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BBA - Biomembranes (v.1818, #9)

Editorial Board (pp. i).

E2→ E1 transition and Rb⁺ release induced by Na⁺ in the Na⁺/K⁺-ATPase. Vanadate as a tool to investigate the interaction between Rb⁺ and E2 by Mónica R. Montes; José L.E. Monti; Rolando C. Rossi (pp. 2087-2093).

This work presents a detailed kinetic study that shows the coupling between the E2→ E1 transition and Rb⁺ deocclusion stimulated by Na⁺ in pig-kidney purified Na,K-ATPase. Using rapid mixing techniques, we measured in parallel experiments the decrease in concentration of occluded Rb⁺ and the increase in eosin fluorescence (the formation of E1) as a function of time. The E2→ E1 transition and Rb⁺ deocclusion are described by the sum of two exponential functions with equal amplitudes, whose rate coefficients decreased with increasing [Rb⁺]. The rate coefficient values of the E2→ E1 transition were very similar to those of Rb⁺-deocclusion, indicating that both processes are simultaneous. Our results suggest that, when ATP is absent, the mechanism of Na⁺-stimulated Rb⁺ deocclusion would require the release of at least one Rb⁺ ion through the extracellular access prior to the E2→ E1 transition. Using vanadate to stabilize E2, we measured occluded Rb⁺ in equilibrium conditions. Results show that, while Mg2⁺ decreases the affinity for Rb⁺, addition of vanadate offsets this effect, increasing the affinity for Rb⁺. In transient experiments, we investigated the exchange of Rb⁺ between the E2-vanadate complex and the medium. Results show that, in the absence of ATP, vanadate prevents the E2→ E1 transition caused by Na⁺ without significantly affecting the rate of Rb⁺ deocclusion. On the other hand, we found the first evidence of a very low rate of Rb⁺ occlusion in the enzyme–vanadate complex, suggesting that this complex would require a change to an open conformation in order to bind and occlude Rb⁺. ► E2→ E1 transition and Rb⁺ release induced by Na⁺ are coupled processes. ► At zero ATP, Na⁺-stimulated E2→ E1 transition would occur after the release of Rb⁺. ► Vanadate stabilizes E2 without significantly affecting the rate of Rb⁺ deocclusion. ► The enzyme–vanadate complex occludes Rb⁺ with very low velocity.

Keywords: Na; ⁺; /K; ⁺; -ATPase; Conformational transition; Rb; ⁺; -deocclusion; Vanadate

Role of molecular architecture on the relative efficacy of aurein 2.5 and modelin 5 by Sarah R. Dennison; Leslie H.G. Morton; David A. Phoenix (pp. 2094-2102).

In order to gain an insight into the mechanism of antimicrobial peptide action, aurein 2.5 and modelin-5 were studied. When tested against Staphylococcus aureus, aurein 2.5 showed approximately 5-fold greater efficacy even though the higher net positive charge and higher helix stability shown by modelin-5 would have predicated modelin-5 to be the more effective antimicrobial. However, in the presence of S. aureus membrane mimics, aurein 2.5 showed greater helical content (75% helical) relative to modelin-5 (51% helical) indicative of increase in membrane association. This was supported by monolayer data showing that aurein 2.5 (6.6mNm−¹) generated greater pressure changes than modelin-5 (5.3mNm−¹). Peptide monolayers indicted that modelin-5 formed a helix horizontal to the plane of an asymmetric interface which would be supported by the even distribution of charge and hydrophobicity along the helical long axis and facilitate lysis by non-specific membrane binding. In contrast, a groove structure observed on the surface of aurein 2.5 was predicted to be the cause of enhanced lipid binding (K_d=75μM) relative to modelin-5 (K_d=118μM) and the balance of hydrophobicity along the aurein 2.5 long axis supported deep penetration into the membrane in a tilt formation. This oblique orientation generates greater lytic efficacy in high anionic lipid (71%) compared to modelin-5 (32%).► Aurein 2.5 and modelin-5 membrane interactions is compared. ► Aurein 2.5 was more helical than modelin-5 in Staphylococcus aureus membranes. ► Modelin-5 formed a helix horizontal to the plane of an asymmetric interface. ► Aurein 2.5 penetrates into the membrane using a possible in tilt formation. ► Hydrophobic groove and tilt orientation enhances efficacy.

Keywords: Abbreviations; AMP; antimicrobial peptide; CD; circular dichroism; CL; cardiolipin; DOPE; dioleoylphosphatidylethanolamine; DOPG; dioleoylphosphatidylglycerol; FPE; fluorescein-phosphatidylethanolamine; ΔG; _mix; Gibbs free energy of mixing; <μ; _H; >; mean hydrophobic moment; ; mean hydrophobicity; K; _d; binding coefficient; MLC; minimum lethal concentrationAntimicrobial peptide; Oblique orientation; Hydrophobic mismatch; Hydrophobic groove; Monolayer

A Ser residue influences the structure and stability of a Pro-kinked transmembrane helix dimer by Mathias Weber; Lydia Tome; Daniel Otzen; Dirk Schneider (pp. 2103-2107).

When localized adjacent to a Pro-kink, Thr and Ser residues can form hydrogen bonds between their polar hydroxyl group and a backbone carbonyl oxygen and thereby modulate the actual bending angle of a distorted transmembrane α-helix. We have used the homo-dimeric transmembrane cytochrome b₅₅₉′ to analyze the potential role of a highly conserved Ser residue for assembly and stabilization of transmembrane proteins. Mutation of the conserved Ser residue to Ala resulted in altered heme binding properties and in increased stability of the holo-protein, most likely by tolerating subtle structural rearrangements upon heme binding. The results suggest a crucial impact of an intrahelical Ser hydrogen bond in defining the structure of a Pro-kinked transmembrane helix dimer.Display Omitted► PsbF forms a dimeric TM structure. ► A pro-kink in the TM helix is crucial. ► The geometry of the pro-kink is influenced by an adjacent serine residue. ► Replacement of the serine residue influences the stability of the holo-protein.

Keywords: Cofactor binding; Helix interaction; Membrane protein; Protein folding; Protein stability; Transmembrane helix

Monensin A acid complexes as a model of electrogenic transport of sodium cation by Huczynski Adam Huczyński; Jan Janczak; Lowicki Daniel Łowicki; Bogumil Brzezinski (pp. 2108-2119).

New Monensin A acid complexes with water molecule, sodium chloride and sodium perchlorate were obtained and studied by X-ray and¹H,¹³C NMR and FT-IR methods as well as ab initio calculations. The crystal structure of the complexes indicates the complexation of the water molecule and Na⁺ cation in the pseudo-cycle conformation of the Monensin acid molecule stabilised by intramolecular hydrogen bonds. Important for stabilisation of this structure is also the intermolecular hydrogen bonds with water molecule or the coordination bonds with Na⁺ cation. It is demonstrated that the counterions forming intermolecular hydrogen bonds with OH groups influence the strength of the intramolecular hydrogen bonds, but they have no influence on the formation of pseudo-cyclic structure. Spectroscopic studies of the complexes in dichloromethane solution have shown that the pseudo-cyclic structure of the compounds is conserved. As follows from the ab initio calculations, the interactions between the Na⁺ cation and the electronegative oxygen atoms of Monensin acid totally change the molecular electrostatic potential around the supramolecular Monensin acid–Na⁺ cationic complex relative to that of the neutral Monensin acid molecule.Display Omitted► Complexes of Monensin acid with H₂O, NaCl and NaClO₄ were synthesised and characterised. ► Crystal structure of complexes shows a pseudo-cyclic structure. ► The pseudo-cyclic structure of complexes is conserved in the solution. ► The role of counterions in hydrogen bond formation was demonstrated.

Keywords: Ionophore; Cation transport; Crystal structure; Hydrogen bond; Ab-initio; calculation; Molecular electrostatic potential

Hinge-bending motions in the pore domain of a bacterial voltage-gated sodium channel by Annika F. Barber; Vincenzo Carnevale; S.G. Raju; Cristiano Amaral; Werner Treptow; Michael L. Klein (pp. 2120-2125).

