Grafting of polylysine with polyethylenoxide prevents demixing of O-pyromellitylgramicidin in lipid membranes

Both natural and synthetic polycations can induce demixing of negatively charged components in artificial and possibly in natural membranes. This process can result in formation of clusters (binding of several components to a polycation chain) and/or domains (aggregation of clusters and formation of a separate phase enriched in some particular component). In order to distinguish between these two phenomena, a model lipid membrane system containing ion channels, formed by a negatively charged peptide, O-pyromellitylgramicidin, and polycations of different structures was used. Microelectrophoresis of liposomes, changes in boundary potential of planar bilayers, the shape of compression curves and potentials of lipid and lipid/peptide monolayers were used to monitor the electrostatic factors in polymer adsorption to the membrane and peptide-polymer interactions. The synthesized PEO-grafted polylysine, PLL-PEO20000, did not induce peptide demixing monitored by stabilization of the gramicidin channels, in contrast to parent polylysine (PLL). Both polymers were shown to bind effectively to negatively charged liposomes and lipid monolayers, suggesting that the ineffectiveness of PLL-PEO20000 was not due to reduction of its binding. It was hypothesized that PLL-PEO20000 could not induce domain formation due to steric hindrance of long PEO chains preventing lateral fusion of clusters. Another copolymer, PLL-PEO4000, having four PEO chains of 4000 Da, exhibited intermediate effect between PLL and PLL-PEO20000, which shows the importance of the copolymer architecture for the effect on the lateral distribution of OPg channels. The model system can be relevant to regulation of lateral organization of ion channels and other components in natural membrane systems.     

Interaction of the antimicrobial peptide pheromone Plantaricin A with model membranes: Implications for a novel mechanism of action

Plantaricin A (plA) is a 26-residue bacteria-produced peptide pheromone with membrane-permeabilizing antimicrobial activity. In this study the interaction of plA with membranes is shown to be highly dependent on the membrane lipid composition. PlA bound readily to zwitterionic 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) monolayers and liposomes, yet without significantly penetrating into these membranes. The presence of cholesterol attenuated the intercalation of plA into SOPC monolayers. The association of plA to phosphatidylcholine was, however, sufficient to induce membrane permeabilization, with nanomolar concentrations of the peptide triggering dye leakage from SOPC liposomes. The addition of the negatively charged phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol POPG (SOPC/POPG; molar ratio 8:2) enhanced the membrane penetration of the peptide, as revealed by (i) peptide-induced increment in the surface pressure of lipid monolayers, (ii) increase in diphenylhexatriene (DPH) emission anisotropy measured for bilayers, and (iii) fluorescence characteristics of the two Trps of plA in the presence of liposomes, measured as such as well as in the presence of different quenchers. Despite deeper intercalation of plA into the SOPC/POPG lipid bilayer, much less peptide-induced dye leakage was observed for these liposomes than for the SOPC liposomes. Further changes in the mode of interaction of plA with lipids were evident when also the zwitterionic phospholipid, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphoethanolaminne (POPE) was present (SOPC/POPG/POPE, molar ratio 3:2:5), thus suggesting increase in membrane spontaneous negative curvature to affect the mode of association of this peptide with lipid bilayer. PlA induced more efficient aggregation of the SOPC/POPG and SOPC/POPG/POPE liposomes than of the SOPC liposomes, which could explain the attenuated peptide-induced dye leakage from the former liposomes. At micromolar concentrations, plA killed human leukemic T-cells by both necrosis and apoptosis. Interestingly, plA formed supramolecular protein-lipid amyloid-like fibers upon binding to negatively charged phospholipid-containing membranes, suggesting a possible mechanistic connection between fibril formation and the cytotoxicity of plA.     

