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BBA - Biomembranes (v.1798, #6)

Editorial Board (pp. i).

The antibacterial activity of phospholipase A₂ type IIA is regulated by the cooperative lipid chain melting behavior in Staphylococcus aureus by Jackson Ocampo; Nicolas Afanador; Martha J. Vives; Juan C. Moreno; Chad Leidy (pp. 1021-1028).

As one of its primary physiological functions, sPLA₂-IIA appears to act as an antibacterial agent. In particular, sPLA₂-IIA shows high activity towards Gram-positive bacteria such as Staphylococcus aureus ( S. aureus). This antibacterial activity results from the preference of the enzyme towards membranes enriched in anionic lipids, which is a common feature of bacterial membranes. An intriguing aspect observed in a variety of bacterial membranes is the presence of a broad but cooperative lipid chain melting event where the lipids in the membrane transition from a solid-ordered ( so) into a liquid-disordered ( ld) state close to physiological temperatures. It is known that the enzyme is sensitive to the level of lipid packing, which changes sharply between the so and the ld states. Therefore, it would be expected that the enzyme activity is regulated by the bacterial membrane thermotropic behavior. We determine by FTIR the thermotropic lipid chain melting behavior of S. aureus and find that the activity of sPLA₂-IIA drops sharply in the so state. The activity of the enzyme is also evaluated in terms of its effects on cell viability, showing that cell survival increases when the bacterial membrane is in the so state during enzyme exposure. These results point to a mechanism by which bacteria can develop increased resistance towards antibacterial agents that act on the membrane through a cooperative increase in the order of the lipid chains. These results show that the physical behavior of the bacterial membrane can play an important role in regulating physiological function in an in vivo system.

Keywords: Staphylococcus aureus; Phospholipase; Antibacterial; Lipid phase behavior; Gram-positive; FTIR

Interaction between CFTR and prestin (SLC26A5) by Kazuaki Homma; Katharine K. Miller; Charles T. Anderson; Soma Sengupta; Guo-Guang Du; Aguinaga Salvador Aguiñaga; MaryAnn Cheatham; Peter Dallos; Jing Zheng (pp. 1029-1040).

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated chloride channel that is present in a variety of epithelial cell types, and usually expressed in the luminal membrane. In contrast, prestin (SLC26A5) is a voltage-dependent motor protein, which is present in the basolateral membrane of cochlear outer hair cells (OHCs), and plays an important role in the frequency selectivity and sensitivity of mammalian hearing. By using in situ hybridization and immunofluorescence, we found that both mRNA and protein of CFTR are present in OHCs, and that CFTR localizes in both the apical and the lateral membranes. CFTR was not detected in the lateral membrane of inner hair cells (IHCs) or in that of OHCs derived from prestin-knockout mice, i.e., in instances where prestin is not expressed. These results suggest that prestin may interact physically with CFTR in the lateral membrane of OHCs. Immunoprecipitation experiments confirmed a prestin–CFTR interaction. Because chloride is important for prestin function and for the efferent-mediated inhibition of cochlear output, the prestin-directed localization of CFTR to the lateral membrane of OHCs has a potential physiological significance. Aside from its role as a chloride channel, CFTR is known as a regulator of multiple protein functions, including those of the solute carrier family 26 (SLC26). Because prestin is in the SLC26 family, several members of which interact with CFTR, we explored the potential modulatory relationship associated with a direct, physical interaction between prestin and CFTR. Electrophysiological experiments demonstrated that cAMP-activated CFTR is capable of enhancing voltage-dependent charge displacement, a signature of OHC motility, whereas prestin does not affect the chloride conductance of CFTR.

Keywords: Abbreviations; CFTR; cystic fibrosis transmembrane conductance regulator; Fsk; forskolin; IBMX; 3-isobutyl-1-methylxanthine; IHC; inner hair cell; NLC; nonlinear capacitance; OHC; outer hair cell; SLC26; solute carrier protein 26CFTR; Prestin; SLC26A; Chloride; Outer hair cell; Nonlinear capacitance

Tuning the insertion properties of pHLIP by Monika Musial-Siwek; Alexander Karabadzhak; Oleg A. Andreev; Yana K. Reshetnyak; Donald M. Engelman (pp. 1041-1046).

The pH (low) insertion peptide (pHLIP) has exceptional characteristics: at neutral pH it is an unstructured monomer in solution or when bound to lipid bilayer surfaces, and it inserts across a lipid bilayer as a monomeric alpha-helix at acidic pH. The peptide targets acidic tissues in vivo and may be useful in cancer biology for delivery of imaging or therapeutic molecules to acidic tumors. To find ways to vary its useful properties, we have designed and analyzed pHLIP sequence variants. We find that each of the Asp residues in the transmembrane segment is critical for solubility and pH-dependent membrane insertion of the peptide. Changing both of the Asp residues in the transmembrane segment to Glu, inserting an additional Asp into the transmembrane segment, or replacing either of the Asp residues with Ala leads to aggregation and/or loss of pH-dependent membrane insertion of the peptide. However, variants with either of the Asp residues changed to Glu remained soluble in an aqueous environment and inserted into the membrane at acidic pH with a higher pK_app of membrane insertion.

Keywords: pHLIP; Transmembrane helix; Membrane insertion; Helix formation

Tuning the insertion properties of pHLIP by Monika Musial-Siwek; Alexander Karabadzhak; Oleg A. Andreev; Yana K. Reshetnyak; Donald M. Engelman (pp. 1041-1046).

Keywords: pHLIP; Transmembrane helix; Membrane insertion; Helix formation

Erythrocyte death in vitro induced by starvation in the absence of Ca²⁺ by Giuliano Pompeo; Marco Girasole; Antonio Cricenti; Giovanna Boumis; Andrea Bellelli; Simona Amiconi (pp. 1047-1055).

Human erythrocytes (RBCs), stored at 4°C under nominal absence of external energy sources and calcium ions, show a gradual decrease in membrane roughness ( R_rms) at the end of which the appearance of morphological phenomena (spicules, vesicles and spherocytes) is observed on the cell membrane, phenomena that can mainly be ascribed to the ATP-dependent disconnection of the cortical cytoskeleton from the lipid bilayer. After depletion of the intracellular energy sources obtained under the extreme conditions chosen, treatment with a minimal rejuvenation solution makes the following remarks possible: (i) RBCs are able to regenerate adenosine triphosphate (ATP) and 2,3-bisphosphoglycerate only up to 4days of storage at 4°C, whereas from the eighth day energy stocks cannot be replenished because of a disorder in the transmembrane mechanisms of transport; (ii) the RBCs' roughness may be restored to the initial value ( i.e. that observed in fresh RBCs) only in samples stored up to 4–5days, whereas after the eighth day of storage the rejuvenation procedure appears to be inefficient; (iii) membrane physical properties – as measured by R_rms – are actually controlled by the metabolic production of ATP, necessary to perform the RBCs' basic functions; (iv) once energy stores cannot be replenished, a regulated sequence of the morphological events (represented by local buckles that lead to formation of spicules and vesicles of the lipid bilayer with generation of spherocytes) is reminiscent of the RBCs' apoptotic final stages; (v) the morphological phenomenology of the final apoptotic stages is passive ( i.e. determined by simple mechanical forces) and encoded in the mechanical properties of the membrane-skeleton; and (vi) necrotic aspects ( e. g. disruption of cell membrane integrity, so that intracellular protein content is easily released) ensue when RBCs are almost totally (≥90%) depleted in an irreversible way of the energetic stores.

