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BBA - Biomembranes (v.1818, #11)

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Microfluidity mapping using fluorescence correlation spectroscopy: A new way to investigate plasma membrane microorganization of living cells by Pascale Winckler; Aurélie Cailler; Régis Deturche; Pierre Jeannesson; Hamid Morjani; Rodolphe Jaffiol (pp. 2477-2485).

Diffusion time distribution analysis has been employed to highlight the microfluidity fingerprint of plasma membrane of living cells. Diffusion time measurements were obtained through fluorescence correlation spectroscopy performed at the single cell level, over various eukaryotic cell lines (MCF7, LR73, KB3.1, MESSA and MDCKII). The nonsymmetric profile of the diffusion time distributions established experimentally, is discussed according to Monte Carlo simulations, which reproduce the diffusion of the fluorescent probe in heterogeneous membrane.Display Omitted► The microfluidity of plasma membrane has been assessed with fluorescence correlation spectroscopy (FCS). ► The resulting diffusion-time distribution allows to highlight the fluidity fingerprint of the membrane. ► Experimental diffusion-time distributions were discussed according to Monte-Carlo simulations.

Keywords: Abbreviations; FCS; fluorescence correlation spectroscopy; GUV; giant unilamellar vesicle; FRAP; fluorescence recovery after photobleaching; ACF; autocorrelation function; DDA; diffusion-time distribution analysisFluorescence spectroscopy; Fluorescence correlation spectroscopy; Plasma membrane organization; Microdomain; Membrane fluidity

Modulated phases of phospholipid bilayers induced by tocopherols by Md. Arif Kamal; V.A. Raghunathan (pp. 2486-2493).

The influence of α-, γ‐ and δ-tocopherols on the structure and phase behavior of dipalmitoyl phosphatidylcholine (DPPC) bilayers has been determined from X-ray diffraction studies on oriented multilayers. In all the three cases the main-transition temperature ( T_m) of DPPC was found to decrease with increasing tocopherol concentration up to around 25mol%. Beyond this the main transition is suppressed in the case of γ‐tocopherol, whereas T_m becomes insensitive to composition in the other two cases. The pre-transition is found to be suppressed over a narrow tocopherol concentration range between 7.5 and 10mol% in DPPC‐γ-tocopherol and DPPC-δ‐tocopherol bilayers, and the ripple phase occurs down to the lowest temperature studied. In all the three cases a modulated phase is observed above a tocopherol concentration of about 10mol%, which is similar to the P_β phase reported in DPPC–cholesterol bilayers. This phase is found to occur even in excess water conditions at lower tocopherol concentrations, and consists of bilayers with periodic height modulation. These results indicate the ability of tocopherols to induce local curvature in membranes, which could be important for some of their biological functions.Display Omitted► Phase behavior of DPPC-tocopherol membranes is similar to that of DPPC-cholesterol membranes. ► Tocopherols induce a modulated phase in DPPC membranes below the main-transition. ► Tocopherols are not as efficient as cholesterol in inducing chain ordering in DPPC membranes. ► Tocopherols abolish the pre-transition of DPPC membranes over a narrow composition range around 10 mol%.

Keywords: Phospholipid bilayer; Tocopherol; X-ray diffraction

A stochastic model for DNA translocation through an electropore by Miao Yu; Wenchang Tan; Hao Lin (pp. 2494-2501).

A 1D Fokker–Planck simulation of DNA translocation through an electropore under finite pulses is presented. This study is motivated by applications relevant to DNA electrotransfer into biological cells via electroporation. The results review important insights. The translocation may occur on two disparate time scales, the electrophoretic time (~ms), and the diffusive time (~s), depending on the pulse length. Furthermore, a power-law correlation is observed, F-PST~( V_m t_p)^a/ N^b, where F-PST is the final probability of successful translocation, V_m is the transmembrane potential, t_p is the pulse length, and N is the DNA length in segments. The values for a and b are close to 1 and 1.5, respectively. The simulated results are compared with previous data to interpret the trends. In particular, the diffusive time scale is used to explain the frequency dependence observed in electroporation experiments with uni- and bi-polar pulse trains. The predictions from the current model can be harnessed to help design experiments for the further understanding and quantification of DNA electrotransfer.► We present a 1D Fokker–Planck simulation on DNA translocation through an electropore under finite pulses. ► Translocation may occur on the electrophoretic or the diffusive time scale. ► We find a power-law correlation between the probability of successful translocation and the governing parameters. ► The diffusive time scale is used to explain the frequency dependence in AC/DC electroporation experiments. ► This work provides quantitative predictions for further experimental validation and understanding.

Keywords: Electroporation; DNA electrotransfer; Fokker–Planck equation; Translocation; Electrophoresis; Diffusion

Polyphenolic compounds are novel protective agents against lipid membrane damage by α-synuclein aggregates in vitro by Mario Caruana; Johanna Neuner; Hogen Tobias Högen; Felix Schmidt; Frits Kamp; Charles Scerri; Armin Giese; Neville Vassallo (pp. 2502-2510).

Cumulative evidence now suggests that the abnormal aggregation of the protein α-synuclein (αS) is a critical factor in triggering neurodegeneration in Parkinson's disease (PD). In particular, a fundamental pathogenetic mechanism appears to involve targeting of neuronal membranes by soluble oligomeric intermediates of αS, leading to their disruption or permeabilisation. Therefore, a model assay was developed in which fluorophore-loaded unilamellar vesicles were permeabilised by soluble oligomers, the latter formed by aggregation of human recombinant αS protein. The αS oligomers induced an impairment of membrane integrity similar to that of the pore-forming bacterial peptide gramicidin. The lipid vesicle permeabilisation assay was then utilised to screen 11 natural polyphenolic compounds, 8 synthetic N′-benzylidene-benzohydrazide compounds and black tea extract for protection against membrane damage by wild-type and mutant (A30P, A53T) synuclein aggregates. A select group of potent inhibitory compounds included apigenin, baicalein, morin, nordihydroguaiaretic acid, and black tea extract. Structure–activity analysis further suggests that a 5,7-dihydroxy-chromen-4-one moiety appears to be favourable for the inhibition reaction. In conclusion, we have identified a group of polyphenols that can effectively hinder membrane damage by αS aggregates. These may serve as a viable source of lead compounds for the development and design of novel therapeutic agents in PD.► α-Synuclein oligomers efficiently permeabilise lipid membranes. ► A group of polyphenols and black tea extract can effectively hinder membrane damage. ► The 5,7-dihydroxy-chromen-4-one moiety is the likely bioactive core structure.

Keywords: Abbreviations; αS; α-synuclein; AFM; atomic force microscopy; Api; apigenin; Baic; baicalein; BTE; black tea extract; EGCG; (−)-epigallocatechin gallate; Gen; genistein; Gink; ginkgolide B; Mor; morin; NBB; N′-benzylidene-benzohydrazide; NDGA; nordihydroguaiaretic acid; ns; not significant; OGB-1; Oregon Green® 488 BAPTA-1; PD; Parkinson's disease; PropylG; propyl gallate; Purp; purpurogallin trimethyl ether; Resv; resveratrol; Scut; scutellarein; SIFT; scanning for intensely fluorescent targets; SUVs; small unilamellar vesicles; ThT; Thioflavin T; WT; wild-typeα-Synuclein; Parkinson's disease; Oligomers; Membrane permeabilisation; Lipid vesicle; Polyphenols

The position of the double bond in monounsaturated free fatty acids is essential for the inhibition of the nicotinic acetylcholine receptor by Vanesa L. Perillo; Fernandez-Nievas Gaspar A. Fernández-Nievas; Valles Ana S. Vallés; Francisco J. Barrantes; Silvia S. Antollini (pp. 2511-2520).

Free fatty acids (FFAs) are non-competitive antagonists of the nicotinic acetylcholine receptor (AChR). Their site of action is supposedly located at the lipid-AChR interface. To elucidate the mechanism involved in this antagonism, we studied the effect that FFAs with a single double-bond at different positions (ω6, ω9, ω11 and ω13 cis-18:1) have on different AChR properties. Electrophysiological studies showed that only two FFAs (ω6 and ω9) reduced the duration of the channel open-state. The briefest component of the closed-time distribution remained unaltered, suggesting that ω6 and ω9 behave as allosteric blockers. Fluorescence resonance energy transfer studies indicated that all FFAs locate at the lipid-AChR interface, ω6 being restricted to annular sites and all others occupying non-annular sites. The perturbation of the native membrane order by FFAs was evaluated by DPH (1,6-diphenyl-1,3,5-hexatriene) and Laurdan fluorescence polarization studies, with the greatest decrease observed for ω9 and ω11. AChR conformational changes produced by FFAs present at the lipid bilayer were evaluated by fluorescence quenching studies of pyrene-labeled AChR and also using the AChR conformational-sensitive probe crystal violet. All cis-FFAs produced AChR conformational changes at the transmembrane level, but only ω9, ω11 and ω13 perturbed the resting state. Thus, the position and isomerism of the torsion angle of unsaturated FFAs are probably a key factor in terms of AChR blockage, suggesting that FFAs with a unique cis double bond at a superficial position inside the membrane directly inhibit AChR function by perturbing a potential conserved core structure for AChR gating at that level.► We studied AChR function inhibition by monounsaturated free fatty acids (FFA). ► Fluorescence spectroscopy and patch-clamp techniques were used. ► AChR function was inhibited by only two of the FFAs studied. ► Double bond position in FFAs was directly related to AChR inhibition.

Keywords: Abbreviations; ACh; acetylcholine; AChR; nicotinic acetylcholine receptor; Carb; carbamylcholine; Chol; cholesterol; CrV; crystal violet; DPH; 1,6-diphenyl-1,3,5-hexatriene; FFAs; free fatty acids; sFFAS; saturated free fatty acids; cis; -FFAs; cis; -unsaturated free fatty acids; trans; -FFAs; trans; -unsaturated free fatty acids; GP; generalized polarization; NCA; non-competitive antagonists; N-PyrM; N-(1-pyrenyl)maleimide; POPA; palmitoyl-oleoyl phosphatidic acid; POPC; palmitoyl-oleoyl phosphatidylcholine; 5-SLFA; 5-spin labeled fatty acid; TM; transmembrane; FRET; Förster-type resonance energy transferNicotinic acetylcholine receptor; Lipid–protein interaction; Free fatty acid; Fluorescence spectroscopy; Electrophysiology

Mechanism of membrane perturbation by the HIV-1 gp41 membrane-proximal external region and its modulation by cholesterol by Andrey Ivankin; Apellaniz Beatriz Apellániz; David Gidalevitz; José L. Nieva (pp. 2521-2528).

Membrane-activity of the glycoprotein 41 membrane-proximal external region (MPER) is required for HIV-1 membrane fusion. Consequently, its inhibition results in viral neutralization by the antibody 4E10. Previous studies suggested that MPER might act during fusion by locally perturbing the viral membrane, i.e., following a mechanism similar to that proposed for certain antimicrobial peptides. Here, we explore the molecular mechanism of how MPER permeates lipid monolayers containing cholesterol, a main component of the viral envelope, using grazing incidence X-ray diffraction and X-ray reflectivity. Our studies reveal that helical MPER forms lytic pores under conditions not affecting the lateral packing order of lipids. Moreover, we observe an increment of the surface area occupied by MPER helices in cholesterol-enriched membranes, which correlates with an enhancement of the 4E10 epitope accessibility in lipid vesicles. Thus, our data support the view that curvature generation by MPER hydrophobic insertion into the viral membrane is functionally more relevant than lipid packing disruption.► MPER is a membrane-transferring domain implied in the fusogenic activity of HIV gp41. ► The molecular mechanism underlying its membrane activity remains to be elucidated. ► We have studied this mechanism by X-ray scattering techniques in peptide–lipid monolayers. ► Our results support the idea that the hydrophobic insertion of MPER domain generates curvature within the viral membrane.

Keywords: HIV gp41; MPER–lipid interaction; X-ray scattering; Lytic pore; Membrane curvature

Ion solvation and structural stability in a sodium channel investigated by molecular dynamics calculations by Hu Qiu; Rong Shen; Wanlin Guo (pp. 2529-2535).