Interaction of the antimicrobial peptide pheromone Plantaricin A with model membranes: implications for a novel mechanism of action

Plantaricin A (plA) is a 26-residue bacteria-produced peptide pheromone with membrane-permeabilizing antimicrobial activity. In this study the interaction of plA with membranes is shown to be highly dependent on the membrane lipid composition. PlA bound readily to zwitterionic 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) monolayers and liposomes, yet without significantly penetrating into these membranes. The presence of cholesterol attenuated the intercalation of plA into SOPC monolayers. The association of plA to phosphatidylcholine was, however, sufficient to induce membrane permeabilization, with nanomolar concentrations of the peptide triggering dye leakage from SOPC liposomes. The addition of the negatively charged phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol POPG (SOPC/POPG; molar ratio 8:2) enhanced the membrane penetration of the peptide, as revealed by (i) peptide-induced increment in the surface pressure of lipid monolayers, (ii) increase in diphenylhexatriene (DPH) emission anisotropy measured for bilayers, and (iii) fluorescence characteristics of the two Trps of plA in the presence of liposomes, measured as such as well as in the presence of different quenchers. Despite deeper intercalation of plA into the SOPC/POPG lipid bilayer, much less peptide-induced dye leakage was observed for these liposomes than for the SOPC liposomes. Further changes in the mode of interaction of plA with lipids were evident when also the zwitterionic phospholipid, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphoethanolaminne (POPE) was present (SOPC/POPG/POPE, molar ratio 3:2:5), thus suggesting increase in membrane spontaneous negative curvature to affect the mode of association of this peptide with lipid bilayer. PlA induced more efficient aggregation of the SOPC/POPG and SOPC/POPG/POPE liposomes than of the SOPC liposomes, which could explain the attenuated peptide-induced dye leakage from the former liposomes. At micromolar concentrations, plA killed human leukemic T-cells by both necrosis and apoptosis. Interestingly, plA formed supramolecular protein-lipid amyloid-like fibers upon binding to negatively charged phospholipid-containing membranes, suggesting a possible mechanistic connection between fibril formation and the cytotoxicity of plA.     

Interaction of poly(l-lysines) with negatively charged membranes: an FT-IR and DSC study

The influence of the binding of poly(l-lysine) (PLL) to negatively charged membranes containing phosphatidylglycerols (PG) was studied by DSC and FT-IR spectroscopy. We found a general increase in the main transition temperature as well as increase in hydrophobic order of the membrane upon PLL binding. Furthermore we observed stronger binding of hydration water to the lipid head groups after PLL binding. The secondary structure of the PLL after binding was studied by FT-IR spectroscopy. We found that PLL binds in an α-helical conformation to negatively charged DPPG membranes or membranes with DPPG-rich domains. Moreover we proved that PLL binding induces domain formation in the gel state of mixed DPPC/DPPG or DMPC/DPPG membranes as well as lipid remixing in the liquid–crystalline state. We studied these effects as a function of PLL chain length and found a significant dependence of the secondary structure, phase transition temperature and domain formation capacity on PLL chain length and also a correlation between the peptide secondary structure and the phase transition temperature of the membrane. We present a system in which the membrane phase transition triggers a highly cooperative secondary structure transition of the membrane-bound peptide from α-helix to random coil. Dedicated to Prof. K. Arnold on the occasion of his 65th birthday.     

Interaction of cationic antimicrobial peptides with model membranes

A series of natural and synthetic cationic antimicrobial peptides from various structural classes, including alpha-helical, beta-sheet, extended, and cyclic, were examined for their ability to interact with model membranes, assessing penetration of phospholipid monolayers and induction of lipid flip-flop, membrane leakiness, and peptide translocation across the bilayer of large unilamellar liposomes, at a range of peptide/lipid ratios. All peptides were able to penetrate into monolayers made with negatively charged phospholipids, but only two interacted weakly with neutral lipids. Peptide-mediated lipid flip-flop generally occurred at peptide concentrations that were 3- to 5-fold lower than those causing leakage of calcein across the membrane, regardless of peptide structure. With the exception of two alpha-helical peptides V681(n) and V25(p,) the extent of peptide-induced calcein release from large unilamellar liposomes was generally low at peptide/lipid molar ratios below 1:50. Peptide translocation across bilayers was found to be higher for the beta-sheet peptide polyphemusin, intermediate for alpha-helical peptides, and low for extended peptides. Overall, whereas all studied cationic antimicrobial peptides interacted with membranes, they were quite heterogeneous in their impact on these membranes.     