Computational methods and experimental data are used to provide structural models for NaChBac, the homo-tetrameric voltage-gated sodium channel from the bacterium Bacillus halodurans, with a closed and partially open pore domain. Molecular dynamic (MD) simulations on membrane-bound homo-tetrameric NaChBac structures, each comprising six helical transmembrane segments (labeled S1 through S6), reveal that the shape of the lumen, which is defined by the bundle of four alpha-helical S6 segments, is modulated by hinge bending motions around the S6 glycine residues. Mutation of these glycine residues into proline and alanine affects, respectively, the structure and conformational flexibility of the S6 bundle. In the closed channel conformation, a cluster of stacked phenylalanine residues from the four S6 helices hinders diffusion of water molecules and Na⁺ ions. Activation of the voltage sensor domains causes destabilization of the aforementioned cluster of phenylalanines, leading to a more open structure. The conformational change involving the phenylalanine cluster promotes a kink in S6, suggesting that channel gating likely results from the combined action of hinge-bending motions of the S6 bundle and concerted reorientation of the aromatic phenylalanine side-chains.► We build structural models of NaChBac inserted in a phospholipid bilayer. ► We investigate the properties of three conformations of NaChBac via MD simulations. ► Hinge-bending motions around two glycine residues affect the shape of the pore. ► Mutations of the glycine residues affect the structure and the conformational flexibility of the S6 bundle. ► A cluster of Phe residues undergoes a structural transition upon activation.

Keywords: Voltage gated; Sodium channel; Gating; NaChBac; Bacterial channel

Ato protein interactions in yeast plasma membrane revealed by fluorescence lifetime imaging (FLIM) by Strachotova Dita Strachotová; Aleš Holoubek; Kucerova Helena Kučerová; Aleš Benda; Humpolickova Jana Humpolíčková; Vachova Libuše Váchová; Palkova Zdena Palková (pp. 2126-2134).

Each of the three plasma membrane Ato proteins is involved in ammonium signalling and the development of yeast colonies. This suggests that although these proteins are homologous, they do not functionally substitute for each other, but may form a functional complex. Here, we present a detailed combined FRET, FLIM and photobleaching study, which enabled us to detect interactions between Ato proteins found in distinct compartments of yeast cells. We thus show that the proteins Ato1p and Ato2p interact and can form complexes when present in the plasma membrane. No interaction was detected between Ato1p and Ato3p or Ato2p and Ato3p. In addition, using specially prepared strains, we were able to detect an interaction between molecules of the same Ato protein, namely Ato1p–Ato1p and Ato3p–Ato3p, but not Ato2p–Ato2p.► New techniques of combined FRET, FLIM and photobleaching revealed protein interaction. ► Ato1p physically interacts with Ato2p and form complexes in the membrane. ► Ato3p forms complexes in plasma membrane.

Keywords: Abbreviations; DRLP; dichroic long pass; FLIM; fluorescence lifetime imaging; FP; fluorescent protein; FRET; fluorescence resonance energy transfer; GM; glycerol medium; LDH; laser diode heads; NA; numerical aperture; PCR; polymerase chain reaction; SPAD; single-photon avalanche diode; TCSPC; time-correlated single-photon counting; tdimer2; tandem dimer of DsRedAmmonium exporters Ato1p, Ato2p and Ato3p; FLIM-photobleaching technique; Homo/hetero di(oligo)mers; Plasma membrane protein interaction; Donor lifetime

Molecular structures of fluid phase phosphatidylglycerol bilayers as determined by small angle neutron and X-ray scattering by Jianjun Pan; Frederick A. Heberle; Stephanie Tristram-Nagle; Michelle Szymanski; Mary Koepfinger; John Katsaras; Kucerka Norbert Kučerka (pp. 2135-2148).

We have determined the molecular structures of commonly used phosphatidylglycerols (PGs) in the commonly accepted biologically relevant fluid phase. This was done by simultaneously analyzing small angle neutron and X-ray scattering data, with the constraint of measured lipid volumes. We report the temperature dependence of bilayer parameters obtained using the one-dimensional scattering density profile model – which was derived from molecular dynamics simulations – including the area per lipid, the overall bilayer thickness, as well as other intrabilayer parameters (e.g., hydrocarbon thickness). Lipid areas are found to be larger than their phosphatidylcholine (PC) counterparts, a result likely due to repulsive electrostatic interactions taking place between the charged PG headgroups even in the presence of sodium counterions. In general, PG and PC bilayers show a similar response to changes in temperature and chain length, but differ in their response to chain unsaturation. For example, compared to PC bilayers, the inclusion of a first double bond in PG lipids results in a smaller incremental change to the area per lipid and bilayer thickness. However, the extrapolated lipid area of saturated PG lipids to infinite chain length is found to be similar to that of PCs, an indication of the glycerol–carbonyl backbone's pivotal role in influencing the lipid–water interface.

Keywords: Lipid bilayer; Bilayer structure; Area per lipid; Bilayer thickness; Molecular dynamics simulations; Fluid phase

Mitochondrial membrane permeabilization upon interaction with lysozyme fibrillation products: Role of mitochondrial heterogeneity by Ali Akbar Meratan; Mohsen Nemat-Gorgani (pp. 2149-2157).

Mitochondrial dysfunction is a common feature of many neurodegenerative disorders, although the relative degree of this functional impairment and the mechanism of selective vulnerability in different regions of the brain related to various neurological diseases are not completely understood. In a recent study, we reported on brain mitochondrial membrane permeabilization upon interaction with hen egg white lysozyme (HEWL) protofibrils, and came to the conclusion that mitochondrial heterogeneity could offer an explanation for some of our observations. Accordingly, the first part of the present investigation was devoted on studies involving interaction of HEWL fibrillation products with mitochondria isolated from various areas of the brain, known to be affected in a number of well-characterized neurodegenerative conditions. This was followed by looking at heart and liver mitochondria. Membrane permeabilization was investigated by monitoring release of mitochondrial enzymes. Mitochondria isolated from cortex and hippocampus showed greater sensitivities than those prepared from substantia nigra. Results clearly demonstrate heterogeneity in brain mitochondria together with a higher resistance to permeabilization in these organelles in comparison with those isolated from liver and heart. Calcium and spermine were found to be more effective in preventing permeabilization in brain mitochondria, as compared to liver and heart. The structure–function aspects and physiological significance of the observations in relation to differences in composition, biophysical nature and morphological properties of mitochondria are discussed. It is argued that studies on heterogeneity in cellular membranes, the primary targets of toxic protofibrils, may provide important insights into mechanism of toxicity, with clinical and pathological manifestations.Display Omitted►We describe interaction of HEWL fibrillation products with mitochondria. ►Heterogeneity in brain mitochondria was explored. ►Lower permeabilization in brain mitochondria compared to those from liver and heart ►Calcium and spermine inhibited mitochondrial membrane permeabilization. ►Membrane heterogeneity may play an important role in protofibril cytotoxicity.

Keywords: Abbreviations; HEWL; hen egg white lysozyme; OS; oxidative stress; ROS; reactive oxygen species; DLS; dynamic light scattering; MDH; malate dehydrogenase; GDH; glutamate dehydrogenase; CS; citrate synthase; AK; adenylate kinase; EDTA; ethylenediaminetetraacetic acidHEWL protofibril; Mitochondrial heterogeneity; Membrane permeabilization; Cytotoxicity; Neurodegenerative disorder

Structural and thermodynamic insight into the process of “weak” dimerization of the ErbB4 transmembrane domain by solution NMR by Eduard V. Bocharov; Konstantin S. Mineev; Marina V. Goncharuk; Alexander S. Arseniev (pp. 2158-2170).

Specific helix–helix interactions between the single-span transmembrane domains of receptor tyrosine kinases are believed to be important for their lateral dimerization and signal transduction. Establishing structure–function relationships requires precise structural-dynamic information about this class of biologically significant bitopic membrane proteins. ErbB4 is a ubiquitously expressed member of the HER/ErbB family of growth factor receptor tyrosine kinases that is essential for the normal development of various adult and fetal human tissues and plays a role in the pathobiology of the organism. The dimerization of the ErbB4 transmembrane domain in membrane-mimicking lipid bicelles was investigated by solution NMR. In a bicellar DMPC/DHPC environment, the ErbB4 membrane-spanning α-helices (651–678)₂ form a right-handed parallel dimer through the N-terminal double GG4-like motif A⁶⁵⁵GxxGG⁶⁶⁰ in a fashion that is believed to permit proper kinase domain activation. During helix association, the dimer subunits undergo a structural adjustment (slight bending) with the formation of a network of inter-monomeric polar contacts. The quantitative analysis of the observed monomer–dimer equilibrium provides insights into the kinetics and thermodynamics of the folding process of the helical transmembrane domain in the model environment that may be directly relevant to the process that occurs in biological membranes. The lipid bicelles occupied by a single ErbB4 transmembrane domain behave as a true (“ideal”) solvent for the peptide, while multiply occupied bicelles are more similar to the ordered lipid microdomains of cellular membranes and appear to provide substantial entropic enhancement of the weak helix–helix interactions, which may be critical for membrane protein activity.Display Omitted► Structure-thermodynamics insight into weak dimerization of transmembrane helices. ► Structural adjustment of transmembrane helices upon specific interactions. ► Substantial entropy-driven enhancement of helix–helix interactions in lipid bicelles. ► High apparent activation barrier is present for transmembrane helix association. ► Microcompartmentalization can affect association of transmembrane domains of proteins.