The stability and ion binding properties of the homo-tetrameric pore domain of a prokaryotic, voltage-gated sodium channel are studied by extensive all-atom molecular dynamics simulations, with the channel protein being embedded in a fully hydrated lipid bilayer. It is found that Na⁺ ion presents in a mostly hydrated state inside the wide pore of the selectivity filter of the sodium channel, in sharp contrast to the nearly fully dehydrated state for K⁺ ions in potassium channels. Our results also indicate that Na⁺ ions make contact with only one or two out of the four polypeptide chains forming the selectivity filter, and surprisingly, the selectivity filter exhibits robust stability for various initial ion configurations even in the absence of ions. These findings are quite different from those in potassium channels. Furthermore, an electric field above 0.5V/nm is suggested to be able to induce Na⁺ permeation through the selectivity filter.► We study the stability and ion binding properties of a sodium ion channel (NavAb). ► Sodium ions in the selectivity filter are highly hydrated. ► Sodium ions bind with the channel protein in an asymmetric manner. ► Robust stability of the NavAb channel ► Ion permeation through NavAb can be induced by an electric field above 0.5V/nm.

Keywords: Sodium channels; Molecular dynamics; Structural stability; Ion hydration

Interaction with membranes of the full C-terminal domain of protein NS4B from Hepatitis C virus by M. Francisca Palomares-Jerez; Henrique Nemesio; Villalain José Villalaín (pp. 2536-2549).

Hepatitis C virus (HCV) NS4B protein is a transmembrane highly hydrophobic protein responsible for many key aspects of the viral replication process. The C-terminal part of NS4B is essential for replication and is a potential target for HCV replication inhibitors. In this work we have carried out a study of the binding to and interaction with model biomembranes of a peptide corresponding to the C-terminal domain of NS4B, NS4B_Cter. We show that NS4B_Cter partitions into phospholipid membranes, is capable of rupturing membranes even at very low peptide-to-lipid ratios and its membrane-activity is modulated by lipid composition. NS4B_Cter is located in a shallow position in the membrane but it is able to affect the lipid environment from the membrane surface down to the hydrophobic core. Our results identify the C-terminal region of the HCV NS4B protein as a membrane interacting domain, and therefore directly implicated in the HCV life cycle and possibly in the formation of the membranous web.Display Omitted► We have studied the binding and interaction of peptide NS4B_Cter from HCV NS4B with model membranes. ► Membrane lipid biophysical properties are modulated by NS4B_Cter. ► NS4B_Cter partitions into and interacts with membrane phospholipid membranes. ► NS4B_Cter structure is modulated by membrane binding. ► NS4B_cter region might be implicated in HCV replication process.

Keywords: Abbreviations; 5-NS; 5-doxyl-stearic acid; 16-NS; 16-doxyl-stearic acid; 14BMP; bis(monomyristoylglycero)phosphate; 18BMP; bis(monooleoylglycero)phosphate; BPI; bovine brain; l; -α-phosphatidylinositol; BPS; bovine brain; l; -α-phosphatidylserine; CF; 5-carboxyfluorescein; Chol; cholesterol; CL; bovine heart cardiolipin; DMPA; 1,2-dimyristoyl-; sn; -glycero-3-phosphatidic acid; DMPC; 1,2-dimyristoyl-; sn; -glycero-3-phosphatidylcholine; DMPG; 1,2-dimyristoyl-; sn; -glycero-3-[phospho-rac-glycerol]; 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; EPG; egg; l; -α-phosphatidylglycerol; ER; endoplasmic reticulum; ESM; egg sphingomyelin; HCV; Hepatitis C virus; LEM; late endosome membrane; LUV; large unilamellar vesicles; MAS; magic angle spinning; MLV; multilamellar vesicles; NMR; nuclear magnetic resonance; NS; non-structural protein; PSM; N-palmitoyl-; d; -erythro-sphingosylphosphorylcholine; TFE; trifluoroethanol; T; _m; temperature of the gel-to‐liquid crystalline phase transition; TMA-DPH; 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene; TPE; egg transphosphatidylated; l; -α-phosphatidylethanolamineHCV replication; HCV; Lipid–peptide interaction; Membraneous web; NS4B

Role of lipid ordered/disordered phase coexistence in pulmonary surfactant function by Cristina Casals /; Canadas Olga Cañadas (pp. 2550-2562).

The respiratory epithelium has evolved to produce a complicated network of extracellular membranes that are essential for breathing and, ultimately, survival. Surfactant membranes form a stable monolayer at the air–liquid interface with bilayer structures attached to it. By reducing the surface tension at the air–liquid interface, surfactant stabilizes the lung against collapse and facilitates inflation. The special composition of surfactant membranes results in the coexistence of two distinct micrometer-sized ordered/disordered phases maintained up to physiological temperatures. Phase coexistence might facilitate monolayer folding to form three-dimensional structures during exhalation and hence allow the film to attain minimal surface tension. These folded structures may act as a membrane reserve and attenuate the increase in membrane tension during inspiration. The present review summarizes what is known of ordered/disordered lipid phase coexistence in lung surfactant, paying attention to the possible role played by domain boundaries in the monolayer-to-multilayer transition, and the correlations of biophysical inactivation of pulmonary surfactant with alterations in phase coexistence.► Surfactant membranes are characterized by ordered/disordered phase coexistence. ► Lateral phase segregation aids surfactant film to attain minimal surface tension. ► Some surfactant inhibitors reduce or abolish phase coexistence. ► These inhibitors interact with surfactant membranes, altering their structure.

Keywords: Abbreviations; AFM; atomic force microscopy; DPPC; dipalmitoylphosphatidylcholine; L; _d; liquid‐crystalline disordered; L; _o; liquid‐crystalline ordered; LE; liquid expanded; LO; liquid expanded ordered; TC; tilted condensed; PL; Phospholipid; PC; phosphatidylcholine; ToF-SIMS; time-of-flight secondary ion mass spectroscopy; γ; surface tension; γ; _e; equilibrium surface tension; π; surface pressure; π; _e; equilibrium surface pressureLateral phase separation; SP-A; SP-B; SP-C; Surface activity; Surfactant inhibition

Mechanism for translocation of fluoroquinolones across lipid membranes by Oana Cramariuc; Tomasz Rog; Matti Javanainen; Luca Monticelli; Anna V. Polishchuk; Ilpo Vattulainen (pp. 2563-2571).

Classical atom-scale molecular dynamics simulations, constrained free energy calculations, and quantum mechanical (QM) calculations are employed to study the diffusive translocation of ciprofloxacin (CPFX) across lipid membranes. CPFX is considered here as a representative of the fluoroquinolone antibiotics class. Neutral and zwitterionic CPFX coexist at physiological pH, with the latter being predominant. Simulations reveal that only the neutral form permeates the bilayer, and it does so through a novel mechanism that involves dissolution of concerted stacks of zwitterionic ciprofloxacins. Subsequent QM analysis of the observed molecular stacking shows the important role of partial charge neutralization in the stacks, highlighting how the zwitterionic form of the drug is neutralized for translocation. The findings propose a translocation mechanism in which zwitterionic CPFX molecules approach the membrane in stacks, but they diffuse through the membrane as neutral CPFX monomers due to intermolecular transfer of protons favored by partial solvation loss. The mechanism is expected to be of importance in the permeation and translocation of a variety of ampholitic drugs with stacking tendencies.Display Omitted► Ciprofloxacin diffuses in water as concerted stacks. ► Stacks of ciprofloxacin dissolve as they enter lipid membranes. ► Translocation of ciprofloxacin through lipid membranes takes place in monomeric form. ► Ciprofloxacin diffuses through a lipid membrane in its neutral form.

Keywords: Abbreviations; FQ; fluoroquinolone; CPFX; ciprofloxacin; MD; molecular dynamics; PC; 1,2‐Dilinoleoyl‐sn‐Glycero‐3‐phosphocholine; QM; quantum mechanical; OPLS; optimized parameters for liquid simulations; CHelpG; CHarges from ELectrostatic Potentials using a Grid based method; BLYP; Becke–Lee–Yang–Parr; DZVP; double zeta valence with polarization; COSMO; conductor‐like screening modelFluoroquinolone; Ciprofloxacin; Lipid membrane; Translocation; Molecular dynamics simulations; Density functional theory

Lipid interactions of the malaria antigen merozoite surface protein 2 by Christopher A. MacRaild; Marie Ø. Pedersen; Robin F. Anders; Raymond S. Norton (pp. 2572-2578).

With more than half the world's population living at risk of malaria infection, there is a strong demand for the development of an effective malaria vaccine. One promising vaccine candidate is merozoite surface protein 2 (MSP2), which is among the most abundant antigens of the blood stage of the Plasmodium falciparum parasite. In solution, MSP2 is intrinsically unstructured, but little is known about the conformation of native MSP2, which is GPI-anchored to the merozoite surface, or of the implications of that conformation for the immune response induced by MSP2. Initial NMR studies have shown that MSP2 interacts with lipid micelles through a highly conserved N-terminal domain. We have further developed these findings by investigating how different lipid environments affect the protein structure. All of the tested lipid preparations perturbed only the N-terminal part of MSP2. In DPC micelles this region adopts an α-helical structure which we have characterized in detail. Our findings suggest a possible mechanism by which lipid interactions might modulate immune recognition of the conserved N-terminus of MSP2, potentially explaining the apparent immunodominance of the central variable region of this important malaria antigen.Display Omitted► MSP2 is a clinically validated candidate for inclusion in a vaccine against malaria. ► We present a detailed structural characterisation of the lipid interactions of MSP2. ► Lipid interactions induce helical structure in the conserved N-terminal region. ► May modulate immune recognition and the immunodominance of the variable region. ► Highlights implications of GPI anchoring for properties of malaria antigens.

Keywords: Abbreviations; MSP2; merozoite surface protein 2; DPC; dodecyl phosphocholine; LPPG; lyso-palmatoyl phosphatidylglycerol; DLPC; dilauryl phosphatidylcholine; SUVs; small unilamellar vesiclesMalaria; Plasmodium falciparum; Merozoite surface protein 2; Structure; NMR

Magic angle spinning NMR study of interaction of N-terminal sequence of dermorphin (Tyr-d-Ala-Phe-Gly) with phospholipids by Agata Jeziorna; Tomasz Pawlak; Katarzyna Trzeciak-Karlikowska; Piotr Paluch; Marek J. Potrzebowski (pp. 2579-2587).

Two modifications of the Tyr-d-Ala-Phe-Gly tetrapeptide with different C-terminal groups (Tyr-d-Ala-Phe-Gly-OH1 and Tyr-d-Ala-Phe-Gly-NH₂2) were investigated by various nuclear magnetic resonance sequences under magic angle spinning. The structural constraints obtained from the magic angle spinning nuclear magnetic resonance measurements suggest that both peptides are aligned on the surface of the membrane and that the sandwich-like π-CH₃-π arrangement of the pharmacophore is preserved. The influence of the chemical modification of the C-terminal residue of1 and2 on their interaction with phosphate group of the phospholipid in the subgel phase L_c and the conformation of the peptides in the liquid crystalline phase L_α are discussed. The correlation between the X-ray structure of1 in the solid state and1 embedded into a membrane in the L_c phase is presented on the basis of the comparative analysis of the two‐dimensional¹³C–¹³C dipolar-assisted rotational resonance cross-peaks and the¹³C isotropic chemical shifts.Display Omitted► We introduced magic angle spinning NMR experiments to study soft matter. ► We investigated interaction of opioid peptide with phospholipids. ► We compared changes of peptide conformation in the solid state in the L_c and L_α phases. ► We proposed the alignment and conformation of peptide bonded to phospholipid.

Keywords: NMR spectroscopy; Peptide–phospholipid interaction; Opioid peptide; Peptide alignment; Bioactive conformation; ¹³; C labeled peptide

Identification of a crab gill FXYD2 protein and regulation of crab microsomal Na,K-ATPase activity by mammalian FXYD2 peptide by Elias C.C. Silva; Douglas C. Masui; Rosa P. Furriel; John C. McNamara; Hector Barrabin; Helena M. Scofano; Jonas Perales; André Teixeira-Ferreira; Francisco A. Leone; Carlos Frederico L. Fontes (pp. 2588-2597).

