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BBA - Biomembranes (v.1768, #1)

A word of farewell and a word of welcome (pp. 1-2).

BBA in the year 2007 by Dennis E. Vance Editor-in-Chief, BBA (pp. 3-4).

Oligomeric states of the SecA and SecYEG core components of the bacterial Sec translocon by Sharyn L. Rusch; Debra A. Kendall (pp. 5-12).

Many proteins synthesized in the cytoplasm ultimately function in non-cytoplasmic locations. In Escherichia coli, the general secretory (Sec) pathway transports the vast majority of these proteins. Two fundamental components of the Sec transport pathway are the SecYEG heterotrimeric complex that forms the channel through the cytoplasmic membrane, and SecA, the ATPase that drives the preprotein to and across the membrane. This review focuses on what is known about the oligomeric states of these core Sec components and how the oligomeric state might change during the course of the translocation of a preprotein.

Keywords: Oligomer; Protein transport; SecA; SecYEG; Translocon

Quantifying the effects of melittin on liposomes by J.F. Popplewell; M.J. Swann; N.J. Freeman; C. McDonnell; R.C. Ford (pp. 13-20).

Melittin, the soluble peptide of bee venom, has been demonstrated to induce lysis of phospholipid liposomes. We have investigated the dependence of the lytic activity of melittin on lipid composition. The lysis of liposomes, measured by following their mass and dimensions when immobilised on a solid substrate, was close to zero when the negatively charged lipids phosphatidyl glycerol or phosphatidyl serine were used as the phospholipid component of the liposome. Whilst there was significant binding of melittin to the liposomes, there was little net change in their diameter with melittin binding reversed upon salt injection. For the zwitterionic phosphatidyl choline the lytic ability of melittin is dependent on the degree of acyl chain unsaturation, with melittin able to induce lysis of liposomes in the liquid crystalline state, whilst those in the gel state showed strong resistance to lysis. By directly measuring the dimensions and mass changes of liposomes on exposure to melittin using Dual Polarisation Interferometry, rather than following the florescence of entrapped dyes we attained further information about the initial stages of melittin binding to liposomes.

Keywords: Liposome; Phospholipid; Bilayer; Melittin; Lysis

Expression and purification of two anti-CD19 single chain Fv fragments for targeting of liposomes to CD19-expressing cells by W.W.K. Cheng; D. Das; M. Suresh; T.M. Allen (pp. 21-29).

Antibody-targeted liposomal anticancer drugs combine the specificity of antibodies with large payloads of entrapped drugs. We previously showed that liposomal doxorubicin (DXR) targeted via anti-CD19 monoclonal antibodies (mAb) or their Fab' fragments against the B-cell antigen CD19 led to improved therapeutic effects in murine B-cell lymphoma models relative to non-targeted liposomal DXR. We now are examining the use of anti-CD19 single chain fragments of the antibody variable region (scFv) as a targeting moiety, to test the hypothesis that scFv have advantages over full-sized mAb or Fab' fragments. We expressed two different anti-CD19 scFv constructs, HD37-C and HD37-CCH in E. coli, and purified the scFvs using two different methods. The HD37-CCH construct was selected for coupling studies due to its relative stability and activity in comparison to HD37-C. When coupled to liposomes, the HD37-CCH scFv showed increased binding in vitro to CD19-positive Raji cells, compared to non-targeted liposomes. Cytotoxicity data showed that HD37-CCH scFv-targeted liposomes loaded with DXR were more cytotoxic than non-targeted liposomal DXR. Our results suggest that anti-CD19 scFv constructs should be explored further for their potential in treating B-lymphoid leukemias and lymphomas.

Keywords: Anti-CD19; scFv; Liposome; Doxorubicin; B-lymphoid cell; Immunoliposome

Configuration of influenza hemagglutinin fusion peptide monomers and oligomers in membranes by M. Sammalkorpi; T. Lazaridis (pp. 30-38).