Keywords: Abbreviations; ErbB; epidermal growth factor receptor; TM; transmembrane; NOE; nuclear Overhauser effect; NOESY; NOE spectroscopy; MHP; molecular hydrophobicity potential; DMPC; dimyristoylphosphatidylcholine; DHPC; dihexanoylphosphatidylcholineReceptor tyrosine kinase; Transmembrane domain; Dimerization; Spatial structure; Thermodynamics; NMR

Enhancing membrane disruption by targeting and multivalent presentation of antimicrobial peptides by Cristina Chamorro; Marcel A. Boerman; Christopher J. Arnusch; Eefjan Breukink; Roland J. Pieters (pp. 2171-2174).

In order to enhance the membrane disruption of antimicrobial peptides both targeting and multivalent presentation approaches were explored. The antimicrobial peptides anoplin and temporin L were conjugated via click chemistry to vancomycin and to di- and tetravalent dendrimers. The vancomycin unit led to enhanced membrane disruption of large unilamellar vesicles (LUVs) displaying the vancomycin target lipid II, but only for temporin L and not for anoplin. The multivalent presentation led to enhanced LUV membrane disruption in the case of anoplin but not for temporin L.Display Omitted► A targeting unit was attached to antimicrobial peptides. ► Antimicrobial peptides were attached to a dendrimer. ► The targeting unit made temporin L more active but not anaoplin. ► The dendimer linkage made anoplin more active but not temporin L.

Keywords: Antimicrobial peptide; Targeting; Multivalency; Membrane disruption; Anoplin; Temporin L

Interaction of GAPR-1 with lipid bilayers is regulated by alternative homodimerization by Josse van Galen; Nick K. Olrichs; Arie Schouten; Ramon L. Serrano; Esther N.M. Nolte-'t Hoen; Ruud Eerland; Dora Kaloyanova; Piet Gros; J. Bernd Helms (pp. 2175-2183).

Golgi-Associated Plant Pathogenesis-Related protein 1 (GAPR-1) is a mammalian protein that belongs to the superfamily of plant pathogenesis related proteins group 1 (PR-1). GAPR-1 is a peripheral membrane-binding protein that strongly associates with lipid-enriched microdomains at the cytosolic leaflet of Golgi membranes. Little is known about the mechanism of GAPR‐1 interaction with membranes. We previously suggested that dimerization plays a role in the function of GAPR‐1 and here we report that phytic acid (inositol hexakisphosphate) induces dimerization of GAPR‐1 in solution. Elucidation of the crystal structure of GAPR‐1 in the presence of phytic acid revealed that the GAPR‐1 dimer differs from the previously published GAPR‐1 dimer structure. In this structure, one of the monomeric subunits of the crystallographic dimer is rotated by 28.5°. To study the GAPR‐1 dimerization properties, we investigated the interaction with liposomes in a light scattering assay and by flow cytometry. In the presence of negatively charged lipids, GAPR‐1 caused a rapid and stable tethering of liposomes. [D81K]GAPR‐1, a mutant predicted to stabilize the IP6‐induced dimer conformation, also caused tethering of liposomes. [A68K]GAPR‐1 however, a mutant predicted to stabilize the non‐rotated dimer conformation, is capable of binding to liposomes but did not cause liposome tethering. Our combined data suggest that the charge properties of the lipid bilayer can regulate GAPR‐1 dynamics as a potential mechanism to modulate GAPR‐1 function.► Phytic acid induces dimerization of GAPR-1 in solution. ► The GAPR-1 dimer crystal structure in the presence of IP6 reveals a rotation of the monomers. ► Negatively charged lipids cause a rapid and stable tethering of liposomes by GAPR-1.

Keywords: GAPR-1; GLIPR-2; Phytic acid; Dimerization; Protein–lipid interaction; Crystal structure

Externalization of phosphatidylserine from inner to outer layer may alter the effect of plant sterols on human erythrocyte membrane — The Langmuir monolayer studies by Hac-Wydro Katarzyna Hąc-Wydro; Dynarowicz-Latka Patrycja Dynarowicz-Łątka (pp. 2184-2191).

One of the factors, which can strongly modify the cell membrane composition, is disordering in membrane asymmetry, resulting from redistribution of lipids from inner to outer layer. Such a disturbance may affect the behavior of various biologically active compounds incorporating into membranes. In this contribution, the relationship between the amounts of phosphatidylserine (PS) in the model outer layer of human erythrocyte (RBC) membrane and the effect induced by a plant sterol (β-sitosterol) was verified. The experiments were performed on multicomponent Langmuir films imitating red blood cell (RBC) membrane, differing in the contents of PS (0%; 5% and 10%) into which the plant sterol was incorporated in various concentrations. The analysis of experimental results (surface pressure–area isotherms complemented with Brewster Angle Microscopy (BAM) proved that the presence of phosphatidylserine molecules, depending on their contents in the mixed monolayer mimicking RBC membrane, changes its properties and exerts influence on the effect of plant sterol on the model system. The addition of phytosterol into the monolayer that lacks or contains only 5% of PS was found to be of rather weak effect on the properties of the system. However, in the case of the model membrane containing the increased amount (10%) of PS, the incorporation of plant sterol strongly affects the interactions between molecules and caused thermodynamic destabilization of the monolayer imitating RBC membrane. These results allow one to suggest that externalization of phosphatidyserine to the outer membrane leaflet may differentiate the effect of plant sterols on cell membranes of various origins.Display Omitted► Phosphatidylserine modifies the effect of phytosterol on model outer membrane layer. ► Phytosterol destabilizes only the systems of increased phosphatidylserine level. ► Phosphatidylserine determines solubility limit of sterols in the mixed film. ► Disturbances in lipid asymmetry may change the effect of plant sterols on membrane.

Keywords: Model outer layer of human erythrocyte (RBC) membrane; Plant sterol; Phosphatidylserine; Langmuir film; Brewster Angle Microscopy

Antimicrobial selectivity based on zwitterionic lipids and underlying balance of interactions by Carola I.E. von Deuster; Volker Knecht (pp. 2192-2201).

An important feature of antimicrobial peptides is their ability to distinguish pro- from eukaryotic membranes. In vitro experiments on the antimicrobial peptide NK-2 indicate that the discrimination between zwitterionic phosphatidylethanolamine lipids exposed by prokaryotes and phosphatidylcholine lipids exposed by eukaryotes plays an important role. The underlying mechanism is not understood. Here we present molecular dynamics simulations in conjunction with a coarse grained model and thermodynamic integration showing that NK-2 binds more strongly to palmitoyloleoylphosphatidylethanolamine (POPE) than to palmitoyloleoylphosphatidylcholine (POPC) bilayers. Finite size effects on the relative free energy have been corrected for with a method that may also be useful in future studies of the affinities of macromolecules for lipid membranes. Our results support the previous hypothesis that the stronger binding to PE compared to PC arises from a better accessibility of the phosphates of the lipids to the cationic peptide in a sense that a similar number of peptide-lipid salt bridges requires to break more favorable electrostatic headgroup-headgroup interactions for PC relative to PE. The transfer of NK-2 from POPC to POPE is found to lead to a decrease in electrostatic peptide-lipid but an increase in lipid-lipid and ion-lipid interactions, correlating with a dehydration of the lipids and the ions but an increased hydration of the peptide. The increase in affinity of NK-2 for POPE compared to POPC hence arises from a complex interplay of competing interactions. This work opens the perspective to study how the affinity of antimicrobial peptides changes with amino acid sequence and lipid composition.► Antimicrobial peptide (AMP) NK-2 binds more strongly to POPE than to POPC bilayer. ► Stronger binding to PE opposed by electrostatic peptide-lipid interactions. ► Binding selectivity arises from subtle balance of interactions. ► Selective binding is crucial for recognition of prokaryotic membranes. ► Understanding AMPs is important for development of new antibiotical drugs.

Keywords: Antimicrobial peptide; MD simulation; Selectivity; Membrane; Thermodynamic integration

Functional reconstitution and characterization of the Arabidopsis Mg²⁺ transporter AtMRS2-10 in proteoliposomes by Sumio Ishijima; Zenpei Shigemi; Hiroaki Adachi; Nana Makinouchi; Ikuko Sagami (pp. 2202-2208).