This investigation discloses the recognition of an FXYD2 protein in a microsomal Na,K-ATPase preparation from the posterior gills of the blue crab, Callinectes danae, by a mammalian (rabbit) FXYD2 peptide specific antibody (γC³³) and MALDI-TOF–TOF mass spectrometry techniques. This is the first demonstration of an invertebrate FXYD2 protein. The addition of exogenous pig FXYD2 peptide to the crab gill microsomal fraction stimulated Na,K-ATPase activity in a dose-dependent manner. Exogenous pig FXYD2 also considerably increased enzyme affinity for K⁺, ATP and NH₄⁺. K_0.5 for Na⁺ was unaffected. Exogenous pig FXYD2 increased the V_max for stimulation of gill Na,K-ATPase activity by Na⁺, K⁺ and ATP, by 30% to 40%. The crab gill FXYD2 is phosphorylated by PKA, suggesting a regulatory function similar to that known for the mammalian enzyme. The PKA-phosphorylated pig FXYD2 peptide stimulated the crab gill Na,K-ATPase activity by 80%, about 2-fold greater than did the non-phosphorylated peptide. Stimulation by the PKC-phosphorylated pig FXYD2 peptide was minimal. These findings confirm the presence of an FXYD2 peptide in the crab gill Na,K-ATPase and demonstrate that this peptide plays an important role in regulating enzyme activity.► This work describes the first evidence of the occurrence of an FXYD in invertebrates. ► This FXYD can be phosphorylated by endogenous PKA. ► The mammalian FXYD2 (from pig kidney) readily regulates the C. danae Na,K-ATPase. ► FXYD increased the V_max of the ATP hydrolysis and the NH₄⁺ affinity of C. danae ATPase. ► C. danae enzyme activation was sensitive to PKA phosphorylation of the mammalian FXYD2.

Keywords: Na,K-ATPase; FXYD2 peptide; Gill microsome; Endogenous kinase; Phosphorylation; Euryhaline crab

Preparation of giant unilamellar vesicles from damp lipid film for better lipid compositional uniformity by Eda Baykal-Caglar; Ebrahim Hassan-Zadeh; Bahar Saremi; Juyang Huang (pp. 2598-2604).

Giant unilamellar vesicles (GUVs) containing cholesterol often have a wide distribution in lipid composition. In this study, GUVs of 1,2-dioleoyl-sn-glycero-3-phosphocholine(DOPC)/1,2-distearoyl-sn-glycero-3-phosphocholine(DSPC)/cholesterol and 1,2-diphytanoyl-sn-glycero-3-phosphocholine(diPhyPC)/1,2-dipalmitoyl-sn-glycero-3-phosphocholine(DPPC)/cholesterol were prepared from dry lipid films using the standard electroformation method as well as a modified method from damp lipid films, which are made from compositional uniform liposomes prepared using the Rapid Solvent Exchange (RSE) method. We quantified the lipid compositional distributions of GUV by measuring the miscibility transition temperature of GUVs using fluorescence microscopy, since a narrower distribution in the transition temperature should correspond to a more uniform distribution in GUV lipid composition. Cholesterol molecules can demix from other lipids in dry state and form cholesterol crystals. Using optical microscopy, micron-sized crystals were observed in some dry lipid films. Thus, a major cause of GUV lipid compositional heterogeneity is the demixing of lipids in the dry film state. By avoiding the dry film state, GUVs prepared from damp lipid films have a better uniformity in lipid composition, and the standard deviations of miscibility transition temperature are about 2.5 times smaller than that of GUVs prepared from dry lipid films. Comparing the two ternary systems, diPhyPC/DPPC/cholesterol GUVs has a larger cholesterol compositional heterogeneity, which directly correlates with the low maximum solubility of cholesterol in diPhyPC lipid bilayers (40.2±0.5mol%) measured by light scattering. Our data indicate that cholesterol interacts far less favorably with diPhyPC than it does with other PCs. The damp lipid film method also has a potential of preparing GUVs from cell membranes containing native proteins without going through a dry state.► GUVs made from dry lipid films have wide distributions in lipid composition. ► The main cause of the wide distribution is the demixing of lipids in the dry state. ► A new GUV procedure has been developed to overcome the demixing problem. ► The maximum solubility of cholesterol in diPhyPC lipid bilayer is low (40mol%). ► Cholesterol interacts unfavorably with the bulky chains of diPhyPC.

Keywords: Miscibility transition temperature; Electroformation; Cholesterol; Umbrella Model; Lipid demixing

Reorganization of lipid domain distribution in giant unilamellar vesicles upon immobilization with different membrane tethers by M.J. Sarmento; M. Prieto; Fábio Fernandes (pp. 2605-2615).

Characterization of phase coexistence in biologically relevant lipid mixtures is often carried out through confocal microscopy of giant unilamellar lipid vesicles (GUVs), loaded with fluorescent membrane probes. This last analysis is generally limited to the vesicle hemisphere further away from the coverslip, in order to avoid artifacts induced by the interaction with the solid surface, and immobilization of vesicles is in many cases required in order to carry out intensity, lifetime or single‐molecule based microscopy. This is generally achieved through the use of membrane tethers adhering to a coverslip surface. Here, we aimed to determine whether GUV immobilization through membrane tethers induces changes in lipid domain distribution within liposomes displaying coexistence of lipid lamellar phases. Confocal imaging and a Förster resonance energy transfer (FRET) methodology showed that biotinylated phospholipids present significantly different membrane phase partition behavior upon protein binding, depending on the presence or absence of a linker between the lipid headgroup and the biotinyl moiety. Membrane phases enriched in a membrane tether displayed in some cases a dramatically increased affinity for the immobilization surface, effectively driving sorting of lipid domains to the adherent membrane area, and in some cases complete sequestering of a lipid phase to the interaction surface was observed. On the light of these results, we conclude that tethering of lipid membranes to protein surfaces has the potential to drastically reorganize the distribution of lipid domains, and this reorganization is solely dictated by the partition properties of the protein–tether complex.Display Omitted► Different membrane tethers present distinct membrane phase partition properties. ► Some membrane tethers drastically changed domain distribution of immobilized GUVs. ► Protein surfaces tethered to the membrane stabilize membrane lipid domains. ► Domain stabilization in the tethered surface is due to phase segregation of tether.

Keywords: Giant unilamellar vesicles; Liposome immobilization; Membrane tether; Biotinylated lipids; Confocal microscopy; Förster resonance energy transfer

Mutual recognition of sphingolipid molecular species in membranes by Andreas Max Ernst; Francesc-Xabier Contreras; Christoph Thiele; Felix Wieland; Brugger Britta Brügger (pp. 2616-2622).

In membranes liquid disordered (l_d) and liquid ordered (l_o) domains can exist that differ in fluidity and function. L_o areas are predominantly composed of cholesterol and sphingomyelin (SM). Study of the formation of such domains is hampered by a lack of methods to analyze specific lipid–lipid interactions at low concentrations of individual molecular lipid species in membranes. Here, we developed a simple biophysical method to experimentally assess the affinity of various molecular species of SM for cholesterol, and for their endogenous counterparts (kin) at physiological concentrations. Fluorescent SM ( flc SM) molecular species with a conjugated pentaene system in their fatty acids are employed to monitor their affinity to either cholesterol or their kin by fluorescence unquenching. With this novel method we show that specific interactions of individual SMs with cholesterol or their kin exist, indicating the presence of SM nano‐domains in l_d-phases, strictly based on kin/cholesterol recognition.► Fluorescent pentaene lipids allow to analyze lipid–lipid interactions in vitro. ► SM species differ in their concentration-dependent affinities for their kin and for cholesterol. ► Concentration-dependent affinities of SMs with kin/cholesterol can drive nano-domain formation.

Keywords: Lipid–Lipid Interaction; Sphingomyelin; Cholesterol; Nano-domain; Kin recognition; Fluorescent polyene-lipid

Synergistic action of Galleria mellonella anionic peptide 2 and lysozyme against Gram-negative bacteria by Agnieszka Zdybicka-Barabas; Pawel Mak; Anna Klys; Krzysztof Skrzypiec; Ewaryst Mendyk; Fiolka Marta J. Fiołka; Cytrynska Małgorzata Cytryńska (pp. 2623-2635).

Lysozyme and antimicrobial peptides are key factors of the humoral immune response in insects. In the present work lysozyme and anionic defense peptide (GMAP2) were isolated from the hemolymph of the greater wax moth Galleria mellonella and their antibacterial activity was investigated. Adsorption of G. mellonella lysozyme on the cell surface of Gram-positive and Gram-negative bacteria was demonstrated using immunoblotting with anti- G. mellonella lysozyme antibodies. Lysozyme effectively inhibited the growth of selected Gram-positive bacteria, which was accompanied by serious alterations of the cell surface, as revealed by atomic force microscopy (AFM) imaging. G. mellonella lysozyme used in concentrations found in the hemolymph of naive and immunized larvae, perforated also the Escherichia coli cell membrane and the level of such perforation was considerably increased by GMAP2. GMAP2 used alone did not perforate E. coli cells nor influence lysozyme muramidase activity. However, the peptide induced a decrease in the turgor pressure of the bacterial cell. Moreover, in the samples of bacteria treated with a mixture of lysozyme and GMAP2 the sodium chloride crystals were found, suggesting disturbance of ion transport across the membrane leading to cell disruption. These results clearly indicated the synergistic action of G. mellonella lysozyme and anionic peptide 2 against Gram-negative bacteria. The reported results suggested that, thanks to immune factors constitutively present in hemolymph, G. mellonella larvae are to some extent protected against infection caused by Gram-negative bacteria.(A)The effect of anionic peptide 2 and lysozyme on the level of E. coli cells' perforation.(B)The bacterial cell surface alterations caused by lysozyme and anionic peptide 2 imaged by AFM. The black arrows indicate the extracellular rectangular structures containing sodium chloride.Display Omitted ► G. mellonella lysozyme caused alterations of Gram-positive bacteria cell surface. ► G. mellonella anionic peptide 2 increased E. coli membrane perforation by lysozyme. ► G. mellonella anionic peptide 2 did not influence lysozyme muramidase activity. ► G. mellonella anionic peptide 2 decreased a turgor pressure of the bacterial cells. ► E. coli treatment by Gm lysozyme and anionic peptide2 induced NaCl crystallization.

Keywords: Defense peptide; Lysozyme; Anionic peptide 2; Galleria mellonella; Atomic force microscopy

Lateral self-assembly of 18.5-kDa myelin basic protein (MBP) charge component-C1 on membranes by Daniel R. Kattnig; Timo Bund; Joan M. Boggs; George Harauz; Dariush Hinderberger (pp. 2636-2647).

Myelin basic protein (MBP), particularly the classic 18.5-kDa isoform, is a major structural protein of the myelin sheath of the central nervous system. It is an intrinsically disordered, peripheral membrane protein that shows structural polymorphism in combination with several overlapping interaction sites. Here, double electron–electron resonance (DEER) spectroscopy, in combination with a simplified, semi-quantitative analysis based on Monte Carlo simulations, is used to determine the distance distribution of murine 18.5-kDa MBP, unmodified charge component-C1, on large unilamellar vesicles of a lipid composition mimicking the cytoplasmic leaflet of myelin. Three singly spin-labeled MBP variants and a mixture of singly-labeled MBP variants are used. The MBPs, each bearing only one spin label, exhibit average intermolecular distances that are significantly shorter than the distances expected when assuming a random distribution at the employed lipid-to-protein ratios, indicating self-assembly on the membrane. The distribution of elliptical pervaded areas (hard ellipses) on a two-dimensional surface can serve as a model of the nonspecific self-assembly process. The corresponding pair correlation functions g( r) are determined from Monte Carlo simulations with variation of various parameters such as the ellipses' aspect ratios. Comparing the g( r) values with the DEER-derived distance distributions, the pervaded volume is best characterized by a nearly elliptical projection onto the membrane, with an aspect ratio of approximately 1.5, and with the longer semi-axis of approximately 1.4nm. The approach of using local information from DEER with low-resolution models derived from Monte Carlo simulations can be applied to study the lateral self-assembly properties of other protein complexes on membranes.► DEER and Monte Carlo (MC) simulations reveal self-assembly of MBP on membranes. ► Singly spin-labeled MBPs exhibit short intermolecular distances on the membranes. ► Distances are shorter than for random distribution indicating membrane self-assembly. ► DEER data are analyzed using MC simulations of ellipses on a surface. ► Generalized approach: combine local DEER data and low-resolution MC interpretation.