The 20 N-terminal residues of the HA2 subunit of influenza hemagglutinin (HA), known as the fusion peptide, play a crucial role in membrane fusion. Molecular dynamics simulations with implicit solvation are employed here to study the structure and orientation of the fusion peptide in membranes. As a monomer the α-helical peptide adopts a shallow, slightly tilted orientation along the lipid tail–head group interface. The average angle of the peptide with respect to membrane plane is 12.4 °. We find that the kinked structure proposed on the basis of NMR data is not stable in our model because of the high energy cost related to the membrane insertion of polar groups. Because hemagglutinin-mediated membrane fusion is promoted by low pH, we examined the effect of protonation of the Glu and Asp residues. The configurations of the protonated peptides were slightly deeper in the membrane but at similar angles. Finally, because HA is a trimer, we modeled helical fusion peptide trimers. We find that oligomerization affects the insertion depth of the peptide and its orientation with respect to the membrane: a trimer exhibits equally favorable configurations in which some or all of the helices in the bundle insert obliquely deep into the membrane.

Keywords: Influenza; Hemagglutinin; Fusion peptide; Membrane configuration; Simulation

Gating of the ATP-sensitive K⁺ channel by a pore-lining phenylalanine residue by Asheebo Rojas; Jianping Wu; Runping Wang; Chun Jiang (pp. 39-51).

ATP-sensitive K⁺ (K_ATP) channels are gated by intracellular ATP, proton and phospholipids. The pore-forming Kir6.2 subunit has all essential machineries for channel gating by these ligands. It is known that channel gating involves the inner helix bundle of crossing in which a phenylalanine residue (Phe168) is found in the TM2 at the narrowest region of the ion-conduction pathway in the Kir6.2. Here we present evidence that Phe168–Kir6.2 functions as an ATP- and proton-activated gate via steric hindrance and hydrophobic interactions. Site-specific mutations of Phe168 to a small amino acid resulted in losses of the ATP- and proton-dependent gating, whereas the channel gating was well maintained after mutation to a bulky tryptophan, supporting the steric hindrance effect. The steric hindrance effect, though necessary, was insufficient for the gating, as mutating Phe168 to a bulky hydrophilic residue severely compromised the channel gating. Single-channel kinetics of the F168W mutant resembled the wild-type channel. Small residues increased P_open, and displayed long-lasting closures and long-lasting openings. Kinetic modeling showed that these resulted from stabilization of the channel to open and long-lived closed states, suggesting that a bulky and hydrophobic residue may lower the energy barrier for the switch between channel openings and closures. Thus, it is likely that the Phe168 acts as not only a steric hindrance gate but also potentially a facilitator of gating transitions in the Kir6.2 channel.

Keywords: Kir6.2; K; _ATP; channel; Gating; Hydrophobicity; Steric hindrance

Conformational and functional studies of gomesin analogues by CD, EPR and fluorescence spectroscopies by Luis G.M. Moraes; Marcos A. Fázio; Renata F.F. Vieira; Clovis R. Nakaie; M. Terêsa M. Miranda; Shirley Schreier; Sirlei Daffre; Antonio Miranda (pp. 52-58).

The aim of this work was to examine the bioactivity and the conformational behavior of some gomesin ( Gm) analogues in different environments that mimic the biological membrane/water interface. Thus, manual peptide synthesis was performed by the solid-phase method, antimicrobial activity was evaluated by a liquid growth inhibition assay, and conformational studies were performed making use of several spectroscopic techniques: CD, fluorescence and EPR. [TOAC¹] -Gm; [TOAC¹, Ser^2,6,11,15] -Gm; [Trp⁷] -Gm; [Ser^2,6,11,15, Trp⁷] -Gm; [Trp⁹] -Gm; and [Ser^2,6,11,15, Trp⁹] -Gm were synthesized and tested. The results indicated that incorporation of TOAC or Trp caused no significant reduction of antimicrobial activity; the cyclic analogues presented a β-hairpin conformation similar to that of Gm. All analogues interacted with negatively charged SDS both above and below the detergent's critical micellar concentration (cmc). In contrast, while Gm and [TOAC¹] -Gm required higher LPC concentrations to bind to micelles of this zwitterionic detergent, the cyclic Trp derivatives and the linear derivatives did not seem to interact with this membrane-mimetic system. These data corroborate previous results that suggest that electrostatic interactions with the lipid bilayer of microorganisms play an important role in the mechanism of action of gomesin. Moreover, the results show that hydrophobic interactions also contribute to membrane binding of this antimicrobial peptide.

