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BBA - Biomembranes (v.1818, #7)

Editorial Board (pp. i).

Adaptation to low body temperature influences pulmonary surfactant composition thereby increasing fluidity while maintaining appropriately ordered membrane structure and surface activity by Lakshmi N.M. Suri; Lynda McCaig; Maria V. Picardi; Olga L. Ospina; Ruud A.W. Veldhuizen; James F. Staples; Fred Possmayer; Li-Juan Yao; Jesus Perez-Gil; Sandra Orgeig (pp. 1581-1589).

The interfacial surface tension of the lung is regulated by phospholipid-rich pulmonary surfactant films. Small changes in temperature affect surfactant structure and function in vitro. We compared the compositional, thermodynamic and functional properties of surfactant from hibernating and summer-active 13-lined ground squirrels ( Ictidomys tridecemlineatus) with porcine surfactant to understand structure-function relationships in surfactant membranes and films. Hibernating squirrels had more surfactant large aggregates with more fluid monounsaturated molecular species than summer-active animals. The latter had more unsaturated species than porcine surfactant. Cold-adapted surfactant membranes displayed gel-to-fluid transitions at lower phase transition temperatures with reduced enthalpy. Both hibernating and summer-active squirrel surfactants exhibited lower enthalpy than porcine surfactant. LAURDAN fluorescence and DPH anisotropy revealed that surfactant bilayers from both groups of squirrels possessed similar ordered phase characteristics at low temperatures. While ground squirrel surfactants functioned well during dynamic cycling at 3, 25, and 37°C, porcine surfactant demonstrated poorer activity at 3°C but was superior at 37°C. Consequently the surfactant composition of ground squirrels confers a greater thermal flexibility relative to homeothermic mammals, while retaining tight lipid packing at low body temperatures. This may represent the most critical feature contributing to sustained stability of the respiratory interface at low lung volumes. Thus, while less effective than porcine surfactant at 37°C, summer-active surfactant functions adequately at both 37°C and 3°C allowing these animals to enter hibernation. Here further compositional alterations occur which improve function at low temperatures by maintaining adequate stability at low lung volumes and when temperature increases during arousal from hibernation.► Hibernation in ground squirrels increases monounsaturated surfactant phospholipids. ► These surfactant membranes have lower phase transition temperatures and enthalpy. ► However, they maintain similar ordered phase characteristics at low temperatures. ► Unlike porcine, both squirrel surfactants function well between 3 and 37°C. ► This thermal flexibility enables a balance between dynamic behaviour and stability.

Keywords: Phase transition; Differential scanning calorimetry; LAURDAN fluorescence spectroscopy; Anisotropy; Electrospray ionization mass spectrometry; Captive bubble surfactometry

Indole prevents Escherichia coli cell division by modulating membrane potential by Catalin Chimerel; Christopher M. Field; Pinero-Fernandez Silvia Piñero-Fernandez; Ulrich F. Keyser; David K. Summers (pp. 1590-1594).

Indole is a bacterial signalling molecule that blocks E. coli cell division at concentrations of 3–5mM. We have shown that indole is a proton ionophore and that this activity is key to the inhibition of division. By reducing the electrochemical potential across the cytoplasmic membrane of E. coli, indole deactivates MinCD oscillation and prevents formation of the FtsZ ring that is a prerequisite for division. This is the first example of a natural ionophore regulating a key biological process. Our findings have implications for our understanding of membrane biology, bacterial cell cycle control and potentially for the design of antibiotics that target the cell membrane.► Indole induces charge transport across reconstituted lipid membranes. ► Indole reduces the E. coli membrane potential. ► Membrane potential is required for the formation of the macromolecular divisome. ► Indole blocks the formation of the macromolecular divisome. ► Indole reversibly blocks Escherichia coli cell division.

Keywords: Membrane potential; Proton ionophore; Indole; Bacteria; Cell division; Escherichia coli

Interactions of the intact FsrC membrane histidine kinase with its pheromone ligand GBAP revealed through synchrotron radiation circular dichroism by Simon G. Patching; Shalini Edara; Pikyee Ma; Jiro Nakayama; Rohanah Hussain; Giuliano Siligardi; Mary K. Phillips-Jones (pp. 1595-1602).

FsrC is the membrane-bound histidine kinase component of the Fsr two-component signal transduction system involved in quorum sensing in the hospital-acquired infection agent Enterococcus faecalis. Synchrotron radiation circular dichroism spectroscopy was used here to study the intact purified protein solubilised in detergent micelles. Conditions required for FsrC stability in detergent were firstly determined and tested by prolonged exposure of stabilised protein to far-ultraviolet radiation. Using stabilised purified protein, far-ultraviolet synchrotron radiation circular dichroism revealed that FsrC is 61% α-helical and that it is relatively thermostable, retaining at least 57% secondary structural integrity at 90°C in the presence or absence of gelatinase biosynthesis-activating pheromone (GBAP). Whilst binding of the quorum pheromone ligand GBAP did not significantly affect FsrC secondary structure, near-ultraviolet spectra revealed that the tertiary structure in the regions of the Tyr and Trp residues was significantly affected. Titration experiments revealed a calculated k_d value of 2μM indicative of relatively loose binding of gelatinase biosynthesis-activating pheromone to FsrC. Although use of synchrotron radiation circular dichroism has been applied to membrane proteins previously, to our knowledge this is the first report of its use to determine a k_d value for an intact membrane protein. Based on our findings, we suggest that synchrotron radiation circular dichroism will be a valuable technique for characterising ligand binding by other membrane sensor kinases and indeed other membrane proteins in general. It further provides a valuable screening tool for membrane protein stability under a range of detergent conditions prior to downstream structural methods such as crystallisation and NMR experiments particularly when lower detergent concentrations are used.► The intact purified FsrC membrane histidine kinase was studied by SRCD spectroscopy. ► Far-UV spectra revealed that FsrC is 61% α-helical and relatively thermostable. ► Tertiary structure was found to be significantly affected upon GBAP ligand binding. ► Titration experiments revealed a calculated k_d value of 2μM. ► SRCD provides a valuable screening tool for membrane protein stability under a range of detergent conditions.

Keywords: Abbreviations; DDM; n-dodecyl-β-; d; -maltoside; SRCD; synchrotron radiation circular dichroism; CD; circular dichroism; GBAP; gelatinase biosynthesis-activating pheromone; IPTG; iso-propylthiogalactoside; HT; high tension; UV; ultraviolet; MRW; mean residue weight; CMC; critical micelle concentration; ESD; extracellular sensory domain; TM; transmembrane domain; SDS-PAGE; sodium dodecyl sulphate-polyacrylamide gel electrophoresis; k; _d; dissociation constantMembrane histidine kinase; FsrC; Gelatinase biosynthesis-activating pheromone (GBAP); Synchrotron radiation circular dichroism; Ligand interactions; k; _d

Physicochemical characterization of anionic lipid-based ternary siRNA complexes by Mamta Kapoor; Diane J. Burgess (pp. 1603-1612).

