1-palmitoyl-2-oleoylphosphatidylcholine and 1-2-dioleoyl-sn-glycero-3-phosphoglycerol

Amyloid formation by islet amyloid polypeptide (IAPP) contributes to β-cell dysfunction in type 2 diabetes. Perturbation of the β-cell membrane may contribute to IAPP-induced toxicity. We examine the effects of lipid composition, salt, and buffer on IAPP amyloid formation and on the ability of IAPP to induce leakage of model membranes. Even low levels of anionic lipids promote amyloid formation and membrane permeabilization. Increasing the percentage of the anionic lipids, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS) or 1,2-dioleoyl-sn-glycero-3-phospho(1'-rac-glycerol), enhances the rate of amyloid formation and increases the level of membrane permeabilization. The choice of zwitterionic lipid has no noticeable effect on membrane-catalyzed amyloid formation but in most cases affects leakage, which tends to decrease in the following order: 1,2-dioleoyl-sn-glycero-3-phosphocholine > 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine > sphingomyelin. Uncharged lipids that increase the level of membrane order weaken the ability of IAPP to induce leakage. Leakage is due predominately to pore formation rather than complete disruption of the vesicles under the conditions used in these studies. Cholesterol at or below physiological levels significantly reduces the rate of vesicle-catalyzed IAPP amyloid formation and decreases the susceptibility to IAPP-induced leakage. The effects of cholesterol on amyloid formation are masked by 25 mol % POPS. Overall, there is a strong inverse correlation between the time to form amyloid and the extent of vesicle leakage. NaCl reduces the rate of membrane-catalyzed amyloid formation by anionic vesicles, but accelerates amyloid formation in solution. The implications for IAPP membrane interactions are discussed, as is the possibility that the loss of phosphatidylserine asymmetry enhances IAPP amyloid formation and membrane damage in vivo via a positive feedback loop.

Topics: Amino Acid Sequence; Amyloid; Cell Membrane; Cell Membrane Permeability; Cholesterol; Glycerylphosphorylcholine; Humans; Insulin-Secreting Cells; Islet Amyloid Polypeptide; Kinetics; Lipid Bilayers; Phosphatidylcholines; Phosphatidylglycerols; Phosphatidylserines; Sodium Chloride; Sphingomyelins

Membrane perturbation induced by cysteine-rich peptides is a crucial biological phenomenon but scarcely investigated, in particular with effective biophysical-chemical methodologies. Hanatoxin (HaTx), a 35-residue polypeptide from spider venom, works as an inhibitor of drk1 (Kv2.1) channels, most likely by interacting with the voltage-sensor. However, how this water-soluble peptide modifies the gating remains poorly understood, as the voltage sensor was proposed to be deeply embedded within the bilayer. To see how HaTx interacts with phospholipid bilayers, we observe the toxin-induced perturbation on POPC/DOPG-membranes through measurements of the change in membrane thickness. Lamellar X-ray diffraction (LXD) was applied on stacked planar bilayers in the near-fully hydrated state. The results provide quantitative evidence for the membrane thinning in a concentration-dependent manner, leading to novel and direct combinatory approaches by discovering how to investigate such a biologically relevant interaction between gating-modifier toxins and phospholipid bilayers.

Topics: Peptides; Phosphatidylcholines; Phosphatidylglycerols; Spider Venoms; X-Ray Diffraction

We present a structural and functional study of a sodium channel activation inhibitor from crab spider venom. Hm-3 is an insecticidal peptide toxin consisting of 35 amino acid residues from the spider Heriaeus melloteei (Thomisidae). We produced Hm-3 recombinantly in Escherichia coli and determined its structure by NMR spectroscopy. Typical for spider toxins, Hm-3 was found to adopt the so-called "inhibitor cystine knot" or "knottin" fold stabilized by three disulfide bonds. Its molecule is amphiphilic with a hydrophobic ridge on the surface enriched in aromatic residues and surrounded by positive charges. Correspondingly, Hm-3 binds to both neutral and negatively charged lipid vesicles. Electrophysiological studies showed that at a concentration of 1 μm Hm-3 effectively inhibited a number of mammalian and insect sodium channels. Importantly, Hm-3 shifted the dependence of channel activation to more positive voltages. Moreover, the inhibition was voltage-dependent, and strong depolarizing prepulses attenuated Hm-3 activity. The toxin is therefore concluded to represent the first sodium channel gating modifier from an araneomorph spider and features a "membrane access" mechanism of action. Its amino acid sequence and position of the hydrophobic cluster are notably different from other known gating modifiers from spider venom, all of which are described from mygalomorph species. We hypothesize parallel evolution of inhibitor cystine knot toxins from Araneomorphae and Mygalomorphae suborders.

