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Raquel F Epand

Publications and source records attributed to Raquel F Epand.

At least 37 records · Page 2Linked to original sources
PubMed2004

An apolipoprotein AI mimetic peptide: membrane interactions and the role of cholesterol.

The 18-amino acid amphipathic helical peptide Ac-DWFKAFYDKVAEKFKEAF-NH(2) promotes the separation of cholesterol from the phospholipid, resulting in the formation of cholesterol crystallites, even at mole fractions of cholesterol as low as 0.3. The peptide exerts a greater degree of penetration into membranes of pure phosphatidylcholine in the absence of cholesterol than into bilayers of phosphatidylcholine and cholesterol. The circular dichroism spectrum of the peptide in buffer indicates that it self-associates, leading to the formation of structures with higher helical content. However, in the presence of lipid, the peptide remains helical over a larger concentration range. The peptide undergoes a thermal transition on heating. Cholesterol has little effect on the secondary structure of the peptide; however, increased Trp emission intensity in the absence of cholesterol indicates a deeper penetration of the helix upon removal of cholesterol from the membrane. The results with these model systems demonstrate changes in peptide-lipid interactions that may be related to the observed biological properties of this peptide.

Amino Acid Sequence↗
PubMed2004

Cholesterol-dependent partitioning of PtdIns(4,5)P2 into membrane domains by the N-terminal fragment of NAP-22 (neuronal axonal myristoylated membrane protein of 22 kDa).

A myristoylated peptide corresponding to the N-terminus of NAP-22 (neuronal axonal myristoylated membrane protein of 22 kDa) causes the quenching of the fluorescence of BODIPY-TMR-labelled PtdIns(4,5) P2 in bilayers of 1-palmitoyl-2-oleoyl phosphatidylcholine containing 40 mol% cholesterol and 0.1 mol% BODIPY-PtdIns(4,5)2. Both fluorescence spectroscopy and total internal reflectance fluorescence microscopy revealed the cholesterol-dependent nature of PtdIns(4,5) P2-enriched membrane-domain formation.

Cholesterol↗
PubMed2004

Aromatic residue position on the nonpolar face of class a amphipathic helical peptides determines biological activity.

The apolipoprotein A-I mimetic peptide 4F (Ac-DWFKAFYDKVAEKFKEAF-NH(2)), with four Phe residues on the nonpolar face of the amphipathic alpha-helix, is strongly anti-inflammatory, whereas two 3F analogs (3F(3) and 3F(14)) are not. To understand how changes in helix nonpolar face structure affect function, two additional 3F analogs, Ac-DKLKAFYDKVFEWAKEAF-NH(2) (3F-1) and Ac-DKWKAVYDKFAEAFKEFL-NH(2) (3F-2), were designed using the same amino acid composition as 3F(3) and 3F(14). The aromatic residues in 3F-1 and 3F-2 are near the polar-nonpolar interface and at the center of the nonpolar face of the helix, respectively. Like 4F, but in contrast to 3F(3) and 3F(14), these peptides effectively inhibited lytic peptide-induced hemolysis, oxidized phospholipid-induced monocyte chemotaxis, and scavenged lipid hydroperoxides from low density lipoprotein. High pressure liquid chromatography retention times and monolayer exclusion pressures indicated that there is no direct correlation of peptide function with lipid affinity. Fluorescence studies suggested that, although the peptides bind phospholipids similarly, the Trp residue in 4F, 3F-1, and 3F-2 is less motionally restricted than in 3F(3) and 3F(14). Based on these results and molecular modeling studies, we propose that the arrangement of aromatic residues in class A amphipathic helical molecules regulates entry of reactive oxygen species into peptide-phospholipid complexes, thereby reducing the extent of monocyte chemotaxis, an important step in atherosclerosis.

Amino Acid Sequence↗
PubMed2004

Fatty acids enhance membrane permeabilization by pro-apoptotic Bax.

Fatty acids are known promoters of apoptosis. In the present study, the direct role of fatty acids with regard to their ability to cause membrane permeabilization by Bax was explored. Addition of fatty acids to liposomes in the presence of cations greatly enhanced the permeabilizing activity of Bax, a pro-apoptotic Bcl-2 protein. This provides a putative mechanism for the role of fatty acids in apoptosis. It is not a result of detergent-like properties of fatty acids, since a different micelle-forming amphiphile, dilysocardiolipin, was strongly inhibitory. We also demonstrate that there is a synergistic effect on Bax-induced permeabilization between Ca(2+) and Mg(2+), both on the binding of Bax to liposomes as well as on the induction of the leakage of liposomal contents. Micromolar concentrations of Ca(2+) added externally or submicromolar concentrations of free Ca(2+) present in the medium were sufficient to promote Bax-induced permeabilization synergistically with externally added Mg(2+). These results indicate that Bax can induce leakage from liposomes at ion concentrations resembling those found physiologically. The synergistic effects of Ca(2+) and Mg(2+) were observed with liposomes with different lipid compositions. Thus the action of Bax is strongly modulated by the presence of bivalent cations that can act synergistically, as well as by micelle-forming lipid components that can be either stimulatory or inhibitory.

Apoptosis↗
PubMed2004

The biological potency of a series of analogues of human calcitonin correlates with their interactions with phospholipids.