Keywords: Atomic force microscopy; Surface roughness; Erythrocyte ageing

Erythrocyte death in vitro induced by starvation in the absence of Ca²⁺ by Giuliano Pompeo; Marco Girasole; Antonio Cricenti; Giovanna Boumis; Andrea Bellelli; Simona Amiconi (pp. 1047-1055).

Keywords: Atomic force microscopy; Surface roughness; Erythrocyte ageing

Membrane interaction of the N-terminal domain of chemokine receptor CXCR1 by Sourav Haldar; H. Raghuraman; Trishool Namani; Krishna Rajarathnam; Amitabha Chattopadhyay (pp. 1056-1061).

The N-terminal domain of chemokine receptors constitutes one of the two critical ligand binding sites, and plays important roles by mediating binding affinity, receptor selectivity, and regulating function. In this work, we monitored the organization and dynamics of a 34-mer peptide of the CXC chemokine receptor 1 (CXCR1) N-terminal domain and its interaction with membranes by utilizing a combination of fluorescence-based approaches and surface pressure measurements. Our results show that the CXCR1 N-domain 34-mer peptide binds vesicles of 1,2-dioleoyl- sn-glycero-3-phosphocholine (DOPC) and upon binding, the tryptophan residues of the peptide experience motional restriction and exhibit red edge excitation shift (REES) of 19nm. These results are further supported by increase in fluorescence anisotropy and mean fluorescence lifetime upon membrane binding. These results constitute one of the first reports demonstrating membrane interaction of the N-terminal domain of CXCR1 and gain relevance in the context of the emerging role of cellular membranes in chemokine signaling.

Keywords: Abbreviations; CXCR1; CXC chemokine receptor 1; DMPC; 1,2-dimyristoyl-; sn; -glycero-3-phosphocholine; DOPC; 1,2-dioleoyl-; sn; -glycero-3-phosphocholine; DPC; dodecylphosphocholine; GPCR; G-protein coupled receptor; LUV; large unilamellar vesicle; MOPS; 3-(N-morpholino)propanesulfonic acid; REES; red edge excitation shiftChemokine receptor; G-protein coupled receptor; Surface pressure; Red edge excitation shift; Membrane vesicle; Lipid monolayer

Membrane interaction of the N-terminal domain of chemokine receptor CXCR1 by Sourav Haldar; H. Raghuraman; Trishool Namani; Krishna Rajarathnam; Amitabha Chattopadhyay (pp. 1056-1061).

Structure–function studies of chemokine-derived carboxy-terminal antimicrobial peptides by Leonard T. Nguyen; David I. Chan; Laura Boszhard; Sebastian A.J. Zaat; Hans J. Vogel (pp. 1062-1072).

Recent reports which show that several chemokines can act as direct microbicidal agents have drawn renewed attention to these chemotactic signalling proteins. Here we present a structure–function analysis of peptides derived from the human chemokines macrophage inflammatory protein-3α (MIP-3α/CCL20), interleukin-8 (IL-8), neutrophil activating protein-2 (NAP-2) and thrombocidin-1 (TC-1). These peptides encompass the C-terminal α-helices of these chemokines, which have been suggested to be important for the direct antimicrobial activities. Far-UV CD spectroscopy showed that the peptides are unstructured in aqueous solution and that a membrane mimetic solvent is required to induce a helical secondary structure. A co-solvent mixture was used to determine solution structures of the peptides by two-dimensional¹H-NMR spectroscopy. The highly cationic peptide, MIP-3α_51–70, had the most pronounced antimicrobial activity and displayed an amphipathic structure. A shorter version of this peptide, MIP-3α_59–70, remained antimicrobial but its structure and mechanism of action were unlike that of the former peptide. The NAP-2 and TC-1 proteins differ in their sequences only by the deletion of two C-terminal residues in TC-1, but intact TC-1 is a very potent antimicrobial while NAP-2 is inactive. The corresponding C-terminal peptides, NAP-2_50–70 and TC-1_50–68, had very limited and no bactericidal activity, respectively. This suggests that other regions of TC-1 contribute to its bactericidal activity. Altogether, this work provides a rational structural basis for the biological activities of these peptides and proteins and highlights the importance of experimental characterization of peptide fragments as distinct entities because their activities and structural properties may differ substantially from their parent proteins.

Keywords: Chemokine; antimicrobial peptide; host defence peptide; innate immunity; peptide structure; peptide-membrane interactions; NMR spectroscopy

Structure–function studies of chemokine-derived carboxy-terminal antimicrobial peptides by Leonard T. Nguyen; David I. Chan; Laura Boszhard; Sebastian A.J. Zaat; Hans J. Vogel (pp. 1062-1072).

Keywords: Chemokine; antimicrobial peptide; host defence peptide; innate immunity; peptide structure; peptide-membrane interactions; NMR spectroscopy

Hemolysis induced by Bacillus cereus sphingomyelinase by Masataka Oda; Masaya Takahashi; Takayuki Matsuno; Kana Uoo; Masahiro Nagahama; Jun Sakurai (pp. 1073-1080).

Bacillus cereus sphingomyelinase ( Bc-SMase) induces hemolysis of sheep erythrocytes which contain large amounts of sphingomyelin. We investigated the mechanism of this hemolysis in comparison to that induced by Clostridium perfringens alpha-toxin. Pertussis toxin, a Gi-specific inhibitor, N-oleoylethernolamine, a ceramidase inhibitor, and dihydrosphingosine, a sphingosine kinase inhibitor, did not inhibit the hemolysis by Bc-SMase, but did inhibit that by alpha-toxin. Bc-SMase broadly bound to whole membranes, and alpha-toxin specifically bound to the detergent-resistant membrane fractions, lipid rafts. The level of ceramide production induced by Bc-SMase in sheep erythrocytes was 6- to 15-fold that induced by alpha-toxin, when the extent of the hemolysis by Bc-SMase was the same as that by the toxin. However, the level of ceramide production induced by Bc-SMase in SM-liposomes was equal to that triggered by the toxin, when the carboxyl fluorescein-release from liposomes induced by Bc-SMase was the same as that induced by alpha-toxin. Confocal laser microscopy showed that treatment of the cells with Bc-SMase resulted in the formation of ceramide-rich domains. A photobleaching analysis suggested that treatment of the cells with Bc-SMase leads to a reduction in membrane fluidity. These results show that Bc-SMase-induced hemolysis of sheep erythrocytes is related to the formation of interface between ceramide-rich domains and ceramide-poor domains through production of ceramide from SM.

Keywords: Abbreviations; B. cereus; Bacillus cereus; Bc; -SMase; Bacillus cereus; sphingomyelinase; C. perfringens; Clostridium perfringens; DRM; detergent-resistant membranes; DSF; detergent-soluble fractions; SM; sphingomyelin; S1P; sphingosine 1-phosphate; MβCD; methyl-β-cyclodextrin; TBS; tris-buffered saline; PT; pertussis toxin; DHS; dihydrosphingosine; CF; carboxyl fluorescein; FRAP; fluorescence recovery after photobleaching Bacillus cereus; sphingomyelinase; Clostridium perfringens; alpha-toxin; Pertussis toxin; Detergent-resistance membranes; Ceramide-rich domain; Fluorescence recovery after photobleaching

Hemolysis induced by Bacillus cereus sphingomyelinase by Masataka Oda; Masaya Takahashi; Takayuki Matsuno; Kana Uoo; Masahiro Nagahama; Jun Sakurai (pp. 1073-1080).

Fluorescence correlation spectroscopy reveals topological segregation of the two tumor necrosis factor membrane receptors by Margarita Gerken; Anja Krippner-Heidenreich; Steffen Steinert; Sylvia Willi; Felix Neugart; Andrea Zappe; Jörg Wrachtrup; Carsten Tietz; Peter Scheurich (pp. 1081-1089).