Keywords: Abbreviations; CW; continuous-wave; DEER; double electron–electron resonance; DSPC; distearoylphosphatidylcholine; DSPG; distearoylphosphatidylglycerol; EPR; electron paramagnetic resonance; ESE; electron-spin-echo; IDP; intrinsically disordered protein; LPR; lipid-to-protein ratio; LUV; large unilamellar vesicle; MLV; multilamellar vesicle; MBP; myelin basic protein; MS; multiple sclerosis; MTS-SL; [1-oxyl-2,2,5,5-tetramethyl-; d; -pyrroline-3-methyl]methanethiosulfonate; rmC1; recombinant murine 18.5-kDa MBP charge component C1; X-band; microwave frequency range of ~; 9.1–9.8; GHzElectron paramagnetic/spin resonance; Myelin basic protein; Myelin; Intrinsically disordered protein; Multiple sclerosis; Membrane protein

Impact of a model synovial fluid on supported lipid membranes by M. Kreuzer; M. Strobl; M. Reinhardt; M.C. Hemmer; Hauss T. Hauß; R. Dahint; R. Steitz (pp. 2648-2659).

The interaction of a model synovial fluid, here a solution of 3mg/mL hyaluronic acid (HA) in heavy water (D₂O), with an oligolamellar stack of lipid (DMPC) membranes on silicon support has been studied by neutron reflectometry and infrared spectroscopy on the molecular scale at non-physiological and physiological conditions. The system under investigation represents a simple model for lipid-coated mammalian joints and other artificial implant surfaces. When exposed to pure D₂O at 21°C, i.e. below the main phase transition of the system, the lipid membranes show a lamellar spacing of 65Å. Heating to 26°C results in detachment of all lipid bilayers except for the innermost lipid lamella directly adsorbed to the surface of the silicon support. On the contrary, when incubated in the solution of HA in D₂O the oligolamellar lipid system starts swelling. In addition, heating to 39°C does not result in loss of the lipid membranes into the liquid phase. The interfacial lipid coating adopts a new stable lamellar state with an increase in d-spacing by 380% to 247Å measured after 43days of incubation with the model synovial fluid. Potential consequences for joint lubrication and protective wear functionality are considered.► Combined neutron reflectivity and infrared spectroscopy study on lipid membrane stacks at solid–liquid interface. ► Membrane stacks behave differently in hyaluronic acid, HA, in D₂O as compared to behavior in pure D₂O. ► HA in D₂O: new stable state of lipid films at physiological temperature. ► D₂O: lipid films detach from support at physiological temperature. ► Findings relevant to joint lubrication in mammals and protective wear functionality.

Keywords: DMPC; Hyaluronic acid; Interface; Neutron reflectivity; FTIR

Membrane adsorption and binding, cellular uptake and cytotoxicity of cell-penetrating peptidomimetics with α-peptide/β-peptoid backbone: Effects of hydrogen bonding and α-chirality in the β-peptoid residues by Xiaona Jing; Mingjun Yang; Marina R. Kasimova; Martin Malmsten; Henrik Franzyk; Lene Jorgensen; Camilla Foged; Hanne M. Nielsen (pp. 2660-2668).

Cell-penetrating peptides (CPPs) provide a promising approach for enhancing intracellular delivery of therapeutic biomacromolecules by increasing transport through membrane barriers. Here, proteolytically stable cell-penetrating peptidomimetics with α-peptide/β-peptoid backbone were studied to evaluate the effect of α-chirality in the β-peptoid residues and the presence of guanidinium groups in the α-amino acid residues on membrane interaction. The molecular properties of the peptidomimetics in solution (surface and intramolecular hydrogen bonding, aqueous diffusion rate and molecular size) were studied along with their adsorption to lipid bilayers, cellular uptake, and toxicity. The surface hydrogen bonding ability of the peptidomimetics reflected their adsorbed amounts onto lipid bilayers as well as with their cellular uptake, indicating the importance of hydrogen bonding for their membrane interaction and cellular uptake. Ellipsometry studies further demonstrated that the presence of chiral centers in the β-peptoid residues promotes a higher adsorption to anionic lipid bilayers, whereas circular dichroism results showed that α-chirality influences their overall mean residue ellipticity. The presence of guanidinium groups and α-chiral β-peptoid residues was also found to have a significant positive effect on uptake in living cells. Together, the findings provide an improved understanding on the behavior of cell-penetrating peptidomimetics in the presence of lipid bilayers and live cells.► Novel proteolytically stable peptidomimetics with α-peptide/β-peptoid structure exhibit promising cell-penetrating potential. ► Hydrogen bonding determined by molecular simulations reflects the level of membrane adsorption and cellular uptake. ► α-chirality stabilizes the folded conformation of the peptidomimetics upon interaction with bilayer membranes. ► α-chirality in the β-peptoid residue correlate with higher levels of membrane adsorption and cellular uptake.

Keywords: Chirality; Hydrogen bonding; Cell-penetrating peptide; Peptidomimetic; Adsorption; Binding constant

Cell surface binding and uptake of arginine- and lysine-rich penetratin peptides in absence and presence of proteoglycans by Amand Helene L. Åmand; Hanna A. Rydberg; Louise H. Fornander; Per Lincoln; Norden Bengt Nordén; Esbjorner Elin K. Esbjörner (pp. 2669-2678).

Cell surface proteoglycans (PGs) appear to promote uptake of arginine-rich cell-penetrating peptides (CPPs), but their exact functions are unclear. To address if there is specificity in the interactions of arginines and PGs leading to improved internalization, we used flow cytometry to examine uptake in relation to cell surface binding for penetratin and two arginine/lysine substituted variants (PenArg and PenLys) in wildtype CHO-K1 and PG-deficient A745 cells. All peptides were more efficiently internalized into CHO-K1 than into A745, but their cell surface binding was independent of cell type. Thus, PGs promote internalization of cationic peptides, irrespective of the chemical nature of their positive charges. Uptake of each peptide was linearly dependent on its cell surface binding, and affinity is thus important for efficiency. However, the gradients of these linear dependencies varied significantly. Thus each peptide's ability to stimulate uptake once bound to the cell surface is reliant on formation of specific uptake-promoting interactions. Heparin affinity chromatography and clustering experiments showed that penetratin and PenArg binding to sulfated sugars is stabilized by hydrophobic interactions and result in clustering, whereas PenLys only interacts through electrostatic attraction. This may have implications for the molecular mechanisms behind arginine-specific uptake stimulation as penetratin and PenArg are more efficiently internalized than PenLys upon interaction with PGs. However, PenArg is also least affected by removal of PGs. This indicates that an increased arginine content not only improve PG-dependent uptake but also that PenArg is more adaptable as it can use several portals of entry into the cell.► We studied cell surface binding and cellular uptake of penetratin variants. ► Proteoglycans increase uptake of membrane-bound peptide, but not peptide binding. ► Arginines increases both affinity and ability to stimulate uptake once a peptide is bound.

Keywords: Abbreviations; 7-AAD; 7-aminoactinomycin D; A745; CHO-K1 clone pgsA-745, defective in glycosaminoglycan synthesis; CF; carboxyfluorescein; CHO-K1; Chinese hamster ovary cell line K1; CPP; cell-penetrating peptide; GAG; glycosaminoglycan; HEPES; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HS; heparan sulfate; PG; proteoglycanCell-penetrating peptide; Penetratin; Proteoglycan; Cellular uptake; Membrane affinity; Arginine

Improving transmembrane protein consensus topology prediction using inter-helical interaction by Han Wang; Chao Zhang; Xiaohu Shi; Li Zhang; You Zhou (pp. 2679-2686).

Alpha helix transmembrane proteins (αTMPs) represent roughly 30% of all open reading frames (ORFs) in a typical genome and are involved in many critical biological processes. Due to the special physicochemical properties, it is hard to crystallize and obtain high resolution structures experimentally, thus, sequence-based topology prediction is highly desirable for the study of transmembrane proteins (TMPs), both in structure prediction and function prediction. Various model-based topology prediction methods have been developed, but the accuracy of those individual predictors remain poor due to the limitation of the methods or the features they used. Thus, the consensus topology prediction method becomes practical for high accuracy applications by combining the advances of the individual predictors. Here, based on the observation that inter-helical interactions are commonly found within the transmembrane helixes (TMHs) and strongly indicate the existence of them, we present a novel consensus topology prediction method for αTMPs, CNTOP, which incorporates four top leading individual topology predictors, and further improves the prediction accuracy by using the predicted inter-helical interactions. The method achieved 87% prediction accuracy based on a benchmark dataset and 78% accuracy based on a non-redundant dataset which is composed of polytopic αTMPs. Our method derives the highest topology accuracy than any other individual predictors and consensus predictors, at the same time, the TMHs are more accurately predicted in their length and locations, where both the false positives (FPs) and the false negatives (FNs) decreased dramatically. The CNTOP is available at:http://ccst.jlu.edu.cn/JCSB/cntop/CNTOP.html.Display Omitted► We developed a consensus alpha helix transmembrane protein topology prediction method CNTOP. ► Transmembrane inter-helical interaction is first time introduced into the topology prediction. ► This improved the topology prediction precision, especially for the polytopic alpha helix transmembrane proteins. ► High accurate topology prediction will benefit the studies on transmembrane protein structure.

Keywords: Transmembrane; Topology; Consensus prediction; Contact

Membrane transport metabolons by Trevor F. Moraes; Reinhart A.F. Reithmeier (pp. 2687-2706).

In this review evidence from a wide variety of biological systems is presented for the genetic, functional, and likely physical association of membrane transporters and the enzymes that metabolize the transported substrates. This evidence supports the hypothesis that the dynamic association of transporters and enzymes creates functional membrane transport metabolons that channel substrates typically obtained from the extracellular compartment directly into their cellular metabolism. The immediate modification of substrates on the inner surface of the membrane prevents back-flux through facilitated transporters, increasing the efficiency of transport. In some cases products of the enzymes are themselves substrates for the transporters that efflux the products in an exchange or antiport mechanism. Regulation of the binding of enzymes to transporters and their mutual activities may play a role in modulating flux through transporters and entry of substrates into metabolic pathways. Examples showing the physical association of transporters and enzymes are provided, but available structural data is sparse. Genetic and functional linkages between membrane transporters and enzymes were revealed by an analysis of Escherichia coli operons encoding polycistronic mRNAs and provide a list of predicted interactions ripe for further structural studies. This article supports the view that membrane transport metabolons are important throughout Nature in organisms ranging from bacteria to humans.

Keywords: Abbreviations; ABC; ATP-binding cassette; AE1; anion exchanger 1; CAII; carbonic anhydrase II; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; GLUT; glucose transporter; MFS; major facilitator superfamily of transporters; PFK; phosphofructokinase; SLC; solute carrier; STAS; sulfate transporter and anti-sigma factor antagonist; TM; transmembraneChanneling; Enzyme; Membrane protein; Metabolic pathways; Metabolon; Operons, protein interactions; Transporter

Molecular characterization of the interaction of crotamine-derived nucleolar targeting peptides with lipid membranes by Margarida Rodrigues; Andrea Santos; Beatriz G. de la Torre; Radis-Baptista Gandhi Rádis-Baptista; David Andreu; Nuno C. Santos (pp. 2707-2717).

A novel class of cell-penetrating, nucleolar-targeting peptides (NrTPs), was recently developed from the rattlesnake venom toxin crotamine. Based on the intrinsic fluorescence of tyrosine or tryptophan residues, the partition of NrTPs and crotamine to membranes with variable lipid compositions was studied. Partition coefficient values (in the 10²–10⁵ range) followed essentially the compositional trend POPC:POPG≤POPGvia different entry mechanisms, reinforcing the applicability of this class of peptide as therapeutic tools for the delivery of molecular cargoes.Display Omitted► Crotamine and NrTPs strongly interact with membrane model systems. ► Translocation is independent of specific receptors or electrochemical gradient. ► The same new microscopy methodology may test the translocation of other molecules. ► NrTPs acquire a partial helical structure upon binding to membranes. ► NrTPs are promising therapeutic tools for delivery of biomolecules or nanoprobes.

