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BBA - Biomembranes (v.1778, #4)

Editorial Board (pp. ii).

Guanidinium group: A versatile moiety inducing transport and multicompartmentalization in complementary membranes by Alexandros Pantos; Ioannis Tsogas; Constantinos M. Paleos (pp. 811-823).

Guanidinium groups present in peptides and dendritic polymers induce their efficient transport through liposomal and cell membranes. Transmembrane crossing of these polymers is affected by their structural features and is critically dependent on the number of guanidinium groups present. Furthermore, the interaction of the guanidinium groups with phosphate groups, both located on liposomal surfaces, triggers a series of processes involving a reorganization of the self-assembled lipids and inducing the formation of multicompartment systems. These observations consistent throughout a diversity of interacting complementary liposomes, support a hypothesis that molecular recognition of liposomes induces the formation of multicompartment structures.

Keywords: Guanidinium; Membrane transport; Multicompartmentalization; Molecular recognition; Dendrimer

Comparison of rat epidermal keratinocyte organotypic culture (ROC) with intact human skin: Lipid composition and thermal phase behavior of the stratum corneum by Sari Pappinen; Martin Hermansson; Judith Kuntsche; Pentti Somerharju; Philip Wertz; Arto Urtti; Marjukka Suhonen (pp. 824-834).

The present report is a part of our continuing efforts to explore the utility of the rat epidermal keratinocyte organotypic culture (ROC) as an alternative model to human skin in transdermal drug delivery and skin irritation studies of new chemical entities and formulations. The aim of the present study was to compare the stratum corneum lipid content of ROC with the corresponding material from human skin. The lipid composition was determined by thin-layer chromatography (TLC) and mass-spectrometry, and the thermal phase transitions of stratum corneum were studied by differential scanning calorimetry (DSC). All major lipid classes of the stratum corneum were present in ROC in a similar ratio as found in human stratum corneum. Compared to human skin, the level of non-hydroxyacid-sphingosine ceramide (NS) was increased in ROC, while α-hydroxyacid-phytosphingosine ceramide (AP) and non-hydroxyacid-phytosphingosine ceramides (NP) were absent. Also some alterations in fatty acid profiles of ROC ceramides were noted, e.g., esterified ω-hydroxyacid-sphingosine contained increased levels of oleic acid instead of linoleic acid. The fraction of lipids covalently bound to corneocyte proteins was distinctly lower in ROC compared to human skin, in agreement with the results from DSC. ROC underwent a lipid lamellar order to disorder transition ( T₂) at a slightly lower temperature (68 °C) than human skin (74 °C). These differences in stratum corneum lipid composition and the thermal phase transitions may explain the minor differences previously observed in drug permeation between ROC and human skin.

Keywords: Abbreviations; AS; α-hydroxyacid-sphingosine; s; AP; α-hydroxyacid-phytosphingosine; s; AH; α-hydroxyacid-6-OH-sphingosine; s; DSC; differential scanning calorimetry; EOS; ω-; O; -acyl-hydroxyacid-sphingosine; EOP; ω-; O; -acyl-hydroxyacid-phytosphingosine; EOH; ω-; O; -acyl-hydroxyacid-6-OH-sphingosine; FFA; free fatty acid; NS; non-hydroxyacid-sphingosine; s; NP; non-hydroxyacid-phytosphingosine; s; NH; non-hydroxyacid-6-OH-sphingosine; s; REK; rat epidermal keratinocyte; ROC; REK organotypic culture; TLC; thin-layer chromatographyROC; Reconstructed epidermis; Stratum corneum; Lipid composition; DSC; TLC

Stabilization of Na,K–ATPase by ionic interactions by Elfrieda Fodor; Natalya U. Fedosova; Csilla Ferencz; Derek Marsh; Tibor Pali; Mikael Esmann (pp. 835-843).

The effect of ions on the thermostability and unfolding of Na,K–ATPase from shark salt gland was studied and compared with that of Na,K–ATPase from pig kidney by using differential scanning calorimetry (DSC) and activity assays. In 1 mM histidine at pH 7, the shark enzyme inactivates rapidly at 20 °C, as does the kidney enzyme at 42 °C (but not at 20 °C). Increasing ionic strength by addition of 20 mM histidine, or of 1 mM NaCl or KCl, protects both enzymes against this rapid inactivation. As detected by DSC, the shark enzyme undergoes thermal unfolding at lower temperature ( T_m≈45 °C) than does the kidney enzyme ( T_m≈55 °C). Both calorimetric endotherms indicate multi-step unfolding, probably associated with different cooperative domains. Whereas the overall heat of unfolding is similar for the kidney enzyme in either 1 mM or 20 mM histidine, components with high mid-point temperatures are lost from the unfolding transition of the shark enzyme in 1 mM histidine, relative to that in 20 mM histidine. This is attributed to partial unfolding of the enzyme due to a high hydrostatic pressure during centrifugation of DSC samples at low ionic strength, which correlates with inactivation measurements. Addition of 10 mM NaCl to shark enzyme in 1 mM histidine protects against inactivation during centrifugation of the DSC sample, but incubation for 1 h at 20 °C prior to addition of NaCl results in loss of components with lower mid-point temperatures within the unfolding transition. Cations at millimolar concentration therefore afford at least two distinct modes of stabilization, likely affecting separate cooperative domains. The different thermal stabilities and denaturation temperatures of the two Na,K–ATPases correlate with the respective physiological temperatures, and may be attributed to the different lipid environments.

Keywords: Abbreviations; CDTA; trans; -1,2-cyclohexylenedinitrilo-tetraacetic acid; E; ₂; (K); the protein conformation in the presence of K; ⁺; SDS; sodium dodecyl sulphate; DSC; differential scanning calorimetryNa,K–ATPase; Denaturation; Pig kidney; Shark salt gland; Differential scanning calorimetry; Electrostatic screening

Cationic amphiphiles and the solubilization of cholesterol crystallites in membrane bilayers by Carlos R. Benatti; M. Teresa Lamy; Richard M. Epand (pp. 844-853).

Cationic amphiphiles used for transfection can be incorporated into biological membranes. By differential scanning calorimetry (DSC), cholesterol solubilization in phospholipid membranes, in the absence and presence of cationic amphiphiles, was determined. Two different systems were studied: 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC)+cholesterol (1:3, POPC:Chol, molar ratio) and 1-palmitoyl-2-oleoyl- sn-glycero-3-[phospho-l-serine] (POPS)+cholesterol (3:2, POPS:Chol, molar ratio), which contain cholesterol in crystallite form. For the zwitterionic lipid POPC, cationic amphiphiles were tested, up to 7 mol%, while for anionic POPS bilayers, which possibly incorporate more positive amphiphiles, the fractions used were higher, up to 23 mol%. 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and DOTAP in methyl sulfate salt form (DOTAP^mss) were found to cause a small decrease on the enthalpy of the cholesterol transition of pure cholesterol aggregates, possibly indicating a slight increase on the cholesterol solubilization in POPC vesicles. With the anionic system POPS:Chol, the cationic amphiphiles dramatically change the cholesterol crystal thermal transition, indicating significant changes in the cholesterol aggregates. For structural studies, phospholipids spin labeled at the 5th or 16th carbon atoms were incorporated. In POPC, at the bilayer core, the cationic amphiphiles significantly increase the bilayer packing, decreasing the membrane polarity, with the cholesterol derivative 3β-[ N-( N′, N′-dimethylaminoethane)-carbamoyl]-cholesterol (DC-chol) displaying a stronger effect. In POPS and POPS:Chol, DC-chol was also found to considerably increase the bilayer packing. Hence, exogenous cationic amphiphiles used to deliver nucleic acids to cells can change the bilayer packing of biological membranes and alter the structure of cholesterol crystals, which are believed to be the precursors to atherosclerotic lesions.

Keywords: Cationic amphiphiles; Cholesterol; Spin labels; DSC; DOTAP; DC-chol

VCAM-1 directed immunoliposomes selectively target tumor vasculature in vivo by Sara Gosk; Torben Moos; Claudia Gottstein; Gerd Bendas (pp. 854-863).

Targeting the tumor vasculature and selectively modifying endothelial functions is an attractive anti-tumor strategy. We prepared polyethyleneglycol modified immunoliposomes (IL) directed against vascular cell adhesion molecule 1 (VCAM-1), a surface receptor over-expressed on tumor vessels, and investigated the liposomal targetability in vitro and in vivo. In vitro, anti-VCAM-1 liposomes displayed specific binding to activated endothelial cells under static conditions, as well as under simulated blood flow conditions. The in vivo targeting of IL was analysed in mice bearing human Colo 677 tumor xenografts 30 min and 24 h post i.v. injection. Whereas biodistribution studies using [³H]-labelled liposomes displayed only marginal higher tumor accumulation of VCAM-1 targeted versus unspecific ILs, fluorescence microscopy evaluation revealed that their localisations within tumors differed strongly. VCAM-1 targeted ILs accumulated in tumor vessels with increasing intensities from 30 min to 24 h, while control ILs accumulated in the tumor tissue by passive diffusion. ILs that accumulated in non-affected organs, mainly liver and spleen, primarily co-localised with macrophages. This is the first morphological evidence for selective in vivo targeting of tumor vessels using ILs. VCAM-directed ILs are candidate drug delivery systems for therapeutic anti-cancer approaches designed to alter endothelial function.