Structural investigation on the adsorption of the MARCKS peptide on anionic lipid monolayers - effects beyond electrostatic

The presence of charged lipids in the cell membrane constitutes the background for the interaction with numerous membrane proteins. As a result, the valence of the lipids plays an important role concerning their lateral organization in the membrane and therefore the very manner of this interaction. This present study examines this aspect, particularly regarding to the interaction of the anionic lipid DPPS with the highly basic charged effector domain of the MARCKS protein, examined in monolayer model systems. Film balance, fluorescence microscopy and X-ray reflection/diffraction measurements were used to study the behavior of DPPS in a mixture with DPPC for its dependance on the presence of MARCKS (151-175). In the mixed monolayer, both lipids are completely miscible therefore DPPS is incorporated in the ordered crystalline DPPC domains as well. The interaction of MARCKS peptide with the mixed monolayer leads to the formation of lipid/peptide clusters causing an elongation of the serine group of the DPPS up to 7? in direction to surface normal into the subphase. The large cationic charge of the peptide pulls out the serine group of the interface which simultaneously causes an elongation of the phosphodiester group of the lipid fraction too. The obtained results were used to compare the interaction of MARCKS peptide with the polyvalent PIP(2) in mixed monolayers. On this way we surprisingly find out, that the relative small charge difference of the anionic lipids causes a significant different interaction with MARCKS (151-175). The lateral arrangement of the anionic lipids depends on their charge values and determines the diffusion of the electrostatic binding clusters within the membrane.     

Large unselective pore in lipid bilayer membrane formed by positively charged peptides containing a sequence of gramicidin A

Ion-channel activity of a series of gramicidin A analogues carrying charged amino-acid sequences on the C-terminus of the peptide was studied on planar bilayer lipid membranes and liposomes. It was found that the analogue with the positively charged sequence GSGRRRRSQS forms classical cationic pores at low concentrations and large unselective pores at high concentrations. The peptide was predominantly in the right-handed beta(6.3)-helical conformation in liposomes as shown by circular dichroism spectroscopy. The single-channel conductance of the large pore was estimated to be 320pS in 100mM choline chloride as judged from the fluctuation analysis of the multi-channel current. The analogue with the negatively charged sequence GSGEEEESQS exhibited solely classical cationic channel activity. The ability of a peptide to form different type of channels can be used in the search for broad-spectrum antibiotics.     

Coupled changes between lipid order and polypeptide conformation at the membrane surface. A 2H NMR and Raman study of polylysine-phosphatidic acid systems

Thermotropism and segmental chain order parameters of sn-2-perdeuteriated dimyristoyl-phosphatidic acid (DMPA)-water dispersions, with and without poly(L-lysine) (PLL) of different molecular weights, have been investigated by solid-state deuterium NMR spectroscopy. The segmental chain order parameter profile of this negatively charged lipid is similar to that already found for other lipids. Addition of long PLL (MW = 200,000) increases the temperature, Tc, of the lipid gel-to-fluid phase transition, whereas short PLL (MW = 4000) has practically no effect on Tc. In the fluid phase both varieties of PLL increase the "plateau" character of segmental order parameters up to carbon position 10. At the same reduced temperature, long PLL more significantly increases the segmental ordering, especially at the methyl terminal position. This leads to the conclusion that polar head-group capping and charge neutralization by PLL induce severe changes in lipid chain ordering, even down to the bilayer core. The structure of PLL bound to the lipid bilayer surface was monitored by Raman spectroscopy, following the amide I bands. Results show that the lipid gel-to-fluid phase transition triggers a conformational transition from ordered beta-sheet to random structure of short PLL, while it does not affect the strongly stabilized beta-sheet structure of long PLL. It is concluded that both short and long PLL can efficiently cap and neutralize lipid head groups, whatever their structure, and that peptide length is a key parameter in whether lipids or peptides are the driving force in conformationally coupled changes of both partners in the membrane.     