Keywords: Abbreviations; Ahx; 6-aminohexanoic acid; AMP; antimicrobial peptide; CD; circular dichroism; CF; 5,(6)-carboxyfluorescein; CPPs; cell penetrating peptides; DLS; dynamic light scattering; DPPE; 1,2-dipalmitoyl-; sn; -glycero-3-phosphoethanolamine; Fmoc; 9-fluorenylmethoxycarbonyl; GUV; giant unilamellar vesicles; HEPES; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HPLC; High-performance liquid chromatography; LUV; large unilamellar vesicles; NBD; nitro-2-1,3-benzoxadiazol-4-yl; NMR; nuclear magnetic resonance; NrTPs; nucleolar-targeting peptides; 5-NS; 5-doxyl-stearic acid; 16-NS; 16-doxyl-stearic acid; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; POPG; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phospho-(19-; sn; -glycerol); RhB; rhodamine BNucleolar-targeting peptide; Crotamine; Cell-penetrating peptide; Membrane partition; Membrane translocation; Fluorescence spectroscopy

The oxidized phospholipid PazePC modulates interactions between Bax and mitochondrial membranes by Marcus Wallgren; Martin Lidman; Quoc Dat Pham; Konrad Cyprych; Grobner Gerhard Gröbner (pp. 2718-2724).

Activation of the pro-apoptotic protein Bax under intracellular oxidative stress is closely related to its association with the mitochondrial outer membrane (MOM) system, ultimately resulting in cell death. The precise mechanism by which this activation and the subsequent structural changes in the protein occur is currently unknown. In addition to triggering the onset of apoptosis, oxidative stress generates oxidized lipids whose impact on mitochondrial membrane integrity and the activity of membrane-associated Bax is unclear. We therefore devised a model system that mimics oxidative stress conditions by incorporating oxidized phospholipids (OxPls) into mitochondria-like liposomes, and studied the OxPls' impact on Bax-membrane interactions. Differential scanning calorimetry (DSC) was used to study membrane organization and protein stability, while conformational changes in the protein upon contact with lipid vesicles were monitored using far-UV circular dichroism (CD) spectroscopy. The thermograms for liposomes containing the OxPl 1-palmitoyl-2-azelaoyl- sn-glycero-3-phosphocholine (PazePC) differed dramatically from those for unmodified liposomes. Moreover, Bax exhibited enhanced thermal stability in the presence of the modified liposomes, indicating that it interacted strongly with PazePC-containing membranes. The presence of PazePC also increased the α-helical character of Bax compared to the protein alone or with PazePC-free vesicles, at 10°C, 20°C, and 37°C. Presumably, the presence of PazePC-like OxPls a) increases the population of membrane-associated Bax and b) facilitates the protein's insertion into the membrane by distorting the bilayer's organization, as seen by solid-state high-resolution¹H and³¹P magic angle spinning nuclear magnetic resonance (MAS NMR) spectroscopy.► The interaction between Bax and mitochondrial membranes are investigated. ► Oxidative stress conditions are mimicked by using the OxPl PazePC. ► Presence of PazePC enhances Bax association with the membranes. ► We propose OxPls can increase the population of membrane-associated Bax in vivo. ► The distorted bilayer organization presumably facilitates insertion of the protein.

Keywords: Abbreviations; CD; circular dichroism; DSC; differential scanning calorimetry; IMM; inner mitochondrial membrane; MAS; magic angle spinning; MLVs; multilamellar vesicles; MOM; mitochondrial outer membrane; MOMP; mitochondrial outer membrane permeabilization; NMR; nuclear magnetic resonance; OxPls; oxidized phospholipids; PazePC; 1-palmitoyl-2-azelaoyl-; sn; -glycero-3-phosphocholine; PC; phosphatidylcholine; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; POPE; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phospho-ethanolamine; ROS; reactive oxygen species; SUVs; small unilamellar vesicles; T; _m; melting temperature; CL; cardiolipin; TMCL; 1,1′,2,2′-tetra-myristoyl cardiolipinApoptosis; Bax; CD; DSC; MAS NMR; PazePC

The DNA–DNA spacing in gemini surfactants–DOPE–DNA complexes by Pullmannova Petra Pullmannová; Sergio S. Funari; Devinsky Ferdinand Devínsky; Uhrikova Daniela Uhríková (pp. 2725-2731).

Gemini surfactants from the homologous series of alkane-α,ω-diyl-bis(dodecyldimethylammonium bromide) (CnCS12, number of spacer carbons n=2–12) and dioleoylphosphatidylethanolamine (DOPE) were used for cationic liposome (CL) preparation. CLs condense highly polymerized DNA creating complexes. Small-angle X-ray diffraction identified them as condensed lamellar phase L_α^C in the studied range of molar ratios CnGS12/DOPE in the temperature range 20–60°C. The DNA–DNA distance (d_DNA) is studied in dependence to CnGS12 spacer length and membrane surface charge density. The high membrane surface charge densities (CnGS12/DOPE=0.35 and 0.4mol/mol) lead to the linear dependence of d_DNA vs. n correlating with the interfacial area of the CnGS12 molecule.Display Omitted► Cationic liposomes composed of CnGS12 gemini surfactant and DOPE condense DNA. ► We used CnGS12 with number of spacer carbons n=2–12. ► The complexes CnGS12–DOPE–DNA form condensed lamellar phase L_α^C. ► The DNA–DNA spacing shows a linear dependence on n at CnGS12/DOPE=0.35 and 0.4mol/mol.

Keywords: Abbreviations; A; _CnGS12; CnGS12 surfactant headgroup area; C12TMA; dodecyltrimethylammonium bromide; CL; cationic liposomes; CnGS12; alkane-α,ω-diyl-bis(dodecyldimethylammonium bromide); DNA; deoxyribonucleic acid; DOPE; dioleoylphosphatidylethanolammine; GS; gemini surfactant; H; _II; ^C; condensed inverse hexagonal phase; L; _α; ^C; condensed lamellar phase; R; ₄; N; ⁺; quaternary ammonium groupDNA; Dioleoylphosphatidylethanolamine; Gemini surfactant; Small angle X-ray diffraction

Difference in lipid packing sensitivity of exchangeable apolipoproteins apoA-I and apoA-II: An important determinant for their distinctive role in lipid metabolism by Chieze Lionel Chièze; Victor Martin Bolanos-Garcia; Le Caer Gérard Le Caër; Anne Renault; Vie Véronique Vié; Sylvie Beaufils (pp. 2732-2741).

Exchangeable apolipoproteins A-I and A-II play distinct roles in reverse cholesterol transport. ApoA-I interacts with phospholipids and cholesterol of the cell membrane to make high density lipoprotein particles whereas apolipoprotein A-II interacts with high density lipoprotein particles to release apolipoprotein A-I. The two proteins show a high activity at the aqueous solution/lipid interface and are characterized by a high content of amphipathic α-helices built upon repetition of the same structural motif. We set out to investigate to what extent the number of α-helix repeats of this structural motif modulates the affinity of the protein for lipids and the sensitivity to lipid packing. To this aim we have compared the insertion of apolipoproteins A-I and A-II in phospholipid monolayers formed on a Langmuir trough in conditions where lipid packing, surface pressure and charge were controlled. We also used atomic force microscopy to obtain high resolution topographic images of the surface at a resolution of several nanometers and performed statistical image analysis to calculate the spatial distribution and geometrical shape of apolipoproteins A-I and A-II clusters. Our data indicate that apolipoprotein A-I is sensitive to packing of zwitterionic lipids but insensitive to the packing of negatively charged lipids. Interestingly, apolipoprotein A-II proved to be insensitive to the packing of zwitterionic lipids. The different sensitivity to lipid packing provides clues as to why apolipoprotein A-II barely forms nascent high density lipoprotein particles while apolipoprotein A-I promotes their formation. We conclude that the different interfacial behaviors of apolipoprotein A-I and apolipoprotein A-II in lipidic monolayers are important determinants of their distinctive roles in lipid metabolism.Display Omitted► ApoA-I is more sensitive to lipid packing than ApoA-II. ► The analysis of apoA‐I and apoA‐II clusters reveals a similar mode of self-assembling. ► Spatial distribution analyses of apolipoprotein oligomers in phospholipid monolayers. ► Apolipoprotein size affects the formation of HDL particles.

Keywords: Abbreviations; AFM; atomic force microscopy; ALPS; amphipathic lipid packing sensor; apoA-I; apolipoprotein A-I; CE; esterified cholesterol; DPPC; dipalmitoyl-phosphatidylcholine; DPPG; dipalmitoyl-phosphatidylglycerol; DOPC; dioleoyl-phosphatidylcholine; HDL; high density lipoprotein; LE; liquid-expanded; LC; liquid-condensed; LCAT; lecithin: cholesterol acyltransferase; RCT; reverse cholesterol transportExchangeable apolipoprotein; Lipid packing sensitivity; Phospholipid monolayer; AFM measurement; Statistical analysis of planar point process

Molecular dynamics simulation of cation–phospholipid clustering in phospholipid bilayers: Possible role in stalk formation during membrane fusion by Hui-Hsu Gavin Tsai; Wei-Xiang Lai; Hong-Da Lin; Jian-Bin Lee; Wei-Fu Juang; Wen-Hsin Tseng (pp. 2742-2755).

In this study, we performed all-atom long-timescale molecular dynamics simulations of phospholipid bilayers incorporating three different proportions of negatively charged lipids in the presence of K⁺, Mg2⁺, and Ca2⁺ ions to systemically determine how membrane properties are affected by cations and lipid compositions. Our simulations revealed that the binding affinity of Ca2⁺ ions with lipids is significantly stronger than that of K⁺ and Mg2⁺ ions, regardless of the composition of the lipid bilayer. The binding of Ca2⁺ ions to the lipids resulted in bilayers having smaller lateral areas, greater thicknesses, greater order, and slower rotation of their lipid head groups, relative to those of corresponding K⁺- and Mg2⁺-containing systems. The Ca2⁺ ions bind preferentially to the phosphate groups of the lipids. The complexes formed between the cations and the lipids further assembled to form various multiple-cation-centered clusters in the presence of anionic lipids and at higher ionic strength—most notably for Ca2⁺. The formation of cation–lipid complexes and clusters dehydrated and neutralized the anionic lipids, creating a more-hydrophobic environment suitable for membrane aggregation. We propose that the formation of Ca2⁺–phospholipid clusters across apposed lipid bilayers can work as a “cation glue” to adhere apposed membranes together, providing an adequate configuration for stalk formation during membrane fusion.Display Omitted► We characterized the properties of lipid bilayers with different compositions. ► Binding of Ca2⁺ ions to lipids forms various complexes involving several lipids. ► We observed the formation of large-scale multiple-cation-centered clusters. ► Large-scale clusters dehydrated and neutralized the anionic bilayer surfaces. ► Multiple-Ca2⁺-centered clusters are adequate configurations for membrane fusion.

Keywords: Cation–phospholipid clustering; Lipid bilayers property; Membrane fusion; Microdomain formation; Cation effect; Molecular dynamics simulation

Interfacial behavior and structural properties of a clinical lung surfactant from porcine source by Odalys Blanco; Antonio Cruz; Olga L. Ospina; Elena López-Rodriguez; Luis Vázquez; Perez-Gil Jesús Pérez-Gil (pp. 2756-2766).

Surfacen® is a clinical surfactant preparation of porcine origin, partly depleted of cholesterol, which is widely used in Cuba to treat pre-term babies at risk or already suffering neonatal respiratory distress. In the present study we have characterized the interfacial behavior of Surfacen in several in vitro functional models, including spreading and compression–expansion cycling isotherms in surface balances and in a captive bubble surfactometer, in comparison with the functional properties of whole native surfactant purified from porcine lungs and its reconstituted organic extract, the material from which Surfacen is derived. Surfacen exhibited similar properties to native porcine surfactant or its organic extract to efficiently form stable surface active films at the air–liquid interface, able to consistently reach surface tensions below 5mN/m upon repetitive compression–expansion cycling. Surfacen films, however, showed a substantially larger and stable compression-driven segregation of condensed lipid phases than exhibited by films formed by native surfactant or its organic extract. In spite of structural differences observed at microscopic level, Surfacen membranes showed a similar thermotropic behavior to membranes from native surfactant or its organic extract, characterized by calorimetry or fluorescence spectroscopy of samples doped with the Laurdan probe. On the other hand, analysis by atomic force microscopy of films formed by Surfacen or by the organic extract of native porcine surfactant revealed a similar network of interconnected condensed nanostructures, suggesting that the organization of the films at the submicroscopic level is the essential feature to support the proper stability and mechanical properties permitting the interfacial surfactant films to facilitate the work of breathing.Display Omitted► Surfacen is a cholesterol-depleted clinical pulmonary surfactant of porcine origin. ► Interfacial adsorption, compressibility and stability of Surfacen mimic those of native surfactant. ► Surfacen films exhibit a particularly marked and stable segregation of ordered phases. ► A network of interconnected nanostructures sustains the surface properties of Surfacen.