Keywords: Immunoliposome; Tumor; Vascular targeting; VCAM-1; Tumor xenograft model

Role of negatively charged amino acids in β4 F-loop in activation and desensitization of α3β4 rat neuronal nicotinic receptors by Jiří Lindovský; Martina Kaniaková; Lucie Svobodová; František Vyskočil; Jan Krůšek (pp. 864-871).

The role of negatively charged amino acids in the F-loop of the β4 subunit in channel activation and desensitization was studied using the patch-clamp technique. The selected amino acids were changed to their neutral analogs via point mutations. Whole-cell currents were recorded in COS cells transiently transfected with the α3β4 nicotinic acetylcholine receptor. The application of acetylcholine (ACh), nicotine (Nic), cytisine (Cyt), carbamylcholine (CCh) and epibatidine (Epi) to cells clamped at −40 mV produced inward currents which displayed biphasic desensitization. The EC₅₀ of Epi and Nic were increased by a factor of 3–6 due to mutations D191N or D192N. Only Epi remained an agonist in the double-mutated receptors with EC₅₀ increased 17-fold. The interaction of the receptors with the competitive antagonist (+)tubocurarine (TC) was weakened almost 3-fold in the double-mutated receptors. The mutations increased the proportion of the slower desensitization component and increased the response plateau, resulting in decreased receptor desensitization. The double mutation substantially accelerated the return from long-term desensitization induced by Epi.

Keywords: Nicotinic acetylcholine receptor; α3β4; Epibatidine; F-loop

Secondary structure and orientation of the pore-forming toxin lysenin in a sphingomyelin-containing membrane by Monika Hereć; Mariusz Gagoś; Magdalena Kulma; Katarzyna Kwiatkowska; Andrzej Sobota; Wiesław I. Gruszecki (pp. 872-879).

Lysenin is a sphingomyelin-recognizing toxin which forms stable oligomers upon membrane binding and causes cell lysis. To get insight into the mechanism of the transition of lysenin from a soluble to a membrane-bound form, surface activity of the protein and its binding to lipid membranes were studied using tensiometric measurements, Fourier-transform infrared spectroscopy (FTIR) and FTIR-linear dichroism. The results showed cooperative adsorption of recombinant lysenin-His at the argon–water interface from the water subphase which suggested self-association of lysenin-His in solution. An assembly of premature oligomers by lysenin-His in solution was confirmed by blue native gel electrophoresis. When a monolayer composed of sphingomyelin and cholesterol was present at the interface, the rate of insertion of lysenin-His into the monolayer was considerably enhanced. Analysis of FTIR spectra of soluble lysenin-His demonstrated that the protein contained 27% β-sheet, 28% aggregated β-strands, 10% α-helix, 23% turns and loops and 12% different kinds of aggregated forms. In membrane-bound lysenin-His the total content of α-helices, turns and loops, and β-structures did not change, however, the 1636cm⁻¹ β-sheet band increased from 18% to 31% at the expense of the 1680cm⁻¹ β-sheet structure. Spectral analysis of the amide I band showed that the α-helical component was oriented with at 41° to the normal to the membrane, indicating that this protein segment could be anchored in the hydrophobic core of the membrane.

Keywords: Lysenin; Toxin; Sphingomyelin; Monolayer; Secondary structure determination by FTIR spectroscopy; FTIR-linear dichroism

Detection of lipid phase coexistence and lipid interactions in sphingomyelin/cholesterol membranes by ATR-FTIR spectroscopy by Zoran Arsov; Luca Quaroni (pp. 880-889).

The phase behavior of binary mixtures of egg sphingomyelin and cholesterol has been inspected by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy in the amide I′ band reion of the spectrum. Because cholesterol does not have any major absorption bands in this region, effects seen in the spectra of mixtures of sphingomyelin and cholesterol can be attributed to the change in the lipid phase and to the interaction with cholesterol. It is shown that the temperature dependence of the overall bandwidth of the amide I′ band displays a phase-specific behavior. In addition, it is observed that the amide I′ band for a sample exhibiting phase coexistence can be described by a linear combination of the spectra of the individual lipid phases. Description of changes in the amide I′ band shape and by that the study of possible hydrogen bonding interactions of sphingomyelin with cholesterol was assisted by the use of curve fitting. It turns out that the presence of hydrogen bonding between hydroxyl group of cholesterol and carbonyl group of sphingomyelin is obscured by the complexity of different possible hydrogen bonding and coupling between the N–H (N–D) and the CO group vibrations.

Keywords: Lipid phase coexistence; Cholesterol; ATR-FTIR spectroscopy; Amide I band; Hydrogen bonding

Osmotic shrinkage and reswelling of giant vesicles composed of dioleoylphosphatidylglycerol and cholesterol by M.M.A.E. Claessens; F.A.M. Leermakers; F.A. Hoekstra; M.A. Cohen Stuart (pp. 890-895).

The osmotic shrinkage of giant unilamellar dioleoylphosphatidylglycerol (DOPG) vesicles in a hypertonic osmotic solution is investigated. The volume reduction for given membrane area leads to a vesiculation of the bilayer into the interior of the giant. The size of the daughter vesicles that appear inside the giant is uniform and an increasing function of the cholesterol content, but independent of the osmotic gradient applied. The radius of the daughter vesicles increases from 0.2 μm to 3.0 μm when the cholesterol content is changed from 0 to 40%. It is argued that the size of the daughter vesicles is regulated by the membrane persistence length, which is an exponential function of the mean bending modulus. From the kinetics of shrinkage it follows that approximately 14% of the daughter vesicles remain attached to the mother giant. This is in reasonable agreement with osmotic swelling experiments which show that approximately 11% of the daughter vesicles is available for area expansion.

Keywords: Dehydration; Vesiculation

Delivery of the Cu-transporting ATPase ATP7B to the plasma membrane in Xenopus oocytes by Éva Lörinczi; Ruslan Tsivkovskii; Winfried Haase; Ernst Bamberg; Svetlana Lutsenko; Thomas Friedrich (pp. 896-906).

Cu-transporting ATPase ATP7B (Wilson disease protein) is essential for the maintenance of intracellular copper concentration. In hepatocytes, ATP7B is required for copper excretion, which is thought to occur via a transient delivery of the ATP7B- and copper-containing vesicles to the apical membrane. The currently available experimental systems do not allow analysis of ATP7B at the cell surface. Using epitope insertion, we identified an extracellular loop into which the HA-epitope can be introduced without inhibiting ATP7B activity. The HA-tagged ATP7B was expressed in Xenopus oocytes and the presence of ATP7B at the plasma membrane was demonstrated by electron microscopy, freeze-fracture experiments, and surface luminescence measurements in intact cells. Neither the deletion of the entire N-terminal copper-binding domain nor the inactivating mutation of catalytic Asp1027 affected delivery to the plasma membrane of oocytes. In contrast, surface targeting was decreased for the ATP7B variants with mutations in the ATP-binding site or the intra-membrane copper-binding site, suggesting that ligand-stabilized conformation(s) are important for ATP7B trafficking. The developed system provides significant advantages for studies that require access to both sides of ATP7B in the membrane.

Keywords: Abbreviations; AP; alkaline phosphatase; ATP-BD; ATP-binding domain; ER; endoplasmic reticulum; HA; haemagglutinin A of influenza virus; HRP; horseradish peroxidase; MBS; metal-binding sites; MNKP; Menkes disease protein; NBD; nucleotide-binding domain; N-MBD; amino-terminal metal-binding domain; WNDP; Wilson disease protein; TGN; trans; -Golgi network; TMS; transmembrane segmentATP7B; Copper; Wilson disease protein; Oocyte; Luminescence; Plasma membrane

Influence of salt on the structure of DMPG studied by SAXS and optical microscopy by Roberto M. Fernandez; Karin A. Riske; Lia Q. Amaral; Rosangela Itri; M. Teresa Lamy (pp. 907-916).