The proinflammatory cytokine tumor necrosis factor (TNF) binds two distinct plasma membrane receptors, TNFR1 and TNFR2. We have produced different receptor mutants fused with enhanced green fluorescent protein to study their membrane dynamics by fluorescence correlation spectroscopy (FCS). TNFR1 mutants show diffusion constants of approximately 1.2×10−⁹cm²/s and a broad distribution of diffusion times, which is hardly affected by ligand binding. However, cholesterol depletion enhances their diffusion, suggesting a constitutive affinity to cholesterol rich membrane microdomains. In contrast, TNFR2 and mutants thereof diffuse rather fast ( D̄=3.1×10−⁹cm²/s) with a marked reduction after 30min of TNF treatment ( D̄=0.9×10−⁹cm²/s). This reduction cannot be explained by the formation of higher ordered receptor clusters, since the fluorescence intensity of TNF treated receptors indicate the presence of a few receptor molecules per complex only. Together, these data point to a topological segregation of the two TNF receptors in different microcompartments of the plasma membrane independent of the cytoplasmic signaling domains of the receptors.

Keywords: Tumor necrosis factor (TNF); Plasma membrane receptor; Receptor complex; Fluorescence correlation spectroscopy; TNF receptor associated factor (TRAF); Microdomains

Surface-induced phase separation of a sphingomyelin/cholesterol/ganglioside GM1-planar bilayer on mica surfaces and microdomain molecular conformation that accelerates Aβ oligomerization by Yanli Mao; Zhiguo Shang; Yosuke Imai; Tyuji Hoshino; Ryugo Tero; Motohiko Tanaka; Naoki Yamamoto; Katsuhiko Yanagisawa; Tsuneo Urisu (pp. 1090-1099).

Ganglioside GM1 mediates the amyloid beta (Aβ) aggregation that is the hallmark of Alzheimer's disease (AD). To investigate how ganglioside-containing lipid bilayers interact with Aβ, we examined the interaction between Aβ40 and supported planar lipid bilayers (SPBs) on mica and SiO₂ substrates by using atomic force microscopy, fluorescence microscopy, and molecular dynamics computer simulations. These SPBs contained several compositions of sphingomyelin, cholesterol, and GM1 and were treated at physiological salt concentrations. Surprisingly high-speed Aβ aggregation of fibril formations occurred at all GM1 concentrations examined on the mica surface, but on the SiO₂ surface, only globular agglomerates formed and they formed slowly. At a GM1 concentration of 20mol%, unique triangular regions formed on the mica surface and the rapidly formed Aβ aggregations were observed only outside these regions. We have found that some unique surface-induced phase separations are induced by the GM1 clustering effects and the strong interactions between the GM1 head group and the water layer adsorbed in the ditrigonal cavities on the mica surface. The speed of Aβ40 aggregation and the shape of the agglomerates depend on the molecular conformation of GM1, which varies depending on the substrate materials. We identified the conformation that significantly accelerates Aβ40 aggregation, and we think that the detailed knowledge about the GM1 molecular conformation obtained in this work will be useful to those investigating Aβ–GM1 interactions.

Keywords: Lipid bilayer; Sphingomyelin; Cholesterol; Ganglioside GM1; Molecular dynamics; Phase separation

Stability study of the human G-protein coupled receptor, Smoothened by Nehme Rony Nehmé; Olivier Joubert; Michel Bidet; Benoît Lacombe; Ange Polidori; Bernard Pucci; Isabelle Mus-Veteau (pp. 1100-1110).

Smoothened is a member of the G-protein coupled receptor (GPCR) family responsible for the transduction of the Hedgehog signal to the intracellular effectors of the Hedgehog signaling pathway. Aberrant regulation of this receptor is implicated in many cancers but also in neurodegenerative disorders. Despite the pharmacological relevance of this receptor, very little is known about its functional mechanism and its physiological ligand. In order to characterize this receptor for basic and pharmacological interests, we developed the expression of human Smoothened in the yeast Saccharomyces cerevisiae and Smoothened was then purified. Using Surface Plasmon Resonance technology, we showed that human Smoothened was in a native conformational state and able to interact with its antagonist, the cyclopamine, both at the yeast plasma membrane and after purification. Thermostability assays on purified human Smoothened showed that this GPCR is relatively stable in the classical detergent dodecyl-β-d-maltoside (DDM). The fluorinated surfactant C₈F₁₇TAC, which has been proposed to be less aggressive towards membrane proteins than classical detergents, increased Smoothened thermostability in solution. Moreover, the replacement of a glycine by an arginine in the third intracellular loop of Smoothened coupled to the use of the fluorinated surfactant C₈F₁₇TAC during the mutant purification increased Smoothened thermostability even more. These data will be very useful for future crystallization assays and structural characterization of the human receptor Smoothened.

Keywords: Abbreviations; GPCR; G-Protein Coupled Receptor; hSmo; Smoothened; MAP; Multitag Affinity Purification; DDM; dodecyl-β-D-maltoside; FS; fluorinated surfactant; SPR; surface plasmon resonance; RU; resonance unit; Hh; Hedgehog; HA; hemagglutinin; CPN; cyclopamineHeterologous expression; GPCR; Smoothened; Hedgehog pathway; Fluorinated surfactant; Thermostability

Pore-forming properties of the Bacillus thuringiensis toxin Cry9Ca in Manduca sexta brush border membrane vesicles by Jean-Frédéric Brunet; Vincent Vachon; Marc Juteau; Jeroen Van Rie; Geneviève Larouche; Charles Vincent; Jean-Louis Schwartz; Raynald Laprade (pp. 1111-1118).

The toxicity and pore-forming ability of the Bacillus thuringiensis Cry9Ca insecticidal toxin, its single-site mutants, R164A and R164K, and the 55-kDa fragment resulting from its proteolytic cleavage at residue 164 were investigated using Manduca sexta neonate larvae and fifth-instar larval midgut brush border membrane vesicles, respectively. Neither the mutations nor the proteolytic cleavage altered Cry9Ca toxicity. Compared with Cry1Ac, Cry9Ca and its mutants formed large poorly selective pores in the vesicles. Pore formation was highly dependent on pH, however, especially for wild-type Cry9Ca and both mutants. Increasing pH from 6.5 to 10.5 resulted in an irregular step-wise decrease in membrane permeabilization that was not related to a change in the ionic selectivity of the pores. Pore formation was much slower with Cry9Ca and its derivatives, including the 55-kDa fragment, than with Cry1Ac and its rate was not influenced by the presence of protease inhibitors or a reducing agent.

Keywords: Insecticidal toxin; Pore formation; Micro-environment; Osmotic swelling assay; Bacillus thuringiensis; Manduca sexta

Secondary structure of cell-penetrating peptides controls membrane interaction and insertion by Eiriksdottir Emelía Eiríksdóttir; Karidia Konate; Ülo Langel; Gilles Divita; Sébastien Deshayes (pp. 1119-1128).

The clinical use of efficient therapeutic agents is often limited by the poor permeability of the biological membranes. In order to enhance their cell delivery, short amphipathic peptides called cell-penetrating peptides (CPPs) have been intensively developed for the last two decades. CPPs are based either on protein transduction domains, model peptide or chimeric constructs and have been used to deliver cargoes into cells through either covalent or non-covalent strategies. Although several parameters are simultaneously involved in their internalization mechanism, recent focuses on CPPs suggested that structural properties and interactions with membrane phospholipids could play a major role in the cellular uptake mechanism. In the present work, we report a comparative analysis of the structural plasticity of 10 well-known CPPs as well as their ability to interact with phospholipid membranes. We propose a new classification of CPPs based on their structural properties, affinity for phospholipids and internalization pathways already reported in the literature.