Magnesium (Mg²⁺) plays critical role in many physiological processes. The mechanism of Mg²⁺ transport has been well documented in bacteria; however, less is known about Mg²⁺ transporters in eukaryotes. The AtMRS2 family, which consists of 10 Arabidopsis genes, belongs to a eukaryotic subset of the CorA superfamily proteins. Proteins in this superfamily have been identified by a universally conserved GlyMetAsn motif and have been characterized as Mg²⁺ transporters. Some members of the AtMRS2 family, including AtMRS2-10, may complement bacterial mutants or yeast mutants that lack Mg²⁺ transport capabilities. Here, we report the purification and functional reconstitution of AtMRS2-10 into liposomes. AtMRS2-10, which contains an N-terminal His-tag, was expressed in Escherichia coli and solubilized with sarcosyl. The purified AtMRS2-10 protein was reconstituted into liposomes. AtMRS2-10 was inserted into liposomes in a unidirectional orientation. Direct measurement of Mg²⁺ uptake into proteoliposomes revealed that reconstituted AtMRS2-10 transported Mg²⁺ without any accessory proteins. Mutation in the GMN motif, M400 to I, inactivated Mg²⁺ uptake. The AtMRS2-10-mediated Mg²⁺ influx was blocked by Co(III)hexamine, and was independent of the external pH from 5 to 9. The activity of AtMRS2-10 was inhibited by Co²⁺ and Ni²⁺; however, it was not inhibited by Ca²⁺, Fe²⁺, or Fe³⁺. While these results indicate that AtMRS2-10 has similar properties to the bacterial CorA proteins, unlike bacterial CorA proteins, AtMRS2-10 was potently inhibited by Al³⁺. These studies demonstrate the functional capability of the AtMRS2 proteins in proteoliposomes to study structure–function relationships.► AtMRS2-10 transports Mg²⁺ without any accessory proteins. ► Mutation in the GMN motif inactivates Mg²⁺ transport through AtMRS2-10. ► Mg²⁺ transport through AtMRS2-10 is inhibited by Co²⁺, Ni²⁺ and Al³⁺. ► Mg²⁺ transport through AtMRS2-10 is independent of the external pH from 5 to 9.

Keywords: Abbreviations; IPTG; isopropyl-β-; d; -thiogalactopyranoside; DDM; n; -dodecyl-β-; d; -maltoside; NMDG-Cl; N; -methyl-; d; -glucamine chloride; WT; wild typeAtMRS2; Magnesium transport; Proteoliposome; Arabidopsis thaliana; CorA superfamily

siRNA carriers based on carbosilane dendrimers affect zeta potential and size of phospholipid vesicles by Maksim Ionov; Zuzana Garaiova; Iveta Waczulikova; Wrobel Dominika Wróbel; Pedziwiatr-Werbicka Elżbieta Pędziwiatr-Werbicka; Rafael Gomez-Ramirez; Francisco Javier de la Mata; Barbara Klajnert; Tibor Hianik; Maria Bryszewska (pp. 2209-2216).

One of the major limitations in gene therapy is an inability of naked siRNA to passively diffuse through negatively charged cell membranes. Therefore, the siRNA transport into a cell requires efficient carriers. In this work we analyzed the charge-dependent interaction of the complexes of cationic carbosilane dendrimers (CBD) and anti-HIV siRNA (dendriplexes) with the model membranes — large unilamellar vesicles (LUV). We used the second generation of branched with CBD carbon–silicon bonds (CBD-CS) which are water-stable and that of oxygen–silicon bonds (CBD-OS) which are slowly hydrolyzed in aqueous solutions. The LUVs were composed of zwitterionic dimyristoylphosphatidylcholine (DMPC), negatively charged dipalmitoylphosphatidylglycerol (DPPG) and their mixture (DMPC/DPPG, molar ratio 7:3). The interaction of dendriplexes with LUVs affected both zeta potential and size of the vesicles. The changes of these values were larger for the negatively charged LUV. CBD-CS resulted in the decrease of zeta potential values to more negative ones, whereas an opposite effect took place for CBD-OS suggesting a different kind of interaction between LUVs and the dendriplexes. The results indicate that both CBD-CS and CBD-OS can be used for transport of siRNA into the cells. However, CBD-CS are preferred due to a better stability in water and improved bioavailability of siRNA on their surface.Display Omitted► Carbosilane dendrimers as carriers for anti-HIV siRNA (siGAG1) were evaluated. ► siRNA/dendrimer complexes were formed. ► Formed dendriplexes were interacting with lipid vesicles. ► Carbosilane dendrimers can be considered for delivery of siRNA into the target cells.

Keywords: Abbreviations; PBS; phosphate-buffered saline; CBD; carbosilane dendrimers; DMPC; 1,2-dimyristoyl-; sn; -glycero-3-phosphocholine; DPPG; dipalmitoylphosphatidylglycerol; LUVs; large unilamellar vesiclesCarbosilane dendrimers; siRNA; Unilamellar vesicles; Zeta potential; Surface charge

Effect of phosphorylation of phosphatidylinositol on myelin basic protein-mediated binding of actin filaments to lipid bilayers in vitro by Joan M. Boggs; Godha Rangaraj; Awa Dicko (pp. 2217-2227).

Myelin basic protein (MBP) binds to negatively charged lipids on the cytosolic surface of oligodendrocytes and is believed to be responsible for adhesion of these surfaces in the multilayered myelin sheath. It can also assemble actin filaments and tether them to lipid bilayers through electrostatic interactions. Here we investigate the effect of increased negative charge of the lipid bilayer due to phosphorylation of phosphatidylinositol (PI) on MBP-mediated binding of actin to the lipid bilayer, by substituting phosphatidylinositol 4-phosphate or phosphatidylinositol 4,5-bisphosphate for PI in phosphatidylcholine/phosphatidylglycerol lipid vesicles. Phosphorylation of PI caused dissociation of the MBP/actin complex from the lipid vesicles due to repulsion of the negatively charged complex from the negatively charged membrane surface. An effect of phosphorylation could be detected even if the inositol lipid was only 2mol% of the total lipid. Calcium–calmodulin dissociated actin from the MBP–lipid vesicles and phosphorylation of PI increased the amount dissociated. These results show that changes to the lipid composition of myelin, which could occur during signaling or other physiological events, could regulate the ability of MBP to act as a scaffolding protein and bind actin filaments to the lipid bilayer.Display Omitted► Myelin basic protein (MBP) binds actin filaments to negatively charged lipid vesicles. ► Phosphorylation of phosphatidylinositol dissociated the actin from the vesicles. ► The negatively charged complex is repelled from the negatively charged membrane surface. ► MBP may act as a scaffolding protein in vivo. ► This function may be regulated by changes in lipid composition during cell signaling.

Keywords: Abbreviations; CaM; calmodulin; C1; unmodified, most highly charged isomer of 18.5; kDa MBP; DTT; dithiothreitol; ECL; enhanced chemiluminescence; HRP; horseradish peroxidase; LUVs; large unilamellar vesicles; MARCKS; myristoylated alanine-rich C kinase substrate; MBP; myelin basic protein; MLVs; multilamellar vesicles; MRP; MARCKS-related protein; OLs; oligodendrocytes; PC; phosphatidylcholine; PG; phosphatidylglycerol; PI; phosphatidylinositol; PIP; phosphatidylinositol 4-phosphate; PIP; ₂; phosphatidylinositol 4,5-bisphosphate; TBS; tris-buffered saline; TLC; thin-layer chromatography; TPI; triphosphoinositidesPhosphoinositide; Signal transduction; Cytoskeleton; Oligodendrocyte; Calmodulin; Lipid vesicle

The role of cholesterol on the activity and stability of neurotensin receptor 1 by Joanne Oates; Belinda Faust; Helen Attrill; Peter Harding; Marcella Orwick; Anthony Watts (pp. 2228-2233).

Understanding the role of specific bilayer components in controlling the function of G-protein coupled receptors (GPCRs) will be a key factor in the development of novel pharmaceuticals. Cholesterol-dependence in particular has become an area of keen interest with respect to GPCR function; not least since the 2.6Å crystal structure of the β2 adrenergic receptor revealed a putative cholesterol binding motif conserved throughout class-A GPCRs. Furthermore, experimental evidence for cholesterol-dependent GPCR function has been demonstrated in a limited number of cases. This modulation of receptor function has been attributed to both direct interactions between cholesterol and receptor, and indirect effects caused by the influence of cholesterol on bilayer order and lateral pressure. Despite the widespread occurrence of cholesterol binding motifs, available experimental data on the functional involvement of cholesterol on GPCRs are currently limited to a small number of receptors. Here we investigate the role of cholesterol in the function of the neurotensin receptor 1 (NTS1) a class‐A GPCR. Specifically we show how cholesterol, and the analogue cholesteryl hemisuccinate, influence activity, stability, and oligomerisation of both purified and reconstituted NTS1. The results caution against using such motifs as indicators of cholesterol‐dependent GPCR activity.► NTS1 is stabilised by cholesteryl hemisuccinate during detergent mediated purification. ► Ligand binding is unaffected by the presence of cholesterol in NTS1 reconstituted systems, despite changes in bilayer order. ► Dimerisation of NTS1 can be promoted in the presence of cholesterol. ► The presence of a cholesterol binding motif in NTS1 is not indicative of cholesterol-dependent function.