Physicochemical characterization is a useful tool in understanding lipoplex assemblies and their correlation to biological activity. Anionic lipid-based ternary siRNA complexes composed of anionic liposomes (DOPG/DOPE), calcium ions and siRNA, have recently been shown to be safe and efficient in a breast cancer cell culture model. In the present work, the effects of various formulation parameters such as liposome composition (DOPG/DOPE ratio) and anionic lipid/Ca2⁺/siRNA molar charge ratio, on the physicochemical attributes (particle size, surface charge, siRNA loading efficiency and serum stability) of these ternary anionic lipoplexes were evaluated. Particle size, siRNA loading efficiency and serum stability correlated with the in vitro silencing efficiency of these lipoplexes. For example, large lipoplex particles (5/2.5/1 anionic lipid/Ca2⁺/siRNA molar charge ratio) showed less efficient silencing while absolute serum stability and high siRNA loading (1.3/2.5/1 anionic lipid/Ca2⁺/siRNA molar charge ratio), exhibited maximum silencing in breast cancer cells. The physicochemical properties also indicated that the siRNA exists in the complexed and/or encapsulated form within the lipoplexes, depending on the anionic lipid/siRNA charge ratio. Based on these studies a model representing lipid–siRNA association within the anionic lipoplexes prepared under various formulation conditions is proposed. Physicochemical attributes can be utilized to estimate in vitro activity of lipid–siRNA complexes and understand their morphology.Schematic representation of ternary anionic siRNA lipoplexes prepared with: (a) low anionic lipid/siRNA ratios indicating the ‘complexed’ siRNA form; and (b) high anionic lipid/siRNA molar charge ratios indicating both ‘encapsulated’ as well as ‘complexed’ siRNA forms.Display Omitted► Physicochemical properties and biological activity of anionic lipoplexes correlated. ► Physicochemical studies were pivotal in formulation optimization for high silencing. ► Formulation components (lipid, Ca) contributed to serum stability and siRNA loading. ► Structural model of lipoplexes correlated to activity for different formulations. ► Information generated will be useful for product optimization and consistency.

Keywords: Physicochemical characterization; Anionic lipoplex; siRNA delivery; Lipoplex morphology; Silencing efficiency; Loading efficiency

Physicochemical characterization of anionic lipid-based ternary siRNA complexes by Mamta Kapoor; Diane J. Burgess (pp. 1603-1612).

Keywords: Physicochemical characterization; Anionic lipoplex; siRNA delivery; Lipoplex morphology; Silencing efficiency; Loading efficiency

Structure, activity and interactions of the cysteine deleted analog of tachyplesin-1 with lipopolysaccharide micelle: Mechanistic insights into outer-membrane permeabilization and endotoxin neutralization by Rathi Saravanan; Harini Mohanram; Mangesh Joshi; Prerna N. Domadia; Jaume Torres; Christiane Ruedl; Surajit Bhattacharjya (pp. 1613-1624).

Tachyplesin-1, a disulfide stabilized β-hairpin antimicrobial peptide, can be found at the hemocytes of horse shoe crab Tachypleus tridentatus. A cysteine deleted linear analog of tachyplesin-1 or CDT (KWFRVYRGIYRRR-NH₂) contains a broad spectrum of bactericidal activity with a reduced hemolytic property. The bactericidal activity of CDT stems from selective interactions with the negatively charged lipids including LPS. In this work, CDT–LPS interactions were investigated using NMR spectroscopy, optical spectroscopy and functional assays. We found that CDT neutralized LPS and disrupted permeability barrier of the outer membrane. Zeta potential and ITC studies demonstrated charge compensation and hydrophobic interactions of CDT with the LPS-outer membrane, respectively. Secondary structure of the peptide was probed by CD and FT-IR experiments indicating β-strands and/or β-turn conformations in the LPS micelle. An ensemble of structures, determined in LPS micelle by NMR, revealed a β-hairpin like topology of the CDT peptide that was typified by an extended cationic surface and a relatively shorter segment of hydrophobic region. Interestingly, at the non-polar face, residue R11 was found to be in a close proximity to the indole ring of W2, suggesting a cation–π type interactions. Further, saturation transfer difference (STD) NMR studies established intimate contacts among the aromatic and cationic residues of CDT with the LPS micelle. Fluorescence and dynamic light scattering experiments demonstrated that CDT imparted structural destabilization to the aggregated states of LPS. Collectively, atomic resolution structure and interactions of CDT with the outer membrane-LPS could be exploited for developing potent broad spectrum antimicrobial and anti-sepsis agents.Display Omitted► β-hairpin like structure of cysteine deleted analog of tachyplesin-1 (CDT) in lipopolysaccharide (LPS). ► Structure shows cation–π interactions between W4 and R11 at the non-polar surface. ► Structure and interactions of CDT in LPS may provide insights into antimicrobial activity and endotoxin neutralization.

Keywords: Abbreviations; AMPs; antimicrobial peptides; CDT; cysteine deleted analog of tachyplesin-1; LPS; lipopolysaccharide; NMR; nuclear magnetic resonance; Tr-NOE; transferred nuclear Overhauser effect; NOESY; nuclear Overhauser effect spectroscopy; TOCSY; total correlation spectroscopy; CD; circular dichroism; NOE; nuclear Overhauser enhancement; STD-NMR; saturation transfer difference NMRAntimicrobial peptide; Lipopolysaccharide; NMR; Structure; Endotoxin; STD-NMR

Determining the mechanism of membrane permeabilizing peptides: Identification of potent, equilibrium pore-formers by Aram J. Krauson; Jing He; William C. Wimley (pp. 1625-1632).

To enable selection and characterization of highly potent pore-forming peptides, we developed a set of novel assays to probe 1) the potency of peptide pores at very low peptide concentration; 2) the presence or absence of pores in membranes after equilibration; 3) the interbilayer exchangeability of pore-forming peptides; and 4) the degree to which pore-forming peptides disrupt the bilayer organization at equilibrium. Here, we use these assays to characterize, in parallel, six membrane-permeabilizing peptides belonging to multiple classes. We tested the antimicrobial peptides LL37 and dermaseptin S1, the well-known natural lytic peptides melittin and alamethicin, and the very potent lentivirus lytic peptides LLP1 and LLP2 from the cytoplasmic domain of HIV GP41. The assays verified that that the antimicrobial peptides are not potent pore formers, and form only transient permeabilization pathways in bilayers which are not detectable at equilibrium. The other peptides are far more potent and form pores that are still detectable in vesicles after many hours. Among the peptides studies, alamethicin is unique in that it is very potent, readily exchanges between vesicles, and disturbs the local bilayer structure even at very low concentration. The equally potent LLP peptides do not exchange readily and do not perturb the bilayer at equilibrium. Comparison of these classes of pore forming peptides in parallel using the set of assays we developed demonstrates our ability to detect differences in their mechanism of action. Importantly, these assays will be very useful in high-throughput screening where highly potent pore-forming peptides can be selected based on their mechanism of action.► The mechanisms of six membrane permeabilizing peptides were studied in parallel. ► Novel assays reveal differences in mechanism, potency and dynamics at equilibrium. ► High-throughput selection of stable, equilibrium pores is possible.

Keywords: Pore-forming peptide; Alamethicin; Melittin; LL-37; Dermaseptin

Determining the mechanism of membrane permeabilizing peptides: Identification of potent, equilibrium pore-formers by Aram J. Krauson; Jing He; William C. Wimley (pp. 1625-1632).

Keywords: Pore-forming peptide; Alamethicin; Melittin; LL-37; Dermaseptin

Asymmetric liposome particles with highly efficient encapsulation of siRNA and without nonspecific cell penetration suitable for target-specific delivery by Amir Abbas Mokhtarieh; Sinyoung Cheong; Semi Kim; Bong Hyun Chung; Myung Kyu Lee (pp. 1633-1641).