Phospholipid interactions of synthetic peptides representing the N-terminus of HIV gp41

Peptides representing the N-terminal 23 residues of the surface protein gp41 of LAV1a and LAVmal strains of the human immunodeficiency virus were synthesized and their interactions with phospholipid vesicles studied. The peptides are surface-active and penetrate lipid monolayers composed of negatively charged but not neutral lipids. Similarly, the peptides induce lipid mixing and solute (6-carboxyfluorescein) leakage of negatively charged, but not neutral, vesicles. Circular dichroism and infrared spectroscopy show that at low peptide:lipid ratios (approximately 1:200), the peptides bind to negatively charged vesicles as alpha-helices. At higher peptide:lipid ratios (1:30), a beta conformation is observed for the LAV1a peptide, accompanied by a large increase in light scattering. The LAVmal peptide showed less beta-structure and induced less light scattering. With neutral vesicles, only the beta conformation and a peptide:lipid ratio-dependent increase in vesicle suspension light scattering were observed for both peptides. We hypothesize that the inserted alpha-helical form causes vesicle membrane disruption whereas the surface-bound beta form induces aggregation.     

Cationic carbosilane dendrimers-lipid membrane interactions

The aim of this work was to study interactions between cationic carbosilane dendrimers (CBS) and lipid bilayers or monolayers. Two kinds of second generation carbosilane dendrimers were used: NN16 with Si-O bonds and BDBR0011 with Si-C bonds. The results show that cationic carbosilane dendrimers interact both with liposomes and lipid monolayers. Interactions were stronger for negatively charged membranes and high concentration of dendrimers. In liposomes interactions were studied by measuring fluorescence anisotropy changes of fluorescent labels incorporated into the bilayer. An increase in fluorescence anisotropy was observed for both fluorescent probes when dendrimers were added to lipids that means the decreased membrane fluidity. Both the hydrophobic and hydrophilic parts of liposome bilayers became more rigid. This may be due to dendrimers' incorporation into liposome bilayer. For higher concentrations of both dendrimers precipitation occurred in negatively charged liposomes. NN16 dendrimer interacted stronger with hydrophilic part of bilayers whereas BDBR0011 greatly modified the hydrophobic area. Monolayers method brought similar results. Both dendrimers influenced lipid monolayers and changed surface pressure. For negatively charged lipids the monitored parameter changed stronger than for uncharged DMPC lipids. Moreover, NN16 dendrimer interacted stronger than the BDBR0011.     

Lateral dynamics of proteins with polybasic domain on anionic membranes: a dynamic Monte-Carlo study

Positively charged polybasic domains are essential for recruiting multiple signaling proteins, such as Ras GTPases and Src kinase, to the negatively charged cellular membranes. Much less, however, is known about the influence of electrostatic interactions on the lateral dynamics of these proteins. We developed a dynamic Monte-Carlo automaton that faithfully simulates lateral diffusion of the adsorbed positively charged oligopeptides as well as the dynamics of mono- (phosphatidylserine) and polyvalent (PIP₂) anionic lipids within the bilayer. In agreement with earlier results, our simulations reveal lipid demixing that leads to the formation of a lipid shell associated with the peptide. The computed association times and average numbers of bound lipids demonstrate that tetravalent PIP₂ interacts with the peptide much more strongly than monovalent lipid. On the spatially homogeneous membrane, the lipid shell affects the behavior of the peptide only by weakly reducing its lateral mobility. However, spatially heterogeneous distributions of monovalent lipids are found to produce peptide drift, the velocity of which is determined by the total charge of the peptide-lipid complex. We hypothesize that this predicted phenomenon may affect the spatial distribution of proteins with polybasic domains in the context of cell-signaling events that alter the local density of monovalent anionic lipids.     

Binding of polylysine to charged bilayer membranes: molecular organization of a lipid.peptide complex.