Topics: Amino Acid Sequence; Animals; Cell Membrane; Escherichia coli; Evolution, Molecular; Gene Expression; Hydrophobic and Hydrophilic Interactions; Ion Channel Gating; Membrane Potentials; Models, Molecular; Molecular Sequence Data; Phosphatidylcholines; Phosphatidylglycerols; Protein Structure, Secondary; Protein Structure, Tertiary; Recombinant Proteins; Sodium Channel Blockers; Spider Venoms; Spiders; Unilamellar Liposomes; Voltage-Gated Sodium Channels

Single-amino acid mutations in the human α-synuclein (αS) protein are related to early onset Parkinson's disease (PD). In addition to the well-known A30P, A53T, and E46K mutants, recently a number of new familial disease-related αS mutations have been discovered. How these mutations affect the putative physiological function of αS and the disease pathology is still unknown. Here we focus on the H50Q and G51D familial mutants and show that like wild-type αS, H50Q and G51D monomers bind to negatively charged membranes, form soluble partially folded oligomers with an aggregation number of ~30 monomers under specific conditions, and can aggregate into amyloid fibrils. We systematically studied the ability of these isolated oligomers to permeabilize membranes composed of anionic phospholipids (DOPG) and membranes mimicking the mitochondrial phospholipid composition (CL:POPE:POPC) using a calcein release assay. Small-angle X-ray scattering studies of isolated oligomers show that oligomers formed from wild-type αS and the A30P, E46K, H50Q, G51D, and A53T disease-related mutants are composed of a similar number of monomers. However, although the binding affinity of the monomeric protein and the aggregation number of the oligomers formed under our specific protocol are comparable for wild-type αS and H50Q and G51D αS, G51D oligomers cannot disrupt negatively charged and physiologically relevant model membranes. Replacement of the membrane-immersed glycine with a negatively charged aspartic acid at position 51 apparently abrogates membrane destabilization, whereas a mutation in the proximal but solvent-exposed part of the membrane-bound α-helix such as that found in the H50Q mutant has little effect on the bilayer disrupting properties of oligomers.

Topics: alpha-Synuclein; Cell Membrane Permeability; Fluoresceins; Humans; Membranes, Artificial; Multiprotein Complexes; Mutation, Missense; Parkinson Disease; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Protein Binding; Scattering, Small Angle; X-Ray Diffraction

Solid-state {(1)H}(13)C cross-polarization/magic angle spinning (CP/MAS) NMR spectroscopy has been applied to 17β-estradiol (E2) and 17α-estradiol (E2α), to analyze the steroidal ring conformations of the two isomers in the absence and presence of lipids at the atomic level. In the absence of lipid, the high-resolution (13)C NMR signals of E2 in a powdered form show only singlet patterns, suggesting a single ring conformation. In contrast, the (13)C signals of E2α reveal multiplet patterns with splittings of 20-300Hz, implying multiple ring conformations. In the presence of a mimic of the lipid environment, made by mixing 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC) in a molar ratio 3:1, E2 and E2α revealed multiplet patterns different from those seen in the absence of lipids, indicating that the two isomers adopt multiple conformations in the lipid environment. In this work, on the basis of chemical shift isotropy and anisotropy analysis, we demonstrated that E2 and E2α prefer to adopt multiple steroidal ring conformations in the presence of a lipid environment, distinct from that observed in solution phase and powdered form.

Topics: Anisotropy; Dimyristoylphosphatidylcholine; Estradiol; Humans; Isomerism; Lipids; Magnetic Resonance Spectroscopy; Molecular Conformation; Phosphatidylcholines; Phosphatidylglycerols; X-Ray Diffraction

We present an overview of the application of dual-focus fluorescence correlation spectroscopy (2f-FCS) for the measurement of diffusion coefficients within free-standing lipid membranes. The first part gives a detailed theoretical analysis of the expected performance of 2f-FCS, in particular about the sensitivity of the method with regard to precise focus position and to aberrations caused by refractive index mismatch or cover slide thickness deviation. After describing the experimental details of the 2f-FCS setup and the preparation of free-standing black lipid membranes (BLMs), we apply the method to study the diffusion of lipids within BLMs as a function of lipid composition and of ion valency and ionic strength of the surrounding buffer.

Topics: Diffusion; Ions; Lipid Bilayers; Lipids; Osmolar Concentration; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Spectrometry, Fluorescence

Aurelin is a 40-residue cationic antimicrobial peptide isolated from the mezoglea of a scyphoid jellyfish Aurelia aurita. Aurelin and its (15)N-labeled analogue were overexpressed in Escherichia coli and purified. Antimicrobial activity of the recombinant peptide was examined, and its spatial structure was studied by NMR spectroscopy. Aurelin represents a compact globule, enclosing one 3(10)-helix and two α-helical regions cross-linked by three disulfide bonds. The peptide binds to anionic lipid (POPC/DOPG, 3:1) vesicles even at physiological salt concentration, it does not interact with zwitterionic (POPC) vesicles and interacts with the DPC micelle surface with moderate affinity via two α-helical regions. Although aurelin shows structural homology to the BgK and ShK toxins of sea anemones, its surface does not possess the "functional dyad" required for the high-affinity interaction with the K(+)-channels. The obtained data permit to correlate the modest antibacterial properties and membrane activity of aurelin.