Aqueous dispersions of 50 mM dimyristoylphosphatidylglycerol (DMPG) in the presence of increasing salt concentrations (2–500 mM NaCl) were studied by small angle X-ray scattering (SAXS) and optical microscopy between 15 and 35 °C. SAXS data show the presence of a broad peak around q∼0.12 Å−¹ at all temperatures and conditions, arising from the electron density contrasts within the bilayer. Up to 100 mM NaCl, this broad peak is the main feature observed in the gel and fluid phases. At higher ionic strength (250–500 mM NaCl), an incipient lamellar repeat distance around d=90–100 Å is detected superimposed to the bilayer form factor. The data with high salt were fit and showed that the emergent Bragg peak is due to loose multilamellar structures, with the local order vanishing after ∼4 d. Optical microscopy revealed that up to 20 mM NaCl, DMPG is arranged in submicroscopic vesicles. Giant (loose) multilamellar vesicles (MLVs) start to appear with 50 mM NaCl, although most lipids are arranged in small vesicles. As the ionic strength increases, more and denser MLVs are seen, up to 500 mM NaCl, when MLVs are the prevailing structure. The DLVO theory could account for the experimentally found interbilayer distances .

Keywords: Gel–fluid phase transition; Intermediate phase; Charged phospholipid; DLVO theory

Ceramides modulate cell-surface acetylcholine receptor levels by C.E. Gallegos; M.F. Pediconi; F.J. Barrantes (pp. 917-930).

The effects of ceramides (Cer) on the trafficking of the nicotinic acetylcholine receptor (AChR) to the plasma membrane were studied in CHO-K1/A5 cells, a clonal cell line that heterologously expresses the adult murine form of the receptor. When cells were incubated with short- (C₆-Cer) or long- (brain-Cer) chain Cer at low concentrations, an increase in the number of cell-surface AChRs was observed concomitant with a decrease in intracellular receptor levels. The alteration in AChR distribution by low Cer treatment does not appear to be a general mechanism since the surface expression of the green fluorescent protein derivative of the vesicular stomatitis virus protein (VSVG-GFP) was not affected. High Cer concentrations caused the opposite effects, decreasing the number of cell-surface AChRs, which exhibited higher affinity for [¹²⁵I]-α-bungarotoxin, and increasing the intracellular pool, which colocalized with trans-Golgi/TGN specific markers. The generation of endogenous Cer by sphingomyelinase treatment also decreased cell-surface AChR levels. These effects do not involve protein kinase Cζ or protein phosphatase 2A activation. Taken together, the results indicate that Cer modulate trafficking of AChRs to and stability at the cell surface.

Keywords: Abbreviations; AChR; nicotinic acetylcholine receptor; Cer; ceramides; CHO; Chinese hamster ovary; Chol; cholesterol; ER; endoplasmic reticulum; Gal-T; galactosyltransferase; GFP; green fluorescence protein; GlcCer; glucosylceramide; LDH; lactate dehydrogenase; RFP; red fluorescent protein; SLs; sphingolipids; SM; sphingomyelin; SMase; sphingomyelinase; TGN; trans; -Golgi network; VSVG; vesicular stomatitis virus protein; YFP; yellow fluorescent protein; αBTX; α-bungarotoxinNicotinic receptor; Ceramide; Sphingosine; Protein traffic; Sphingomyelinase

Cyclooxygenase inhibition by diclofenac formulated in bioadhesive carriers by Inbar Elron-Gross; Yifat Glucksam; Dina Melikhov; Rimona Margalit (pp. 931-936).

Adverse effects and gastrointestinal toxicity limit the use of Diclofenac, a frequently-used NSAID for treatments of rheumatic disorders and other chronic inflammatory diseases. Diclofenac-carrier formulations may alleviate adverse effects, increase efficacy and allow local administration. We report here our first step, biophysical and biochemical investigations of Diclofenac formulated in our previously-developed bioadhesive liposomes carrying hyaluronan (HA-BAL) or collagen (COL-BAL) on their surface. Both liposome types encapsulated Diclofenac at high efficiency, encapsulated doses reaching 13mg drug/ml, and performed as sustained-release Diclofenac depots, half-lives of drug release (under fastest conditions) ranging from 1 to 3days. Therapeutic activity of liposomal Diclofenac was evaluated in CT-26 cells that possess the CD44 hyaluronan receptors and integrins, and are a bench-mark for intracellular COX enzymes. HA-BAL and COL-BAL showed high cellular-affinity that was 40 fold and 6 fold over that of regular liposomes. Free, and liposome-encapsulated, Diclofenac showed similar activities. For example: 2–3nM Diclofenac given to intact cells generated COX-inhibition levels in the range of 60–70% for free drug and for encapsulated drug in COL-BAL and in HA-BAL. We propose these novel Diclofenac formulations possess key physicochemical and biochemical attributes for task performance, meriting the next step into in vivo studies.

Keywords: Osteoarthritis; Diclofenac; Cyclooxygenase; Drug carriers; Hyaluronan; Collagen

Solution NMR of signal peptidase, a membrane protein by Monika Musial-Siwek; Debra A. Kendall; Philip L. Yeagle (pp. 937-944).

Useful solution nuclear magnetic resonance (NMR) data can be obtained from full-length, enzymatically active type I signal peptidase (SPase I), an integral membrane protein, in detergent micelles. Signal peptidase has two transmembrane segments, a short cytoplasmic loop, and a 27-kD C-terminal catalytic domain. It is a critical component of protein transport systems, recognizing and cleaving amino-terminal signal peptides from preproteins during the final stage of their export. Its structure and interactions with the substrate are of considerable interest, but no three-dimensional structure of the whole protein has been reported. The structural analysis of intact membrane proteins has been challenging and only recently has significant progress been achieved using NMR to determine membrane protein structure. Here we employ NMR spectroscopy to study the structure of the full-length SPase I in dodecylphosphocholine detergent micelles. HSQC–TROSY spectra showed resonances corresponding to approximately 3/4 of the 324 residues in the protein. Some sequential assignments were obtained from the 3D HNCACB, 3D HNCA, and 3D HN(CO) TROSY spectra of uniformly²H,¹³C,¹⁵N-labeled full-length SPase I. The assigned residues suggest that the observed spectrum is dominated by resonances arising from extramembraneous portions of the protein and that the transmembrane domain is largely absent from the spectra. Our work elucidates some of the challenges of solution NMR of large membrane proteins in detergent micelles as well as the future promise of these kinds of studies.

Keywords: Abbreviations; DPC; dodecylphosphocholine; HRMAS; high resolution magic angle spinning; HSQC; heteronuclear single-quantum correlation; NMR; nuclear magnetic resonance; OG; n; -octyl-β-; d; -glucopyranoside; SPase I; type I signal peptidaseSignal; peptidase; NMR; Membrane; protein

Membrane curvature and surface area per lipid affect the conformation and oligomeric state of HIV-1 fusion peptide: A combined FTIR and MD simulation study by Bogdan Barz; Tuck C. Wong; Ioan Kosztin (pp. 945-953).

Fourier-transformed infrared spectroscopy (FTIR) and molecular dynamics (MD) simulation results are presented to support our hypothesis that the conformation and the oligomeric state of the HIV-1 gp41 fusion domain or fusion peptide (gp41-FP) are determined by the membrane surface area per lipid (APL), which is affected by the membrane curvature. FTIR of the gp41-FP in the Aerosol-OT (AOT) reversed micellar system showed that as APL decreases from ∼50 to 35 Å² by varying the AOT/water ratio, the FP changes from the monomeric α-helical to the oligomeric β-sheet structure. MD simulations in POPE lipid bilayer systems showed that as the APL decreases by applying a negative surface tension, helical monomers start to unfold into turn-like structures. Furthermore, an increase in the applied lateral pressure during nonequilibrium MD simulations favored the formation of β-sheet structure. These results provide better insight into the relationship between the structures of the gp41-FP and the membrane, which is essential in understanding the membrane fusion process. The implication of the results of this work on what is the fusogenic structure of the HIV-1 FP is discussed.

Keywords: HIV; gp41 fusion peptide; Membrane fusion; Conformation; Fourier-transformed infrared spectroscopy (FTIR); Molecular dynamics (MD) simulation

N-palmitoyl-sulfatide participates in lateral domain formation in complex lipid bilayers by Y.J.E. Björkqvist; S. Nybond; T.K.M. Nyholm; J.P. Slotte; B. Ramstedt (pp. 954-962).

Sulfatides (galactosylceramidesulfates) are negatively charged glycosphingolipids that are important constituents of brain myelin membranes. These membranes are also highly enriched in galactosylceramide and cholesterol. It has been implicated that sulfatides, together with other sphingolipids, take part in lateral domain formation in biological membranes. This study was conducted to characterize the lateral phase behavior of N-palmitoyl-sulfatide in mixed bilayer membranes. Going from simple lipid mixtures with sulfatide as the only sphingolipid in a fluid matrix of POPC, to more complex membranes including other sphingolipids, we have examined 1) ordered domain formation with sulfatide, 2) sterol enrichment in such domains and 3) stabilization of the domains against temperature by the addition of calcium. Using two distinct phase selective fluorescent probes, trans-parinaric acid and cholestatrienol, together with a quencher in the fluid phase, we were able to distinguish between ordered domains in general and ordered domains enriched in sterol. We found that N-palmitoyl-sulfatide formed ordered domains when present as the only sphingolipid in a fluid phospholipid bilayer, but these domains did not contain sterol and their stability was unaffected by calcium. However, at low, physiologically relevant concentrations, sulfatide partitioned favorably into domains enriched in other sphingolipids and cholesterol. These domains were stabilized against temperature in the presence of divalent cations. We conclude that sulfatides are likely to affect the lateral organization of biomembranes.