The discovery of siRNA has been an important step in gene therapy, but the problem of delivering siRNA to a target organ limits its use as a therapeutic drug. Liposomes can be used as a nonviral vector to deliver siRNA to target cells. In this study we developed a novel method of producing asymmetric liposome particles (ALPs) with highly efficient siRNA encapsulation. Two kinds of lipid inverted micelles were prepared for the purpose of obtaining ALPs. The inner one is composed of ionizable cationic 1,2-dioleoyl-3-dimethylammonium-propane (DODAP) and 1,2-dioleoyl- sn-glycero-3-phosphoethanolamine (DOPE), which entrap siRNA, and the outer one is composed of 1,2-distearoyl- sn-glycero-3-phosphocholine (DSPC), DOPE, polyethylene glycol-1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine (PEG-PE), and cholesterol. After mixing the inverted micelles, ALPs encapsulating siRNA were obtained by solvent evaporation and dialysis. This process allowed more than 90% siRNA encapsulation as well as the negatively charged surface. The ALPs protected siRNA from ribonuclease A degradation. ALPs without any surface modification elicited almost no uptake into cells, while the surface-modified ALPs with a polyarginine peptide (R12) induced nonspecific cell penetration. The conjugation of the anti-human epidermal growth factor receptor antibody (anti-EGFR) to ALPs induces an EGFR-mediated uptake into the non-small cell lung cancer cell lines but not into NIH-3T3 cells without the receptor. The siRNA encapsulated in ALPs showed the R12- or anti-EGFR-dependent target gene silencing in NCI-H322 cells. These properties of ALPs are useful for target-specific delivery of siRNA after modification of ALPs with a target-specific ligand.► Asymmetric liposome particles (ALPs) with highly efficient siRNA encapsulation and negatively charged surface. ► The ALPs protect the encapsulated siRNA from RNase A. ► The cellular uptake of ALPs is only induced by attachment of ligands interacting with cell surface receptors. ► The internalized siRNA successfully inhibits the target gene expression.

Keywords: Asymmetric liposome particle; siRNA encapsulation; Target specific delivery; Gene silencing

Estimation of the subunit stoichiometry of the membrane-associated daptomycin oligomer by FRET by Jawad K. Muraih; Michael Palmer (pp. 1642-1647).

Daptomycin is a lipopeptide antibiotic that kills Gram-positive bacteria by depolarizing their cell membranes. This antibacterial action of daptomycin is correlated with the formation of membrane-associated oligomers. We here examine the number of subunits contained in one oligomer using fluorescence resonance energy transfer (FRET). The results suggest that the oligomer contains approximately 6 to 7 subunits, or possibly twice this number if it spans both membrane monolayers.► Hybrid oligomers of daptomycin and NBD-labeled daptomycin were generated. ► FRET between daptomycin and NBD-labeled daptomycin was analyzed. ► The extent of FRET indicates an oligomer subunit stoichiometry of 6–7 subunits.

Keywords: Daptomycin; Lipopeptide antibiotics; Oligomer stoichiometry; Fluorescence resonance energy transfer

Estimation of the subunit stoichiometry of the membrane-associated daptomycin oligomer by FRET by Jawad K. Muraih; Michael Palmer (pp. 1642-1647).

Keywords: Daptomycin; Lipopeptide antibiotics; Oligomer stoichiometry; Fluorescence resonance energy transfer

Antifungal property of dihydrodehydrodiconiferyl alcohol 9′-O-β-d-glucoside and its pore-forming action in plasma membrane of Candida albicans by Hyemin Choi; Jaeyong Cho; Qinglong Jin; Eun-Rhan Woo; Dong Gun Lee (pp. 1648-1655).

The aims of this study were to investigate the antifungal activity as a bioactive property of dihydrodehydrodiconiferyl alcohol 9′-O-β-d-glucoside (DDDC9G) and the mode of action(s) involved in its effect. Antifungal susceptibility testing showed that DDDC9G possessed potent antifungal activities toward various fungal strains with almost no hemolytic effect. To understand the antifungal mechanism(s) of DDDC9G, we conducted the following experiments in this study using Candida albicans. Fluorescence experiments using the probes, 1, 6-diphenyl-1, 3, 5-hexatriene (DPH) and propidium iodide suggested that DDDC9G perturbed the fungal plasma membrane. Consecutively, the analysis of the transmembrane electrical potential (ΔΨ) with 3, 3′-dipropylthiadicarbocyanine iodide [DiSC₃(5)] and bis-(1, 3-dibutylbarbituric acid) trimethine oxonol [DiBAC₄(3)] indicated that DDDC9G induced membrane-depolarization. Furthermore, model membrane studies were performed with rhodamine-labeled giant unilamellar vesicles (GUVs), calcein encapsulating large unilamellar vesicles (LUVs), and FITC-dextran (FD) loaded LUVs. These results demonstrated that the antifungal effects of DDDC9G upon the fungal plasma membrane were through the formation of pores with the radii between 0.74nm and 1.4nm. Finally, in three dimensional (3D) flow cytometric contour plots, a reduced cell size was observed as a result of osmolarity changes from DDDC9G-induced structural and functional membrane damages. Therefore, the present study suggests that DDDC9G exerts its antifungal effect by damaging the membrane through pore formation in the fungal plasma membrane.► This study was aimed to examine the antifungal activity of DDDC9G. ► DDDC9G showed potent antifungal activities with almost no hemolytic effect. ► We investigated the mode of action(s) involved in this effect using Candida albicans. ► DDDC9G exerted the activity on the fungal plasma membrane. ► The cause of the membrane damages was pore formation in the plasma membrane.

Keywords: Abbreviations; DDDC9G; dihydrodehydrodiconiferyl alcohol 9′-O-β-; d; -glucoside; MeOH; methanol; MIC; minimum inhibitory concentration; CLSI; Clinical and Laboratory Standards Institute; MTT; 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2; H; -tetrazolium bromide; DPH; 1,6-diphenyl-1,3,5-hexatriene; diSC; ₃; (5); 3,3′-dipropylthiacarbocyanine; DiBAC; ₄; (3); bis-(1,3-dibutylbarbituric acid) trimethine oxonol; GUV; giant unilamellar vesicle; PC; phosphatidylcholine; PE; phosphatidylethanolamine; PI; phosphatidylinositol; ITO; indium tin oxide; LUVs; large unilamellar vesicles; FITC; fluorescein isothiocyanate; FD; FITC-dextran; FS; forward scatter; SS; side scatterDDDC9G; Antifungal effect; Plasma membrane; Antifungal mechanism; Antifungal compound

Probing membrane permeabilization by the antibiotic lipopeptaibol trichogin GA IV in a tethered bilayer lipid membrane by Lucia Becucci; Flavio Maran; Rolando Guidelli (pp. 1656-1662).

The lipopeptaibol trichogin GA IV (TCG) can be incorporated in the lipid bilayer moiety of a mercury-supported tethered bilayer lipid membrane (tBLM) at a non-physiological transmembrane potential of about −240mV, negative on the trans side of the bilayer. Once incorporated in the tBLM, TCG is stable over the range of physiological transmembrane potentials and permeabilizes the membrane at transmembrane potentials negative of −80÷−90mV. The chronocoulometric behavior is consistent with a kinetics of nucleation and growth of bundles of TCG building blocks with ion-channel properties. The TCG building blocks also permeabilize the lipid bilayer, albeit at more negative transmembrane potentials, and can be tentatively regarded as dimers of aligned TCG helical monomers. The cyclic voltammograms of tBLMs incorporating TCG point to a voltage-gated behavior of the TCG channel, similar to that exhibited by the peptaibol alamethicin.► Trichogin GA IV forms ion channels in lipid bilayers following a kinetics of nucleation and growth. ► Trichogin GA IV ion channels exhibit a voltage-gated behavior in lipid bilayers. ► Trichogin GA IV ion channels permeabilizes membranes at trans-negative transmembrane potentials of −80÷−90mV.