Keywords: Abbreviations; AMP; antimicrobial peptide; CD; circular dichroism; cmc; critical micelle concentration; EPR; electron paramagnetic resonance; LC/ESI-MS; liquid chromatography electrospray ionization mass spectrometry; LPC; lysophosphatidylcholine; MBHAR; 4-methylbenzhydrylamine-resin; NMR; nuclear magnetic resonance; PB; poor broth; PDB; potato dextrose broth; RP-HPLC; reversed phase high performance liquid chromatography; SDS; sodium dodecyl sulfate; t-Boc; tert-butyloxycarbonyl; TEAP; triethylammonium phosphate; TFA; trifluoroacetic acid; TFE; 2,2,2-trifluoroethanol; TOAC; 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acidGomesin; Antimicrobial peptide; Solid-phase peptide synthesis; Electron paramagnetic resonance; Circular dichroism; Fluorescence

Membrane localization and dynamics of Nile Red: Effect of cholesterol by Soumi Mukherjee; H. Raghuraman; Amitabha Chattopadhyay (pp. 59-66).

The organization and dynamics of the hydrophobic fluorescent probe Nile Red incorporated in DOPC vesicles containing varying amounts of cholesterol has been monitored utilizing fluorescence-based approaches which include the red edge excitation shift (REES) approach and the parallax method for depth determination. Our results show that the fluorescence emission maximum, intensity, polarization, and lifetime of Nile Red vary with the cholesterol content of the membrane. Interestingly, Nile Red exhibits significant REES independent of the presence of cholesterol. This indicates that Nile Red is localized in a motionally restricted environment in the membrane. This is supported by analysis of membrane penetration depth of Nile Red using the parallax method which points out to a membrane interfacial localization of Nile Red. These results could be useful in analyzing membrane organization and heterogeneity in natural membranes using Nile Red.

Keywords: Cholesterol; Nile Red; Membrane heterogeneity; Fluorescence lifetime; Parallax method; Red edge excitation shiftAbbreviations; 2-AS; 2-(9-anthroyloxy)stearic acid; 12-AS; 12-(9-anthroyloxy)stearic acid; DMPC; dimyristoyl-; sn; -glycero-3-phosphocholine; DOPC; dioleoyl-; sn; -glycero-3-phosphocholine; LUV; large unilamellar vesicle; Nile Red; 9-diethylamino-5H-benzo[α]phenoxazine-5-one; 5-PC; 1-palmitoyl-2-(5-doxyl)stearoyl-; sn; -glycero-3-phosphocholine; Tempo-PC; dioleoyl-; sn; -glycero-3-phosphotempocholine

Lipid dependence of diadinoxanthin solubilization and de-epoxidation in artificial membrane systems resembling the lipid composition of the natural thylakoid membrane by Reimund Goss; Dariusz Latowski; Joanna Grzyb; Astrid Vieler; Martin Lohr; Christian Wilhelm; Kazimierz Strzalka (pp. 67-75).