Keywords: Cell-penetrating peptides; Structure; Membrane interactions; Conformation; Versatility

Combined effects of polymers and KL₄ peptide on surface activity of pulmonary surfactant lipids by Karen W. Lu; H. William Taeusch (pp. 1129-1134).

Addition of ionic and nonionic polymers can improve the function of therapeutic surfactants in vitro and in vivo, especially under conditions that tend to inhibit surfactant activity. Since surfactant proteins also act to reduce surfactant inhibition, we studied the relative effects of a synthetic peptide (that mimics some of the properties of a surfactant protein), polymers, and their combination on function of surfactant phospholipid activity in vitro. We evaluated surface activity after adding polymers—polyethylene glycol or hyaluronan—to a lipid mixture with or without the synthetic peptide, sinapultide (KL₄). Using a pulsating bubble surfactometer, we measured peptide/polymer effects separately or combined at two peptide concentrations. Phospholipid mixtures, with or without KL₄ or polymers, all demonstrated good surface activity. With serum present as an inhibiting agent, adding either concentration of KL₄ reduced inhibition. Mixtures containing the higher concentration of KL₄ required higher concentrations of serum for inhibition to occur. Adding either polymer to mixtures with KL₄ further decreased susceptibility to inhibition (required higher serum concentrations). In the presence of serum, high molecular weight hyaluronan with KL₄ at 0.4mg/ml improved surface activity to a greater degree than 0.8mg/ml KL₄ without polymer. If the beneficial effects of adding polymer to KL₄–lipid mixtures are also borne out in the treatment of experimental lung injury, these peptide–polymer surfactant combinations may eventually prove useful in the treatment of some forms of acute lung injury in humans.

Keywords: Pulmonary surfactant; KL; ₄; Surface activity; Polyethylene glycol; Hyaluronan

Combined effects of polymers and KL₄ peptide on surface activity of pulmonary surfactant lipids by Karen W. Lu; H. William Taeusch (pp. 1129-1134).

Keywords: Pulmonary surfactant; KL; ₄; Surface activity; Polyethylene glycol; Hyaluronan

P-glycoprotein ATPase from the resistant pest, Helicoverpa armigera: Purification, characterization and effect of various insecticides on its transport function by Ravindra M. Aurade; Senigala K. Jayalakshmi; Kuruba Sreeramulu (pp. 1135-1143).

Helicoverpa armigera is a major pest of agricultural crops and has developed resistance to various insecticides. A P-glycoprotein (Pgp) with ATPase activity likely to be involved in insecticide resistance was purified and characterized from insecticide-resistant H. armigera. The purification was 18-fold with 3% yield. The optimum pH and temperature were found to be 7.4 and 30–40°C, respectively. Kinetic studies indicated that this enzyme had a K_m value of 1.2mM for ATP. Pgp from H. armigera was partially sequenced and found to be homologous to conserved sequences of mammalian Pgps. Pesticides stimulated H. armigera Pgp ATPase activity with a maximum stimulation of up to 40%. Quenching of the intrinsic tryptophan fluorescence of purified Pgp was used to quantitate insecticide binding. Using the high-affinity fluorescent substrate, tetramethylrosamine, transport was monitored in real time in proteoliposomes containing H. armigera Pgp. The presence of Pgp could be one of the reasons for insecticide resistance in this pest.

Keywords: Abbreviations; ABC; ATP-binding cassette; CHAPS; 3[(3-cholamidopropyl) dimethylammonio]-propanesulfonic acid; DMPC; dimyristoyl-; l; -α-phosphatidylcholine; DTT; dithiothreitol; Ha-Pgp; Helicoverpa armigera; P-glycoprotein; MDR; multidrug resistance/resistant; NBD; nucleotide-binding domain; Pgp; P-glycoprotein; p-NPP; p-nitrophenylphosphate; PMSF; phenylmethanesulfonyl fluoride; TMD; transmembrane domain; TMR; tetramethylrosamine Helicoverpa armigera; P-glycoprotein ATPase; Proteoliposomes; Insecticides; Tryptophan quenching; Drug transport

Influence of the nature of the sterol on the behavior of palmitic acid/sterol mixtures and their derived liposomes by Zhong-Kai Cui; Guillaume Bastiat; Chester Jin; Amir Keyvanloo; Michel Lafleur (pp. 1144-1152).

The phase behavior of mixtures formed with palmitic acid (PA) and one of the following sterols (dihydrocholesterol, ergosterol, 7-dehydrocholesterol, stigmasterol and stigmastanol), in a PA/sterol molar ratio of 3/7, has been characterized by IR and²H NMR spectroscopy at different pH. Our study shows that it is possible to form liquid-ordered (lo) lamellar phases with these binary non-phospholipid mixtures. The characterization of alkyl chain dynamics of PA in these systems revealed the large ordering effect of the sterols. It was possible to extrude these systems, using standard extrusion techniques, to form large unilamellar vesicles (LUVs), except in the case of ergosterol-containing mixture. The resulting LUVs displayed a very limited passive permeability consistent with the high sterol concentration. In addition, the stability of these PA/sterol self-assembled bilayers was also found to be pH-sensitive, therefore, potentially useful as nanovectors. By examining different sterols, we could establish some correlations between the structure of these bilayers and their permeability properties. The structure of the side chain at C17 of the sterol appears to play a prime role in the mixing properties with fatty acid.

Keywords: Palmitic acid; Sterol; Liquid-ordered phase; Liposome; Permeability; pH-sensitive

Interaction of 18-methoxycoronaridine with nicotinic acetylcholine receptors in different conformational states by Hugo R. Arias; Avraham Rosenberg; Dominik Feuerbach; Katarzyna M. Targowska-Duda; Ryszard Maciejewski; Krzysztof Jozwiak; Ruin Moaddel; Stanley D. Glick; Irving W. Wainer (pp. 1153-1163).

The interaction of 18-methoxycoronaridine (18-MC) with nicotinic acetylcholine receptors (AChRs) was compared with that for ibogaine and phencyclidine (PCP). The results established that 18-MC: (a) is more potent than ibogaine and PCP inhibiting (±)-epibatidine-induced AChR Ca²⁺ influx. The potency of 18-MC is increased after longer pre-incubation periods, which is in agreement with the enhancement of [³H]cytisine binding to resting but activatable Torpedo AChRs, (b) binds to a single site in the Torpedo AChR with high affinity and inhibits [³H]TCP binding to desensitized AChRs in a steric fashion, suggesting the existence of overlapping sites. This is supported by our docking results indicating that 18-MC interacts with a domain located between the serine (position 6′) and valine (position 13′) rings, and (c) inhibits [³H]TCP, [³H]ibogaine, and [³H]18-MC binding to desensitized AChRs with higher affinity compared to resting AChRs. This can be partially attributed to a slower dissociation rate from the desensitized AChR compared to that from the resting AChR. The enthalpic contribution is more important than the entropic contribution when 18-MC binds to the desensitized AChR compared to that for the resting AChR, and vice versa. Ibogaine analogs inhibit the AChR by interacting with a luminal domain that is shared with PCP, and by inducing desensitization.

Keywords: Nicotinic acetylcholine receptor; Conformational state; Noncompetitive antagonist; Ibogaine analog; 18-Methoxycoronaridine

Sodium-coupled electrogenic transport of pyroglutamate (5-oxoproline) via SLC5A8, a monocarboxylate transporter by Seiji Miyauchi; Elangovan Gopal; Ellappan Babu; Sonne R. Srinivas; Yoshiyuki Kubo; Nagavedi S. Umapathy; Santoshanand V. Thakkar; Vadivel Ganapathy; Puttur D. Prasad (pp. 1164-1171).