Keywords: Antimicrobial peptide; Ion channel; Tethered bilayer lipid membrane; Nucleation and growth

Probing membrane permeabilization by the antibiotic lipopeptaibol trichogin GA IV in a tethered bilayer lipid membrane by Lucia Becucci; Flavio Maran; Rolando Guidelli (pp. 1656-1662).

Keywords: Antimicrobial peptide; Ion channel; Tethered bilayer lipid membrane; Nucleation and growth

Binding of cationic pentapeptides with modified side chain lengths to negatively charged lipid membranes: Complex interplay of electrostatic and hydrophobic interactions by Maria Hoernke; Christian Schwieger; Andreas Kerth; Alfred Blume (pp. 1663-1672).

Basic amino acids play a key role in the binding of membrane associated proteins to negatively charged membranes. However, side chains of basic amino acids like lysine do not only provide a positive charge, but also a flexible hydrocarbon spacer that enables hydrophobic interactions. We studied the influence of hydrophobic contributions to the binding by varying the side chain length of pentapeptides with ammonium groups starting with lysine to lysine analogs with shorter side chains, namely ornithine (Orn), α,γ-diaminobutyric acid (Dab) and α, β-diaminopropionic acid (Dap). The binding to negatively charged phosphatidylglycerol (PG) membranes was investigated by calorimetry, FT-infrared spectroscopy (FT-IR) and monolayer techniques. The binding was influenced by counteracting and sometimes compensating contributions. The influence of the bound peptides on the lipid phase behavior depends on the length of the peptide side chains. Isothermal titration calorimetry (ITC) experiments showed exothermic and endothermic effects compensating to a different extent as a function of side chain length. The increase in lipid phase transition temperature was more significant for peptides with shorter side chains. FTIR-spectroscopy revealed changes in hydration of the lipid bilayer interface after peptide binding. Using monolayer techniques, the contributions of electrostatic and hydrophobic effects could clearly be observed. Peptides with short side chains induced a pronounced decrease in surface pressure of PG monolayers whereas peptides with additional hydrophobic interactions decreased the surface pressure much less or even lead to an increase, indicating insertion of the hydrophobic part of the side chain into the lipid monolayer.Display Omitted► Binding of Lys₅ analogs with shorter side chains to anionic lipid membranes. ► ITC, DSC, IR_spectroscopy and monolayer techniques were used for binding studies. ► Hydrophobic spacers of the side chains contribute to binding. ► Discrimination between electrostatic and hydrophobic contributions to binding.

Keywords: Abbreviations; Lys; lysine; Orn; ornithine; Dab; α, γ-diaminobutyric acid; Dap; α, β-diaminopropionic acid; FT-IR; Fourier-transform infrared; ITC; isothermal titration calorimetry; DSC; differential scanning calorimetry; PG; phosphatidyl glycerol; DPPG; 1,2-palmitoyl-; sn; -glycero-3-phosphoglycerol; DMPG; 1,2-myristoyl-; sn; -glycero-3-phosphoglycerol; POPG; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphoglycerol; TFA; Trifluoroacetic acid; Arg; arginine; PLL; poly-; l; lysine; MARCKS; myristoylated alanine-rich C-kinase substratePentapeptide; Peptide binding; DPPG; Hydrophobic interaction; Electrostatic interaction

A comparison of the behavior of cholesterol, 7-dehydrocholesterol and ergosterol in phospholipid membranes by Changfeng Chen; Carl P. Tripp (pp. 1673-1681).

A molecular description of the effect of incorporation of cholesterol (CHOL), 7-dehydrocholesterol (7DHC) and ergosterol (ERGO) on the structure of DPPC or EggPC liposomes is provided. Data obtained from ATR-IR spectroscopy, detergent solubility and zeta potential measurements show that the insertion of the various sterols alters the packing arrangement of the tails and headgroup of the PC lipids and may lead to lipid domain formation. On a molecular basis, the differences in lipid packing architecture are traced to differences between the ring and tail structure of the three sterols and these differences in structure produce different effects in DPPC liposomes in the gel phase and EggPC liposomes in the fluid phase. Specifically, CHOL has a relatively flat and linear structure and among the three sterols, shows the strongest molecular interactions with DPPC and EggPC lipids. An extra double bond in the fused ring of 7DHC hinders a tightly packing arrangement with DPPC lipids and leads to less domain formation than CHOL whereas 7DHC clearly produces more lipid domain formation in EggPC. ERGO produces similar structural changes to 7DHC in the tail and headgroup region of DPPC. Nevertheless, ERGO incorporation into DPPC liposomes produces more domain formation than 7DHC.► We utilize three approaches to study phospholipids–sterol interaction. ► Sterol insertion alters the packing arrangement of PC tails and headgroup. ► Insertion of various sterols behaves differently in DPPC and EggPC liposomes. ► Different effects of sterols attribute to their different ring and tail structures. ► 7-Dehydrocholesterol produces lipid domain formation in EggPC liposomes.

Keywords: ATR-IR; Detergent insolubility; Zeta potential; Sterol structure; Liposome; DPPC and EggPC

A comparison of the behavior of cholesterol, 7-dehydrocholesterol and ergosterol in phospholipid membranes by Changfeng Chen; Carl P. Tripp (pp. 1673-1681).

Keywords: ATR-IR; Detergent insolubility; Zeta potential; Sterol structure; Liposome; DPPC and EggPC

K⁺ channels regulate ENaC expression via changes in promoter activity and control fluid clearance in alveolar epithelial cells by Olivier Bardou; Prive Anik Privé; Francis Migneault; Karl Roy-Camille; André Dagenais; Yves Berthiaume; Emmanuelle Brochiero (pp. 1682-1690).

Active Na⁺ absorption by alveolar ENaC is the main driving force of liquid clearance at birth and lung edema resorption in adulthood. We have demonstrated previously that long-term modulation of KvLQT1 and K_ATP K⁺ channel activities exerts sustained control in Na⁺ transport through the regulation of ENaC expression in primary alveolar type II (ATII) cells. The goal of the present study was: 1) to investigate the role of the α-ENaC promoter, transfected in the A549 alveolar cell line, in the regulation of ENaC expression by K⁺ channels, and 2) to determine the physiological impact of K⁺ channels and ENaC modulation on fluid clearance in ATII cells. KvLQT1 and K_ATP channels were first identified in A549 cells by PCR and Western blotting. We showed, for the first time, that KvLQT1 activation by R-L3 (applied for 24h) increased α-ENaC expression, similarly to K_ATP activation by pinacidil. Conversely, pharmacological KvLQT1 and K_ATP inhibition or silencing with siRNAs down-regulated α-ENaC expression. Furthermore, K⁺ channel blockers significantly decreased α-ENaC promoter activity. Our results indicated that this decrease in promoter activity could be mediated, at least in part, by the repressor activity of ERK1/2. Conversely, KvLQT1 and K_ATP activation dose-dependently enhanced α-ENaC promoter activity. Finally, we noted a physiological impact of changes in K⁺ channel functions on ERK activity, α-, β-, γ-ENaC subunit expression and fluid absorption through polarized ATII cells. In summary, our results disclose that K⁺ channels regulate α-ENaC expression by controlling its promoter activity and thus affect the alveolar function of fluid clearance.

Keywords: KvLQT1 channels; k; _ATP; channels; α-ENaC; MAPK; Alveolar cells; Fluid clearance

Similar structures but different mechanisms by Zamarreno Fernando Zamarreño; Fernando E. Herrera; Corsico Betina Córsico; Marcelo D. Costabel (pp. 1691-1697).