Keywords: Abbreviations; 7SLPC; 1-palmitoyl-2-stearoyl-(7-doxyl)-; sn; -glycero-3-phosphocholine; AFM; atomic force microscopy; CTL; cholestatrienol; DPH; diphenylhexatriene; DSPC; di-stearoyl-; sn; -glycero-3-phosphocholine; FTIR; fourier transform infrared spectroscopy; PGalCer; N; -palmitoyl-galactosylceramide; PGlcCer; N; -palmitoyl-glucosylceramide; PLacCer; N; -palmitoyl-lactosylceramide; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; PSM; N; -palmitoyl-sphingomyelin; tPA; trans; -parinaric acid; tPar-SM; N-trans; parinoyl-sphingomyelin; tPar-sulfatide; 3-; O; -sulfo-; d; -galactosyl-β1-1′-; N-trans; parinoyl-; d; -; erythro; -sphingosineGalactosylceramidesulfate; Calcium; Cholesterol; Cholestatrienol; trans; -parinaric acid; Raft

Transmembrane topology of the Acr3 family arsenite transporter from Bacillus subtilis by Emil K.J. Aaltonen; Maria Silow (pp. 963-973).

The transmembrane topology of the Acr3 family arsenite transporter Acr3 from Bacillus subtilis was analysed experimentally using translational fusions with alkaline phosphatase and green fluorescent protein and in silico by topology modelling. Initial topology prediction resulted in two models with 9 and 10 TM helices respectively. 32 fusion constructs were made between truncated forms of acr3 and the reporter genes at 17 different sites throughout the acr3 sequence to discriminate between these models. Nine strong reporter protein signals provided information about the majority of the locations of the cytoplasmic and extracellular loops of Acr3 and showed that both the N- and the C-termini are located in the cytoplasm. Two ambiguous data points indicated the possibility of an alternative 8 helix topology. This possibility was investigated using another 10 fusion variants, but no experimental support for the 8 TM topology was obtained. We therefore conclude that Acr3 has 10 transmembrane helices. Overall, the loops which connect the membrane spanning segments are short, with cytoplasmic loops being somewhat longer than the extracellular loops. The study provides the first ever experimentally derived structural information on a protein of the Acr3 family which constitutes one of the largest classes of arsenite transporters.

Keywords: Abbreviations; PhoA; Alkaline Phosphatase from; E. coli; GFP; Green Fluorescent Protein; TM; Transmembrane; SCAM; Substituted Cysteine Accessibility MethodArsenite; Transmembrane topology; Acr3; Translational fusion; Alkaline phosphatase; GFP

Interaction with dopamine D2 receptor enhances expression of transient receptor potential channel 1 at the cell surface by Meredith A. Hannan; Nadine Kabbani; Constantinos D. Paspalas; Robert Levenson (pp. 974-982).

Receptor signaling is mediated by direct protein interaction with various types of cytoskeletal, adapter, effector, and additional receptor molecules. In brain tissue and in cultured neurons, activation of dopamine D2 receptors (D2Rs) has been found to impact cellular calcium signaling. Using a yeast two-hybrid approach, we have uncovered a direct physical interaction between the D2R and the transient receptor potential channel (TRPC) subtypes 1, 4 and 5. The TRPC/D2R interaction was further validated by GST-pulldown assays and coimmunoprecipitation from mammalian brain. Ultrastructural analysis of TRPC1 and D2R expression indicates colocalization of the two proteins within the cell body and dendrites of cortical neurons. In cultured cells, expression of D2Rs was found to increase expression of TRPC1 at the cell surface by 50%. These findings shed new light on the constituents of the D2R signalplex, and support the involvement of D2Rs in cellular calcium signaling pathways via a novel link to TRPC channels.

Keywords: Dopamine receptor; Transient receptor potential; TRPC1; G-protein coupled receptor; Interacting proteins; Calcium signaling

Binding of LL-37 to model biomembranes: Insight into target vs host cell recognition by Rohit Sood; Yegor Domanov; Milla Pietiäinen; Vesa P. Kontinen; Paavo K.J. Kinnunen (pp. 983-996).

Pursuing the molecular mechanisms of the concentration dependent cytotoxic and hemolytic effects of the human antimicrobial peptide LL-37 on cells, we investigated the interactions of this peptide with lipids using different model membranes, together with fluorescence spectroscopy for the Trp-containing mutant LL-37(F27W). Minimum concentrations inhibiting bacterial growth and lipid interactions assessed by dynamic light scattering and monolayer penetration revealed the mutant to retain the characteristics of native LL-37. Although both LL-37 and the mutant intercalated effectively into zwitterionic phosphatidylcholine membranes the presence of acidic phospholipids caused augmented membrane binding. Interestingly, strongly attenuated intercalation of LL-37 into membranes containing both cholesterol and sphingomyelin (both at X=0.3) was observed. Accordingly, the distinction between target and host cells by LL-37 is likely to derive from i) acidic phospholipids causing enhanced association with the former cells as well as ii) from attenuated interactions with the outer surface of the plasma membrane of the peptide secreting host, imposed by its high content of cholesterol and sphingomyelin. Our results further suggest that LL-37 may exert its antimicrobial effects by compromising the membrane barrier properties of the target microbes by a mechanism involving cytotoxic oligomers, similarly to other peptides forming amyloid-like fibers in the presence of acidic phospholipids.

Keywords: Abbreviations; AMPs; antimicrobial peptides; Chol; cholesterol; CD; circular dichroism; DPPC; 1,2-dipalmitoyl-; sn; -glycero-3-phosphocholine; DPPE; 1,2-dipalmitoyl-; sn; -glycero-3-phosphoethanolamine; DPPG; 1,2-dipalmitoyl-; sn; -glycero-3-[phospho-; rac; -(1-glycerol)]; DPPDns; 1,2-dipalmitoyl-; sn; -glycero-3-phosphoethanolamino; N; -(5-dimethylaminonaphthalene-1-sulfonyl)triethylammonium salt; ET; energy transfer efficiencies; EDTA; ethylenediaminetetraacetic acid; FRET; fluorescence resonance energy transfer; K; _SV; Stern–Volmer quenching constant; k; _q; bimolecular quenching constant; K; _d; apparent dissociation constant; LL-37; native LL-37; LL-37(F27W) with Phe27 replaced by Trp; LUV; large unilamellar vesicles; L/P; lipid to peptide ratio; LB; Luria–Bertani; MIC; minimal inhibitory concentration; NBD-PC; 1-oleoyl-2-[6-[7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-; sn; -glycero-3-phosphocholine; POPG; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phospho-rac-glycerol; POPS; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phospho-; l; -serine; (6,7)-Br; ₂; -PC; 1-palmitoyl-2-(6,7-dibromostearoyl)phosphocholine; (9,10)-Br; ₂; -PC; 1-palmitoyl-2-(9,10-dibromostearoyl)phosphocholine; (11,12)-Br; ₂; -PC,-palmitoyl-2-(11,12-dibromostearoyl)phosphocholine; PC; phosphatidylcholine; PG; phosphatidylglycerol; PS; phosphatidylserine; P/L; peptide to lipid ratio; Q; quencher; RT; room temperature; Spm; sphingomyelin; SLBs; supported lipid bilayers; SOPC; 1-stearoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; λ; _max; fluorescence emission maximum; τ; _f; fluorescence lifetime; ζ; zeta potentialLL-37(F27W); Fluorescence spectroscopy; Acidic phospholipid; Phase separation; Cytotoxic oligomer; Amyloid-like fiber

Phase behavior of phosphatidylglycerol in spinach thylakoid membranes as revealed by³¹P-NMR by Sashka B. Krumova; Cor Dijkema; Pieter de Waard; Henk Van As; Győző Garab; Herbert van Amerongen (pp. 997-1003).