Pyroglutamate, also known as 5-oxoproline, is a structural analog of proline. This amino acid derivative is a byproduct of glutathione metabolism, and is reabsorbed efficiently in kidney by Na⁺-coupled transport mechanisms. Previous studies have focused on potential participation of amino acid transport systems in renal reabsorption of this compound. Here we show that it is not the amino acid transport systems but instead the Na⁺-coupled monocarboxylate transporter SLC5A8 that plays a predominant role in this reabsorptive process. Expression of cloned human and mouse SLC5A8 in mammalian cells induces Na⁺-dependent transport of pyroglutamate that is inhibitable by various SLC5A8 substrates. SLC5A8-mediated transport of pyroglutamate is saturable with a Michaelis constant of 0.36±0.04mM. Na⁺-activation of the transport process exhibits sigmoidal kinetics with a Hill coefficient of 1.8±0.4, indicating involvement of more than one Na⁺ in the activation process. Expression of SLC5A8 in X enopus laevis oocytes induces Na⁺-dependent inward currents in the presence of pyroglutamate under voltage-clamp conditions. The concentration of pyroglutamate necessary for induction of half-maximal current is 0.19±0.01mM. The Na⁺-activation kinetics is sigmoidal with a Hill coefficient of 2.3±0.2. Ibuprofen, a blocker of SLC5A8, suppressed pyroglutamate-induced currents in SLC5A8-expressing oocytes; the concentration of the blocker necessary for causing half-maximal inhibition is 14±1μM. The involvement of SLC5A8 can be demonstrated in rabbit renal brush border membrane vesicles by showing that the Na⁺-dependent uptake of pyroglutamate in these vesicles is inhibitable by known substrates of SLC5A8. The Na⁺ gradient-driven pyroglutamate uptake was stimulated by an inside-negative K⁺ diffusion potential induced by valinomycin, showing that the uptake process is electrogenic.

Keywords: Pyroglutamate; Renal reabsorption; SLC5A8; Monocarboxylate transporter

Interaction of recombinant analogs of spider silk proteins 1F9 and 2E12 with phospholipid membranes by Yuri N. Antonenko; Irina V. Perevoshchikova; Lyubov I. Davydova; Igor A. Agapov; Vladimir G. Bogush (pp. 1172-1178).

Recombinant analogs of spider dragline silk proteins 1F9 and 2E12 are characterized by numerous repeats consisting of hydrophobic poly-Ala blocks and Gly-rich sequences with a substantial number of positively charged amino acid residues which suggest a pronounced ability to interact with negatively charged phospholipid membranes. Actually both proteins displayed substantial binding affinity towards lipid vesicles formed of acidic lipids as measured by fluorescence correlation spectroscopy (FCS) using rhodamine-labeled conjugates of the proteins. Both proteins did not induce liposome leakage, fusion or breakdown, but were able to bring about liposome aggregation. 1F9 was more active in the induction of liposome aggregation compared to 2E12. Interestingly, 2E12 markedly decreased the rate of calcium-induced liposome fusion. Circular dichroism data showed that binding of the proteins to negatively charged phosphatidylserine liposomes provoked transition from the left-handed helix of polyproline II (PPII) type to β-structures and α-helices. The data suggested predominantly surface location of membrane bound proteins without significant perturbation of their hydrophobic core.

Keywords: Abbreviations; 1F9; recombinant analog of spidroin 1; 2E12; recombinant analog of spidroin 2; PPII; polyproline II; GRAVY; total hydropathicity index; CD; circular dichroism; TRITC; tetramethylrhodamine-5-(and-6)-isothiocyanate; SRB; sulforhodamine B; CF; carboxyfluorescein; R18; octadecylrhodamine B; BLM; bilayer lipid membrane; FCS; fluorescence correlation spectroscopy; N; number of particles; D; diffusion coefficient; G(τ); autocorrelation functionSpidroin; Bilayer lipid membrane; Liposome; Biomaterial; Protein structure; Aggregation; Fusion; Leakage; FCS; Circular dichroism

Expression of functional mammal flavocytochrome b₅₅₈ in yeast: Comparison with improved insect cell system by Mariano A. Ostuni; Leila B. Lamanuzzi; Tania Bizouarn; Marie-Claire Dagher; Laura Baciou (pp. 1179-1188).

Activity of phagocyte NADPH-oxidase relies on the assembly of five proteins, among them the transmembrane flavocytochrome b₅₅₈ (Cyt b₅₅₈) which consists of a heterodimer of the gp91^phox and p22^phox subunits. The Cyt b₅₅₈ is the catalytic core of the NADPH-oxidase that generates a superoxide anion from oxygen by using a reducing equivalent provided by NADPH via FAD and two hemes. We report a novel strategy to engineer and produce the stable and functional recombinant Cyt b₅₅₈ (rCyt b₅₅₈). We expressed the gp91^phox and p22^phox subunits using the baculovirus insect cell and, for the first time, the highly inducible Pichia pastoris system. In both hosts, the expression of the full-length proteins reproduced native electrophoretic patterns demonstrating that the two polypeptides are present and, that gp91^phox undergoes co-translational glycosylation. Spectroscopic analyses showed that the rCyt b₅₅₈ displayed comparable spectral properties to neutrophil Cyt b₅₅₈. In contrast to rCyt b₅₅₈ produced in the insect cells with higher yield, the enzyme expressed in yeast displayed a superoxide dismutase-sensitive NADPH-oxidase activity, indicating a superoxide generation activity. It was also blocked by an inhibitor of the respiratory burst oxidase, diphenylene iodonium (DPI). As in neutrophil NADPH-oxidase, activation occurred by the interactions with the soluble regulatory subunits suggesting comparable protein–protein contact patterns. We focus on the stability and function of the protein during solubilisation and reconstitution into liposomes. By comparing oxidase activities in different membrane types, we confirm that the lipid-protein environment plays a key role in the protein function.

Keywords: Abbreviations; rCyt; b; ₅₅₈; recombinant cytochrome; b; ₅₅₈; PL; proteoliposome; DMSO; dimetylsulphoxide; SOD; superoxide dismustase; Cyt; c; cytochrome; c; PMSF; phenylmethanesulfonyl fluoride; DPI; diphenylene iodonium; DDM; n-dodecyl β-; d; -maltoside; OG; octyl β-; d; -glucopyranoside; SM; sucrose monolaurate; δ-ALA; δ-aminolevulinic acid; DOPC; dioleoylphosphatidylcholine; EPC; egg phosphatidylcholine; NADPH; reduced β-nicotinamide adenine dinucleotide phosphate; PNGase-F; peptide N-glycosidase F; DTT; dithiotreitol; FAD; flavin adenine dinucleotideNADPH-oxidase; Flavocytochrome; b; ₅₅₈; Heterologous expression; Membrane protein; Proteoliposome; Superoxide generation

Red blood cell permeabilization by hypotonic treatments, saponin, and anticancer avicins by Mauricio Arias; Jairo C. Quijano; Valsala Haridas; Jordan U. Gutterman; Victor V. Lemeshko (pp. 1189-1196).