The role of fatty acid binding proteins as intracellular fatty acid transporters may require their direct interaction with membranes. In this way different mechanisms have been previously characterized through experimental studies suggesting different models for FABPs–membrane association, although the process in which the molecule adsorbs to the membrane remains to be elucidated. To estimate the importance of the electrostatic energy in the FABP–membrane interaction, we computationally modeled the interaction of different FABPs with both anionic and neutral membranes. Free Electrostatic Energy of Binding (dE), was computed using Finite Difference Poisson Boltzmann Equation (FDPB) method as implemented in APBS (Adaptive Poisson Boltzmann Solver). Based on the computational analysis, it is found that recruitment to membranes is facilitated by non-specific electrostatic interactions. Also energetic analysis can quantitatively differentiate among the mechanisms of membrane association proposed and determinate the most energetically favorable configuration for the membrane-associated states of different FABPs. This type of calculations could provide a starting point for further computational or experimental analysis.► Fast in silico method used to differentiate FABPs–membrane interaction mechanisms. ► Exhaustive sampling of interaction conformations. ► Electrostatic free energy is a driving force for FABPs–membrane interaction. ► Anionic membranes repel not favorable conformations for FABPs–membrane interactions.

Keywords: Fatty acid binding protein; Electrostatic interaction; Protein–membrane interaction; Molecular dynamics simulation; Biomolecular modeling; Structure–function relation

Similar structures but different mechanisms by Zamarreno Fernando Zamarreño; Fernando E. Herrera; Corsico Betina Córsico; Marcelo D. Costabel (pp. 1691-1697).

Keywords: Fatty acid binding protein; Electrostatic interaction; Protein–membrane interaction; Molecular dynamics simulation; Biomolecular modeling; Structure–function relation

Autonomous transmembrane segment S4 of the voltage sensor domain partitions into the lipid membrane by Venkataswarup Tiriveedhi; Melissa Miller; Peter Butko; Min Li (pp. 1698-1705).

The S4 transmembrane segment in voltage-gated ion channels, a highly basic α helix, responds to changes in membrane potential and induces channel opening. Earlier work by others indicates that the S4 segment interacts with lipids in plasma membrane, but its mechanism is unclear. Working with synthetic tryptophan-labeled S4 peptides, we characterized binding of autonomous S4 to lipid membranes. The binding free energy (5.2±0.2kcal/mol) of the peptide–lipid interaction was estimated from the apparent dissociation constants, determined from the changes in anisotropy of tryptophan fluorescence induced by addition of lipid vesicles with 30mol% phosphatidylglycerol. The results are in good agreement with the prediction based on the Wimley–White hydrophobicity scale for interfacial (IF) binding of an alpha-helical peptide to the lipid bilayer (6.98kcal/mol). High salt inhibited the interaction, thus indicating that the peptide/membrane interaction has both electrostatic and non-electrostatic components. Furthermore, the synthetic S4 corresponding to the Shaker potassium channel was found to spontaneously penetrate into the negatively charged lipid membrane to a depth of about 9Å. Our results revealed important biophysical parameters that influence the interaction of S4 with the membrane: they include fluidity, surface charge, and surface pressure of the membrane, and the α helicity and regular spacing of basic amino-acid residues in the S4 sequence.► Fluorescence spectroscopy approach to study the S4 peptide interaction with model membrane. ► Fluidity, surface charge, and surface pressure of the membrane affect the S4-membrane interaction. ► The α-helicity and the periodicity of basic amino acids in the peptide sequence favors membrane localization.

Keywords: Potassium channel; Voltage sensor domain; S4; Fluorescence spectroscopy; Resonance energy transfer; Small unilamellar vesicle

Autonomous transmembrane segment S4 of the voltage sensor domain partitions into the lipid membrane by Venkataswarup Tiriveedhi; Melissa Miller; Peter Butko; Min Li (pp. 1698-1705).

Keywords: Potassium channel; Voltage sensor domain; S4; Fluorescence spectroscopy; Resonance energy transfer; Small unilamellar vesicle

Flexibility of the PDZ-binding motif in the micelle-bound form of Jagged-1 cytoplasmic tail by Matija Popovic; Ventsislav Zlatev; Vesna Hodnik; Gregor Anderluh; Isabella C. Felli; Sándor Pongor; Alessandro Pintar (pp. 1706-1716).

Human Jagged-1, one of the ligands of Notch receptors, is a transmembrane protein composed of a large extracellular region and a 125-residue cytoplasmic tail which bears a C-terminal PDZ recognition motif. To investigate the interaction between Jagged-1 cytoplasmic tail and the inner leaflet of the plasma membrane we determined, by solution NMR, the secondary structure and dynamics of the recombinant protein corresponding to the intracellular region of Jagged-1, J1_tmic, bound to negatively charged lysophospholipid micelles. NMR showed that the PDZ binding motif is preceded by four α-helical segments and that, despite the extensive interaction between J1_tmic and the micelle, the PDZ binding motif remains highly flexible. Binding of J1_tmic to negatively charged, but not to zwitterionic vesicles, was confirmed by surface plasmon resonance. To study the PDZ binding region in more detail, we prepared a peptide corresponding to the last 24 residues of Jagged-1, J1C24, and different phosphorylated variants of it. J1C24 displays a marked helical propensity and undergoes a coil–helix transition in the presence of negatively charged, but not zwitterionic, lysophospholipid micelles. Phosphorylation at different positions drastically decreases the helical propensity of the peptides and abolishes the coil–helix transition triggered by lysophospholipid micelles. We propose that phosphorylation of residues upstream of the PDZ binding motif may shift the equilibrium from an ordered, membrane-bound, interfacial form of Jagged-1 C-terminal region to a more disordered form with an increased accessibility of the PDZ recognition motif, thus playing an indirect role in the interaction between Jagged-1 and the PDZ-containing target protein.Display Omitted► Jagged-1 cytoplasmic tail binds to negatively charged micelles and liposomes. ► Binding is associated with the formation of four helical regions, as mapped by NMR. ► NMR shows that the C-terminal PDZ binding motif remains highly flexible. ► Phoshorylation abolishes the helical propensity in the C-terminal region. ► PDZ recognition may be tuned by phosphorylation upstream of the PDZ binding motif.

Keywords: Abbreviations; DMPC; 1,2-dimyristoyl-; sn; -glycero-3-phosphocholine; DMPG; 1,2-dimyristoyl-; sn; -glycero-3-[phospho-; rac; -(1-glycerol)] sodium salt; DMPS; 1,2-dimyristoyl-sn-glycero-3-[phospho-; l; -serine] sodium salt; DSS; 2,2-dimethyl-2-silapentane-5-sulfonate-d; ₆; sodium salt; HSQC; heteronuclear single quantum correlation; LMPC; 1-myristoyl-2-hydroxy-; sn-; glycero-3-phosphocholine; LMPG; 1-myristoyl-2-hydroxy-; sn-; glycero-3-[phospho-; rac-; (1-glycerol)] sodium salt; LPPC; 1-palmitoyl-2-hydroxy-; sn-; glycero-3-phosphocholine; LPPG; 1-palmitoyl-2-hydroxy-; sn-; glycero-3-[phospho-; rac-; (1-glycerol)] sodium salt; PDZ; domain present in PSD-95, Dlg, and ZO-1/2; POPG; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phospho-(1′-; rac; -glycerol) sodium salt; SDS; sodium dodecyl sulfate; SPR; surface plasmon resonance; TFE; 2,2,2-TrifluoroethanolNotch signaling; Membrane/cytoplasm interface; Phosphorylation; NMR; Surface plasmon resonance; Circular dichroism

Topology and lipid selectivity of pulmonary surfactant protein SP-B in membranes: Answers from fluorescence by Cabre Elisa J. Cabré; Luís M.S. Loura; Alexander Fedorov; Jesus Perez-Gil; Manuel Prieto (pp. 1717-1725).