Non-bilayer lipids account for about half of the total lipid content in chloroplast thylakoid membranes. This lends high propensity of the thylakoid lipid mixture to participate in different phases which might be functionally required. It is for instance known that the chloroplast enzyme violaxanthin de-epoxidase (VDE) requires a non-bilayer phase for proper functioning in vitro but direct evidence for the presence of non-bilayer lipid structures in thylakoid membranes under physiological conditions is still missing.In this work, we used phosphatidylglycerol (PG) as an intrinsic bulk lipid label for³¹P-NMR studies to monitor lipid phases of thylakoid membranes. We show that in intact thylakoid membranes the characteristic lamellar signal is observed only below 20 °C. But at the same time an isotropic phase is present, which becomes even dominant between 14 and 28 °C despite the presence of fully functional large membrane sheets that are capable of generating and maintaining a transmembrane electric field. Tris-washed membranes show a similar behavior but the lamellar phase is present up to higher temperatures. Thus, our data show that the location of the phospholipids is not restricted to the bilayer phase and that the lamellar phase co-exists with a non-bilayer isotropic phase.

Keywords: Non-bilayer lipid phases; Isotropic lipid phase; Phosphatidylglycerol; ³¹; P-NMR; Thylakoid membranes

Thermodynamics of the interactions of tryptophan-rich cathelicidin antimicrobial peptides with model and natural membranes by Valery V. Andrushchenko; Mohammed H. Aarabi; Leonard T. Nguyen; Elmar J. Prenner; Hans J. Vogel ⁎ (pp. 1004-1014).

Tritrpticin and indolicidin are short 13-residue tryptophan-rich antimicrobial peptides that hold potential as future alternatives for antibiotics. Isothermal titration calorimetry (ITC) has been applied as the main tool in this study to investigate the thermodynamics of the interaction of these two cathelicidin peptides as well as five tritrpticin analogs with large unilamellar vesicles (LUVs), representing model and natural anionic membranes. The anionic LUVs were composed of (a) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPE/POPG) (7:3) and (b) natural E. coli polar lipid extract. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was used to make model zwitterionic membranes. Binding isotherms were obtained to characterize the antimicrobial peptide binding to the LUVs, which then allowed for calculation of the thermodynamic parameters of the interaction. All peptides exhibited substantially stronger binding to anionic POPE/POPG and E. coli membrane systems than to the zwitterionic POPC system due to strong electrostatic attractions between the highly positively charged peptides and the negatively charged membrane surface, and results with tritrpticin derivatives further revealed the effects of various amino acid substitutions on membrane binding. No significant improvement was observed upon increasing the Tritrp peptide charge from +4 to +5. Replacement of Arg residues with Lys did not substantially change peptide binding to anionic vesicles but moderately decreased the binding to zwitterionic LUVs. Pro to Ala substitutions in tritrpticin, allowing the peptide to adopt an α-helical structure, resulted in a significant increase of the binding to both anionic and zwitterionic vesicles and therefore reduced the selectivity for bacterial and mammalian membranes. In contrast, substitution of Trp with other aromatic amino acids significantly decreased the peptide's ability to bind to anionic LUVs and essentially eliminated binding to zwitterionic LUVs. The ITC results were consistent with the outcome of fluorescence spectroscopy membrane binding and perturbation studies. Overall, our work showed that a natural E. coli polar lipid extract as a bacterial membrane model was advantageous compared to the simpler and more widely used POPE/POPG lipid system.

Keywords: Abbreviations; POPC; 1-palmitoyl-2oleoyl-sn-glycero-3-phosphocholine; POPE; 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; POPG; 1-palmitoyl-2oleoyl-sn-glycero-3-phosphoglycerol; ePC; egg α-phosphatidylcholine; ePE; egg α-phosphatidylethanolamine; ePG; egg α-phosphatidylglycerol; ITC; isothermal titration calorimetry; LUVs; large unilamellar vesiclesAntimicrobial peptide; ITC; Indolicidin; Lipid vesicle; Tritrpticin

Phospholipid membranes affect tertiary structure of the soluble cytochrome b₅ heme-binding domain by Liana V. Basova; Elisaveta I. Tiktopulo; Victor P. Kutyshenko; A. Grant Mauk; Valentina E. Bychkova (pp. 1015-1026).

The influence of charged phospholipid membranes on the conformational state of the water-soluble fragment of cytochrome b₅ has been investigated by a variety of techniques at neutral pH. The results of this work provide the first evidence that aqueous solutions with high phospholipid/protein molar ratios (pH 7.2) induce the cytochrome to undergo a structural transition from the native conformation to an intermediate state with molten-globule like properties that occur in the presence of an artificial membrane surface and that leads to binding of the protein to the membrane. At other phospholipid/protein ratios, equilibrium was observed between cytochrome free in solution and cytochrome bound to the surface of vesicles. Inhibition of protein binding to the vesicles with increasing ionic strength indicated for the most part an electrostatic contribution to the stability of cytochrome b₅vesicle interactions at pH 7.2. The possible physiological role of membrane-induced conformational change in the structure of cytochrome b₅ upon the interaction with its redox partners is discussed.

Keywords: Cytochrome; b; ₅; water-soluble fragment; Artificial membranes; Protein denaturation; Intermediate state

The influence of phase transitions in phosphatidylethanolamine models on the activity of violaxanthin de-epoxidase by Astrid Vieler; Holger A. Scheidt; Peter Schmidt; Cindy Montag; Janine F. Nowoisky; Martin Lohr; Christian Wilhelm; Daniel Huster; Reimund Goss (pp. 1027-1034).

In the present study, the influence of the phospholipid phase state on the activity of the xanthophyll cycle enzyme violaxanthin de-epoxidase (VDE) was analyzed using different phosphatidylethanolamine species as model lipids. By using³¹P NMR spectroscopy, differential scanning calorimetry and temperature dependent enzyme assays, VDE activity could directly be related to the lipid structures the protein is associated with. Our results show that the gel (L_β) to liquid-crystalline (L_α) phase transition in these single lipid component systems strongly enhances both the solubilization of the xanthophyll cycle pigment violaxanthin in the membrane and the activity of the VDE. This phase transition has a significantly stronger impact on VDE activity than the transition from the L_α to the inverted hexagonal (H_II) phase. Especially at higher temperatures we found increased VDE reaction rates in the presence of the L_α phase compared to those in the presence of H_II phase forming lipids. Our data furthermore imply that the H_II phase is better suited to maintain high VDE activities at lower temperatures.

Keywords: Abbreviations; Ax; Antheraxanthin; DOPE; dioleoylphosphatidylethanolamine; DSC; differential scanning calorimetry; DTT; dithiothreitol; EDTA; ethylenediaminetetraacetate; eggPE; phosphatidylethanolamine from egg yolk; GdmHCl; guanidinium hydrochloride; GSH; reduced glutathione; GSSG; oxidized glutathione; H; _II; inverted hexagonal phase; IPTG; isopropylthiogalactoside; L; _α; liquid-crystalline phase; L; _β; gel phase; MGDG; monogalactosyldiacylglycerol; POPE; palmitoyloleoylphosphatidylethanolamine; VDE; violaxanthin de-epoxidase; Vx; Violaxanthin; Zx; ZeaxanthinPhospholipid structures; ³¹; P NMR; Inverted hexagonal phase; Arrhenius; De-epoxidation; Monogalactosyldiacylglycerol

Electrostatic interactions and complement activation on the surface of phospholipid vesicle containing acidic lipids: Effect of the structure of acidic groups by Keitaro Sou; Eishun Tsuchida (pp. 1035-1041).

Anionic vesicles containing acidic phospholipids are known complement activators. To clarify which negative physicochemical electrostatic charges on vesicles and structural specificities of acidic lipids are critical to complement activation, the electrostatic properties and activity to complement of two anionic vesicles modified with a carboxylic acid derivative or a conventional acidic phospholipid were compared. Electrophoretic mobility measurements indicated that the negative zeta potential and the electrostatic interactivity of these two anionic vesicles were equal at pH 7.4. However, the infusion of vesicles containing acidic phospholipid induced significant complement activation, while vesicles containing the carboxylic acid derivative failed to activate complement. These results indicate that the negative charge on the surface of vesicles is not critical for the activation complement, suggesting that complement activation is specific to the structure of acidic groups. This finding is likely to be important to the design of anionic biointerfaces and may support the promising medical applications of this anionic vesicle modified with a carboxylic acid derivative.

Keywords: Liposome; Acidic lipid; Anionic surface; Electrostatic interaction; Complement activation

Mutational analysis of histidine residues in the human proton-coupled amino acid transporter PAT1 by Linda Metzner; Kristin Natho; Katja Zebisch; Madlen Dorn; Eva Bosse-Doenecke; Vadivel Ganapathy; Matthias Brandsch (pp. 1042-1050).

The proton-coupled amino acid transporter 1 (PAT1) represents a major route by which small neutral amino acids are absorbed after intestinal protein digestion. The system also serves as a novel route for oral drug delivery. Having shown that H⁺ affects affinity constants but not maximal velocity of transport, we investigated which histidine residues are obligatory for PAT1 function. Three histidine residues are conserved among the H⁺-coupled amino acid transporters PAT1 to 4 from different animal species. We individually mutated each of these histidine residues and compared the catalytic function of the mutants with that of the wild type transporter after expression in HRPE cells. His-55 was found to be essential for the catalytic activity of hPAT1 because the corresponding mutants H55A, H55N and H55E had no detectablel-proline transport activity. His-93 and His-135 are less important for transport function since H93N and H135N mutations did not impair transport function. The loss of transport function of His-55 mutants was not due to alterations in protein expression as shown both by cell surface biotinylation immunoblot analyses and by confocal microscopy. We conclude that His-55 might be responsible for binding and translocation of H⁺ in the course of cellular amino acid uptake by PAT1.