Keywords: Neurotensin; GPCR; Cholesterol; Reconstitution; POPE; FRET

Structural features of aquaporin 4 supporting the formation of arrays and junctions in biomembranes by Hofinger Siegfried Höfinger; Eiji Yamamoto; Yoshinori Hirano; Francesco Zerbetto; Tetsu Narumi; Kenji Yasuoka; Masato Yasui (pp. 2234-2243).

A limited class of aquaporins has been described to form regular arrays and junctions in membranes. The biological significance of these structures, however, remains uncertain. Here we analyze the underlying physical principles with the help of a computational procedure that takes into account protein–protein as well as protein–membrane interactions. Experimentally observed array/junction structures are systematically (dis)assembled and major driving forces identified. Aquaporin 4 was found to be markedly different from the non-junction forming aquaporin 1. The environmental stabilization resulting from embedding into the biomembrane was identified as the main driving force. This highlights the role of protein–membrane interactions in aquaporin 4. Analysis of the type presented here can help to decipher the biological role of membrane arrays and junctions formed by aquaporin.Display Omitted► Computational analysis of association free energies for array/junction-forming aquaporins. ► Tetrameric assemblies and combinations thereof are analyzed. ► Major driving forces are identified.

Keywords: Abbreviations; AMBER; a force field and molecular modeling package; AQ; aqueous domain; AQP1/4; aquaporin type 1 or 4; ESP; electrostatic potential; ff99SB; improved AMBER force field; GAFF; general AMBER force field; GPU; graphics processing unit; HC; hydrophobic core domain; JAM; junctional adhesion molecule; MD; molecular dynamics; MM; molecular mechanics; MM/PBSA; molecular mechanics, Poisson–Boltzmann, surface area; MM/PB; +; ⁺; a variant of MM/PBSA analysis; NPT; ensemble with constant number of particles, constant pressure and constant temperature; parm99; standard AMBER force field; PB; Poisson–Boltzmann; PDB; protein data bank; PH; polar headgroup domain; PME; particle mesh Ewald summation; POPE; phosphatidylethanolamine; PTRAJ; process trajectories, a program from the AMBER package; SANDER; central program from the AMBER package; SHAKE; standard algorithm to accelerate MD by neglecting C–H vibrations; TIP3P; popular parameterization to describe water moleculesAquaporin 4; Junction; Array; Biomembrane mimicry; Free energies; MM/PBSA

Membrane and lipopolysaccharide interactions of C-terminal peptides from S1 peptidases by Shalini Singh; Gopinath Kasetty; Artur Schmidtchen; Martin Malmsten (pp. 2244-2251).

The mechanisms underlying antimicrobial and anti-endotoxic effects were investigated for a series of structurally related peptides derived from the C-terminal region of S1 peptidases. For this purpose, results on bacterial killing were compared to those on peptide-induced liposome leakage, and to ellipsometry and dual polarization interferometry results on peptide binding to, and disordering of, supported lipid bilayers. Furthermore, the ability of these peptides to block endotoxic effects caused by bacterial lipopolysaccharide (LPS), monitored through NO production in macrophages, was compared to the binding of these peptides to LPS, and to secondary structure formation in the peptide/LPS complex. Bacteria killing, occurring through peptide-induced membrane lysis, was found to correlate with liposome rupture, and with the extent of peptide binding to the lipid membrane, no adsorption threshold for peptide insertion being observed. Membrane and LPS binding was found to depend on peptide net charge, illustrated by LPS binding increasing with increasing peptide charge, and peptides with net negative charge being unable to lyse membranes, kill bacteria, and block LPS-induced endotoxic effect. These effects were, however, also influenced by peptide hydrophobicity. LPS binding was furthermore demonstrated to be necessary, but not sufficient, for anti-endotoxic effect of these peptides. Circular dichroism spectroscopy showed that pronounced helix formation occurs in peptide/LPS complexes for all peptides displaying anti-endotoxic effect, hence potentially linked to this functionality. Similarly, ordered secondary structure formation was correlated to membrane binding, lysis, and antimicrobial activity of these peptides. Finally, preferential binding of these peptides to LPS over the lipid membrane was demonstrated.Bacterial membranes present multiple binding sites for C-terminal peptides derived from S1 peptidases. Apart from interacting with phospholipid membranes, thereby causing membrane rupture and resulting in antimicrobial effects, they bind to bacterial lipopolysaccharides (LPS), a necessary but not sufficient criterium for their anti-endotoxic effect. LPS and membrane binding is promoted by electrostatic and hydrophobic interactions. Membrane destabilization and antimicrobial effect, as well as anti-endotoxic effect, is correlated to helix formation in the presence of lipid membranes and LPS, respectively.Display Omitted► S1 peptide interaction with lipid membrane and bacterial lipopolysaccharide (LPS) investigated. ► Membrane rupture increases with peptide adsorption and insertion. ► LPS/membrane binding depends on peptide charge, hydrophobicity, and helix content. ► Preferential binding of S1 peptides to LPS over lipid membranes. ► Helix formation important for both membrane rupture and anti-endotoxic effect.

Keywords: Antimicrobial peptide; Dual polarization interferometry; Ellipsometry; Lipopolysaccharide; Liposome; Membrane

Surface modified liposomes by mannosylated conjugates anchored via the adamantyl moiety in the lipid bilayer by Stimac Adela Štimac; Segota Suzana Šegota; Dutour Sikiric Maja Dutour Sikirić; Ribic Rosana Ribić; Leo Frkanec; Svetlicic Vesna Svetličić; Tomic Srđanka Tomić; Vranesic Branka Vranešić; Ruža Frkanec (pp. 2252-2259).

The aim of the present study was to encapsulate mannosylated 1-aminoadamantane and mannosylated adamantyltripeptides, namely [(2 R)- N-(adamant-1-yl)-3-(α,β-d-mannopyranosyloxy)-2-methylpropanamide and (2 R)- N-[3-(α-d-mannopyranosyloxy)-2-methylpropanoyl]-d,l-(adamant-2-yl)glycyl-l-alanyl-d- isoglutamine] in liposomes. The characterization of liposomes, size and surface morphology was performed using dynamic light scattering (DLS) and atomic force microscopy (AFM). The results have revealed that the encapsulation of examined compounds changes the size and surface of liposomes. After the concanavalin A (ConA) was added to the liposome preparation, increase in liposome size and their aggregation has been observed. The enlargement of liposomes was ascribed to the specific binding of the ConA to the mannose present on the surface of the prepared liposomes. Thus, it has been shown that the adamantyl moiety from mannosylated 1-aminoadamantane and mannosylated adamantyltripeptides can be used as an anchor in the lipid bilayer for carbohydrate moiety exposed on the liposome surface.Schematic presentation of ConA interaction with mannosylated adamantyltripeptides anchored via the adamantyl moiety in the lipid bilayer; it does not imply any stoichiometry of the ConA–liposome complex.Display Omitted► Mannosylated 1-aminoadamantane and adamantylpeptides were incorporated in liposomes. ► DLS and AFM were used for liposome characterization. ► Mannosylated conjugates were anchored via adamantyl moiety in the lipid bilayer. ► The incorporated mannosylated adamantyltripeptides are grouped into domains. ► ConA confirmed exposure of the mannose on the liposome surface.

Keywords: Adamantane; Mannose; Liposome targeting; AFM; DLS

Human erythrocytes and neuroblastoma cells are in vitro affected by sodium orthovanadate by M. Suwalsky; P. Fierro; F. Villena; L.F. Aguilar; C.P. Sotomayor; M. Jemiola-Rzeminska; K. Strzalka; S. Gul-Hinc; A. Ronowska; A. Szutowicz (pp. 2260-2270).

Research on biological influence of vanadium has gained major importance because it exerts potent toxic, mutagenic, and genotoxic effects on a wide variety of biological systems. However, hematological toxicity is one of the less studied effects. The lack of information on this issue prompted us to study the structural effects induced on the human erythrocyte membrane by vanadium (V). Sodium orthovanadate was incubated with intact erythrocytes, isolated unsealed human erythrocyte membranes (IUM) and molecular models of the erythrocyte membrane. The latter consisted of bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. This report presents evidence in order that orthovanadate interacted with red cell membranes as follows: a) in scanning electron microscopy (SEM) studies it was observed that morphological changes on human erythrocytes were induced; b) fluorescence spectroscopy experiments in isolated unsealed human erythrocyte membranes (IUM) showed that an increase in the molecular dynamics and/or water content at the shallow depth of the lipids glycerol backbone at concentrations as low as 50μM was produced; c) X-ray diffraction studies showed that orthovanadate 0.25–1mM range induced increasing structural perturbation to DMPE; d) somewhat similar effects were observed by differential scanning calorimetry (DSC) with the exception of the fact that DMPC pretransition was shown to be affected; and e) fluorescence spectroscopy experiments performed in DMPC large unilamellar vesicles (LUV) showed that at very low concentrations induced changes in DPH fluorescence anisotropy at 18°C. Additional experiments were performed in mice cholinergic neuroblastoma SN56 cells; a statistically significant decrease of cell viability was observed on orthovanadate in low or moderate concentrations.► Vanadates exert toxic effects on a variety of biological systems. ► Hematological toxicity of vanadates has been scarcely studied. ► Sodium orthovanadate interacts with the human erythrocyte membrane. ► It also affects the bilayer structure of lipids present in the erythrocyte membrane.