Keywords: Pulmonary surfactant; Interfacial film; Surface tension; Epifluorescence microscopy; Calorimetry; Surfacen®

Ordered-disordered domain coexistence in ternary lipid monolayers activates sphingomyelinase by clearing ceramide from the active phase by Elisa Carmen Ale; Bruno Maggio; Maria Laura Fanani (pp. 2767-2776).

We explored the action of sphingomyelinase (SMase) on ternary monolayers containing phosphatidylcholine, sphingomyelin (SM) and dihydrocholesterol, which varied along a single tie line of phase coexistence. SMase activity exhibited a higher rate and extent of hydrolysis when the film is within the liquid-expanded (LE)/liquid-ordered (LO) coexistence range, compared to monolayers in the full LO phase. Since Alexa-SMase preferably adsorbs to the LE phase and there was no direct correlation found between enzymatic activity and domain borders, we postulate that the LE phase is the active phase for ceramide (Cer) generation. The enzymatically generated Cer was organized in different ways depending on the initial LE/LO ratio. The action of SMase in Chol-poor monolayers led to the formation of Cer-enriched domains, while in Chol-rich monolayers it resulted in the incorporation of Cer in the LO phase and the formation of new Chol- and Cer-enriched domains. The following novel mechanism is proposed to provide an explanation for the favored action of SMase on interfaces that exhibit an LE–LO phase coexistence: the LO phase sequesters the product Cer causing its depletion from the more enzyme-susceptible LE phase, thus decreasing inhibition by the reaction product. Furthermore, LO domains function as a substrate reservoir by allowing a rapid exchange of the substrate from this phase to the SM-depleted LE phase.Display Omitted► SMase activity is favored by the presence of liquid-ordered domains. ► Fluorescent-labeled SMase adsorbs preferentially to the liquid-expanded phase. ► Cer production occurs mainly in the liquid-expanded phase, rather than at domain borders. ► The SMase-driven changes of surface structure depend on the liq-expanded/liq-ordered proportion. ► The modulation mechanism involves substrate supply and low product in the active phase.

Keywords: Abbreviations; SM; sphingomyelin, pSM, N-palmitoylsphingomyelin; Cer; ceramide; pCer; N-palmitoylceramide; Chol; cholesterol; Dchol; dihydrocholesterol; PC; phosphatidylcholine; dlPC; dilauroylphosphatidylcholine; LE; liquid-expanded; LO; liquid-ordered; LC; liquid-condensed; LD; liquid-disordered; BAM; Brewster angle microscopy; SMase; Bacillus cereus; sphingomyelinaseLiquid-ordered domain; Liquid-expanded phase; Brewster angle microscopy; Sphingomyelin; Lipid domain border; Tie line

Increased salt concentration promotes competitive block of OmpF channel by protons by Antonio Alcaraz; Queralt-Martin María Queralt-Martín; Garcia-Gimenez Elena García-Giménez; Vicente M. Aguilella (pp. 2777-2782).

Porins are channel-forming proteins that are located in the outer membranes (OM) of Gram-negative bacteria and allow the influx of hydrophilic nutrients and the extrusion of waste products. The fine regulation of the ion transport through these wide channels could play an important role in the survival of the bacteria in acidic media. We investigate here the mechanism responsible for the pH sensitivity of the trimeric porin OmpF, of Escherichia coli. Planar lipid bilayer electrophysiology and site-directed mutagenesis were used to study the effect of pH on the ion conductive properties of the OmpF channel in its fully open, “nongated” conformation. At low pH we observe a large drop in the OmpF open channel conductance that is accompanied by a substantial increase of the current noise. These channel features are strongly dependent on the salt concentration and we propose that they are originated by competitive binding of cations and protons occurring in the narrow central constriction of the channel. This subtle mechanism reveals to be capital for the channel function because it not only drives the channel sensitivity to pH but is also indispensable for the particularly efficient permeation mechanism of the channel at physiological conditions (~neutral pH).Display Omitted► We show that OmpF porin displays an unusual salt dependent pH sensitivity. ► A competitive interaction between K⁺ and H⁺ explains the pH-regulated conductance. ► Two acidic residues at the channel eyelet are involved in the proton block. ► These residues seem essential for an efficient ion permeation at neutral pH.

Keywords: OmpF channel; Proton block; pH sensitivity; Channel conductance regulation; Bacterial porin

Structural and functional analysis of extracellular loop 4 of the Nhe1 isoform of the Na⁺/H⁺ exchanger by Brian L. Lee; Yongsheng Liu; Xiuju Li; Brian D. Sykes; Larry Fliegel (pp. 2783-2790).

The mammalian Na⁺/H⁺ exchanger isoform 1 (NHE1) is a ubiquitously expressed plasma membrane protein. It regulates intracellular pH by removing a single intracellular H⁺ in exchange for one extracellular Na⁺. The membrane domain of NHE1 comprises the 500 N-terminal amino acids and is made of 12 transmembrane segments. The extracellular loops of the transmembrane segments are thought to be involved in cation coordination and inhibitor sensitivity. We have characterized the structure and function of amino acids 278–291 representing extracellular loop 4. When mutated to Cys, residues F277, F280, N282 and E284 of EL4 were sensitive to mutation and reaction with MTSET inhibiting NHE1 activity. In addition they were found to be accessible to extracellular applied MTSET. A peptide of the amino acids of EL4 was mostly unstructured suggesting that it does not provide a rigid structured link between TM VII and TM VIII. Our results suggest that EL4 makes an extension upward from TM VII to make up part of the mouth of the NHE1 protein and is involved in cation selectivity or coordination. EL4 provides a flexible link to TM VIII which may either allow movement of TM VII or allow TM VIII to not be adjacent to TM VII.Display Omitted► We characterized extracellular loop 4 of the Na⁺/H⁺ exchanger. ► Residues F277, F280, N282 and E284 of EL4 were reactive with external MTSET. ► EL4 was unstructured and it may serve as a flexible linkage between transmembrane segments. ► EL4 extends from transmembrane segment VII to make up part of the cation channel.

Keywords: Abbreviations; CHO; Chinese hamster ovary; EL; extracellular loop; HA; hemagglutinin; NHE1; Na; ⁺; /H; ⁺; exchanger type 1 isoform; MTSES; ([2-Sulfonatoethyl]methanethiosulfonate); MTSET; ([2-(Trimethylammonium)ethyl] methanethiosulfonate); TM; transmembrane segmentCation coordination; Cysteine scanning mutagenesis; Extracellular loop; Membrane protein; Na; ⁺; /H; ⁺; exchanger

Mouse TSPO in a lipid environment interacting with a functionalized monolayer by David Teboul; Sylvie Beaufils; Jean-Christophe Taveau; Soria Iatmanen-Harbi; Anne Renault; Catherine Venien-Bryan; Véronique Vie; Jean-Jacques Lacapere (pp. 2791-2800).

Translocator protein TSPO is a membrane protein highly conserved in evolution which does not belong to any structural known family. TSPO is involved in physiological functions among which transport of molecules such as cholesterol to form steroids and bile salts in mammalian cells. Membrane protein structure determination remains a difficult task and needs concomitant approaches (for instance X-ray- or Electron-crystallography and NMR). Electron microscopy and two-dimensional crystallization under functionalized monolayers have been successfully developed for recombinant tagged proteins. The difficulty comes from the detergent carried by membrane proteins that disrupt the lipid monolayer.We identified the best conditions for injecting the histidine tagged recombinant TSPO in detergent in the subphase and to keep the protein stable. Reconstituted recombinant protein into a lipid bilayer favors its adsorption to functionalized monolayers and limits the disruption of the monolayer by reducing the amount of detergent. Finally, we obtained the first transmission electron microscopy images of recombinant mouse TSPO negatively stained bound to the lipid monolayer after injection into the subphase of pre-reconstituted TSPO in lipids. Image analysis reveals that circular objects could correspond to an association of at least four monomers of mouse TSPO.The different amino acid compositions and the location of the polyhistidine tag between bacterial and mouse TSPO could account for the formation of dimer versus tetramer, respectively. The difference in the loop between the first and second putative transmembrane domain may contribute to distinct monomer interaction, this is supported by differences in ligand binding parameters and biological functions of both proteins.Display Omitted► Oligomeric structure of mammalian TSPO in membrane under functionalized monolayer. ► Low detergent over TSPO ratio preserves monolayer structure. ► His-tagged TSPO appears like “donuts” adsorbed under Ni-NTA functionalized monolayer. ► Electron microscopy image analysis suggests that mouse TSPO might form a tetramer. ► Bacterial and mammalian TSPO with high homologue sequences form different polymers.

Keywords: Abbreviations; PK 11195; N-butan-2-yl-1-(2-chlorophenyl)-N-methylisoquinoline-3-carboxamide; TSPO; 18; kDa translocator protein; DOPC; 1,2-dioleoyl-sn-glycero-3-phosphocholine; DOGS-NTA-Ni; 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentanyl)iminodiacetic acid)succinyl] (Nickel salt); DMPC; 1,2-dimyristoyl-sn-glycero-3-phosphocholine; DMPE; 1,2-dimyristoyl-sn-glycero-3phosphoethanolamine; BAM; Brewster Angle Microscopy; TEM; Transmission Electron Microscopy; BB; BioBead; PPIX; Protoporphyrin IX; PET; Positron Emission TomographyMembrane protein; Electron microscopy; Protein structure; Lipid membrane

Comparative studies of polyethylene glycol-modified liposomes prepared using different PEG-modification methods by Koji Nakamura; Keiko Yamashita; Yuki Itoh; Keisuke Yoshino; Shigenori Nozawa; Hiroaki Kasukawa (pp. 2801-2807).

To address the issue of excess polyethylene glycol (PEG)-lipid degradation observed when PEG-modified liposomes are prepared using the pH-gradient method, a concept using a novel PEG-modification method, called the post-modification method, was proposed and evaluated. To assess the proof concept, a preservation–stability study and a pharmacokinetic study were performed that compared the conventional PEG-modification method, called the pre-modification method, with the post-modification method. The results show that PEG-lipid degradation could be markedly inhibited in the post-modification method. Furthermore, the post-modification method could be used without any manufacturing process difficulties, especially with high PEG-lipid content. In addition, a higher blood circulation capability was observed in the post-modification method. Through comparative studies, it was found that the post-modification method was advantageous compared to the pre-modification method. In conclusion, the post-modification method has the potential to be a novel PEG-modification method that can achieve a higher preservation stability of PEG-lipid, a greater ease of manufacturing, and a higher blood circulation capability, especially in the manufacturing of pH-gradient liposomal products.Display Omitted► To avoid PEG-lipid degradation, a novel PEG-modification method was proposed. ► PEG-lipid degradation could be markedly inhibited in the proposed method. ► The proposed method could have ease of manufacturing relative to conventional one. ► The higher blood circulation capability was observed in the proposed method. ► The proposed method would be a great advantage relative to conventional one.

Keywords: Polyethylene glycol (PEG); pH-gradient method; PEG modification method; PEG-modified liposomes; PEG-lipid degradation

An aryleneethynylene fluorophore for cell membrane staining by Antonio Cardone; Francesco Lopez; Francesco Affortunato; Giovanni Busco; Aldebaran M. Hofer; Rosanna Mallamaci; Carmela Martinelli; Matilde Colella; Gianluca M. Farinola (pp. 2808-2817).

The use of an amphiphilic aryleneethynylene fluorophore as a plasma membrane marker in fixed and living mammalian cells and liposome model systems is demonstrated. We show here that the optical properties of the novel dye are almost independent on pH, in the range 5.0–8.0. Spectroscopic characterization performed on unilamellar liposomes ascertained that the fluorescence intensity of the aryleneethynylene fluorophore greatly increases after incorporation in lipidic membranes. Experiments performed on different mammalian cells demonstrated that the novel membrane marker exhibits fast staining and a good photostability that make it a suitable tool for live cell imaging. Importantly, the aryleneethynylene fluorophore was also shown to be a fast and reliable blue membrane marker in classical multicolor immunofluorescence experiments. This study adds new important findings to the recent exploitation of the wide class of aryleneethynylene molecules as luminescent markers for biological investigations.The effective staining of fixed and living cell membranes with an aryleneethynylene fluorophore is demonstrated.Display Omitted► An aryleneethynylene-based fluorophore as a suitable cell membrane marker is demonstrated. ► Structural flexibility of the oligo-aryleneethynylene fluorophores. ► Chemical and fluorescence stability in a wide pH range. ► Fast staining and good photostability for live cell imaging. ► A new blue membrane marker for multicolor labeling in immunofluorescence experiments.