Topics: Amino Acid Sequence; Animals; Antimicrobial Cationic Peptides; Bacteria; Escherichia coli; Micelles; Molecular Sequence Data; Phosphatidylcholines; Phosphatidylglycerols; Protein Structure, Secondary; Recombinant Proteins; Scyphozoa; Solutions; Water

Topics: Amino Acid Sequence; Apolipoprotein A-I; Lipid Bilayers; Membrane Proteins; Molecular Sequence Data; Nanostructures; Nuclear Magnetic Resonance, Biomolecular; Peptaibols; Peptides; Phosphatidylcholines; Phosphatidylglycerols; Phospholipids

The N-terminally truncated derivative of salmon calcitonin (sCt) (acetyl-[Asn(30),Tyr(32)]-calcitonin fragment 8-32) (AC 187) lacks hormonal activity and is a potent and selective antagonist of the hormone and amylin receptor. It was investigated for its capability to interact and form channels in palmitoleoylphosphatidylcholine:dioleoylphosphatidylglycerol planar lipid membranes. Interestingly, AC 187 exhibits channel activity, whose parameters, i.e., central conductance (Lambda (c)), occurrence (number of channels/min), voltage-dependence and lifetime, are similar to those found for sCt although, in the same experimental conditions, it takes longer to incorporate into the membrane than sCt. This channel activity can be modulated by changing either the holding potential or the pH of the medium, or by adding picomolar concentrations of SDS. One evident difference between the two peptides is that sCt is unselective (1.03) while AC 187 displays a cationic selectivity (P (K) (+)/P (Cl) (-) = 2.7) at pH 7, increasing to 3.87 when the pH drops to 3.8. The present findings indicate that the 1-7 disulfide bridge is sufficient but not necessary for membrane interaction, in accordance with the observation reported on the interaction with membrane receptors. Furthermore, the remarkable pH dependence of the cationic channel could be taken into consideration for full biotechnological study.

Topics: Animals; Calcitonin; Ion Channel Gating; Ion Channels; Membranes, Artificial; Peptides; Phosphatidylcholines; Phosphatidylglycerols

Human Calcitonin (hCt) is a peptide hormone which has a regulatory action in calcium-phosphorus metabolism. It is currently used as a therapeutic tool in bone pathologies such as osteoporosis and Paget's disease. However, due to its amphiphilic property tends to form a gelatinous solution in water which consists of fibrils that limits its therapeutic use. Here we show that sodium dodecyl sulfate (SDS), an anionic detergent able to induce and stabilize alpha-helices in polypeptides, at a monomeric concentration ranging between 0.26 mM-5 pM (all concentrations are below the CMC), increases the rate and number of hCt channel formation in planar lipid membranes, at both high and low hCt concentrations, with a maximum increase at a molecular hCt/SDS ratio of 1000:1. This effect could be interpreted as a counteraction to the fibrillation process of hCt molecules by removing molecules available for aggregation from the fluid; furthermore, this action, independently of channel formation in the cell membrane, could improve the peptide-receptor interaction. The action of SDS could be attributable to the strength of the sulfate negative charge and the hydrophobic chain; in fact, a similar effect was obtained with lauryl sarcosine and not with a neutral detergent such as n-dodecyl-beta-D-maltoside. The very low molecular ratio between SDS and peptide is suggestive of a possible catalytic action of SDS that could induce alpha-helices, the appropriate structures for interacting with the membrane. Moreover, in the experimental conditions investigated, the addition of SDS does not modify the membrane's electrical properties and most of the channel properties. This finding may contribute to the knowledge of environment-folding diseases due to protein and peptides.

Topics: Calcitonin; Catalysis; Electric Conductivity; Humans; Ion Channel Gating; Ion Channels; Lipid Bilayers; Membrane Potentials; Membranes, Artificial; Microchemistry; Nanotechnology; Phosphatidylcholines; Phosphatidylglycerols; Protein Conformation; Protein Structure, Secondary; Sodium Dodecyl Sulfate