Keywords: Abbreviations; CHDP; cis; -4-hydroxy-; d; -proline; CHLP; cis; -4-hydroxy-; l; -proline; DAPI; 4′,6-diamidino-2-phenylindole dihydrochloride; DEPC; diethylpyrocarbonate; GABA; γ-aminobutyric acid; HA; hemagglutinin; HRPE; human retinal pigment epithelium; IU; infectious units; LACA; l; -azetidine-2-carboxylic acid; MeAIB; α-(methylamino)isobutyric acid; MVA; modified vaccinia virus Ankara; PAT1; proton-coupled amino acid transporter 1; PEPT1; peptide transporter 1; TMD; transmembrane domain;Amino acid transport; PAT1; Histidine residues; Site directed mutagenesis; Drug delivery; Proline

Identification of transport pathways for citric acid cycle intermediates in the human colon carcinoma cell line, Caco-2 by Jittima Weerachayaphorn; Ana M. Pajor (pp. 1051-1059).

Citric acid cycle intermediates are absorbed from the gastrointestinal tract through carrier-mediated mechanisms, although the transport pathways have not been clearly identified. This study examines the transport of citric acid cycle intermediates in the Caco-2 human colon carcinoma cell line, often used as a model of small intestine. Inulin was used as an extracellular volume marker instead of mannitol since the apparent volume measured with mannitol changed with time. The results show that Caco-2 cells contain at least three distinct transporters, including the Na⁺-dependent di- and tricarboxylate transporters, NaDC1 and NaCT, and one or more sodium-independent pathways, possibly involving organic anion transporters. Succinate transport is mediated mostly by Na⁺-dependent pathways, predominantly by NaDC1, but with some contribution by NaCT. RT-PCR and functional characteristics verified the expression of these transporters in Caco-2 cells. In contrast, citrate transport in Caco-2 cells occurs by a combination of Na⁺-independent pathways, possibly mediated by an organic anion transporter, and Na⁺-dependent mechanisms. The non-metabolizable dicarboxylate, methylsuccinate, is also transported by a combination of Na⁺-dependent and -independent pathways. In conclusion, we find that multiple pathways are involved in the transport of di- and tricarboxylates by Caco-2 cells. Since many of these pathways are not found in human intestine, this model may be best suited for studying Na⁺-dependent transport of succinate by NaDC1.

Keywords: Abbreviations; NaDC1; Na; ⁺; /dicarboxylate cotransporter 1; NaCT; Na; ⁺; -coupled citrate transporter; OAT; organic anion transporter; TEER; transepithelial electrical resistanceCaco-2 cells; Transporter; Sodium; Organic anion; Succinate; Citrate; Small intestine

Chemical cleavage of fusion proteins for high-level production of transmembrane peptides and protein domains containing conserved methionines by Jian Hu; Huajun Qin; Mukesh Sharma; Timothy A. Cross; Fei Philip Gao (pp. 1060-1066).

Due to their high hydrophobicity, it is a challenge to obtain high yields of transmembrane peptides for structural and functional characterization. In the present work, a robust method is developed for the expression, purification and reconstitution of transmembrane peptides, especially for those containing conserved methionines. By using a truncated and mutated glutathione- S-transferase construct as the carrier protein and hydroxylamine (which specifically cleaves the peptide bond between Asn and Gly) as the cleavage reagent, 10 mg of the first transmembrane helix of CorA, a Mg²⁺ transporter from Mycobacterium tuberculosis, can be conveniently obtained with high purity from 1 L of M9 minimal media under optimized conditions. The biophysical properties of the peptide were studied by circular dichroism and nuclear magnetic resonance spectroscopy, and the results show that this CorA peptide is well folded in detergent micelles and the secondary structure is very similar to that in recent crystal structures. In addition, this CorA construct is oligomeric in perfluoro-octanoic acid micelles. The compatibility with the transmembrane peptides containing conserved methionines, the high yield and the simple process make the present method competitive with other commonly used methods to produce such peptides for structural and functional studies.

Keywords: Abbreviations; CD; circular dichroism; CorA-TM1; the first transmembrane helix of CorA; CSI; chemical shift index; DPC; dodecyl phosphocholine; ESI-TOF mass; electrospray ionization time-of-flight mass; CNBr; cyanogen bromide; GST; glutathione-; S; -transferase; GuHCl; guanidine chloride; HSQC; heteronuclear single quantum coherence; IPTG; isopropyl-β-; d; -thiogalactopyranoside; LIC; ligation independent cloning; MBP; maltose binding protein; NMR; nuclear magnetic resonance; PFO; perfluoro-octanoic acid; TEV; tobacco etch virus protease; TMPs; transmembrane peptides; TGST; truncated glutathione-; S; -transferase; SDS; sodium dodecyl sulfateHydroxylamine; Transmembrane peptide; Fusion protein expression; Glutathione-; S; -transferase; Membrane protein

Effect of hydrostatic pressure on the bilayer phase behavior of symmetric and asymmetric phospholipids with the same total chain length by Masaki Goto; Masataka Kusube; Nobutake Tamai; Hitoshi Matsuki; Shoji Kaneshina (pp. 1067-1078).

The bilayer phase transitions of palmitoylstearoyl-phosphatidylcholine (PSPC), diheptadecanoyl-PC (C17PC) and stearoylpalmitoyl-PC (SPPC) which have the same total carbon numbers in the two acyl chains were observed by differential scanning calorimetry and high-pressure optical method. As the temperature increased, these bilayers exhibited four phases of the subgel (L_c), lamellar gel (L_β′), ripple gel (P_β′) and liquid crystal (L_α), in turn. The L_c phase was observed only in the first heating scan after cold storage. The temperatures of the phase transitions were almost linearly elevated by applying pressure. The temperature–pressure phase diagrams and the thermodynamic quantities associated with the phase transitions were compared among the lipid bilayers. For all the bilayers studied, the pressure-induced interdigitated gel (L_βI) phase appeared above the critical interdigitation pressure (CIP) between the L_β′ and P_β′ phases. The CIPs for the PSPC, C17PC and SPPC bilayers were found to be 50.6, 79.1 and 93.0 MPa, respectively. Contribution of two acyl chains to thermodynamic properties for the phase transitions of asymmetric PSPC and SPPC bilayers was not even. The sn-2 acyl chain lengths of asymmetric PCs governed primarily the bilayer properties. The fluorescence spectra of Prodan in lipid bilayers showed the emission maxima characteristic of bilayer phases, which were dependent on the location of Prodan in the bilayers. Second derivative of fluorescent spectrum exhibited the original emission spectrum of Prodan to be composed of the distribution of Prodan into multiple locations in the lipid bilayer. The F″₄₉₇/ F″₄₃₀ value, a ratio of second derivative of fluorescence intensity at 497 nm to that at 430 nm, is decisive evidence whether bilayer interdigitation will occur. With respect to the L_β′/L_βI phase transition in the SPPC bilayer, the emission maximum of Prodan exhibited the narrow-range red-shift from 441 to 449 nm, indicating that the L_βI phase in the SPPC bilayer has a less polar “pocket” formed by a space between uneven terminal methyl ends of the sn-1 and sn-2 chains, in which the Prodan molecule remains stably.

Keywords: Asymmetric phospholipid; Bilayer membrane; Fluorescence; Interdigitation; Phase transition; Pressure

Characterization of lipid domains in reconstituted porcine lens membranes using EPR spin-labeling approaches by Marija Raguz; Justyna Widomska; James Dillon; Elizabeth R. Gaillard; Witold K. Subczynski (pp. 1079-1090).

The physical properties of membranes derived from the total lipid extract of porcine lenses before and after the addition of cholesterol were investigated using EPR spin-labeling methods. Conventional EPR spectra and saturation-recovery curves indicate that the spin labels detect a single homogenous environment in membranes before the addition of cholesterol. After the addition of cholesterol (when cholesterol-to-phospholipid mole to mole ratio of 1.55–1.80 was achieved), two domains were detected by the discrimination by oxygen transport method using a cholesterol analogue spin label. The domains were assigned to a bulk phospholipid–cholesterol bilayer made of the total lipid mixture and to a cholesterol crystalline domain. Because the phospholipid analogue spin labels cannot partition into the pure cholesterol crystalline domain, they monitor properties of the phospholipid–cholesterol domain outside the pure cholesterol crystalline domain. Profiles of the order parameter, hydrophobicity, and oxygen transport parameter are identical within experimental error in this domain when measured in the absence and presence of a cholesterol crystalline domain. This indicates that both domains, the phospholipid–cholesterol bilayer and the pure cholesterol crystalline domain, can be treated as independent, weakly interacting membrane regions. The upper limit of the oxygen permeability coefficient across the cholesterol crystalline domain at 35 °C had a calculated value of 42.5 cm/s, indicating that the cholesterol crystalline domain can significantly reduce oxygen transport to the lens center. This work was undertaken to better elucidate the major factors that determine membrane resistance to oxygen transport across the lens lipid membrane, with special attention paid to the cholesterol crystalline domain.