Keywords: Abbreviations; SEM; scanning electron microscopy; IUM; isolated unsealed human erythrocyte membrane; LUV; large unilamellar vesicles; r; anisotropy; GP; generalized polarization; DPH; 1,6-diphenyl-1,3,5-hexatriene; laurdan; 6-dodecanoyl-2-methylaminonaphthalene; DMPC; dimyristoylphosphatidylcholine; DMPE; dimyristoylphosphatidylethanolamine; PDH; pyruvate dehydrogenase; ChAT; choline acetyltransferase; cAMP; dibutyryl cAMP; DC; differentiated cells; NC; nondifferentiated cells; RA; all trans retinoic acidOrthovanadate; Erythrocyte membrane; Lipid bilayer; Neuroblastoma cell

Effect of membrane tension on the physical properties of DOPC lipid bilayer membrane by A. Srinivas Reddy; Dora Toledo Warshaviak; Mirianas Chachisvilis (pp. 2271-2281).

Molecular dynamics simulations of a dioleoylphosphocholine (DOPC) lipid bilayer were performed to explore its mechanosensitivity. Variations in the bilayer properties, such as area per lipid, volume, thickness, hydration depth (HD), hydration thickness (HT), lateral diffusion coefficient, and changes in lipid structural order were computed in the membrane tension range 0 to 15dyn/cm. We determined that an increase in membrane tension results in a decrease in the bilayer thickness and HD of ~5% and ~5.7% respectively, whereas area per lipid, volume, and HT/HD increased by 6.8%, 2.4%, and 5% respectively. The changes in lipid conformation and orientation were characterized using orientational ( S²) and deuterium ( S_CD) order parameters. Upon increase of membrane tension both order parameters indicated an increase in lipid disorder by 10–20%, mostly in the tail end region of the hydrophobic chains. The effect of membrane tension on lipid lateral diffusion in the DOPC bilayer was analyzed on three different time scales corresponding to inertial motion, anomalous diffusion and normal diffusion. The results showed that lateral diffusion of lipid molecules is anomalous in nature due to the non-exponential distribution of waiting times. The anomalous and normal diffusion coefficients increased by 20% and 52% when the membrane tension changed from 0 to 15dyn/cm, respectively. In conclusion, our studies showed that membrane tension causes relatively significant changes in the area per lipid, volume, polarity, membrane thickness, and fluidity of the membrane suggesting multiple mechanisms by which mechanical perturbation of the membrane could trigger mechanosensitive response in cells.Display Omitted► MD simulations were used to study effects of membrane tension on lipid bilayer properties. ► Area per lipid, bilayer volume and polarity (hydration level) increase with membrane tension. ► Lipid bilayer thickness decreases with membrane tension. ► Microscopic fluidity and lateral diffusion coefficient increase with membrane tension. ► Lipid diffusion is anomalous at shorter times (<10ns) and nearly normal on longer time scale.

Keywords: Abbreviations; DOPC; 1,2-dioleoyl-sn-glycero-3-phosphocholine; HD; hydration depth; HT; hydration thickness; MSD; mean square displacement; COM; center of mass; PME; particle mesh Ewald method; MD; molecular dynamics; ns; nanosecondMechanosensing; Lipid diffusion; Membrane tension; Shear stress; Anomalous diffusion; GPCR

Targeting the lateral interactions of transmembrane domain 5 of Epstein–Barr virus latent membrane protein 1 by Xiaohui Wang; Jonel P. Saludes; Tina X. Zhao; Adam Csakai; Zeno Fiorini; Sherry A. Chavez; Jing Li; Gui-in Lee; Krisztina Varga; Hang Yin (pp. 2282-2289).

The lateral transmembrane protein–protein interaction has been regarded as “undruggable” despite its importance in many biological processes. The homo-trimerization of transmembrane domain 5 (TMD-5) of latent membrane protein 1 (LMP-1) is critical for the constitutive oncogenic activation of the Epstein–Barr virus (EBV). Herein, we report a small molecule agent, NSC 259242 (compound1), to be a TMD-5 self-association disruptor. Both the positively charged acetimidamide functional groups and the stilbene backbone of compound1 are essential for its inhibitory activity. Furthermore, cell-based assays revealed that compound1 inhibits full-length LMP-1 signaling in EBV infected B cells. These studies demonstrated a new strategy for identifying small molecule disruptors for investigating transmembrane protein–protein interactions.Display Omitted► Propose a cell based screen strategy for identifying TMDs disruptors ► Identified an LMP-1 TMD-5 self association disruptor, NSC 259242 ► NSC 259242 disrupts TMD-5 homotrimerization and inhibits LMP-1 signaling

Keywords: Abbreviations; EBV; Epstein–Barr virus; LMP-1; latent membrane protein 1; PPI; protein-protein interaction; TMD; transmembrane domain; MBP; maltose binding protein; ONPG; O-nitrophenyl ß-galactoside; SPPS; standard solid phase peptide synthesis; DAGK; diacylglycerol kinase; BSA; bovine serum albumin; HRP; horseradish peroxidase; SDS-PAGE; sodium dodecyl sulfate polyacrylamide gel electrophoresis; POPC; 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; DHPC; dihexanoylphosphadtidylcholine; HMQC; heteronuclear multiple quantum coherence; HSQC; heteronuclear single quantum coherence; CSI; chemical shift index; C14 betaine; 3-(; N,N-; dimethylmyristylammonio)propanesulfonate; TRAF; TNF receptor associated factor; iNOS; inducible NO synthaseEpstein–Barr virus; Latent membrane protein 1; Transmembrane domain; Protein–protein interaction; High throughput screen; Small molecule inhibitor

Mapping of unfolding states of integral helical membrane proteins by GPS-NMR and scattering techniques: TFE-induced unfolding of KcsA in DDM surfactant by Antonello Calcutta; Christian M. Jessen; Manja Annette Behrens; Cristiano L.P. Oliveira; Maria Lourdes Renart; Gonzalez-Ros José M. González-Ros; Daniel E. Otzen; Jan Skov Pedersen; Anders Malmendal; Niels Chr. Nielsen (pp. 2290-2301).

Membrane proteins are vital for biological function, and their action is governed by structural properties critically depending on their interactions with the membranes. This has motivated considerable interest in studies of membrane protein folding and unfolding. Here the structural changes induced by unfolding of an integral membrane protein, namely TFE-induced unfolding of KcsA solubilized by the n-dodecyl β-d-maltoside (DDM) surfactant is investigated by the recently introduced GPS-NMR (Global Protein folding State mapping by multivariate NMR) (Malmendal et al., PlosONE 5, e10262 (2010)) along with dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS). GPS-NMR is used as a tool for fast analysis of the protein unfolding processes upon external perturbation, and DLS and SAXS are used for further structural characterization of the unfolding states. The combination allows addressing detergent properties and protein conformations at the same time. The mapping of the states reveals that KcsA undergoes a series of rearrangements which include expansion of the tetramer in several steps followed by dissociation into monomers at 29% TFE. Supplementary studies of DDM and TFE in the absence of KcsA suggest that the disintegration of the tetramer at 29% TFE is caused by TFE dissolving the surrounding DDM rim. Above 34% TFE, KcsA collapses to a new structure that is fully formed at 44% TFE.Display Omitted► NMR and scattering analysis of membrane protein unfolding. ► TFE-induced unfolding of KcsA in DDM surfactants. ► Fast mapping of folding states is obtained by GPS NMR. ► SAXS and DLS provides structural information about distinct folding states. ► A model for the unfolding states of KcsA is presented.

Keywords: KcsA–surfactant complex; TFE-induced unfolding; PCA; DDM micelle; Dynamic light scattering; Small-angle X-ray scattering

Binding of the three-repeat domain of tau to phospholipid membranes induces an aggregated-like state of the protein by Kunze Georg Künze; Barre Patrick Barré; Holger A. Scheidt; Lars Thomas; David Eliezer; Daniel Huster (pp. 2302-2313).