Keywords: Abbreviations; INS1-E cells; insulin secreting beta cells; BHK-21 cells; Baby Hamster Kidney cells; DMSO; dimethyl sulfoxide; DDAB; dimethyl–dioctadecyl ammonium bromide; HEPES; (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); DPH; 1,6-Diphenyl-1,3,5-hexatriene; DHPE Oregon Green; 1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine Oregon GreenAryleneethynylene; Fluorescent marker; pH-insensitive; Membranes; Cross-coupling

NS4A and NS4B proteins from dengue virus: Membranotropic regions by Nemesio Henrique Nemésio; Francis Palomares-Jerez; Villalain José Villalaín (pp. 2818-2830).

Proteins NS4A and NS4B from Dengue Virus (DENV) are highly hydrophobic transmembrane proteins which are responsible, at least in part, for the membrane arrangements leading to the formation of the viral replication complex, essential for the viral life cycle. In this work we have identified the membranotropic regions of DENV NS4A and NS4B proteins by performing an exhaustive study of membrane rupture induced by NS4A and NS4B peptide libraries on simple and complex model membranes as well as their ability to modulate the phospholipid phase transitions P_β′–L_α of DMPC and L_β–L_α/L_α–H_II of DEPE. Protein NS4A presents three membrane active regions coincident with putative transmembrane segments, whereas NS4B presented up to nine membrane active regions, four of them presumably putative transmembrane segments. These data recognize the existence of different membrane-active segments on these proteins and support their role in the formation of the replication complex and therefore directly implicated in the DENV life cycle.Display Omitted► We have studied the membranotropic regions of DENGUE NS4A and NS4B proteins. ► NA4A displayed three membrane-interacting regions, possibly transmembrane domains. ► NS4B showed nine membranotropic regions, both transmembrane and interacting domains. ► These zones should be involved in the formation of the replication complex.

Keywords: Abbreviations; BMP; S,R-bis(monooleoylglycero)phosphate; BPI; bovine liver L-α-phosphadidylinositol; BPS; bovine brain L-α-phosphatidylserine; CF; 5-carboxyfluorescein; CHOL; cholesterol; CL; bovine heart cardiolipin; DENV; dengue virus; DEPE; 1,2-Dielaidoyl-sn-glycero-3-phosphatidylethanolamine; DMPC; 1,2-dimyristoyl-; sn; -glycero-3-phosphatidylcholine; DPH; 1,6-diphenyl-1,3,5-hexatriene; DSC; differential scanning calorimetry; EPA; egg L-α-phosphatidic acid; EPC; egg L-α-phosphatidylcholine; EPG; egg L-α-phosphatidylglycerol; ER; endoplasmic reticulum; ESM; egg sphingomyelin; HCV; hepatitis C virus; LEM; late endosome membrane; LUV; large unilamellar vesicles; MLV; multilamellar vesicles; NS; non-structural protein; TFE; 2,2,2-Trifluoroethanol; T; _m; temperature of the gel-to‐liquid crystalline phase transition; TM; transmembrane domain; TPE; egg transphosphatidylated L-α-phosphatidylethanolamineDENV replication; DENV; Lipid–peptide interaction; Membranous web; Replication complex

Assessment of the relevance of supported planar bilayers for modeling specific interactions between glycodendrimeric porphyrins and retinoblastoma cells by Ali Makky; Katia Daghildjian; Jean-Philippe Michel; Philippe Maillard; Véronique Rosilio (pp. 2831-2838).

Glycodendrimeric porphyrins seem promising photosensitizers usable in photodynamic therapy. Evidence of their ability to interact with an artificial supported bilayer membrane exhibiting a model sugar receptor has been previously shown. In the present work, the interaction of the glycodendrimeric porphyrins with retinoblastoma cells bearing the actual sugar receptor has been assessed by both classical cell cultures and an original approach using the quartz crystal microbalance (QCM-D). Our results showed that unlike cell cultures, QCM-D allowed analyzing the mechanism of interaction of the glycodendrimeric porphyrins with the sugar receptor. Not only was molecular recognition demonstrated, but our methodology also proved efficient to discriminate between the studied compounds, depending on the presence of carbohydrate, and the spacer length.Display Omitted► Assessment of dendrimeric porphyrin phototoxicity against retinoblastoma (Y79) cells. ► Analysis of dendrimeric porphyrin interaction with fetal calf serum. ► Building of an Y79 cell-sensor for evaluation of specific glycodendrimeric porphyrin–sugar receptor interactions. ► Discrimination between nonglyconjugated and glycoconjugated dendrimeric porphyrins. ► Comparison of the Y79 cell model with an artificial lipid-concanavalin A bilayer.

Keywords: Photodynamic therapy; Retinoblastoma cell; Dendrimeric porphyrins; Liposome; Sugar-receptor interaction; QCM-D

Binding of moesin and ezrin to membranes containing phosphatidylinositol (4,5) bisphosphate: A comparative study of the affinity constants and conformational changes by Ofelia Maniti; Nada Khalifat; Kriti Goggia; Fabien Dalonneau; Guerin Christophe Guérin; Laurent Blanchoin; Laurence Ramos; Catherine Picart (pp. 2839-2849).

The plasma membrane–cytoskeleton interface is a dynamic structure participating in a variety of cellular events. Moesin and ezrin, proteins from the ezrin/radixin/moesin (ERM) family, provide a direct linkage between the cytoskeleton and the membrane via their interaction with phosphatidylinositol 4,5-bisphosphate (PIP₂). PIP₂ binding is considered as a prerequisite step in ERM activation. The main objective of this work was to compare moesin and ezrin interaction with PIP₂-containing membranes in terms of affinity and to analyze secondary structure modifications leading eventually to ERM activation. For this purpose, we used two types of biomimetic model membranes, large and giant unilamellar vesicles. The dissociation constant between moesin and PIP₂-containing large unilamellar vesicles or PIP₂-containing giant unilamellar vesicles was found to be very similar to that between ezrin and PIP₂-containing large unilamellar vesicles or PIP₂-containing giant unilamellar vesicles. In addition, both proteins were found to undergo conformational changes after binding to PIP₂-containing large unilamellar vesicles. Changes were evidenced by an increased sensitivity to proteolysis, modifications in the fluorescence intensity of the probe attached to the C-terminus and in the proportion of secondary structure elements.Display Omitted► We studied the affinity of moesin with PIP2-containing LUV and GUV membranes. ► We investigated the secondary structure of moesin and ezrin in solution by FTIR spectroscopy. ► We proved that ezrin and moesin interaction with PIP2-LUVs increased sensitivity to chymotrypsin. ► We proved that interaction with PIP2-LUVs leads a change in the secondary structure of the proteins.

Keywords: ERM protein; Phosphoinositide; Membrane–protein interaction; Vesicles; Fluorescence spectroscopy; Infrared spectroscopy

Effect of 2-(4-fluorophenylamino)-5-(2,4-dihydroxyphenyl)-1,3,4-thiadiazole on the molecular organisation and structural properties of the DPPC lipid multibilayers by Kaminski Daniel M. Kamiński; Arkadiusz Matwijczuk; Damian Pociecha; Gorecka Ewa Górecka; Andrzej Niewiadomy; Mirosława Dmowska; Gagos Mariusz Gagoś (pp. 2850-2859).

Interactions and complex formation between lipids and biologically active compounds are crucial for better understanding of molecular mechanisms occurring in living cells. In this paper a molecular organisation and complex formation of 2-(4-fluorophenylamino)-5-(2,4-dihydroxybenzeno)-1,3,4-thiadiazole (FABT) in DPPC multibilayers are reported. The simplified pseudo binary phase diagram of this system was created based on the X-ray diffraction study and fourier transform infrared spectroscopic data. The detailed analysis of the refraction effect indicates a much higher concentration of FABT in the polar zones during phase transition. Both the lipid and the complex ripple after cooling. It was found that FABT occupied not only the hydrophilic zones of the lipid membranes but also partly occupied the central part of the non polar zone. The infrared spectroscopy study reveals that FABT strongly interact with hydrophilic (especially PO₂⁻) and hydrophobic (especially “kink” vibrations of CH₂ group). The interactions of FABT molecules with these groups are responsible for changes of lipid multibilayers observed in X-ray diffraction study.Display Omitted► X-ray diffraction study of molecular organisation ofFABT inDPPC lipid multibilayers ► Complex formation betweenFABT andDPPC in multibilayers ► Pseudo binary phase diagram forFABT–DPPC system

Keywords: 1,3,4-Thiadiazole; DPPC lipid; Multibilayer; X-ray diffraction; FTIR spectroscopy

Ganglioside GM1 forces the redistribution of cholesterol in a biomimetic membrane by V. Rondelli; G. Fragneto; S. Motta; E. Del Favero; P. Brocca; S. Sonnino; Cantu L. Cantù (pp. 2860-2867).

Neutron reflectivity has been applied to investigate different mixed asymmetric lipid systems, in the form of single “supported+floating” bilayers, made of phospholipids, cholesterol and GM1 ganglioside (Neu5Acα2-3(Galβ1-3GalNAcβ1-4)Galβ1-4Glcβ1Cer)) in bio-similar mole ratios. Bilayer preparation was carried out layer-by-layer with the Langmuir–Blodgett Langmuir–Schaefer techniques, allowing for compositional asymmetry in the system buildup. It is the first time that such a complex model membrane system is reported. Two important conclusions are drawn. First, it is experimentally shown that the presence of GM1 enforces an asymmetry in cholesterol distribution, opposite to what happens for a GM1-free membrane that, submitted to a similar procedure, results in a full symmetrization of cholesterol distribution. We underline that natural cholesterol has been used. Second, and most interesting, our results suggest that a preferential asymmetric distribution of GM1 and cholesterol is attained in a model membrane with biomimetic composition, revealing that a true coupling between the two molecular species occurs.Display Omitted► Asymmetric floating membranes with mixed individual leaflets have been realized. ► GM1 and cholesterol constitute a collective structural pair across the membrane. ► Floating membrane tolerates incubation with inserting lipids.

Keywords: Floating membrane; GM1 ganglioside; Lipid membrane; Langmuir–Blodgett; Neutron reflectivity; Cholesterol

Modulation of P2X3 receptors by spider toxins by Natalia V. Kabanova; Alexander A. Vassilevski; Olga A. Rogachevskaja; Marina F. Bystrova; Yuliya V. Korolkova; Kirill A. Pluzhnikov; Roman A. Romanov; Eugene V. Grishin; Stanislav S. Kolesnikov (pp. 2868-2875).

Recently, the novel peptide named purotoxin-1 (PT1) has been identified in the venom of the spider Geolycosa sp. and shown to exert marked modulatory effects on P2X3 receptors in rat sensory neurons. Here we studied another polypeptide from the same spider venom, purotoxin-2 (PT2), and demonstrated that it also affected activity of mammalian P2X3 receptors. The murine and human P2X3 receptors were heterologously expressed in cells of the CHO line, and nucleotide-gated currents were stimulated by CTP and ATP, respectively. Both PT1 and PT2 negligibly affected P2X3-mediated currents elicited by brief pulses of the particular nucleotide. When subthreshold CTP or ATP was added to the bath to exert the high-affinity desensitization of P2X3 receptors, both spider toxins strongly enhanced the desensitizing action of the ambient nucleotides. At the concentration of 50nM, PT1 and PT2 elicited 3–4-fold decrease in the IC₅₀ dose of ambient CTP or ATP. In contrast, 100nM PT1 and PT2 negligibly affected nucleotide-gated currents mediated by mP2X2 receptors or mP2X2/mP2X3 heteromers. Altogether, our data point out that the PT1 and PT2 toxins specifically target the fast-desensitizing P2X3 receptor, thus representing a unique tool to manipulate its activity.The spider toxin PT1 enhances the high affinity desensitization of the mouse P2X3 receptor. (A) Time evolution of mP2X3 currents in control (o), in the presence of 50 nM PT1 (●), and in the presence 40 nM CTP and 50 nM PT1 (▲). (B) mP2X3 current elicited by 100 μM CTP versus ambient CTP concentration in control (▲) and in the presence of 50 nM PT1 (Δ).Display Omitted► Peptides, purotoxin-1 and purotoxin-2, were isolated from the venom of the spider Geolycosa sp. ► The sensitivity of recombinant P2X3 receptors to the spider toxins was studied. ► Purotoxin-1 and purotoxin-2 slowed down inactivation of P2X3 currents. ► The spider toxins shifted an inhibitory curve for P2X3 high-affinity desensitization.