In the present study, the solubility and enzymatic de-epoxidation of diadinoxanthin (Ddx) was investigated in three different artificial membrane systems: (1) Unilamellar liposomes composed of different concentrations of the bilayer forming lipid phosphatidylcholine (PC) and the inverted hexagonal phase (H_II phase) forming lipid monogalactosyldiacylglycerol (MGDG), (2) liposomes composed of PC and the H_II phase forming lipid phosphatidylethanolamine (PE), and (3) an artificial membrane system composed of digalactosyldiacylglycerol (DGDG) and MGDG, which resembles the lipid composition of the natural thylakoid membrane. Our results show that Ddx de-epoxidation strongly depends on the concentration of the inverted hexagonal phase forming lipids MGDG or PE in the liposomes composed of PC or DGDG, thus indicating that the presence of inverted hexagonal structures is essential for Ddx de-epoxidation. The difference observed for the solubilization of Ddx in H_II phase forming lipids compared with bilayer forming lipids indicates that Ddx is not equally distributed in the liposomes composed of different concentrations of bilayer versus non-bilayer lipids. In artificial membranes with a high percentage of bilayer lipids, a large part of Ddx is located in the membrane bilayer. In membranes composed of equal proportions of bilayer and H_II phase forming lipids, the majority of the Ddx molecules is located in the inverted hexagonal structures. The significance of the pigment distribution and the three-dimensional structure of the H_II phase for the de-epoxidation reaction is discussed, and a possible scenario for the lipid dependence of Ddx (and violaxanthin) de-epoxidation in the native thylakoid membrane is proposed.

Keywords: Abbreviations; LHC/FCP; light-harvesting complexes of vascular plants or diatoms, respectively; DDE; diadinoxanthin de-epoxidase; VDE; violaxanthin de-epoxidase; Ddx; diadinoxanthin; Dtx; diatoxanthin; Vx; violaxanthin; Zx; zeaxanthin; MGDG; monogalactosyldiacylgycerol; DGDG; digalactosyldiacylglycerol; PC; phosphatidylcholine; PE; phosphatidylethanolamineBilayer lipid; Non-bilayer lipid; Inverted hexagonal phase; Thylakoid membrane; Diadinoxanthin; Xanthophyll cycle

Modulation of proton-induced current fluctuations in the human nicotinic acetylcholine receptor channel by Christophe Danelon; Jörg Grandl; Ruud Hovius; Horst Vogel (pp. 76-89).

The nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel that switches upon activation from a closed state to a full conducting state. We found that the mutation δ S268K, located at 12′ position of the second transmembrane domain of the δ subunit of the human nAChR generates a long-lived intermediate conducting state, from which openings to a wild-type like conductance level occur on a submillisecond time scale. Aiming to understand the interplay between structural changes near the 12′ position and channel gating, we investigated the influence of various parameters: different ligands (acetylcholine, choline and epibatidine), ligand concentrations, transmembrane voltages and both fetal and adult nAChRs. Since sojourns in the high conductance state are not fully resolved in time, spectral noise analysis was used as a complement to dwell time analysis to determine the gating rate constants. Open channel current fluctuations are described by a two-state Markov model. The characteristic time of the process is markedly influenced by the ligand and the receptor type, whereas the frequency of openings to the high conductance state increases with membrane hyperpolarization. Conductance changes are discussed with regard to reversible transfer reaction of single protons at the lysine 12′ side chain.

Keywords: Noise analysis; Current fluctuation; Ionizable site; Ion channel gating; Slow-channel syndrome

Atomic force microscope visualization of lipid bilayer degradation due to action of phospholipase A₂ and Humicola lanuginosa lipase by Konstantin Balashev; N. John DiNardo; Thomas H. Callisen; Allan Svendsen; Thomas Bjørnholm (pp. 90-99).