In patients with Alzheimer's disease, the microtubule-associated protein tau is found aggregated into paired helical filaments (PHFs) in neurofibrillary deposits. In solution, tau is intrinsically unstructured. However, the tubulin binding domain consisting of three or four 31–32 amino acid repeat regions exhibits both helical and β-structure propensity and makes up the proteolysis resistant core of PHFs. Here, we studied the structure and dynamics of the three-repeat domain of tau ( i.e. K19) when bound to membranes consisting of a phosphatidylcholine and phosphatidylserine mixture or phosphatidylserine alone. Tau K19 binds to phospholipid vesicles with submicromolar affinity as measured by fluorescence spectroscopy. The interaction is driven by electrostatic forces between the positively charged protein and the phospholipid head groups. The structure of the membrane-bound state of K19 was studied using CD spectroscopy and solid-state magic-angle spinning NMR spectroscopy. To this end, the protein was selectively¹³C-labeled at all valine and leucine residues. Isotropic chemical shift values of tau K19 were consistent with a β-structure. In addition, motionally averaged¹H–¹³C dipolar couplings indicated a high rigidity of the protein backbone. The structure formation of K19 was also shown to depend on the charge density of the membrane. Phosphatidylserine membranes induced a gain in the α-helix structure along with an immersion of K19 into the phospholipid bilayer as indicated by a reduction of the lipid chain²H NMR order parameter. Our results provide structural insights into the membrane-bound state of tau K19 and support a potential role of phospholipid membranes in mediating the physiological and pathological functions of tau.Display Omitted► The three repeat domain of tau (i.e. K19) forms the core of tau fibrils in AD. ► The structure and dynamics of membrane-bound K19 was studied by solid-state NMR spectroscopy. ► Chemical shifts together with¹H^-13C dipolar couplings indicate a rigid β-structure of K19 in PC/PS membranes. ► The protein conformation was shown to depend on the charge density of the membrane. ► A potential role of biological membranes in tau pathology is suggested.

Keywords: Abbreviations; Aβ; Amyloid β peptide; AD; Alzheimer's disease; CD; circular dichroism; CP; cross polarization; DIPSHIFT; dipolar coupling and chemical shift correlation; DMPC; 1,2-dimyristoyl-; sn; -glycero-3-phosphocholine; DMPS; 1,2-dimyristoyl-; sn; -glycero-3-phosphoserine; DSC; differential scanning calorimetry; FLSG; frequency-switched Lee Goldburg; FTDP-17; frontotemporal dementia and Parkinsonism linked to chromosome 17; HEPES; 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid; HetCor; heteronuclear correlation; IAPP; islet amyloid polypeptide; LUV; large unilamellar vesicle; MAS; magic-angle spinning; NFTs; neurofibrillary tangles; PDSD; proton-driven spin diffusion; PHFs; paired helical filaments; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; POPS; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphoserine; SDS; sodium dodecyl sulfate; SFs; straight filaments; SUV; small unilamellar vesicle; ThT; thioflavin T; TPPM; two-pulse phase modulationTau; Alzheimer's disease; Phospholipid; Membrane; Protein aggregation

trans Arachidonic acid isomers inhibit NADPH-oxidase activity by direct interaction with enzyme components by H. Souabni; V. Thoma; T. Bizouarn; C. Chatgilialoglu; A. Siafaka-Kapadai; L. Baciou; C. Ferreri; Houee-Levin C. Houée-Levin; M.A. Ostuni (pp. 2314-2324).

NADPH-oxidase is an enzyme that represents, when activated, the major source of non-mitochondrial reactive oxygen species. In phagocytes, this production is an indispensable event for the destruction of engulfed pathogens. The functional NADPH-oxidase complex consists of a catalytic membrane flavocytochrome b (Cyt b₅₅₈) and four cytosolic proteins p47^phox, p67^phox, Rac and p40^phox. The NADPH-oxidase activity is finely regulated spatially and temporally by cellular signaling events that trigger the translocation of the cytosolic subunits to its membrane partner involving post-translational modifications and activation by second messengers such as arachidonic acid (AA). Arachidonic acid in its natural cis-poly unsaturated form (C20:4) has been described to be an efficient activator of the enzyme in vivo and in vitro. In this work, we examined in a cell-free system whether a change of the natural cis geometry to the trans configuration, which could occur either by diet or be produced by the action of free radicals, may have consequences on the functioning of NADPH-oxidase. We showed the inability of mono- trans AA isomers to activate the NADPH-oxidase complex and demonstrated the inhibitory effect on the cis-AA-induced NADPH oxidase activation. The inhibition is mediated by a direct effect of the mono- trans AA which targets both the membrane fraction containing the cyt b₅₅₈ and the cytosolic p67^phox. Our results suggest that the loss of the natural geometric feature ( cis-AA) induces substantial structural modifications of p67^phox that prevent its translocation to the complex.Display Omitted►NADPH oxidase is inhibited by trans arachidonic acid ( trans-AA). ►Mono- trans-AA inhibits cis arachidonic acid activated NADPH oxidase. ►trans-AA directly targets cytosolic subunit p67phox. ►trans-AA indirectly targets NOX2 probably by altering physical membrane properties. ►p47phox in its active form preferentially translocates to the membrane associated to p67phox.

Keywords: Abbreviations; cyt; cytochrome; AA; arachidonic acid; Ag-TLC; argentation thin layer chromatography; PMSF; phenylmethanesulfonyl fluoride; SOD; superoxide dismutase; bMF; bovine membrane fractions; yMF; yeast membrane fractions; Me AA; AA methyl ester; GC/MS; gas chromatography–mass spectrometry; NMR; nuclear magnetic resonance; BSA; bovine serum albumin; Cytc; cytochrome c; SH3; Src Homology 3; SDS; sodium dodecyl sulfateNADPH oxidase; trans; Fatty acid; Arachidonic acid; Protein complex assembly; Cell free system; Superoxide production inhibition

A yeast toxic mutant of HET-s amyloid disrupts membrane integrity by Ha Phuong Ta; Karine Berthelot; Bénédicte Coulary-Salin; Sabine Castano; Bernard Desbat; Pierre Bonnafous; Olivier Lambert; Isabel Alves; Christophe Cullin; Sophie Lecomte (pp. 2325-2334).

Many studies have pointed out the interaction between amyloids and membranes, and their potential involvement in amyloid toxicity. Previously, we generated a yeast toxic amyloid mutant (M8) from the harmless amyloid protein by changing a few residues of the Prion Forming Domain of HET-s (PFD HET-s_218–289) and clearly demonstrated the complete different behaviors of the non-toxic Wild Type (WT) and toxic amyloid (called M8) in terms of fiber morphology, aggregation kinetics and secondary structure. In this study, we compared the interaction of both proteins (WT and M8) with membrane models, as liposomes or supported bilayers. We first demonstrated that the toxic protein (M8) induces a significant leakage of liposomes formed with negatively charged lipids and promotes the formation of microdomains inside the lipid bilayer (as potential “amyloid raft”), whereas the non-toxic amyloid (WT) only binds to the membrane without further perturbations. The secondary structure of both amyloids interacting with membrane is preserved, but the anti-symmetric PO₂⁻ vibration is strongly shifted in the presence of M8. Secondly, we established that the presence of membrane models catalyzes the amyloidogenesis of both proteins. Cryo-TEM (cryo‐transmission electron microscopy) images show the formation of long HET-s fibers attached to liposomes, whereas a large aggregation of the toxic M8 seems to promote a membrane disruption. This study allows us to conclude that the toxicity of the M8 mutant could be due to its high propensity to interact and disrupt lipid membranes.►Toxic amyloid promotes the formation of “raft”-like microdomains inside the lipid bilayer. ►The non-toxic amyloid only binds to the membrane without strong perturbations. ►For both proteins, the presence of membrane models catalyzes the amyloidogenesis. ►In interaction with membrane the secondary structure of both amyloids is not affected. ►The toxic amyloid induces a strong variation of the phosphate groups of the lipid.

Keywords: Abbreviations; PFD; Prion-Forming Domain; ATR-FTIR; Attenuated Total Reflection Fourier Transform Infrared; PWR; Plasmon-Waveguide ResonanceAmyloid toxicity; HET-s; Membrane; Leakage; ATR-FTIR; PWR

Transfection efficiency boost of cholesterol-containing lipoplexes by Daniela Pozzi; Cristina Marchini; Francesco Cardarelli; Heinz Amenitsch; Chiara Garulli; Angelo Bifone; Giulio Caracciolo (pp. 2335-2343).