Plasma membrane permeabilization by saponin and anticancer avicins was studied using light dispersion measurements, since high correlation between light dispersion changes and hemolysis has been demonstrated. Nevertheless, we observed that rat red blood cell swelling in moderately hypotonic media was accompanied by up to 20% decrease of light dispersion, when hemolysis was not yet detectable. Avicin G and avicin D were significantly more efficient than saponin in inducing cytotoxicity in PC3 human prostate cancer cells. We found that the preincubation of avicins with the plasma membrane, but not with the cytosolic fraction of previously lysed red blood cells, completely protected fresh cells against permeabilization. The data suggest that the plasma membrane can tightly bind the avicins, but not the saponin. Using the “osmotic protection” method with 100mOsm PEGs of increasing molecular weight in isotonic media, the size of the pores generated by avicin G and avicin D in the plasma membrane was estimated to be higher than the hydrodynamic radius of PEG-8000. The obtained results indicate that the anticancer activity of avicin G and avicin D could be related, at least partially, to their high ability to permeabilize biological membranes. These data might represent interest for possible applications of these anticancer drugs in vivo.

Keywords: Red blood cells; Light dispersion; Hemolysis; Plasma membrane; Saponin; Avicins; Pore size

P66 porins are present in both Lyme disease and relapsing fever spirochetes: A comparison of the biophysical properties of P66 porins from six Borrelia species by Barcena-Uribarri Iván Bárcena-Uribarri; Marcus Thein; Anna Sacher; Ignas Bunikis; Mari Bonde; Bergstrom Sven Bergström; Roland Benz (pp. 1197-1203).

The genus Borrelia is the cause of the two human diseases: Lyme disease (LD) and relapsing fever (RF). Both LD and RF Borrelia species are obligate parasites and are dependent on nutrients provided by their hosts. The first step of nutrient uptake across the outer membrane of these Gram-negative bacteria is accomplished by water-filled channels, so-called porins. The knowledge of the porin composition in the outer membranes of the different pathogenic Borrelia species is limited. Only one porin has been described in relapsing fever spirochetes to date, whereas four porins are known to be present in Lyme disease agents. From these, the Borrelia burgdorferi outer membrane channel P66 is known to act as an adhesin and was well studied as a porin. To investigate if P66 porins are expressed and similarly capable of pore formation in other Borrelia causing Lyme disease or relapsing fever three LD species ( B. burgdorferi, B. afzelii, B. garinii) and three RF species ( B. duttonii, B. recurrentis and B. hermsii) were investigated for outer membrane proteins homologous to P66. A search in current published RF genomes, comprising the ones of B. duttonii, B. recurrentis and B. hermsii, indicated that they all contained P66 homologues. The P66 homologues of the six Borrelia species were purified to homogeneity and their pore-forming abilities as well as the biophysical properties of the pores were analyzed using the black lipid bilayer assay.

Keywords: Borrelia; P66; Porin; Outer membrane

Determination of molecular groups involved in the interaction of annexin A5 with lipid membrane models at the air–water interface by Zahia Fezoua-Boubegtiten; Bernard Desbat; Alain Brisson; Sophie Lecomte (pp. 1204-1211).

Annexin A5 (AnxA5) is a member of a family of homologous proteins sharing the ability to bind to negatively charged phospholipid membranes in a Ca²⁺-dependent manner. In this paper, we used polarization-modulated infrared reflection absorption spectroscopy (PMIRRAS), Brewster angle microscopy (BAM), and ellipsometry to investigate changes both in the structure of AnxA5 and phospholipid head groups associated with membrane binding. We found that the secondary structure of AnxA5 in the AnxA5/Ca²⁺/lipid ternary complex is conserved, mainly in α-helices and the average orientation of the α-helices of the protein is slightly tilted with respect to the normal to the phospholipid monolayer. Upon interaction between AnxA5 and phospholipids, a shift of the ν_as PO₂⁻ band is observed by PMIRRAS. This reveals that the phosphate group is the main group involved in the binding of AnxA5 to phospholipids via Ca²⁺ ions, even when some carboxylate groups are accessible (PS). PMIRRAS spectra also indicate a change of carboxylate orientation in the aspartate and glutamate residues implicated in the association of the AnxA5, which could be linked to the 2D crystallization of protein under the phospholipid monolayer. Finally, we demonstrated that the interaction of AnxA5 with pure carboxylate groups of an oleic acid monolayer is possible, but the orientation of the protein under the lipid is completely different.

Keywords: Abbreviations; AnxA5; Annexin A5; BAM; Brewster angle microscopy; PMIRRAS; polarization modulation infrared reflection absorption spectroscopy; DMPS; dimyristoylphosphatidyl-serineAnnexin A5; Lipid monolayer; PMIRRAS; BAM; Ellipsometry

Interaction of the N-terminal segment of HCV protein NS5A with model membranes by M. Francisca Palomares-Jerez; Guillen Jaime Guillén; Villalain José Villalaín (pp. 1212-1224).

We have identified a membrane-active region in the HCV NS5A protein by performing an exhaustive study of membrane rupture induced by a NS5A-derived peptide library on model membranes having different phospholipid compositions. We report the identification in NS5A of a highly membranotropic region located at the suggested membrane association domain of the protein. We report the binding and interaction with model membranes of two peptides patterned after this segment, peptides 1A and 1B, derived from the strains 1a_H77 and 1b_HC-4J respectively. We show that they insert into phospholipid membranes, interact with them, and are located in a shallow position in the membrane. The NS5A region where this segment resides might have an essential role in the membrane replication and/or assembly of the viral particle through the modulation of the replication complex, and consequently, directly implicated in the HCV life cycle.

Keywords: Abbreviations; 16-NS; 16-doxyl-stearic acid; 5-NS; 5-doxyl-stearic acid; BPI; bovine brain; l; -α-phosphatidylinositol; BPS; bovine brain; l; -α-phosphatidylserine; CF; 5-carboxyfluorescein; Chol; cholesterol; CL; bovine heart cardiolipin; di-8-ANEPPS; 4-(2-(6-(dioctylamino)-2-naphthalenyl)-(ethenyl)-1-(3-sulfopropyl)-pyridinium inner salt; DMPA; 1,2-dimyristoyl-sn-glycero-3-phosphatidic acid; DMPC; 1,2-dimyristoyl-sn-glycero-phosphatidylcholine; DMPG; 1,2-dimyristoyl-sn-glycero-phosphatidylglycerol; DMPS; 1,2-dimyristoyl-sn-glycero-3-phosphatidylserine; DPH; 1,6-diphenyl-1,3,5-hexatriene; DSC; differential scanning calorimetry; EPA; egg; l; -α-phosphatidic acid; EPC; egg; l; -α-phosphatidylcholine; ER; endoplasmic reticulum; ESM; egg sphingomyelin; FD10; fluorescein isothiocyanate dextran with an average molecular weight of 10,000; HCV; hepatitis C virus; LUV; large unilamellar vesicles; MLV; multilamellar vesicles; NS; non-structural protein; TFE; trifluoroethanol; T; _m; temperature of the gel-to-liquid crystalline phase transition; TM; transmembrane domain; TMA-DPH; 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene; TPE; egg trans-esterified; l; -α-phosphatidylethanolamineHCV; NS5A; Model membrane

Interaction of the N-terminal segment of HCV protein NS5A with model membranes by M. Francisca Palomares-Jerez; Guillen Jaime Guillén; Villalain José Villalaín (pp. 1212-1224).

Interdomain Ca²⁺ effects in Escherichia coli α-haemolysin: Ca²⁺ binding to the C-terminal domain stabilizes both C- and N-terminal domains by Sanchez-Magraner Lissete Sánchez-Magraner; Aitziber L. Cortajarena; Garcia-Pacios Marcos García-Pacios; José-Luis R. Arrondo; Jon Agirre; Guerin Diego M.A. Guérin; Goni Félix M. Goñi; Helena Ostolaza (pp. 1225-1233).