Magainin 2, a polycationic peptide, displays bactericidal and tumoricidal activity, presumably interacting with negatively charged phospholipids in the membrane hosts. In this work, we investigate the role played by the lipid head-group in the interactions and self-association of magainin 2 during pore formation in lipid bilayers. Two methods are used: single-channel and macroscopic incorporation into planar lipid membranes. Single-channel incorporation showed that magainin 2 did not interact with zwitterionic membranes, while the addition of negatively charged dioleoylphosphatidylglycerol to the membrane leads to channel formation. On the other hand, magainin 2 did not form channels in membranes made up of dioleoylphosphatidylserine (DOPS), although the addition of ergosterol to DOPS membranes leads to channel formation. This finding could indicate that ergosterol may be a possible target of magainin 2 in fungal membranes. Further support for this hypothesis comes from experiments in which the addition of ergosterol to palmitoyloleoylphosphatidylcholine membranes induced channel formation. Besides the role of negatively charged membranes, this study has shown that magainin 2 also forms channels in membranes lacking heads, such as monoolein and oxidized cholesterol, indicating an interaction of magainin 2 with acyl chains and cholesterol, respectively. This finding provides further evidence that peptide binding and assembly in lipid membranes is a complex process driven by electrostatic and/or hydrophobic interactions, depending on the structure of the peptide and the membrane composition.

Topics: Antimicrobial Cationic Peptides; Electric Conductivity; Electrochemistry; Ergosterol; Hydrophobic and Hydrophilic Interactions; Ion Channel Gating; Ion Channels; Lipid Bilayers; Lipids; Magainins; Membrane Potentials; Membranes, Artificial; Permeability; Phosphatidylcholines; Phosphatidylglycerols; Phosphatidylserines; Porosity; Static Electricity; Xenopus Proteins

Fluorescent-labeled derivatives of the Antennapedia-derived cell-penetating peptide penetratin, and of the simpler but similarly charged peptides R(6)GC-NH(2) and K(6)GC-NH(2), are shown to be able to translocate into large unilamellar lipid vesicles in the presence of a transbilayer potential (inside negative). Vesicles with diverse lipid compositions, and combining physiological proportions of neutral and anionic lipids, are able to support substantial potential-dependent uptake of all three cationic peptides. The efficiency of peptide uptake under these conditions is strongly modulated by the vesicle lipid composition, in a manner that suggests that more than one mechanism of peptide uptake may operate in different systems. Remarkably, peptide uptake is accompanied by only minor perturbations of the overall barrier function of the lipid bilayer, as assessed by assays of vesicle leakiness under the same conditions. Fluorescence microscopy of living CV-1 and HeLa cells incubated with the labeled peptides shows that the peptides accumulate in peripheral vesicular structures at early times of incubation, consistent with an initial endosomal localization as recently reported, but gradually accumulate in the cytoplasm and nucleus during more extended incubations (several hours). Our findings indicate that these relatively hydrophilic, polybasic cell-penetrating peptides can translocate through lipid bilayers by a potential- and composition-dependent pathway that causes only minimal perturbation to the overall integrity and barrier function of the bilayer.

Topics: Amino Acid Sequence; Animals; Antennapedia Homeodomain Protein; Carrier Proteins; Cell Line; Cell Membrane Permeability; Cell-Penetrating Peptides; Chlorocebus aethiops; Drosophila Proteins; HeLa Cells; Homeodomain Proteins; Humans; Lipid Bilayers; Membrane Potentials; Molecular Sequence Data; Nuclear Proteins; Peptide Fragments; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Protein Transport; Time Factors; Transcription Factors

We introduce here a new model to describe the binding of extrinsic membrane proteins to acidic lipid membranes. In this view, macroscopic binding affinity is determined by two processes: nonspecific adsorption of protein to the membrane surface and association of acidic lipids with specific sites on the bound protein. We apply this model here to compare the binding of human prothrombin and factor X/Xa to phosphatidylglycerol (PG)- and phosphatidylserine (PS)-containing small unilamellar vesicles measured via relative light scattering. This comparison was undertaken because model membranes containing PS are much more effective in supporting thrombin formation than are membranes containing PG. Analysis of binding isotherms in terms of a traditional membrane binding model gave apparent dissociation constants systematically varying from 0.1 to 10 microM over a range of 8-65 mol% negatively charged phospholipid. With our new description of membrane binding, the dependence of binding data on the acidic lipid surface concentration revealed that only two or three acidic lipid molecules were associated with each surface-bound factor X/Xa or prothrombin molecule. Assuming four independent and equivalent acidic lipid binding sites per protein, it was possible to adjust the values of only the nonspecific adsorption equilibrium constant and the equilibrium constant describing binding of each species of acidic lipid to individual sites on the protein and thereby obtain a good simulation of log-linear binding isotherms for the full range of acidic lipid surface concentrations. The protein-associated binding sites had a greater affinity for PS than for PG; i.e., a lower surface concentration of PS was required to fill the binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Factor X; Humans; Lipid Bilayers; Membrane Proteins; Models, Biological; Phosphatidylcholines; Phosphatidylglycerols; Phosphatidylserines; Phospholipids; Protein Binding; Prothrombin