The interaction between a positively charged peptide (poly-L-lysine) and model membranes containing charged lipids has been investigated. Conformational changes of the polypeptide as well as changes in the membrane lipid distribution were observed upon lipid-protein agglutination: 1. The strong binding of polylysine is shown directly by the use of spinlabelled polypeptide. Upon binding to phosphatidic acid a shift in the hyperfine coupling constant from 16.5 to 14.6 Oe is observed. The spectrum of the lipid-bound peptide is superimposed on the spectrum of polylysine in solution. Half of the lysine groups are bound to the charged membranes. A change in the conformation of polylysine from a random coil to a partially ordered configuration is suggested. 2. Spin labelling of the lipid component gives evidence concerning the molecular organization of a lipid mixture containing charged phosphatitid acid. Addition of polylysine induces the formation of crystalline patches of bound phosphatidic acid. 3. Excimer forming pyrene decanoic acid has been employed. Addition of positively charged polylysine (pH 9.0) to phosphatidic acid membranes increases the transition temperature of the lipid from Tt = 50 to Tt = 62 degrees C. Thus, a lipid segregation of lipid into regions of phosphatidic acid bound to the peptide which differ in their microviscosity from the surrounding membrane is induced. One lysine group binds one phosphatidic acid molecule, but only half of the phosphatidic acid is bound. 4. Direct evidence for charge induced domain formation in lipid mixtures containing phosphatidic acid is given by electron microscopy. Addition of polylysine leads to a change in the surface curvature of the bound charged lipid. The domain size is estimated from the electron micrographs. The number of domains present is dependent on both the ratio of charged to uncharged lipids as well as on the amount of polylysine added to the vesicles. The size of the domains is not dependent on membrane composition. However, the size seems to increase in a stepwise manner that is correlated with a multiple of the area covered by one polylysine molecule.     

Lipolysis in model membranes in the presence of positively charged soluble proteins]

Phospholipase A2 hydrolysis of neutral and negatively charged lipid membranes modified by positively charged proteins has been studied using liposomes composed of either dioleoylphosphatidylcholine (DOPC) or dioleoylphosphatidylglycerol (DOPG) alone or their equimolar mixture in the presence of cytochrome c, histone H1, cytochrome b5, and polylysine. Twenty minutes after the reaction had been initiated, DOPC hydrolysis was 58%, while that in the equimolar mixture with DOPG was 35%. DOPG hydrolysis was more complete in binary mixtures of liposomes. The same was observed for liposomes in the presence of cytochrome c. Hydrolysis of phospholipids in binary liposomes in the presence of histone H1 was 3 times faster than that in protein-free liposomes. In the presence of polylysine the rate of DOPG hydrolysis was decreased. The results obtained are suggestive of electrostatic interactions between hydrophilic proteins and negatively charged phospholipids, the phospholipase A2 catalytic activity being affected by these interactions.     

Peptide induced demixing in PG/PE lipid mixtures: A mechanism for the specificity of antimicrobial peptides towards bacterial membranes?

Antimicrobial peptides attract a lot of interest as potential candidates to overcome bacterial resistance. So far, nearly all the proposed scenarios for their mechanism of action are associated with perforating and breaking down bacterial membranes after a binding process. In this study we obtained additional information on peptide induced demixing of bacterial membranes as a possible mechanism of specificity of antimicrobial peptides. We used DSC and FT-IR to study the influence of a linear and cyclic arginine- and tryptophan-rich antimicrobial peptide having the same sequence (RRWWRF) on the thermotropic phase transitions of lipid membranes. The cyclization of the peptide was found to enhance its antimicrobial activity and selectivity ( Dathe, M. Nikolenko, H. Klose, J. Bienert, M. Biochemistry 43 (2004) 9140-9150). A particular preference of the binding of the peptides to DPPG headgroups compared to other headgroups of negatively charged phospholipids, namely DMPA, DPPS and cardiolipin was observed. The main transition temperature of DPPG bilayers was considerably decreased by the bound peptides. The peptides caused a substantial down-shift of the transition of DPPG/DMPC. In contrast, they induced a demixing in DPPG/DPPE bilayers and led to the appearance of two peaks in the DSC curves indicating a DPPG-peptide-enriched domain and a DPPE-enriched domain. These results could be confirmed by FT-IR-spectroscopic measurements. We therefore propose that the observed peptide-induced lipid demixing in PG/PE-membranes could be a further specific effect of the antimicrobial peptides operating only on bacterial membranes, which contain appreciable amounts of PE and PG, and which could in principle also occur in liquid-crystalline membranes.     