Keywords: Lens lipids; Cholesterol domain; Oxygen transport; Cholesterol; Membrane; Spin label; EPR

GLUT1 and GLUT9 as major contributors to glucose influx in HepG2 cells identified by a high sensitivity intramolecular FRET glucose sensor by Hitomi Takanaga; Bhavna Chaudhuri; Wolf B. Frommer (pp. 1091-1099).

Genetically encoded FRET glucose nanosensors have proven to be useful for imaging glucose flux in HepG2 cells. However, the dynamic range of the original sensor was limited and thus it did not appear optimal for high throughput screening of siRNA populations for identifying proteins involved in regulation of sugar flux. Here we describe a hybrid approach that combines linker-shortening with fluorophore-insertion to decrease the degrees of freedom for fluorophore positioning leading to improved nanosensor dynamics. We were able to develop a novel highly sensitive FRET nanosensor that shows a 10-fold higher ratio change and dynamic range (0.05–11 mM) in vivo, permitting analyses in the physiologically relevant range. As a proof of concept that this sensor can be used to screen for proteins playing a role in sugar flux and its control, we used siRNA inhibition of GLUT family members and show that GLUT1 is the major glucose transporter in HepG2 cells and that GLUT9 contributes as well, however to a lower extent. GFP fusions suggest that GLUT1 and 9 are preferentially localized to the plasma membrane and thus can account for the transport activity. The improved sensitivity of the novel glucose nanosensor increases the reliability of in vivo glucose flux analyses, and provides a new means for the screening of siRNA collections as well as drugs using high-content screens.

Keywords: Abbreviations; FLIP; fluorescent indicator protein; FLIIP; fluorescent intramolecular indicator protein; FRET; fluorescence resonance energy transfer; Gluc; glucose; CYT; cytosolGLUT; Transporter; Liver; Blood; Homeostasis

Glycosylation induces shifts in the lateral distribution of cholesterol from ordered towards less ordered domains by Katrin K. Halling; Bodil Ramstedt; J. Peter Slotte (pp. 1100-1111).

Several studies have indicated the involvement of steryl glycosides in the cellular stress response. In this work, we have compared the effect of 1- O-cholesteryl-β-d-glucoside, 1- O-cholesteryl-β-d-galactoside and cholesterol on the properties of glycerophospholipid and sphingolipid bilayers. The studies were performed in order to gain insight into the change in membrane properties that would follow upon the glycosylation of cholesterol in cells subjected to stress. DPH anisotropy measurements indicated that the cholesteryl glycosides (10–40 mol%) increased the order of the hydrophobic region of a POPC bilayer almost as efficiently as cholesterol. In a PSM bilayer, the cholesteryl glycosides were however shown to be much less effective compared to cholesterol in ordering the hydrocarbon chain region at temperatures above the gel to liquid-crystalline phase transition. Fluorescence quenching analysis of multicomponent lipid bilayers demonstrated that the cholesteryl glycosides, in contrast to cholesterol, were unable to stabilize ordered domains rich in PSM against temperature-induced dissociation. When the sterols were incorporated into bilayers composed of both POPC and PSM, the cholesteryl glycosides showed a higher propensity, compared to cholesterol, to influence the endothermal component representing the melting of POPC-rich domains, as determined by differential scanning calorimetry. Taken together, the results indicate that the glycosylation of cholesterol diminishes the ability of the sterol to reside in lateral domains constituted by membrane lipids having highly ordered hydrocarbon chains.

Keywords: Model membrane; Cholesteryl glucoside; Cholesteryl galactoside; Cell stress

Dimeric structures of α-synuclein bind preferentially to lipid membranes by Eleni Giannakis; Jessica Pacífico; David P. Smith; Lin Wai Hung; Colin L. Masters; Roberto Cappai; John D. Wade; Kevin J. Barnham (pp. 1112-1119).

There is substantial evidence which implicates α-synuclein and its ability to aggregate and bind vesicle membranes as critical factors in the development of Parkinson's disease. In order to investigate the interaction between α-synuclein wild type (Wt) and its familial mutants, A53T and A30P with lipid membranes, we developed a novel lipid binding assay using surface enhanced laser desorption/ionisation-time of flight-mass spectrometry (SELDI-TOF MS). Wt and A53T exhibited similar lipid binding profiles; monomeric species and dimers bound with high relative affinity to the lipid surface, the latter of which exhibited preferential binding. Wt and A53T trimers and tetramers were also detected on the lipid surface. A30P exhibited a unique lipid binding profile; monomeric A30P bound with a low relative affinity, however, the dimeric species of A30P exhibited a higher binding ability. Larger order A30P oligomers were not detected on the lipid surface. Tapping mode atomic force microscopy (AFM) imaging was conducted to further examine the α-synuclein–lipid interaction. AFM analysis revealed Wt and its familial mutants can penetrate lipid membranes or disrupt the lipid and bind the hydrophobic alkyl self-assembled monolayer (SAM) used to form the lipid layer. The profile of these studied proteins revealed the presence of ‘small features’ consistent with the presence of monomeric and dimeric forms of the protein. These data collectively indicate that the dimeric species of Wt and its mutants can bind and cause membrane perturbations.

Keywords: Parkinson's disease; α-Synuclein; Lipid membrane; SELDI-TOF MS; AFM

CRAC motif peptide of the HIV-1 gp41 protein thins SOPC membranes and interacts with cholesterol by Alexander I. Greenwood; Jianjun Pan; Thalia T. Mills; John F. Nagle; Richard M. Epand; Stephanie Tristram-Nagle (pp. 1120-1130).

This study uses low-angle (LAXS) and wide-angle (WAXS) X-ray synchrotron scattering, volume measurements and thin layer chromatography to determine the structure and interactions of SOPC, SOPC/cholesterol mixtures, SOPC/peptide and SOPC/cholesterol/peptide mixtures. N- acetyl-LWYIK-amide (LWYIK) represents the naturally-occurring CRAC motif segment in the pretransmembrane region of the gp41 protein of HIV-1, and N- acetyl-IWYIK-amide (IWYIK), an unnatural isomer, is used as a control. Both peptides thin the SOPC bilayer by ∼3 Å, and cause the area/unit cell (peptide+SOPC) to increase by ∼9 Å² from the area/lipid of SOPC at 30 °C (67.0±0.9 Å²). Model fitting suggests that LWYIK's average position is slightly closer to the bilayer center than IWYIK's, and both peptides are just inside of the phosphate headgroup. Both peptides increase the wide-angle spacing d of SOPC without cholesterol, whereas with 50% cholesterol LWYIK increases d but IWYIK decreases d. TLC shows that LWYIK is more hydrophobic than IWYIK; this difference persists in peptide/SOPC 1:9 mole ratio mixtures. Both peptides counteract the chain ordering effect of cholesterol to roughly the same degree, and both decrease K_C, the bending modulus, thus increasing the SOPC membrane fluidity. Both peptides nucleate crystals of cholesterol, but the LWYIK-induced crystals are weaker and dissolve more easily.

Keywords: HIV-1; Biomembranes; Lipid bilayers; CRAC motif peptide; Cholesterol crystals; SOPC

Remarkable stability of the proton translocating F₁F_O-ATP synthase from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 by Tina Suhai; Norbert A. Dencher; Ansgar Poetsch; Holger Seelert (pp. 1131-1140).

For functional characterization, we isolated the F₁F_O-ATP synthase of the thermophilic cyanobacterium Thermosynechococcus elongatus. Because of the high content of phycobilisomes, a combination of dye-ligand chromatography and anion exchange chromatography was necessary to yield highly pure ATP synthase. All nine single F₁F_O subunits were identified by mass spectrometry. Western blotting revealed the SDS stable oligomer of subunits c in T. elongatus. In contrast to the mass archived in the database (10,141 Da), MALDI-TOF-MS revealed a mass of the subunit c monomer of only 8238 Da. A notable feature of the ATP synthase was its ability to synthesize ATP in a wide temperature range and its stability against chaotropic reagents. After reconstitution of F₁F_O into liposomes, ATP synthesis energized by an applied electrochemical proton gradient demonstrated functional integrity. The highest ATP synthesis rate was determined at the natural growth temperature of 55 °C, but even at 95 °C ATP production occurred. In contrast to other prokaryotic and eukaryotic ATP synthases which can be disassembled with Coomassie dye into the membrane integral and the hydrophilic part, the F₁F_O-ATP synthase possessed a particular stability. Also with the chaotropic reagents sodium bromide and guanidine thiocyanate, significantly harsher conditions were required for disassembly of the thermophilic ATP synthase.