Keywords: P2X3 receptors; High-affinity desentisitization; Purotoxin; Geolycosa; sp. spider venom

Alpha-synuclein pore forming activity upon membrane association by Laura Tosatto; Alberto O. Andrighetti; Nicoletta Plotegher; Valeria Antonini; Isabella Tessari; Leonardo Ricci; Luigi Bubacco; Mauro Dalla Serra (pp. 2876-2883).

Alpha-synuclein is a natively unfolded protein widely expressed in neurons at the presynaptic level. It is linked to Parkinson's disease by two lines of evidence: amyloid fibrils of the protein accumulate in patients’ brains and three genetic mutants cause autosomal dominant forms of the disease. The biological role of the protein and the mechanisms involved in the etiopathogenesis of Parkinson's disease are still unknown. Membrane binding causes the formation of an amphipathic alpha-helix, which lies on the surface without crossing the bilayer. Recent observations however reported that the application of a voltage induces a pore-like activity of alpha-synuclein. This study aims to characterize the pore forming activity of the protein starting from its monomeric form. In particular, experiments with planar lipid membranes allowed recording of conductance activity bursts with a defined and reproducible fingerprint. Additional experiments with deletion mutants and covalently bound alpha-synuclein dimers were performed to understand both pore assembly and stoichiometry. The information acquired allowed formulation of a model for pore formation at different conductance levels.Alpha-synuclein model for pore formation at different conductance levels.Display Omitted► Monomeric alpha-synuclein causes bursts of conductance in Planar Lipid Membranes.► Three levels of sequentially related conductance levels were identified.► Covalently bound alpha-synuclein dimers allow to explore relative orientation of the monomers in the pore.► Alpha-synuclein forms barrel stave pores with the progressive insertion of four helical units.► A model of pore formation accounting for the conductance levels is proposed.

Keywords: Alpha-synuclein; Parkinson's disease; Planar lipid membrane; Pore forming activity; Pore model

Structure of solid-supported lipid membrane probed by noble metal nanoparticle deposition by Hyeun Hwan An; Seung Jae Lee; Hee-Soo Kim; Won Bae Han; Chong Seung Yoon (pp. 2884-2891).

Direct deposition of a noble metal layer onto a solid-supported membrane was proposed as an indirect microscopy tool to visually observe different lipid phases that may develop in the lipid membrane. The method relied on the different permeability of the lipid membrane towards the incident atoms during deposition. Liquid state or structural defects such as phase boundaries, step ledges in a multi-lamellar stack, and pores permitted the metal atoms to penetrate and nucleate inside the membrane whereas rigid gel state was relatively impermeable to the incident atoms, thus enabling visualization of liquid phase or structural defects inside the gel state. Based on the proposed method, we demonstrated the phase states resulting from thermotropic transitions of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), dioleoylphosphatidylethanolamine (DOPE)/DPPC mixture, and 1,2-dioleoyl-3-trimethylammonium propane (DOTAP). Although the proposed method does not allow in-situ observation of equilibrium states, the method should be an excellent complementary tool for visualizing the lipid phases as the method can resolve fine structural details (up to tens of nanometer scale) as seen in the DPPC membrane while providing macroscopic images (up to several micrometers).Display Omitted► We proposed an indirect microscopy tool to visually observe different lipid phases. ► The method relied on the different permeability of the lipid membrane. ► We demonstrated the phase states resulting from thermotropic transitions of lipids.

Keywords: Lipid membrane; Nanoparticle; Phase transition

Lipid organization regulates annexin A2 Ca2⁺-sensitivity for membrane bridging and its modulator effects on membrane fluidity by Françoise Illien; Hong-Rong Piao; Coue Marine Coué; Chiara di Marco; Jesus Ayala-Sanmartin (pp. 2892-2900).

Annexin A2 (AnxA2) is a phospholipid binding protein that has been implicated in many membrane-related cellular functions. AnxA2 is able to bind different acidic phospholipids such as phosphatidylserine (PS) and phosphatidylinositol-4,5-bisphosphate (PI2P). This binding is mediated by Ca2⁺-dependent and Ca2⁺-independent mechanisms. The specific functions of annexin A2 related to these two phospholipids and the molecular mechanisms involved in their interaction remain obscure. Herein we studied the influence of lipid composition on the Ca2⁺-dependency of AnxA2-mediated membrane bridging and on membrane fluidity. Membrane models of ten different lipid compositions and detergent-resistant membranes from two cellular sources were investigated. The results show that the AnxA2-mediated membrane bridging requires 3 to 50 times less calcium for PS-membranes than for PI2P-membranes. Membrane fluidity was measured by the ratiometric fluorescence parameter generalized polarization method with two fluorescent probes. Compared to controls containing low phospholipid ligand, AnxA2 was found to reduce the membrane fluidity of PI2P-membranes twice as much as the PS-membranes in the presence of calcium. On the contrary, at mild acidic pH in the absence of calcium AnxA2 reduces the fluidity of the PS-membranes more than the PI2P-membranes. The presence of cholesterol on the bilayer reduced the AnxA2 capacity to reduce membrane fluidity. The presented data shed light on the specific roles of PI2P, PS and cholesterol present on membranes related to the action of annexin A2 as a membrane bridging molecule during exocytosis and endocytosis events and as a plasma membrane domain phospholipid packing regulator.Display Omitted► Lipid organization regulates the Ca2⁺ concentration for annexin A2 membrane bridging. ► Annexin A2 reduces differentially the membrane fluidity of PS and PI2P membranes. ► The presence of cholesterol reduces the AnxA2 capacity to rigidify the membranes.

Keywords: Abbreviations; Anx; Annexin; DRM; detergent-resistant membranes; LUV; large unilamellar vesicles; MDCK; Madin Darby canine kidney; PE; phosphatidylethanolamine; PI2P; phosphatidylinositol-4,5-bisphosphate; PS; phosphatidylserineAnionic phospholipid; Annexin A2; Calcium; Cholesterol; Membrane domain; Membrane fluidity

Comparative studies of irinotecan-loaded polyethylene glycol-modified liposomes prepared using different PEG-modification methods by Keisuke Yoshino; Koji Nakamura; Yoko Terajima; Akinobu Kurita; Takeshi Matsuzaki; Keiko Yamashita; Masashi Isozaki; Hiroaki Kasukawa (pp. 2901-2907).

Recently, a polyethylene glycol (PEG)-modification method for liposomes prepared using pH-gradient method has been proposed. The differences in the pharmacokinetics and the impact on the antitumor effect were examined; however the impact of PEG-lipid molar weight has not been investigated yet. The main purpose of this study is to evaluate the impact of PEG-lipid molar weight against the differences in the pharmacokinetics, the drug-release profile, and the antitumor effect between the proposed PEG-modification method, called the post-modification method, and the conventional PEG-modification method, called the pre-modification method. Various comparative studies were performed using irinotecan as a general model drug. The results showed that PEG-lipid degradation could be markedly inhibited in the post-modification method. Furthermore, prolonged circulation time was observed in the post-modification method. The sustained drug-release was observed in the post-modification method by the results of the drug-releasing test in plasma. Moreover, a higher antitumor effect was observed in the post-modification method. It was also confirmed that the same behaviors were observed in all comparative studies even though the PEG molecular weight was lower.In conclusion, the post-modification method has the potential to be a valuable PEG-modification method that can achieve higher preservation stability of PEG-lipid, prolonged circulation time, and higher antitumor effect with only half the amount of PEG-lipid as compared to the pre-modification method. Furthermore, it was demonstrated that PEG₅₀₀₀-lipid would be more desirable than PEG₂₀₀₀-lipid since it requires much smaller amount of PEG-lipid to demonstrate the same performances.Display Omitted

Keywords: Polyethylene glycol (PEG) molar weight; pH-gradient method; PEG-modification method; Irinotecan; Prolonged circulation time; Antitumor effect

Cation selectivity is a conserved feature in the OccD subfamily of Pseudomonas aeruginosa by Jiaming Liu; Aaron J. Wolfe; Elif Eren; Jagamya Vijayaraghavan; Mridhu Indic; Bert van den Berg; Liviu Movileanu (pp. 2908-2916).

To achieve the uptake of small, water-soluble nutrients, Pseudomonas aeruginosa, a pathogenic Gram-negative bacterium, employs substrate-specific channels located within its outer membrane. In this paper, we present a detailed description of the single-channel characteristics of six members of the outer membrane carboxylate channel D (OccD) subfamily. Recent structural studies showed that the OccD proteins share common features, such as a closely related, monomeric, 18-stranded β-barrel conformation and large extracellular loops, which are folded back into the channel lumen. Here, we report that the OccD proteins displayed single-channel activity with a unitary conductance covering an unusually broad range, between 20 and 670pS, as well as a diverse gating dynamics. Interestingly, we found that cation selectivity is a conserved trait among all members of the OccD subfamily, bringing a new distinction between the members of the OccD subfamily and the anion-selective OccK channels. Conserved cation selectivity of the OccD channels is in accord with an increased specificity and selectivity of these proteins for positively charged, carboxylate-containing substrates.Display Omitted► We present detailed single-channel features of six members of the OccD subfamily. ► OccD proteins displayed a unitary conductance covering a broad range. ► Cation selectivity is a conserved trait among the members of the OccD subfamily.

Keywords: Abbreviations; LPS; Lipopolysaccharide; OM; Outer membrane of Gram-negative bacterium; Occ; Outer membrane carboxylate channel family of; P. aeruginosa; OccD; Outer membrane carboxylate channel D subfamily of; P. aeruginosa; OccK; Outer membrane carboxylate channel K subfamily of; P. aeruginosa; OmpC; Outer membrane protein C of; E. coli; OmpF; Outer membrane protein F of; E. coliSpontaneous gating; Single-molecule biophysics; Single-channel electrical recording; The OprD family; The OccD subfamily

Anticancer mechanisms of action of two small amphipathic β^2,2-amino acid derivatives derived from antimicrobial peptides by Dominik Ausbacher; Gunbjørg Svineng; Terkel Hansen; Strom Morten B. Strøm (pp. 2917-2925).

We have recently discovered that small antimicrobial β^2,2-amino acid derivatives (Mw<500) also display activity against cancer cells. To explore their drug potential, we have presently investigated the mechanisms of action of two derivatives BAA-1 (IC₅₀ 8.1μg/ml) and BAA-2 (IC₅₀ 3.8μg/ml) on Ramos human Burkitt's lymphoma cells. Studies using annexin-V-FITC/propidium iodide staining and flow cytometry revealed essential mechanistic differences, which was confirmed by screening for active caspases, investigation of mitochondrial membrane potential, and electron microscopy studies. Our results indicated that BAA-1 killed Ramos cells by destabilizing the cell membrane, whereas BAA-2 caused apoptosis by the mitochondrial-mediated pathway.Display Omitted► Investigation of the anticancer activity of two small β^2,2-amino acid derivatives► Elucidation of the mechanism of action to explore drug potential ► Induction of necrosis and apoptosis via the mitochondria mediated pathway ► Similar cancer cell killing properties as reported for larger anticancer peptides ► Marginal structural differences with considerable impact on cell killing properties

Keywords: Abbreviations; AIF; Apoptosis-inducing factor; AMP; antimicrobial peptide; ACP; anticancer peptide; DMSO; dimethyl sulfoxide; FBS; fetal bovine serum; FITC; fluorescein isothiocyanate; HBSS; Hank's balanced salt solution; Lfcin B; bovine lactoferricin; NCI; National Cancer Institute; PBS; phosphate buffered saline; PI; propidium iodide; SEM; scanning electron microscopy; TBTC; tributyltin chloride; TEM; transmission electron microscopy; TMRE; tetramethylrhodamine ethyl ester perchlorateAntimicrobial peptide; Anticancer peptide; Apoptosis; Beta-amino acid; Caspases; Mitochondria

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BBA - Biomembranes (v.1818, #11)

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BBA - Biomembranes (v.1818, #11)