Fluorescence study of protein-lipid complexes with a new symmetric squarylium probe

The novel symmetric squarylium derivative SQ-1 has been synthesized and tested for its sensitivity to the formation of protein-lipid complexes. SQ-1 binding to the model membranes composed of zwitterionic lipid phosphatidylcholine (PC) and its mixtures with anionic lipid cardiolipin (CL) in different molar ratios was found to be controlled mainly by hydrophobic interactions. Lysozyme (Lz) and ribonuclease A (RNase) exerted an influence on the probe association with lipid vesicles resulting presumably from the competition between SQ-1 and the proteins for bilayer free volume and modification of its properties. The magnitude of this effect was much higher for lysozyme which may stem from the amphipathy of protein alpha-helix involved in the membrane binding. Varying membrane composition provides evidence for the dye sensitivity to both hydrophobic and electrostatic protein-lipid interactions. Fluorescence anisotropy studies uncovered the restriction of SQ-1 rotational mobility in lipid environment in the presence of Lz and RNase being indicative of the incorporation of the proteins into bilayer interior. The results of binding, fluorescence quenching and kinetic experiments suggested lysozyme-induced local lipid demixing upon protein association with negatively charged membranes with threshold concentration of CL for the lipid demixing being 10 mol%.     

Amyloid-β Membrane Binding and Permeabilization are Distinct Processes Influenced Separately by Membrane Charge and Fluidity

The 40 and 42 residue amyloid-β (Aβ) peptides are major components of the proteinaceous plaques prevalent in the Alzheimer's disease-afflicted brain and have been shown to have an important role in instigating neuronal degeneration. Whereas it was previously thought that Aβ becomes cytotoxic upon forming large fibrillar aggregates, recent studies suggest that soluble intermediate-sized oligomeric species cause cell death through membrane permeabilization. The present study examines the interactions between Aβ40 and lipid membranes using liposomes as a model system to determine how changes in membrane composition influence the conversion of Aβ into these toxic species. Aβ40 membrane binding was monitored using fluorescence-based assays with a tryptophan-substituted peptide (Aβ40 [Y10W]). We extend previous observations that Aβ40 interacts preferentially with negatively charged membranes, and show that binding of nonfibrillar, low molecular mass oligomers of Aβ40 to anionic, but not neutral, membranes involves insertion of the peptide into the bilayer, as well as sequential conformational changes corresponding to the degree of oligomerization induced. Significantly, while anionic membranes in the gel, liquid crystalline, and liquid ordered phases induce these conformational changes equally, membrane permeabilization is reduced dramatically as the fluidity of the membrane is decreased. These findings demonstrate that binding alone is not sufficient for membrane permeabilization, and that the latter is also highly dependent on the fluidity and phase of the membrane. We conclude that binding and pore formation are two distinct steps. The differences in Aβ behavior induced by membrane composition may have significant implications on the development and progression of AD as neuronal membrane composition is altered with age.     

The fusogenic effect of synthetic polycations on negatively charged lipid bilayers