Contradictory results have been reported with respect to the depth of penetration and the orientation of pulmonary surfactant protein SP-B in phospholipid membranes and its relative selectivity to interact with anionic over zwitterionic phospholipid species. In the present study we have re-evaluated lipid–protein interactions of SP-B by analysing Förster resonance energy transfer (FRET) efficiencies, obtained from time-resolved measurements, from the single tryptophan in SP-B to different fluorescently labelled phospholipids in matrix bilayers made of either pure phosphatidylcholine (POPC) or the full lipid extract obtained from purified surfactant. In the background of POPC membranes SP-B exhibits a certain level of selectivity for anionic fluorescent phospholipids over the corresponding zwitterionic analogues, but apparently no preference for phosphatidylglycerol over other anionic species such as phosphatidylserine. No selectivity was detected in membranes made of full surfactant lipids, indicating that specific lipid–protein binding sites could already be occupied by endogenous anionic phospholipids. Furthermore, we have analysed the fit of two different models of how SP-B could be orientated with respect to phospholipid membrane surfaces to the FRET data. The FRET results are consistent with topology models in which the protein has a superficial orientation, with no regions of exclusion by the protein to the access of phospholipids, both in POPC membranes and in membranes made of the whole surfactant lipid fraction. This discards a deep penetration of the protein into the core of bilayers and suggests that most hydrophobic segments of SP-B could participate in protein–protein instead of lipid–protein interactions.► Time-resolved fluorescence of SP-B allows studying selectivity and mode of L/P interaction. ► SP-B shows selectivity for anionic lipids but not specifically for NBD-PG. ► A model has been derived to interpret FRET from peripheral membrane protein donors. ► FRET data from SP-B indicates that the protein interacts with membranes in a superficial manner.

Keywords: Protein–lipid interaction; Lipid annulus; Fluorescence spectroscopy; Membrane protein topology; FRET

Coupling between the voltage-sensing and pore domains in a voltage-gated potassium channel by Eric V. Schow; J. Alfredo Freites; Alex Nizkorodov; Stephen H. White; Douglas J. Tobias (pp. 1726-1736).

Voltage-dependent potassium (Kv), sodium (Nav), and calcium channels open and close in response to changes in transmembrane (TM) potential, thus regulating cell excitability by controlling ion flow across the membrane. An outstanding question concerning voltage gating is how voltage-induced conformational changes of the channel voltage-sensing domains (VSDs) are coupled through the S4–S5 interfacial linking helices to the opening and closing of the pore domain (PD). To investigate the coupling between the VSDs and the PD, we generated a closed Kv channel configuration from Aeropyrum pernix (KvAP) using atomistic simulations with experiment-based restraints on the VSDs. Full closure of the channel required, in addition to the experimentally determined TM displacement, that the VSDs be displaced both inwardly and laterally around the PD. This twisting motion generates a tight hydrophobic interface between the S4–S5 linkers and the C-terminal ends of the pore domain S6 helices in agreement with available experimental evidence.►Coupling between voltage-sensing and pore domains in a Kv channel is studied by MD. ►Restraints based on experimental data are used to move the voltage-sensing domains. ►Full channel closure required additional lateral and inward motion of the VSDs. ►An all-atom closed/resting state of the KvAP channel was produced by this work.

Keywords: Ion channel; Voltage-gating; Molecular dynamics simulation; Membrane protein

Role of a conserved glycine triplet in the NSS amino acid transporter KAAT1 by M. Giovanola; F. D'Antoni; M. Santacroce; S.A. Mari; F. Cherubino; E. Bossi; V.F. Sacchi; M. Castagna (pp. 1737-1744).

K⁺-coupled amino acid transporter 1 (KAAT1) belongs to the NSS family of solute transporters and it is expressed in the midgut and in salivary glands of Manduca sexta larvae. As more than 80% of family members, KAAT1 shows a stretch of three glycines (G85–G87) that according to the structure of the prototype transporter LeuT, is located close to the access of the permeation pathway. In this work the role of the triplet has been investigated by alanine and cysteine scanning methods in protein heterologously expressed in Xenopus laevis oocytes. All the mutants were functional but the surface expression level was reduced for G85A and G87A mutants and unaffected for G86A mutant. All presented altered amino acid uptake and transport associated currents in the presence of each of the cations (Na⁺, K⁺, Li⁺) that can be exploited by the wt. G87A mutant induced increased uncoupled fluxes in the presence of all the cations. Cross-linking studies, performed by the treatment of cysteine mutants with the oxidative complex Cu(II)(1,10-phenanthroline)₃, showed that limiting the flexibility of the region by covalent blockage of position 87, causes a significant reduction of amino acid uptake. Na⁺ protected G87C mutant from oxidation, both directly and indirectly. The conserved glycine triplet in KAAT1 plays therefore a complex role that allows initial steps of cation interaction with the transporter.► KAAT1 Gly 85–87 mutants are expressed on the membrane surface although at different level. ► Mutants showed altered interaction with cations. ► Cross-linking of cysteine mutants by oxidative treatment inhibited transport activity. ► Na⁺ protected position 87 from oxidation both directly and indirectly. ► Glycine flexibility is involved in KAAT1 function and its interaction with cations.

Keywords: Abbreviations; KAAT1; K; ⁺; -coupled amino acid transporter 1; CuPh; Cu(II)(1,10-phenanthroline); ₃; DTT; dithiothreitol; TMA; tetramethylammonium; EL; extracellular loop; TM; transmembrane domainStructure-function; Site-directed mutagenesis; Cross-linking; Cation interaction; Flexibility

Role of a conserved glycine triplet in the NSS amino acid transporter KAAT1 by M. Giovanola; F. D'Antoni; M. Santacroce; S.A. Mari; F. Cherubino; E. Bossi; V.F. Sacchi; M. Castagna (pp. 1737-1744).

Molecular organization of bacterial membrane lipids in mixed systems—A comprehensive monolayer study combined with Grazing Incidence X-ray Diffraction and Brewster Angle Microscopy experiments by Paweł Wydro; Flasinski Michał Flasiński; Marcin Broniatowski (pp. 1745-1754).

To properly design and investigate new antibacterial drugs a detailed description of the organization of bacterial membrane is highly important. Therefore in this work we performed a comprehensive characteristic of the Langmuir monolayers composed of phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) mixed in a wide range of composition and treated as an artificial cytoplasmic layer of bacterial membrane. To obtain detailed information on the properties of these films we combined the analysis of the surface pressure–area curves with the surface potential measurements, Brewster Angle Microscopy studies and Grazing Incidence X-ray Diffraction experiments. It was found that the investigated phospholipids mix nonideally in the monolayers and that the most favorable packing of molecules occurs at their equimolar proportion. This is directly connected with the formation of hydrogen bonds between both types of molecules in the system. All the collected experimental data evidenced that dipalmitoylphosphatidylethanolamine (DPPE) and dipalmitoylphosphatidylglycerol (DPPG) form highly ordered associates of fixed (DPPE:DPPG 1:1) stoichiometry. The obtained results allow one to conclude a nonuniform distribution of lipids in bacterial membranes and the existence of domains composed of the investigated phospholipids. The latter seems to be of great importance in the perspective of further studies on the mechanism of action of antibacterial agents.Display Omitted► Molecular organization of bacterial membrane lipids in mixed Langmuir monolayers. ► Favorable packing of phosphatidylethanolamine and phosphatidylglycerol at 1:1 ratio. ► Formation of highly ordered associates between the lipids in model system.