α-Haemolysin (HlyA) is a toxin secreted by pathogenic Escherichia coli, whose lytic activity requires submillimolar Ca²⁺ concentrations. Previous studies have shown that Ca²⁺ binds within the Asp and Gly rich C-terminal nonapeptide repeat domain (NRD) in HlyA. The presence of the NRD puts HlyA in the RTX (Repeats in Toxin) family of proteins. We tested the stability of the whole protein, the amphipathic helix domain and the NRD, in both the presence and absence of Ca²⁺ using native HlyA, a truncated form of HlyAΔN601 representing the C-terminal domain, and a novel mutant HlyA W914A whose intrinsic fluorescence indicates changes in the N-terminal domain. Fluorescence and infrared spectroscopy, tryptic digestion, and urea denaturation techniques concur in showing that calcium binding to the repeat domain of α-haemolysin stabilizes and compacts both the NRD and the N-terminal domains of HlyA. The stabilization of the N-terminus through Ca²⁺ binding to the C-terminus reveals long-range inter-domain structural effects. Considering that RTX proteins consist, in general, of a Ca²⁺-binding NRD and separate function-specific domains, the long-range stabilizing effects of Ca²⁺ in HlyA may well be common to other members of this family.

Keywords: Abbreviations; TC buffer; 150; mM NaCl, 20; mM Tris, pH; =; 7; TH buffer; 150; mM NaCl, 10; mM CaCl; ₂; , 20; mM Tris, pH; =; 7; TCU buffer; 6; M urea, 150; mM NaCl, 20; mM Tris, pH; =; 7Membrane-targeted protein toxins; Calcium-binding domains; RTX toxin family; Protein stability; Urea denaturation; Amphipathic proteins

Unravelling the molecular basis of the selectivity of the HIV-1 fusion inhibitor sifuvirtide towards phosphatidylcholine-rich rigid membranes by Henri G. Franquelim; A. Salomé Veiga; Weissmuller G. Weissmüller; Nuno C. Santos; Miguel A.R.B. Castanho (pp. 1234-1243).

Sifuvirtide, a 36 amino acid negatively charged peptide, is a novel HIV-1 fusion inhibitor with improved antiretroviral activity. In this work we evaluated the physical chemistry foundation of the interaction of sifuvirtide with biomembrane model systems. Since this peptide has aromatic residues, fluorescence spectroscopy techniques were mostly used. The interaction was assessed by partition and quenching experiments. Results showed no significant interaction with large unilamellar vesicles composed by sphingomyelin and ceramide. In contrast, sifuvirtide presented selectivity towards vesicles composed by phosphatidylcholines (PC) in the gel phase, in opposition to fluid phase PC vesicles. The interaction of this peptide with gel phase PC membranes ( K_p=1.2×10²) is dependent on the ionic strength, which indicates the mediation of electrostatic interactions at an interfacial level. The effects of sifuvirtide on the lipid membranes' structural properties were further evaluated using dipole-potential membrane probes, zeta-potential, dynamic light scattering and atomic force microscopy measurements. The results show that sifuvirtide does not cause a noticeable effect on lipid bilayer structure, except for membranes composed by cationic phospholipids. Altogether, we can conclude that sifuvirtide presents a specific affinity towards rigid PC membranes, and the interaction is mediated by electrostatic factors, not affecting the membrane architecture.

Keywords: HIV; Fusion inhibitor; Peptide; Membrane; Gel phase phosphatidylcholine

Signal sequence non-optimal codons are required for the correct folding of mature maltose binding protein by Yaramah M. Zalucki; Christopher E. Jones; Preston S.K. Ng; Benjamin L. Schulz; Michael P. Jennings (pp. 1244-1249).

Non-optimal codons are generally characterised by a low concentration of isoaccepting tRNA and a slower translation rate compared to optimal codons. In a previous study, we reported a 20-fold reduction in maltose binding protein (MBP) level when the non-optimal codons in the signal sequence were optimised. In this study, we report that the 20-fold reduction is rescued when MBP is expressed at 28°C instead of 37°C, suggesting that the signal sequence optimised MBP protein (MBP-opt) may be misfolded, and is being degraded at 37°C. Consistent with this idea, transient induction of the heat shock proteases prior to MBP expression at 28°C restores the 20-fold difference, demonstrating that the difference in production levels is due to post-translational degradation of MBP-opt by the heat-shock proteases. Analysis of the structure of purified MBP-wt and MBP-opt grown at 28°C showed that although they have similar secondary structure content, MBP-opt is more resistant to thermal unfolding than is MBP-wt. The two proteins also exhibit different tryptic fragment profiles, further confirming that they are folded into conformationally different states. This is the first study to demonstrate that signal sequence non-optimal codons can influence the folding of the mature exported protein.

Keywords: Codon usage; Protein folding; Protein export; Maltose binding protein

Studying subunit–subunit interactions in a bacterial ABC transporterby in vitro assembly by Viola Eckey; Heidi Landmesser; Erwin Schneider (pp. 1250-1253).

The thermostable arginine ABC transporter of Geobacillus stearothermophilus consists of a solute binding protein, ArtJ; a transmembrane subunit, ArtM; and a nucleotide-binding subunit, ArtP. An ArtM/His₆–ArtP complex was functionally assembled from separately purified subunits as demonstrated by assaying stimulation of its ATPase activity by arginine-loaded ArtJ in proteoliposomes. Studying in vitro assembly with variants carrying mutations in the conserved Q loop and/or the EAA loop of ArtP and ArtM, respectively, confirmed the predicted roles of both motifs in intersubunit signaling and physical interaction, respectively. In vitro assembly is considered a useful tool for investigating assembly defects of ABC transporters caused by mutations.

Keywords: Abbreviations; ABC; ATP-binding cassette; DDM; n; -dodecyl-β-; d; -maltopyranoside; DS; decanoylsucrose; His-tag; hexahistidine tag; IPTG; isopropyl-β-; d; -thio-galactopyranoside; PMSF; phenylmethylsulfonylfluorideABC transporter; ArtJ-(MP); ₂; Q loop; EAA loop; In vitro assembly; Geobacillus stearothermophilus

Studying subunit–subunit interactions in a bacterial ABC transporterby in vitro assembly by Viola Eckey; Heidi Landmesser; Erwin Schneider (pp. 1250-1253).

Thermodynamics of RTA3 peptide binding to membranes and consequences for antimicrobial activity by Ayman Hawrani; Robin A. Howe; Timothy R. Walsh; Christopher E. Dempsey (pp. 1254-1262).

RTA3 is an α-helical, amphipathic peptide with broad-spectrum activity against Gram-negative bacteria and low mammalian cell toxicity. RTA3 contains a cysteine residue, replacement of which with an alanine or serine (RTA3-C15S) virtually abolishes antimicrobial activity. Much of the activity of RTA3 can be recovered in RTA3-C15L, indicating that the C15 residue functions largely as a bulky hydrophobic side chain promoting target cell membrane interactions. The poorly active RTA3-C15S is a useful variant for assessing the mechanistic aspects of RTA3 activity. Binding and membrane perturbation in vesicles containing different proportions of negative surface charge are analyzed in terms of amino acid-specific free energy contributions to interfacial binding, which likely underlie variations in antimicrobial activity amongst RTA3 variants. Comparison with published free energy scales indicates that the reduced electrostatic contribution to binding to membranes having reduced negative surface charge can be compensated in RTA3 (but not RTA3-C15S) by a slightly deeper insertion of the C-terminus of the peptide to maximize hydrophobic contributions to binding. Analysis of inner membrane (IM)- and outer membrane (OM)-selective permeabilization of E scherichia coli demonstrates a broad similarity between peptide effects on vesicles with low negative surface charge (20% negatively charged lipids), E . coli membrane perturbation, and antimicrobial activity, supporting a role for membrane perturbation in the killing mechanism of RTA3. The results demonstrate that large variations in antimicrobial activity on subtle changes in amino acid sequence in helical amphipathic peptides can be rationalized in terms of the thermodynamics of peptide binding to membranes, allowing a more systematic understanding of antimicrobial activity in these peptides.

