cholesterol was essential for phase separation of the probe. Fluorometry experiments indicate that rafts exist at 23°C and at 37°C, even though the membrane appears to be uniform at the resolution of microscope. The distinct GM₁ patches distributed over entire membrane of the erythrocytes were observed at both 23°C and at 37°C in RBCs stained with Alexa FL 647 cholera toxin subunit B conjugate (CTB-A647 ). Based on both fluorometry and fluorescence microscopy, some rafts clearly exist at 37°C.► Lipid rafts in living erythrocytes exist at physiological temperatures.► Rafts are mobile structures diffusing over entire cell surface.► Cholesterol is critical for erythrocyte raft integrity.► Low temperature promotes the big raft formation.

Keywords: Abbreviations; RBCs; red blood cells; BODIPY-GM1; polar head-labeled ganglioside GM; ₁; BODIPY; ^®; FL C5-GM; ₁; lipid tail labeled ganglioside GM; ₁; DRMs; detergent-resistant membranes; DOPC; 1,2-dioleoyl-sn-glycero-3-phosphocholine; egg-SM; egg sphingomyelin; CTB-A647; Alexa FL 647 cholera toxin subunit B conjugate; GPI; glycosylphosphatidylinositolLipid rafts; Ganglioside GM; ₁; Erythrocytes

Effect of hydrophobic mismatch and interdigitation on sterol/sphingomyelin interaction in ternary bilayer membranes by Shishir Jaikishan; J. Peter Slotte (pp. 1940-1945).

Sphingomyelin (SM) is a major phospholipid in most cell membranes. SMs are composed of a long-chain base (often sphingosine, 18:1^Δ4t), and N-linked acyl chains (often 16:0, 18:0 or 24:1^Δ15c). Cholesterol interacts with SM in cell membranes, but the acyl chain preference of this interaction is not fully elucidated. In this study we have examined the effects of hydrophobic mismatch and interdigitation on cholesterol/sphingomyelin interaction in complex bilayer membranes. We measured the capacity of cholestatrienol (CTL) and cholesterol to form sterol-enriched ordered domains with saturated SM species having different chain lengths (14 to 24 carbons) in ternary bilayer membranes. We also determined the equilibrium bilayer partitioning coefficient of CTL with 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC) membranes containing 20mol% of saturated SM analogs. Ours results show that while CTL and cholesterol formed sterol-enriched domains with both short and long-chain SM species, the sterols preferred interaction with 16:0-SM over any other saturated chain length SM analog. When CTL membrane partitioning was determined with fluid POPC bilayers containing 20mol% of a saturated chain length SM analog, the highest affinity was seen with 16:0-SM (both at 23 and 37°C). These results indicate that hydrophobic mismatch and/or interdigitation attenuate sterol/SM association and thus affect lateral distribution of sterols in the bilayer membrane.► Biologically relevant sphingomyelin (SM) species are often asymmetric molecules. ► It is unclear how asymmetry affects interactions with cholesterol. ► We made SM analogs with saturated acyl chains which are 14 to 24 carbons long. ► We show that SM asymmetry affected sterol interaction. ► The most favorable interaction was seen between cholesterol and 16:0-SM.

Keywords: Abbreviations; 7SLPC; 1-palmitoyl-2-stearoyl-(7-doxyl)-; sn; -glycero-3-phosphocholine; CTL; cholesta-5,7 (11)-trien-3-beta-ol; DPH; 1,6-diphenyl-1,3,5-hexatriene; K; _x; partitioning coefficient; PC; phosphatidylcholine; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; SM; sphingomyelinCholesterol; Cholestatrienol; Sterol partitioning; Lateral domain; Fluorescence quenching

Corrigendum to “The membrane-activity of Ibuprofen, Diclofenac, and Naproxen: A physico-chemical study with lecithin phospholipids” [Biochim. Biophys. Acta 1788 (2009) 1296–1303] by Marcela Manrique-Moreno; Patrick Garidel; Mario Suwalsky; Jörg Howe; Klaus Brandenburg (pp. 1946-1946).

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BBA - Biomembranes (v.1808, #7)