Keywords: Langmuir monolayer; Model bacterial membrane; 1,2-dipalmitoyl-; sn; -glycero-3-phosphoethanolamine; 1,2-dipalmitoyl-; sn; -glycero-3-phospho-(1′-; rac; -glycerol) sodium salt; Grazing Incidence X-ray Diffraction; Brewster Angle Microscopy

Membrane interactions of two arginine-rich peptides with different cell internalization capacities by Astrid Walrant; Alexander Vogel; Isabelle Correia; Olivier Lequin; Bjoern E.S. Olausson; Bernard Desbat; Sandrine Sagan; Isabel D. Alves (pp. 1755-1763).

Cell penetrating peptides (CPPs) can cross cell membranes in a receptor independent manner and transport cargo molecules inside cells. These peptides can internalize through two independent routes: energy dependent endocytosis and energy independent translocation across the membrane, but the exact mechanisms are still unknown. The interaction of the CPP with different membrane components is certainly a preliminary key point that triggers internalization, such as the interaction with lipids to lead to the translocation process. In this study, we used two arginine-rich peptides, RW9 (RRWWRRWRR-NH₂), which is a potent CPP, and RL9 (RRLLRRLRR-NH₂) that, although binding tightly and accumulating on membranes, does not enter into cells. Using a set of experimental and theoretical techniques, we studied the binding, insertion and orientation of the peptides into different model membranes as well as the subsequent membrane reorganization. Herein we show that although the two peptides had rather similar behavior regarding lipid membrane interaction, subtle differences were found concerning the depth of peptide insertion, effect on the lipid chain ordering and kinetics of peptide insertion in the membrane, which altogether might explain their different cell internalization capacities. Molecular dynamics simulation studies show that some peptide molecules flipped their orientation over the course of the simulation such that the hydrophobic residues penetrated deeper in the lipid core region while Arg-residues maintained H-bonds with the lipid headgroups, serving as a molecular hinge in a conformation that appeared to correspond to the equilibrium one.► Two cell penetrating peptides with opposite cell uptake capacities are investigated. ► Their binding, insertion and perturbation of model membranes are studied. ► The peptides share many similarities regarding lipid membrane interaction. ► Subtle differences concern insertion depth, effect on lipid ordering and kinetics. ► Some peptide molecules flipped their orientation with arginines serving as hinges.

Keywords: Abbreviations; Apa; Aminopentanoic Acid; ATR; Attenuated Total Reflectance; COM; center of mass; CPP; cell penetrating peptide; DMPC; Dimyristoyl Phosphatidylcholine; DMPG; Dimyristoyl Phosphatidylglycerol; DPC; Dodecyl Phosphocholine; FTIR; Fourier Transform Infrared Spectroscopy; ITC; isothermal titration calorimetry; LUV; large unilamellar vesicle; MLV; multilamellar vesicle; POPG; 1-palmitoyl-2-oleoyl Phosphatidylglycerol; SDS; Sodium Dodecyl Sulfate; SUV; small unilamellar vesicleCell penetrating peptide; Peptide/lipid interaction; Calorimetry; Attenuated Total Reflectance Infrared Spectroscopy; Molecular dynamics; NMR

Lipid shape is a key factor for membrane interactions of amphipathic helical peptides by Erik Strandberg; Deniz Tiltak; Sebastian Ehni; Parvesh Wadhwani; Anne S. Ulrich (pp. 1764-1776).

The membrane alignment of the amphiphilic α-helical model peptide MSI-103 (sequence [KIAGKIA]₃-NH₂) was examined by solid state²H-NMR in different lipid systems by systematically varying the acyl chain length and degree of saturation, the lipid head group type, and the peptide-to-lipid molar ratio. In liquid crystalline phosphatidylcholine (PC) lipids with saturated chains, the amphiphilic helix changes its orientation from a surface-bound “S-state” to a tilted “T-state” with increasing peptide concentration. In PC lipids with unsaturated chains, on the other hand, the S-state is found throughout all concentrations. Using phosphatidylethanolamine lipids with a small head group or by addition of lyso-lipids with only one acyl chain, the spontaneous curvature of the bilayer was purposefully changed. In the first case with a negative curvature only the S-state was found, whereas in systems with a positive curvature the peptide preferred the obliquely immersed T-state at high concentration. The orientation of MSI-103 thus correlates very well with the shape of the lipid molecules constituting the membrane. Lipid charge, on the other hand, was found to affect only the initial electrostatic attraction to the membrane surface but not the alignment preferences. In bilayers that are “sealed” with 20% cholesterol, MSI-103 cannot bind in a well-oriented manner and forms immobilized aggregates instead. We conclude that the curvature properties of a membrane are a key factor in the interactions of amphiphilic helical peptides in general, whose re-alignment and immersion preferences may thus be inferred in a straightforward manner from the lipid-shape concept.Display Omitted► Amphipathic peptides in membranes change their alignment from surface-bound to tilted. ► The re-alignment of the antimicrobial peptide MSI-103 is compared in many different lipids. ► The accurate²H-NMR data allows for the first time a systematic generalization. ► In lipids with negative spontaneous curvature the peptide always remains surface-bound. ► Only a positive lipid curvature allows the peptide to tilt deeper into the membrane.

Keywords: Abbreviations; AMP; antimicrobial peptide; CHOL; Cholesterol; DLPC; 1,2-dilauroyl-; sn; -glycero-3-phosphatidylcholine; DMPC; 1,2-dimyristoyl-; sn; -glycero-3-phosphatidylcholine; DMPG; 1,2-dimyristoyl-; sn; -glycero-3-phosphatidylglycerol; DMoPC; 1,2-dimyristoleoyl-; sn; -glycero-3-phosphatidylcholine; DOPC; 1,2-dioleoyl-; sn; -glycero-3-phosphatidylcholine; I-state; inserted state; L; _α; phase; liquid crystalline lamellar phase; Lyso-OPC; 1-oleoyl-2-hydroxy-; sn; -glycero-3-phosphatidylglycerol; MLV; multi-lamellar vesicle; OCD; oriented circular dichroism; PC; phosphatidylcholine; PE; phosphatidylethanolamine; PG; phosphatidylglycerol; P/L; peptide-to-lipid molar ratio; P/L*; threshold P/L value for peptide reorientation; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphatidylcholine; POPE; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphatidylethanolamine; POPG; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphatidylglycerol; R; ₀; radius of spontaneous curvature; ρ; azimuthal rotation angle; rmsd; root mean square deviation; σ; _ρ; standard deviation of Gaussian distribution of azimuthal rotation angles; σ; _τ; standard deviation of Gaussian distribution of tilt angles; S; _mol; molecular order parameter; S-state; surface-aligned state; τ; tilt angle; T; _m; gel to liquid crystalline phase transition temperature; T-state; tilted stateMembrane-active amphipathic alpha-helical antimicrobial peptide; MSI-103 [KIAGKIA]; ₃; -NH; ₂; Helix alignment and dynamics; Solid-state; ²; H-NMR; Spontaneous bilayer curvature; Lipid shape concept

Lipid shape is a key factor for membrane interactions of amphipathic helical peptides by Erik Strandberg; Deniz Tiltak; Sebastian Ehni; Parvesh Wadhwani; Anne S. Ulrich (pp. 1764-1776).

Partitioning, diffusion, and ligand binding of raft lipid analogs in model and cellular plasma membranes by Erdinc Sezgin; Ilya Levental; Michal Grzybek; Günter Schwarzmann; Veronika Mueller; Alf Honigmann; Vladimir N. Belov; Christian Eggeling; Ünal Coskun; Kai Simons; Petra Schwille (pp. 1777-1784).