Keywords: Abbreviations; buffer A; 10; mM Tris–HCl, 107; mM NaCl, pH 7.4; CD; circular dichroism; CF; carboxyfluorescein; CH; cholesterol; CL; cardiolipin; POPC; palmitoyloleoylphosphatidylcholine; FIC; fractional inhibitory concentration; FPE; fluorescein phosphatidylethanolamine; IM; inner membrane; LUV; large unilamellar vesicle; MIC; minimum inhibitory concentration; OM; outer membrane; ONPG; ortho-nitrophenyl-β-galactoside; PC; phosphatidylcholine; PE; phosphatidylethanolamine; PG; phosphatidylglycerolAmphipathic peptide; Interfacial binding; Cysteine; Free energy; Commensal organism; Phospholipid bilayer

Thermodynamics of RTA3 peptide binding to membranes and consequences for antimicrobial activity by Ayman Hawrani; Robin A. Howe; Timothy R. Walsh; Christopher E. Dempsey (pp. 1254-1262).

Specific interaction restrains structural transitions of an amphiphilic peptide in pulmonary surfactant model systems: An in situ PM-IRRAS investigation by Hiromichi Nakahara; Sannamu Lee; Osamu Shibata (pp. 1263-1271).

In situ polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) at the air–water interface has been used to determine secondary structure of the pulmonary surfactant model peptide, Hel 13-5, in the absence and the presence of phospholipid monolayers. Herein, fully saturated phospholipids of DPPC and DPPG are utilized to understand the effect of specific interaction between anionic DPPG and cationic Hel 13-5 on the peptide secondary structure. The spectrum frequency in the amide region (1500–1700cm−¹) obtained from PM-IRRAS has been confirmed by comparing with that from ATR-FTIR for the corresponding bulk films. The PM-IRRAS spectra of single Hel 13-5 monolayers indicate the α-helical contour in the amide region, which coincides with the result from CD measurements in aqueous solutions. In the presence of phospholipid monolayers, however, Hel 13-5 changes its conformation from the α-helix to the extended β-sheet as surface pressure increases upon compression at the interface, and this interconversion is found to be irreversible even during expansion process of monolayers. Furthermore, it is notable that the electrostatic interaction between DPPG and Hel 13-5 inhibits to some extent the interconversion to the β-sheet during compression. These features are completely different from the bulk behavior, which demonstrates different roles of native proteins in the bulk phase and at the interface for pulmonary functions. In addition, the conformational variation of Hel 13-5 does not indicate close correlation with surface activity, which is common characteristic even for reversible hysteresis curves in pulmonary surfactant systems. This suggests that the secondary structure of native proteins is not strongly related to the surface activity during respiration. This work contributes to secondary structure determination of Hel 13-5 in the phospholipid domains in situ at the air–water interface and will provide insight into the molecular and physiological mechanism for SP-B and SP-C actions across the interface.

Keywords: RDS; Langmuir monolayer; Lung surfactant; DPPC; DPPG; SP-B

Lipid clustering by three homologous arginine-rich antimicrobial peptides is insensitive to amino acid arrangement and induced secondary structure by Richard M. Epand; Raquel F. Epand; Christopher J. Arnusch; Brigitte Papahadjopoulos-Sternberg; Guangshun Wang; Yechiel Shai (pp. 1272-1280).

Three Arg-rich nonapeptides, containing the same amino acid composition but different sequences, PFWRIRIRR-amide (PR-9), RRPFWIIRR-amide (RR-9) and PRFRWRIRI-amide (PI-9), are able to induce segregation of anionic lipids from zwitterionic lipids, as shown by changes in the phase transition properties of lipid mixtures detected by differential scanning calorimetry and freeze fracture electron microscopy. The relative Minimal Inhibitory Concentration (MIC) of these three peptides against several strains of Gram positive bacteria correlated well with the extent to which the lipid composition of the bacterial membrane facilitated peptide-induced clustering of anionic lipids. The lower activity of these three peptides against Gram negative bacteria could be explained by the retention of these peptides in the LPS layer. The membrane morphologies produced by PR-9 as well as by a cathelicidin fragment, KR-12 that had previously been shown to induce anionic lipid clustering, was directly visualized using freeze fracture electron microscopy. This work shows the insensitivity of phase segregation to the specific arrangement of the cationic charges in the peptide sequence as well as to their tendency to form different secondary structures. It also establishes the role of anionic lipid clustering in the presence of zwitterionic lipids in determining antimicrobial selectivity.

Keywords: Abbreviations; AMP; antimicrobial peptides; MIC; minimal inhibitory concentration; LPS; lipopolysaccharide; LTA; lipoteichoic acid; LUVs; large unilamellar vesicles; SUVs; small unilamellar vesicles; MLVs; multilamellar vesicles; CD; circular dichroism; DSC; differential scanning calorimetry; ITC; isothermal titration calorimetry; POPE; 1-palmitoyl-2-oleoyl phosphatidylethanolamine; TOCL; tetraoleoyl cardiolipin; DOPG; dioleoyl phosphatidylglycerol; TMCL; tetramyristoyl cardiolipin; ANTS; 8-aminonaphthalene-1,3,6-trisulfonic acid; DPX; p; -xylene-bis-pyridinium bromide; DPC; dodecylphosphocholine; LB; Luria Bertani; L/P; lipid/peptide molar ratioLipid clustering; Antibacterial peptides; Membrane domains; Bacterial species specificity; Freeze fracture electron microscopy; Differential scanning calorimetry

Pulmonary surfactant layers accelerate O₂ diffusion through the air-water interface by Bárbara Olmeda; Villen Laura Villén; Antonio Cruz; Guillermo Orellana; Jesus Perez-Gil (pp. 1281-1284).

During respiration, it is accepted that oxygen diffuses passively from the lung alveolar spaces through the respiratory epithelium until reaching the pulmonary capillaries, where blood is oxygenated. It is also widely assumed that pulmonary surfactant, a lipid–protein complex secreted into alveolar spaces, has a main surface active function, essential to stabilize the air–liquid interface, reducing in this way the work of breathing. The results of the present work show that capillary water layers containing enough density of pulmonary surfactant membranes transport oxygen much faster than a pure water phase or a water layer saturated with purely lipidic membranes. Membranes reconstituted from whole pulmonary surfactant organic extract, containing all the lipids plus the hydrophobic surfactant proteins, permit also very rapid oxygen diffusion, substantially faster than achieved by membranes prepared from the surfactant lipid fraction depleted of proteins. A model is proposed suggesting that protein-promoted membrane networks formed by pulmonary surfactant might have important properties to facilitate oxygenation through the thin water layer covering the respiratory surface.

Keywords: Pulmonary surfactant; Lipid–protein interactions; Surfactant proteins; Air–liquid interface; Surface tension; Oxygen transport

Pulmonary surfactant layers accelerate O₂ diffusion through the air-water interface by Bárbara Olmeda; Villen Laura Villén; Antonio Cruz; Guillermo Orellana; Jesus Perez-Gil (pp. 1281-1284).

Keywords: Pulmonary surfactant; Lipid–protein interactions; Surfactant proteins; Air–liquid interface; Surface tension; Oxygen transport

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BBA - Biomembranes (v.1798, #6)