An important application of liquid cell Atomic Force Microscopy (AFM) is the study of enzyme structure and behaviour in organized molecular media that mimic in-vivo systems. In this study we demonstrate the use of AFM as a tool to study the kinetics of lipolytic enzyme reactions occurring at the surface of a supported lipid bilayer. In particular, the time course of the degradation of lipid bilayers by Phospholipase A₂ (PLA₂) and Humicola Lanuginosa Lipase (HLL) has been investigated. Contact mode imaging allows visualization of enzyme activity on the substrate with high lateral resolution. Lipid bilayers were prepared by the Langmuir–Blodgett technique and transferred to an AFM liquid cell. Following injection of the enzyme into the liquid cell, a sequence of images was acquired at regular time intervals to allow the identification of substrate structure, preferred sites of enzyme activation, and enzyme reaction rates.

Keywords: Atomic force microscopy; Phospholipase A; ₂; Humicola lanuginose lipase; Supported bilayers; Enzyme kinetics

Supported planar bilayers from hexagonal phases by Òscar Domènech; Antoni Morros; Miquel E. Cabañas; M. Teresa Montero; Jordi Hernández-Borrell (pp. 100-106).

In this work the presence of inverted hexagonal phases H_II of 1-palmitoy-2-oleoyl- sn-glycero-3-phosphoethanolamine (POPE) and cardiolipin (CL) (0.8:0.2, mol/mol) in the presence of Ca²⁺ were observed via³¹P-NMR spectroscopy. When suspensions of the same composition were extended onto mica, H_II phases transformed into structures which features are those of supported planar bilayers (SPBs). When characterized by atomic force microscopy (AFM), the SPBs revealed the existence of two laterally segregated domains (the interdomain height being ∼1 nm). Cytochrome c (cyt c), which binds preferentially to acidic phospholipids like CL, was used to demonstrate the nature of the domains. We used 1-anilinonaphtalen-8-sulfonate (ANS) to demonstrate that in the presence of cyt c, the fluorescence of ANS decreased significantly in lamellar phases. Conversely, the ANS binding to H_II phases was negligible. When cyt c was injected into AFM fluid imaging cells, where SPBs of POPE:CL had previously formed poorly defined structures, protein aggregates (∼100 nm diameter) were ostensibly observed only on the upper domains, which suggests not only that they are mainly formed by CL, but also provides evidence of bilayer formation from H_II phases. Furthermore, a model for the nanostructure of the SPBs is herein proposed.

Keywords: Liposome; Supported planar bilayers (SPBs); Hexagonal phase (H; _II; ); ³¹; P-NMR; ANS fluorescence; AFM; Cytochrome; c; (cyt; c; )

Comparison of excitation and emission ratiometric fluorescence methods for quantifying the membrane dipole potential by Mark F. Vitha; Ronald J. Clarke (pp. 107-114).

We are interested in developing fluorescence methods for quantifying lateral variations in the dipole potential across cell surfaces. Previous work in this laboratory showed that the ratio of fluorescence intensities of the voltage-sensitive dye di-8-ANEPPS using excitation wavelengths at 420 and 520 nm correlates well with measurements of the dipole potential. In the present work we evaluate the use of di-8-ANEPPS and an emission ratiometric method for measuring dipole potentials, as Bullen and Saggau ( Biophys. J. 65 (1999) 2272–2287) have done to follow changes in the membrane potential in the presence of an externally applied field. Emission ratiometric methods have distinct advantages over excitation methods when applied to fluorescence microscopy because only a single wavelength is needed for excitation. We found that unlike the excitation ratio, the emission ratio does not correlate with the dipole potential of vesicles made from different lipids. A difference in the behaviour of the emission ratio in saturated compared to unsaturated lipid vesicles was noted. Furthermore, the emission ratio did not respond in the same way as the excitation ratio when cholesterol, 6-ketocholestanol, 7-ketocholesterol, and phloretin were added to dimyristoylphosphatidylcholine (DMPC) vesicles. We attribute the lack of correlation between the emission ratio and the dipole potential to simultaneous changes in membrane fluidity caused by changes in membrane composition, which do not occur when the electric field is externally applied as in the work of Bullen and Saggau. Di-8-ANEPPS can, thus, only be used via an excitation ratiometric method to quantify the dipole potential.