Several simplified membrane models featuring coexisting liquid disordered (Ld) and ordered (Lo) lipid phases have been developed to mimic the heterogeneous organization of cellular membranes, and thus, aid our understanding of the nature and functional role of ordered lipid–protein nanodomains, termed “rafts”. In spite of their greatly reduced complexity, quantitative characterization of local lipid environments using model membranes is not trivial, and the parallels that can be drawn to cellular membranes are not always evident. Similarly, various fluorescently labeled lipid analogs have been used to study membrane organization and function in vitro, although the biological activity of these probes in relation to their native counterparts often remains uncharacterized. This is particularly true for raft-preferring lipids (“raft lipids”, e.g. sphingolipids and sterols), whose domain preference is a strict function of their molecular architecture, and is thus susceptible to disruption by fluorescence labeling. Here, we analyze the phase partitioning of a multitude of fluorescent raft lipid analogs in synthetic Giant Unilamellar Vesicles (GUVs) and cell-derived Giant Plasma Membrane Vesicles (GPMVs). We observe complex partitioning behavior dependent on label size, polarity, charge and position, lipid headgroup, and membrane composition. Several of the raft lipid analogs partitioned into the ordered phase in GPMVs, in contrast to fully synthetic GUVs, in which most raft lipid analogs mis-partitioned to the disordered phase. This behavior correlates with the greatly enhanced order difference between coexisting phases in the synthetic system. In addition, not only partitioning, but also ligand binding of the lipids is perturbed upon labeling: while cholera toxin B binds unlabeled GM1 in the Lo phase, it binds fluorescently labeled GM1 exclusively in the Ld phase. Fluorescence correlation spectroscopy (FCS) by stimulated emission depletion (STED) nanoscopy on intact cellular plasma membranes consistently reveals a constant level of confined diffusion for raft lipid analogs that vary greatly in their partitioning behavior, suggesting different physicochemical bases for these phenomena.►Raft lipid analogs show mispartitioning in both synthetic and cell-derived membrane systems. ►The partitioning of an analog is a function of type, size, polarity, charge and position of the dye. ►Acyl-chain labeling directly modulates bioactivity of lipids in a phase-specific manner. ►Partitioning in cell-derived GPMVs more closely follows raft predictions than in synthetic GUVs. ►Nanoscale diffusion in live cell membranes is uncorrelated with partitioning in model membranes.

Keywords: Lipid; Partitioning; GUV; GPMV; Raft; STED-FCS

Localization and interaction of genistein with model membranes formed with dipalmitoylphosphatidylcholine (DPPC) by Pawlikowska-Pawlega Bożena Pawlikowska-Pawlęga; Lucjan E. Misiak; Barbara Zarzyka; Roman Paduch; Antoni Gawron; Wiesław I. Gruszecki (pp. 1785-1793).

The effect of genistein on the liposomes formed with dipalmitoylphosphatidylcholine was studied with the application of Fourier-transform infrared spectroscopy, nuclear magnetic resonance (¹H NMR) and electron paramagnetic resonance techniques. Membranous structures organization of human skin fibroblasts and colon myofibroblasts was also examined using fluorescence and electron microscopy. The strongest rigidifying effect of genistein with respect to polar head groups was concluded on the basis of the effect of the flavonoid on the shape of NMR lines attributed to –N⁺(CH₃)₃ groups. The rigidifying effect of genistein with respect to the hydrophobic core of lipid membranes was also concluded from the genistein-dependent broadening of the NMR lines assigned to –CH₂ groups and terminal –CH₃ groups of alkyl chains. EPR data supported ordering effect of genistein of the hydrophobic core in the liquid–crystalline phase (L_α). The analysis of the FTIR spectra of the two-component liposomes showed that genistein incorporates into DPPC membranes via hydrogen bonding between the lipid polar head groups in the C–O–P–O–C segment and its hydroxyl groups. Both fluorescence microscopy and ultrastructural observation revealed changes in membranous structures organization as aftermath of genistein treatment. In conclusion, genistein localized within membranes changes the properties of membrane that can be followed by the changes inside cells being crucial for pharmacological activity of genistein used in cancer or other disease treatment.► Genistein interacts with DPPC liposomes and changes their dynamic properties. ► Genistein shows the highest propensity to polar head group zone and below it. ► Hydroxyl groups of genistein are involved in hydrogen link formation. ► C–O–P–O–C segment of the lipid takes part in hydrogen bonding creation. ► Genistein changes the organization of natural membranes.

Keywords: Abbreviations; DPPC; dipalmitoylphosphatidylcholine; EPR spectroscopy; electron paramagnetic resonance spectroscopy; FTIR spectroscopy; Fourier-transform infrared spectroscopy; L; _α; lamellar liquid -crystalline phase; NMR spectroscopy; nuclear magnetic resonance spectroscopy; 16-SASL; 2-(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinyloxy free radicalGenistein; DPPC liposome; ¹; H NMR; EPR; FTIR; Electron microscopy

Structure and internal organization of overcharged cationic-lipid/peptide/DNA self-assembly complexes by Jiang Yan; Nikolay V. Berezhnoy; Nikolay Korolev; Chun-Jen Su; Nordenskiold Lars Nordenskiöld (pp. 1794-1800).

The combination of cationic lipids with cationic peptides and DNA vectors can produce synergistic effects in gene delivery to eukaryotic cells. Binary complexes of cationic lipids with DNA are well-studied whereas little information is available about the structure of the ternary lipid/peptide/DNA (LPD) complexes and mechanisms defining DNA protection and delivery. Here we use synchrotron small angle X-ray scattering and dynamic light scattering zeta-potential measurements to determine structure and the net charge of supramolecular aggregates of complexes in mixtures of plasmid DNA, cationic liposomes formed from DOTAP, plus a linear cationic ε-oligolysine with the pendant α-amino acids Leu-Tyr-Arg (LYR), ε-(LYR)K10. These ternary complexes display multilamellar structures with relatively constant separation between DOTAP bilayers, accommodating a hydrated monolayer of parallel DNA rods. The DNA–DNA distance in the complexes varies as a function of the net positive to negative (lipid+peptide)/DNA charge ratio. An explanation for the observed dependence of DNA–DNA distance on charge ratio was proposed based on general polyelectrolyte properties of non-stoichiometric polycation–DNA mixtures.► Lamellar lipid/ε-peptide/DNA complex contains DNA monolayer and lipid bilayer. ► ε-peptides insert between parallel DNA and are present on particle surface. ► DNA–DNA distance (d_DNA) depends on total positive to negative charge ratio. ► The dependence is explained by an electrostatic analysis. ► Variation of surface potential has a similar tendency as the change in d_DNA.

Keywords: Abbreviations; CR; charge ratio; CR; _+/−; charge ratio of positive to negative; d; _DNA; interaxial DNA–DNA distance; d; _interlayer; interlayer spacing; DLS; dynamic light scattering; DOPE; 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine; DOTAP; 1,2-dioleoyl-3-trimethylammonium-propane; HBS; Hepes-buffered saline; L/D; charge ratio of lipids to DNA; Lipoplex; lipid/DNA complexes; LPD; lipid/peptide/DNA complexes; ε-(LYR)K10: α-substituted ε-K10; the amino acid letters in the brackets denote the substitution groups; P/D; charge ratio of peptides to DNA; SAXS; small-angle X-ray scatteringZeta potential; Poisson–Boltzmann model; Polyelectrolyte thermodynamics; Gene delivery

Corrigendum to “An atomic force microscopy study of Galleria mellonella apolipophorin III effect on bacteria” [Biochim. Biophys. Acta 1808 (2011) 1896–1906] by Agnieszka Zdybicka-Barabas; Barbara Januszanis; Pawel Mak; Cytrynska Małgorzata Cytryńska (pp. 1801-1801).

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BBA - Biomembranes (v.1818, #7)