Keywords: Abbreviations; BN-PAGE; Blue-native polyacrylamide gel electrophoresis; CF; ₁; F; _O; chloroplast ATP synthase; chl a; chlorophyll a; DCCD; dicyclohexylcarbodiimide; DDM; n; -dodecyl-β-; d; -maltoside; ESI-MS; electrospray ionisation mass spectrometry; MALDI-TOF-MS; matrix-assisted laser desorption/ionisation time-of-flight mass spectrometryATP synthase; Cyanobacteria; Thermo stability; Chaotropic reagent

H₂O₂ induces rapid biophysical and permeability changes in the plasma membrane of Saccharomyces cerevisiae by Vanderlei Folmer; Nuno Pedroso; Ana C. Matias; Sílvia C.D.N. Lopes; Fernando Antunes; Luísa Cyrne; H. Susana Marinho (pp. 1141-1147).

In Saccharomyces cerevisiae, the diffusion rate of hydrogen peroxide (H₂O₂) through the plasma membrane decreases during adaptation to H₂O₂ by means of a mechanism that is still unknown. Here, evidence is presented that during adaptation to H₂O₂ the anisotropy of the plasma membrane increases. Adaptation to H₂O₂ was studied at several times (15min up to 90min) by applying the steady-state H₂O₂ delivery model. For wild-type cells, the steady-state fluorescence anisotropy increased after 30min, or 60min, when using 2-(9-anthroyloxy) stearic acid (2-AS), or diphenylhexatriene (DPH) membrane probe, respectively. Moreover, a 40% decrease in plasma membrane permeability to H₂O₂ was observed at 15min with a concomitant two-fold increase in catalase activity. Disruption of the ergosterol pathway, by knocking out either ERG3 or ERG6, prevents the changes in anisotropy during H₂O₂ adaptation. H₂O₂ diffusion through the plasma membrane in S. cerevisiae cells is not mediated by aquaporins since the H₂O₂ permeability constant is not altered in the presence of the aquaporin inhibitor mercuric chloride. Altogether, these results indicate that the regulation of the plasma membrane permeability towards H₂O₂ is mediated by modulation of the biophysical properties of the plasma membrane.

Keywords: Abbreviations; 2-AS; 2-(9-anthroyloxy) stearic acid; DMSO; dimethyl sulfoxide; DPH; Diphenylhexatriene; E. coli; Escherichia coli; H; ₂; O; ₂; hydrogen peroxide; OD; ₆₀₀; optical density at 600nm; ROS; reactive oxygen species; S. cerevisiae; Saccharomyces cerevisiae; TMA-DPH; trimethylammonium diphenylhexatriene; wt; BY4741 wild-typeHydrogen peroxide; H; ₂; O; ₂; gradient; H; ₂; O; ₂; adaptation; Catalase; Cytochrome; c; peroxidase; Plasma membrane fluidity

Characterization of changes in the viscosity of lipid membranes with the molecular rotor FCVJ by Matthew E. Nipper; Sheereen Majd; Michael Mayer; James C.-M. Lee; Emmanuel A. Theodorakis; Mark A. Haidekker (pp. 1148-1153).

Membrane viscosity is a key parameter in cell physiology, cell function, and cell signaling. The most common methods to measure changes in membrane viscosity are fluorescence recovery after photobleaching (FRAP) and fluorescence anisotropy. Recent interest in a group of viscosity sensitive fluorophores, termed molecular rotors, led to the development of the highly membrane-compatible (2-carboxy-2-cyanovinyl)-julolidine farnesyl ester (FCVJ). The purpose of this study is to examine the fluorescent behavior of FCVJ in model membranes exposed to various agents of known influence on membrane viscosity, such as alcohols, dimethyl sulfoxide (DMSO), cyclohexane, cholesterol, and nimesulide. The influence of key agents (propanol and cholesterol) was also examined using FRAP, and backcalculated viscosity change from FCVJ and FRAP was correlated. A decrease of FCVJ emission was found with alcohol treatment (with a strong dependency on the chain length and concentration), DMSO, and cyclohexane, whereas cholesterol and nimesulide led to increased FCVJ emission. With the exception of nimesulide, FCVJ intensity changes were consistent with expected changes in membrane viscosity. A comparison of viscosity changes computed from FRAP and FCVJ led to a very good correlation between the two experimental methods. Since molecular rotors, including FCVJ, allow for extremely easy experimental methods, fast response time, and high spatial resolution, this study indicates that FCVJ may be used to quantitatively determine viscosity changes in phospholipid bilayers.

Keywords: Viscosity; Fluidity; FRAP; Molecular rotor; Alcohol; Cholesterol; Fluorescence

Characterization of domain instabilities in lipid bilayers by Karhunen–Loeve analysis by Joshua D. Deetz; Roland Faller; Ahmet Palazoglu (pp. 1154-1180).

We study the stability of lipid bilayers with artificial domains. In investigating different domain structures, we identify scenarios of stable and unstable arrangements of patches of mixed phospholipids. These are then characterized using Karhunen–Loeve Expansion (KLE), a special form of Principal Components Analysis (PCA). The simulation data are interrogated using KLE to reveal spatiotemporal patterns that explain relevant motions in the bilayer system. By projecting the high-dimensional dataset onto a small number of key modes, KLE reveals specific dynamic signatures that can help distinguish and characterize various domain instability mechanisms. We find that typically very few modes are responsible for describing a mechanism of instability to a reasonable extent and can clearly distinguish between stable and unstable arrangements. Different instability modes are characterized as they exhibit unique features like global deformation or local mixing modes.

Keywords: Principal component analysis; Lipid bilayer; Molecular simulation; Pattern formation

Formation of aquaporin-4 arrays is inhibited by palmitoylation of N-terminal cysteine residues by Hiroshi Suzuki; Kouki Nishikawa; Yoko Hiroaki; Yoshinori Fujiyoshi (pp. 1181-1189).

Tetramers of the mammalian water channel aquaporin-4 (AQP4) assemble into square arrays and mediate bidirectional water transport across the blood–brain interface. The aqp4 gene expresses two splicing isoforms. Only the shorter AQP4M23 isoform assembles into square arrays, while the longer AQP4M1 isoform interferes with array formation, presumably due to the additional 22 N-terminal residues. To understand why the N-terminus of AQP4M1 interferes with array formation, we constructed a series of N-terminal deletion mutants and examined their ability to form square arrays in Chinese hamster ovary (CHO) cells using SDS-digested freeze fracture replica labeling. Mutants with deletions of less than seventeen N-terminal residues did not form square arrays and showed dispersed immunogold labels against AQP4 molecules, whereas more deletions led to the formation of square arrays labeled with immunogolds. Furthermore, mutagenic substitution of the two cysteine residues at the position 13 and 17 in the N-terminus of AQP4M1 also resulted in the square array formation. Biochemical analysis and metabolic labeling of transfected CHO cells revealed that the two N-terminal cysteines of AQP4M1 are palmitoylated. These results suggest that palmitoylation of the N-terminal cysteines is the reason for the inability of AQP4M1 to form square arrays.

Keywords: Abbreviations; AQP; aquaporin; CHO; Chinese hamster ovary; IMP; intramembrane particle; SDS-FRL; SDS-digested freeze fracture replica labeling; P-face; protoplasmic fracture face; E-face; exoplasmic fracture face; ABE; acyl-biotinyl exchange; NEM; N; -ethylmaleimide; AP; Alkaline PhosphataseWater channel; Square array; Palmitoylation; Freeze fracture; SDS-FRL; Electron microscopy

Coupling between line tension and domain contact angle in heterogeneous membranes by Kevin B. Towles; Nily Dan (pp. 1190-1195).

The compositional differences between domains in phase-separated membranes are associated with differences in bilayer thickness and moduli. The resulting packing deformation at the phase boundary gives rise to a line tension, the one dimensional equivalent of surface tension. In this paper we calculate the line tension between a large membrane domain and a continuous phase as a function of the thickness mismatch and the contact angle between the phases. We find that the packing-induced line tension is sensitive to the contact angle, reaching a minimum at a specific value. The difference in the line tension between a flat domain (that is within the plane of the continuous phase) and a domain at the optimal contact angle may be of order 40%. This could explain why previous calculations of the thickness mismatch based line tension tend to yield values that are higher than those measured experimentally.

Keywords: Phase separation; Line tension; Vesicle

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BBA - Biomembranes (v.1778, #4)