Keywords: Lipid vesicles; Voltage-sensitive styryl dyes; Emission ratio; Excitation ratio; Cholesterol; Dipole potential

The membrane-induced structure of melittin is correlated with the fluidity of the lipids by August Andersson; Henrik Biverståhl; Jon Nordin; Jens Danielsson; Emma Lindahl; Lena Mäler (pp. 115-121).

The effect of the bee toxin melittin on DMPC dynamics in fast-tumbling bicelles has been investigated. The¹³C R₁ and¹³C–¹H NOE relaxation parameters for DMPC were used to monitor the effect of melittin and cholesterol on lipid dynamics. It was found that melittin has the largest effect on the DMPC mobility in DMPC/DHPC bicelles, while less effect was observed in cholesterol-doped bicelles, or in bicelles made with CHAPS, indicating that the rigidity of the membrane affects the melittin–membrane interaction. CD spectra were analysed in terms of cooperativity of the α-helix to random coil transition in melittin, and these results also indicated similar differences between the bicelles. The study shows that bicelles can be used to investigate lipid dynamics by spin relaxation, and in particular of peptide-induced changes in membrane fluidity.

Keywords: Abbreviations; NMR; nuclear magnetic resonance; CD; circular dichroism; DMPC; 1,2-dimyristoyl-; sn; -glycero-3-phosphatidylcholine; DHPC; 1,2-dihexaoyl-; sn; -glycero-3-phosphatidylcholine; CHAPS; 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonateBicelle; Melittin; Cholesterol; NMR; Relaxation; Helix transition

Inhibitors of amyloid β-protein aggregation mediated by GM1-containing raft-like membranes by Katsumi Matsuzaki; Taeko Noguch; Masaki Wakabayashi; Keisuke Ikeda; Takuma Okada; Yumiko Ohashi; Masaru Hoshino; Hironobu Naiki (pp. 122-130).

The aggregation (fibril formation) of amyloid β-protein (Aβ) is considered to be a crucial step in the etiology of Alzheimer's disease (AD). The inhibition of Aβ aggregation and/or decomposition of fibrils formed in aqueous solution by small compounds have been studied extensively for the prevention and treatment of AD. However, recent studies suggest that Aβ aggregation also occurs in lipid rafts mediated by a cluster of monosialoganglioside GM1. This study examined the effects of representative compounds on Aβ aggregation and fibril destabilization in the presence of GM1-containing raft-like liposomes. Among the compounds tested, nordihydroguaiaretic acid (NDGA), rifampicin (RIF), tannic acid (TA), and quercetin (QUE) showed strong fibrillization inhibitory activity. NDGA and RIF inhibited the binding of Aβ to GM1 liposomes by competitively binding to the membranes and/or direct interaction with Aβ in solution, thus at least partly preventing fibrils from forming. Coincubation of Aβ with NDGA, RIF, and QUE in the presence of GM1 liposomes resulted in elongate particles, whereas the presence of TA yielded protofibrillar structures. TA and RIF also destabilized fibrils. The most potent NDGA prevented Aβ-induced toxicity in PC12 cells by inhibiting Aβ accumulation. Furthermore, a comparison of the inhibitory effects of various compounds between aqueous-phase and GM1-mediated aggregation of Aβ suggested that the two aggregation processes are not identical.

Keywords: Abbreviations; Aβ; amyloid β-protein; AD; Alzheimer's disease; ASP; acetylsalicylic acid; DMEM; Dulbecco's Modified Eagle's Medium; DMSO; dimethylsulfoxide; EM; electron microscopy; GM1; monosialoganglioside GM1; GT1b; trisialoganglioside GT1b; IND; indomethacin; NDGA; nordihydroguaiaretic acid; NGF; nerve growth factor; PC; egg yolk; l; -α-phosphatidylcholine; PG; l; -α-phosphatidyl-; dl; -glycerol enzymatically converted from PC; QUE; quercetin; RIF; rifampicin; RUT; rutin; SM; sphingomyelin; TA; tannic acid; TC; tetracycline hydrochloride; ThT; thioflovin-TAlzheimer's disease; Amyloid β-protein; Lipid raft; Monosialoganglioside GM1; Fibril formation; Protein–lipid interaction

Lack of enantiomeric specificity in the effects of anesthetic steroids on lipid bilayers by Juha-Matti Alakoskela; Douglas F. Covey; Paavo K.J. Kinnunen (pp. 131-145).