The effect of synthetic polycations, polyallylamine, and polyethylenimine, on liposomes containing phosphatidylserine was investigated along with that of polylysine and divalent cations. The addition of polycations caused aggregation of sonicated vesicles composed of phosphatidylserine and phosphatidylcholine (molar ratio 1:4) as determined by measuring the turbidity changes. Liposomal turbidity increased 10 times compared with that of control liposomes at charge ratios of polymer/vesicle from 0.23 (polylysine) to 2.5 (linear polyethylenimine), while the turbidity was unchanged by the addition of Ca2+ or Mg2+ at charge ratios up to 500. These polycations also induced intermixing of liposomal membranes as indicated by resonance energy transfer between fluorescent lipids incorporated in lipid bilayers, without inducing drastic permeability changes as determined from the calcein release. Fifty percent intermixing of liposomes (0.05 mM as lipid concentration) was induced by these polycations at charge ratios of around 1.0. However, the highest resonance energy transfer was produced by the addition of polyallylamine, which caused multicycles of membrane intermixing between vesicles. Polycation-induced membrane intermixing and permeability changes of phosphatidylserine liposomes were also investigated. At charge ratios of around 1.0, these polymers caused resonance energy transfer of fluorescent lipids incorporated in separate vesicles; however, polyallylamine and branched polyethylenimine also caused permeability increases of liposomal membranes. Membrane intermixing and permeability changes of phosphatidylserine vesicles induced by polyallylamine were dependent on the polymer/vesicle charge ratio, and were different from those induced by Ca2+ since the latter caused half-maximal membrane intermixing or permeability change of phosphatidylserine vesicles at about 1 mM at the liposomal concentrations investigated.     

Antioxidant effect of bovine serum albumin on membrane lipid peroxidation induced by iron chelate and superoxide

Albumin is supposed to be the major antioxidant circulating in blood. This study examined the prevention of membrane lipid peroxidation by bovine serum albumin (BSA). Lipid peroxidation was induced by the exposing of enzymatically generated superoxide radicals to egg yolk phosphatidylcholine liposomes incorporating lipids with different charges in the presence of chelated iron catalysts. We used three kinds of Fe³⁺-chelates, which initiated reactions that were dependent on membrane charge: Fe³⁺-EDTA and Fe³⁺-EGTA catalyzed peroxidation in positively and negatively charged liposomes, respectively, and Fe³⁺-NTA, a renal carcinogen, catalyzed the reaction in liposomes of either charge. Fe³⁺-chelates initiated more lipid peroxidation in liposomes with increased zeta potentials, followed by an increase of their availability for the initiation of the reaction at the membrane surface. BSA inhibits lipid peroxidation by preventing the interaction of iron chelate with membranes, followed by a decrease of its availability in a charge-dependent manner depending on the iron-chelate concentration: one is accompanied and the other is unaccompanied by a change in the membrane charge. The inhibitory effect of BSA in the former at high concentrations of iron chelate would be attributed to its electrostatic binding with oppositely charged membranes. The inhibitory effect in the latter at low concentrations of iron chelate would be caused by BSA binding with iron chelates and keeping them away from membrane surface where lipid peroxidation is initiated. Although these results warrant further in vivo investigation, it was concluded that BSA inhibits membrane lipid peroxidation by decreasing the availability of iron for the initiation of membrane lipid peroxidation, in addition to trapping active oxygens and free radicals.     

Structure and dynamics of helix-0 of the N-BAR domain in lipid micelles and bilayers

Bin/Amphiphysin/Rvs-homology (BAR) domains generate and sense membrane curvature by binding the negatively charged membrane to their positively charged concave surfaces. N-BAR domains contain an N-terminal extension (helix-0) predicted to form an amphipathic helix upon membrane binding. We determined the NMR structure and nano-to-picosecond dynamics of helix-0 of the human Bin1/Amphiphysin II BAR domain in sodium dodecyl sulfate and dodecylphosphocholine micelles. Molecular dynamics simulations of this 34-amino acid peptide revealed electrostatic and hydrophobic interactions with the detergent molecules that induce helical structure formation from residues 8-10 toward the C-terminus. The orientation in the micelles was experimentally confirmed by backbone amide proton exchange. The simulation and the experiment indicated that the N-terminal region is disordered, and the peptide curves to adopted the micelle shape. Deletion of helix-0 reduced tubulation of liposomes by the BAR domain, whereas the helix-0 peptide itself was fusogenic. These findings support models for membrane curving by BAR domains in which helix-0 increases the binding affinity to the membrane and enhances curvature generation.