Most lipid formulations require cholesterol for successful transfection, but the precise reason remains to be more clearly understood. Here, we have studied the effect of cholesterol on the transfection efficiency (TE) of lipoplexes in vitro. Addition of cholesterol to highly effective DC-Chol–DOPE/DNA lipoplexes increases TE, with 40mol% cholesterol yielding about 10-fold improvement. The transfection mechanisms of cholesterol-containing lipoplexes have been investigated by combining dynamic light scattering, synchrotron small angle X-ray scattering, laser scanning confocal microscopy and transfection efficiency measurements. Our results revealed that cholesterol-containing lipoplexes enter the cells partially by membrane fusion and this mechanism accounts for efficient endosomal escape. We also found evidence that formulations with high cholesterol content are not specifically targeted to metabolic degradation. These studies will contribute to rationally design novel delivery systems with superior transfection efficiency.Display Omitted► Addition of cholesterol to effective lipoplexes increases transfection efficiency. ► Cholesterol-containing lipoplexes enter the cells partially by membrane fusion. ► Fusion accounts for efficient endosomal escape of cholesterol-containing lipoplexes. ► Cholesterol-containing lipoplexes are not targeted to metabolic degradation.

Keywords: Lipoplex; Cholesterol; Transfection efficiency; Membrane fusion; Endosomal escape

Dynamics of Klebsiella pneumoniae OmpA transmembrane domain: The four extracellular loops display restricted motion behavior in micelles and in lipid bilayers by Iordan Iordanov; Marie Renault; Reat Valérie Réat; Patrick D. Bosshart; Andreas Engel; Olivier Saurel; Alain Milon (pp. 2344-2353).

The transmembrane domain of Klebsiella pneumoniae OmpA (KpOmpA) possesses four long extracellular loops that exhibit substantial sequence variability throughout OmpA homologs in Enterobacteria, in comparison with the highly conserved membrane-embedded β-barrel core. These loops are responsible for the immunological properties of the protein, including cellular and humoral recognition. In addition to key features revealed by structural elucidation of the KpOmpA transmembrane domain in detergent micelles, studies of protein dynamics provide insight into its function and/or mechanism of action. We have investigated the dynamics of KpOmpA in a lipid bilayer, using magic angle spinning solid-state NMR. The dynamics of the β-barrel and loop regions were probed by the spin–lattice relaxation times of the C^α and C^β atoms of the serine and threonine residues, and by cross-polarization dynamics. The β-barrel core of the protein is rigid; the C-terminal halves of two of the four extracellular loops (L1 and L3), which are particularly long in KpOmpA, are highly mobile. The other two loops (L2 and L4), which are very similar to their homologs in Escherichia coli OmpA, and the N-terminal halves of L1 and L3 exhibit more restricted motions. We suggest a correlation between the sequence variability and the dynamics of certain loop regions, which accounts for their respective contributions to the structural and immunological properties of the protein.► KpOmpA loop dynamics were assessed in lipid bilayers. ► T₁, T_1ρ, CP and J-based excitations revealed a variety of motional regimes. ► The L2 and L4 loops display a restricted motional regime. ► The C-terminal halves of L1 and L3 are highly mobile.

Keywords: Abbreviations; KpOmpA; Klebsiella pneumoniae; outer membrane protein A; TM; transmembrane; LPR; lipid to protein ratio; CMC; critical micellar concentration; PLE; E. coli; Polar Lipid Extract; OG; n; -octyl-β-; d; -glucopyranoside; DHPC; 1,2-dihexanoyl-; sn; -glycero-3-phosphocholine; DOPC; 1,2-dioleoyl-sn-glycero-3-phosphocholine; DMPC; 1,2-dimyristoyl-sn-glycero-3-phosphocholine; ssNMR; solid state Nuclear Magnetic Resonance; PDSD; proton-driven spin diffusion; CP; cross-polarizationOuter membrane protein A; Protein reconstitution; Protein dynamics; Spin–lattice relaxation; Solid-state NMR; Electron microscopy

Gly₆ of kalata B1 is critical for the selective binding to phosphatidylethanolamine membranes by Kristopher Hall; Tzong-Hsien Lee; Norelle L. Daly; David J. Craik; Marie-Isabel Aguilar (pp. 2354-2361).

The membrane interaction of the cyclotide kalata B1, an all-d-analogue and a single alanine substituted analogue (G6A), was studied by surface plasmon resonance (SPR) and atomic force microscopy (AFM). Kalata B1 showed a strong binding selectivity for dimyristoyl-phosphatidylethanolamine (DMPE) compared to dimyristoyl-phoshatidylcholine (DMPC)-containing lipids. However, when the interaction was visualized by AFM the peptide interacted with DMPC and DMPE in a similar manner. There was no apparent change in membrane morphology with either lipid, suggesting that kalata B1 does not act via a carpet-like disruption mechanism. Thed-analogue showed similar binding by SPR and the same strong selectivity for DMPE, indicating that the membrane-interaction and lipid selectivity are not stereo-specific. SPR studies of the G6A analogue revealed that it interacted in a similar way to kalata B1 on the DMPC containing lipids, but showed no increased response on the DMPE containing lipids observed for kalata B1 andd-kalata B1. These results indicate that the Gly6 residue directly influences membrane binding as it is located near a putative membrane interacting hydrophobic patch. Overall, the data suggest that very small changes in amino acid composition (with no change in conformation) can influence specific self-association in combination with membrane binding and mediate the activity of kalata B1.Display Omitted► The membrane binding of kalata B1 was studied by surface plasmon resonance. ► Kalata B1 and a D-analogue selectively bound to phosphatidylethanolamine. ► The membrane binding and lipid selectivity are not stereo-specific. ► A single alanine mutant (G6A) showed no increased response on DMPE. ► Small changes in sequence affect membrane binding and the activity of kalata B1.

Keywords: Abbreviations; AMP; antimicrobial peptides; CD; circular dichroism; CHAPS; 3-[(cholamidopropyl)dimethyl-ammonio]-1-propane sulfonate; DMPC; 1,2-dimyristoyl-sn-glycero-3-phosphocholine; DMPG; 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]; DMPE; 1,2-dimyristoyl-; sn; -Glycero-3-phosphoethanolamine; MIC; minimum inhibitory concentration; MLV; multilamellar vesicle; PC; phosphatidylcholine; PG; phospho-rac-(1-glycerol); SPR; surface plasmon resonance; AFM; atomic force microscopySPR; AFM; Peptide–lipid interaction; Antimicrobial peptide; Cyclotide; Phospholipid membrane

Arabidopsis KEA2, a homolog of bacterial KefC, encodes a K⁺/H⁺ antiporter with a chloroplast transit peptide by María Nieves Aranda-Sicilia; Olivier Cagnac; Salil Chanroj; Heven Sze; Rodriguez-Rosales María Pilar Rodríguez-Rosales; Kees Venema (pp. 2362-2371).

KEA genes encode putative K⁺ efflux antiporters that are predominantly found in algae and plants but are rare in metazoa; however, nothing is known about their functions in eukaryotic cells. Plant KEA proteins show homology to bacterial K⁺ efflux (Kef) transporters, though two members in the Arabidopsis thaliana family, AtKEA1 and AtKEA2, have acquired an extra hydrophilic domain of over 500 residues at the amino terminus. We show that AtKEA2 is highly expressed in leaves, stems and flowers, but not in roots, and that an N-terminal peptide of the protein is targeted to chloroplasts in Arabidopsis cotyledons. The full-length AtKEA2 protein was inactive when expressed in yeast; however, a truncated AtKEA2 protein (AtsKEA2) lacking the N-terminal domain complemented disruption of the Na⁺(K⁺)/H⁺ antiporter Nhx1p to confer hygromycin resistance and tolerance to Na⁺ or K⁺ stress. To test transport activity, purified truncated AtKEA2 was reconstituted in proteoliposomes containing the fluorescent probe pyranine. Monovalent cations reduced an imposed pH gradient (acid inside) indicating AtsKEA2 mediated cation/H⁺ exchange with preference for K⁺=Cs⁺>Li⁺>Na⁺. When a conserved Asp⁷²¹ in transmembrane helix 6 that aligns to the cation binding Asp¹⁶⁴ of Escherichia coli NhaA was replaced with Ala, AtsKEA2 was completely inactivated. Mutation of a Glu⁸³⁵ between transmembrane helix 8 and 9 in AtsKEA2 also resulted in loss of activity suggesting this region has a regulatory role. Thus, AtKEA2 represents the founding member of a novel group of eukaryote K⁺/H⁺ antiporters that modulate monovalent cation and pH homeostasis in plant chloroplasts or plastids.► Plant KEA proteins share high homology to bacterial KefC/B antiporters. ► Full‐length AtKEA2 is inactive in yeast. ► AtKEA2 without N-terminal domain complements yeast Nhx1p. ► Purified and reconstituted short AtKEA2 protein has K⁺/H⁺ exchange activity. ► Cation specificity of AtKEA2 is similar to EcKefC.

Keywords: Potassium transport; Arabidopsis; K; ⁺; /H; ⁺; antiport; Yeast; Reconstitution

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BBA - Biomembranes (v.1818, #9)

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BBA - Biomembranes (v.1818, #9)