The most important target protein for many anesthetics, including volatile and steroid anesthetics, appears to be the type A γ-amino butyric acid receptor (GABA_AR), yet direct binding remains to be demonstrated. Hypotheses of lipid-mediated anesthesia suggest that lipid bilayer properties are changed by anesthetics and that this in turn affects the functions of proteins. While other data could equally well support direct or lipid-mediated action, enantiomeric specificity displayed by some anesthetics is not reflected in their interactions with lipids. In the present study, we studied the effects of two pairs of anesthetic steroid enantiomers on bilayers of several compositions, measuring potentially relevant physical properties. For one of the pairs, allopregnanolone and ent-allopregnanolone, the natural enantiomer is 300% more efficacious as an anesthetic, while for the other, pregnanolone and ent-pregnanolone, there is little difference in anesthetic potency. For each enantiomer pair, we could find no differences. This strongly favors the view that the effects of these anesthetics on lipid bilayers are not relevant for the main features of anesthesia. These steroids also provide tools to distinguish in general the direct binding of steroids to proteins from lipid-mediated effects.

Keywords: Pregnanolone; Allopregnanolone; Dipole potential; Lateral pressure profile; Lipid phase transition

Preferential accumulation of Aβ(1−42) on gel phase domains of lipid bilayers: An AFM and fluorescence study by A. Choucair; M. Chakrapani; B. Chakravarthy; J. Katsaras; L.J. Johnston (pp. 146-154).

Peptide–membrane interactions have been implicated in both the toxicity and aggregation of β-amyloid (Aβ) peptides. Recent studies have provided evidence for the involvement of liquid-ordered membrane domains known as lipid rafts in the formation and aggregation of Aβ. As a model, we have examined the interaction of Aβ(1−42) with phase separated DOPC/DPPC lipid bilayers using a combination of atomic force microscopy (AFM) and total internal reflection fluorescence microscopy (TIRF). AFM images show that addition of Aβ to preformed supported bilayers leads to accumulation of small peptide aggregates exclusively on the gel phase DPPC domains. Initial aggregates are observed approximately 90 min after peptide addition and increase in diameter to 45–150 nm within 24 h. TIRF studies with a mixture of Aβ and Aβ–Fl demonstrate that accumulation of the peptide on the gel phase domains occurs as early as 15 min after Aβ addition and is maintained for over 24 h. By contrast, Aβ is randomly distributed throughout both fluid and gel phases when the peptide is reconstituted into DOPC/DPPC vesicles prior to formation of a supported bilayer. The preferential accumulation of Aβ on DPPC domains suggests that rigid domains may act as platforms to concentrate peptide and enhance its aggregation and may be relevant to the postulated involvement of lipid rafts in modulating Aβ activity in vivo.

Keywords: Abbreviations; AD; Alzheimer's disease; Aβ; Amyloid β; APP; amyloid precursor protein; AFM; atomic force microscopy; DOPC; 1,2-dioleoyl-; sn; -glycero-3-phosphocholine; DPPC; 1,2-dipalmitoyl-; sn; -glycero-3-phosphocholine; TIRF; total internal reflection fluorescence; TR-DHPE; Texas Red® 1,2-dihexadecanoyl-; sn; -glycero-3-phosphoethanolamine, triethylammonium saltβ-amyloid peptide; Atomic force microscopy; Bilayers; Fluourescence

Spatial and temporal Ca²⁺, Mg²⁺, and ATP²⁻ dynamics in cardiac dyads during calcium release by Ivan Valent; Alexandra Zahradníková; Jana Pavelková; Ivan Zahradník (pp. 155-166).

We have constructed a three-dimensional reaction-diffusion model of the mammalian cardiac calcium release unit. We analyzed effects of diffusion coefficients, single channel current amplitude, density of RyR channels, and reaction kinetics of ATP²⁻ with Ca²⁺ and Mg²⁺ ions on spatiotemporal concentration profiles of Ca²⁺, Mg²⁺, and ATP²⁻ in the dyadic cleft during Ca²⁺ release. The model revealed that Ca²⁺ concentration gradients persist near RyRs in the steady state. Even with low number of open RyRs, peak [Ca²⁺] in the dyadic space reached values similar to estimates of luminal [Ca²⁺] in ∼1 ms, suggesting that during calcium release the Ca²⁺ gradient moves from the cisternal membrane towards the boundary of the dyadic space with the cytosol. The released Ca²⁺ bound to ATP²⁻, and thus substantially decreased ATP²⁻ concentration in the dyadic space. The released Ca²⁺ could also replace Mg²⁺ in its complex with ATP²⁻ during first milliseconds of release if dissociation of MgATP was fast. The results suggest that concentration changes of Ca²⁺, Mg²⁺, and ATP²⁻ might be large and fast enough to reduce dyadic RyR activity. Thus, under physiological conditions, termination of calcium release may be facilitated by the synergic effect of the construction and chemistry of mammalian cardiac dyads.

Keywords: Reaction-diffusion model; Calcium release unit; Calcium signaling

Differential effects of carotenoids on lipid peroxidation due to membrane interactions: X-ray diffraction analysis by Hyesun P. McNulty; Jungsoo Byun; Samuel F. Lockwood; Robert F. Jacob; R. Preston Mason (pp. 167-174).

The biological benefits of certain carotenoids may be due to their potent antioxidant properties attributed to specific physico-chemical interactions with membranes. To test this hypothesis, we measured the effects of various carotenoids on rates of lipid peroxidation and correlated these findings with their membrane interactions, as determined by small angle X-ray diffraction approaches. The effects of the homochiral carotenoids (astaxanthin, zeaxanthin, lutein, β-carotene, lycopene) on lipid hydroperoxide (LOOH) generation were evaluated in membranes enriched with polyunsaturated fatty acids. Apolar carotenoids, such as lycopene and β-carotene, disordered the membrane bilayer and showed a potent pro-oxidant effect (>85% increase in LOOH levels) while astaxanthin preserved membrane structure and exhibited significant antioxidant activity (40% decrease in LOOH levels). These findings indicate distinct effects of carotenoids on lipid peroxidation due to membrane structure changes. These contrasting effects of carotenoids on lipid peroxidation may explain differences in their biological activity.

Keywords: Abbreviations; ABIN; 2,2′-azobis-isobutyronitrile; AMVN; 2,2′-azobis(2,4′-dimethylvaleronitrile); C/P; cholesterol to phospholipid mole ratio; DLPC; 1,2-dilinoleoyl-3-; sn; -glycero phosphatidylcholine; DMPC; 1,2-dimyristoyl-3-; sn; -glycero phosphatidylcholine; DOPC; 1,2-dioleoyl-3-; sn; -glycero phosphatidylcholine; DPPC; 1,2-dipalmitoy-3-; sn; -glycero phosphatidylcholine; EPR; electron paramagnetic resonance; EYPC; egg-yolk phosphatidylcholine; LOOH; lipid peroxide; POPC; 1-palmitoyl 2-oleoyl-3-; sn; -glycero phosphatidylcholineAstaxanthin; β-carotene; Antioxidant; Lipid peroxidation; Liposome; X-ray diffraction

Acknowledgement (pp. 175-178).

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BBA - Biomembranes (v.1768, #1)