The conformational and lipid binding properties of several calcitonin analogs were compared. These analogs were designed to have the central amphipathic helical region of salmon calcitonin and N- and C-terminal segments similar to human calcitonin. The various analogs differed from one another either by removal of Leu19 from this hybrid analog, replacement of Leu19 with Gly19 or having a carboxyl terminus more closely related to salmon calcitonin. It had been found that replacement of Leu19 with Gly19 caused a marked reduction in the hypocalemic activity of the analog. The ability of the analogs to form helical structures in the presence of dimyristoylphosphatidylglycerol as well as their ability to lower the enthalpy of the calorimetric phase transition of this phospholipid correlates well with the hypocalcemic potency of the peptide.

Amino Acid Sequence↗
PubMed2004

Properties of polyunsaturated phosphatidylcholine membranes in the presence and absence of cholesterol.

Mixtures of cholesterol with phosphatidylcholine species containing the polyunsaturated acyl chains arachidonoyl or docosahexaenoyl were studied by (13)C magic angle spinning (MAS) NMR using both cross-polarization and direct polarization, by (31)P NMR and by differential scanning calorimetry. Several unique features of these systems were observed. The separation of cholesterol in crystalline form occurred at much lower molar fractions than with other forms of phosphatidylcholine. The crystals that were formed were sensitive to the history of the sample. At cholesterol molar fractions below 0.5, they dissolved into the membrane by sequential heating and cooling scans. With higher molar fractions of cholesterol, larger amounts of anhydrous crystals were formed after the first heating. This was accompanied by the formation of non-lamellar phases. The cholesterol crystals that were formed generally were not observed by direct polarization (13)C MAS NMR, even with delay times of 100 s. This suggests that the cholesterol crystals are in a more rigid state in mixtures with these lipids. This is in contrast with the terminal methyl group of the acyl chains that is too mobile to allow cross-polarization using 1 ms contact times.

Calorimetry, Differential Scanning↗
PubMed2004

Non-raft forming sphingomyelin-cholesterol mixtures.

Sphingomyelin from biological membranes forms segregated domains with cholesterol in fluid bilayers. However, a synthetic form of sphingomyelin with an oleoyl chain linked to sphingosine is not incorporated into cholesterol-rich domains. We have studied the properties of mixtures of oleoyl-sphingomyelin and cholesterol as well as mixtures of oleoyl-sphingomyelin with 1-stearoyl-2-oleoyl-phosphatidylcholine by DSC and NMR. Cholesterol has a high miscibility with oleoyl-sphingomyelin and it does not separate in crystalline form until the mol fraction of cholesterol reaches a value above 0.6. A large fraction of the cholesterol crystals that are formed are in the monohydrate form. Furthermore, these crystals rehydrate relatively rapidly compared with pure cholesterol crystals in the absence of phospholipid. The environment of the carbonyl group of the phospholipid indicates that it is similar to other forms of sphingomyelin with saturated acyl chains. Also similar to other forms of sphingomyelin, the quaternary ammonium group of oleoyl-sphingomyelin is more rigid than that of phosphatidylcholines, as indicated by the strong resonance observed with cross-polarization/magic angle spinning. Additionally, oleoyl-sphingomyelin produces a larger alteration than egg sphingomyelin of the phase transition of 1-stearoyl-2-oleoyl-phosphatidylcholine. These studies indicate that oleoyl-sphingomyelin, unlike saturated forms of sphingomyelin, does not form segregated domains with cholesterol because of its greater miscibility with phosphatidylcholine.

Cholesterol↗
PubMed2004

Structural and functional properties of an unusual internal fusion peptide in a nonenveloped virus membrane fusion protein.

The avian and Nelson Bay reoviruses are two of only a limited number of nonenveloped viruses capable of inducing cell-cell membrane fusion. These viruses encode the smallest known membrane fusion proteins (p10). We now show that a region of moderate hydrophobicity we call the hydrophobic patch (HP), present in the small N-terminal ectodomain of p10, shares the following characteristics with the fusion peptides of enveloped virus fusion proteins: (i) an abundance of glycine and alanine residues, (ii) a potential amphipathic secondary structure, (iii) membrane-seeking characteristics that correspond to the degree of hydrophobicity, and (iv) the ability to induce lipid mixing in a liposome fusion assay. The p10 HP is therefore predicted to provide a function in the mechanism of membrane fusion similar to those of the fusion peptides of enveloped virus fusion peptides, namely, association with and destabilization of opposing lipid bilayers. Mutational and biophysical analysis suggested that the internal fusion peptide of p10 lacks alpha-helical content and exists as a disulfide-stabilized loop structure. Similar kinked structures have been reported in the fusion peptides of several enveloped virus fusion proteins. The preservation of a predicted loop structure in the fusion peptide of this unusual nonenveloped virus membrane fusion protein supports an imperative role for a kinked fusion peptide motif in biological membrane fusion.

Amino Acid Sequence↗
PubMed2003

Transbilayer lipid diffusion promoted by Bax: implications for apoptosis.

It is known that the proapoptotic protein Bax facilitates the formation of pores in bilayers, resulting in the release of proteins from the intermitochondrial space. We demonstrate that another consequence of the interaction of Bax with membranes is an increase in the rate of lipid transbilayer diffusion. We use two independent assays for transbilayer diffusion, one involving the formation of asymmetric liposomes by placing a pyrene-labeled lipid into the outer monolayer of preformed vesicles and another assay based on the initial preparation of liposomes having an asymmetric transbilayer distribution of lipids. With both methods we find that oligomeric BaxDeltaC or full-length Bax in the presence of tBid, but not monomeric full-length Bax, strongly promotes the rate of transbilayer diffusion. Although biological membranes exhibit rates of lipid transbilayer diffusion of minutes or less, they are able to maintain an asymmetric distribution of lipids across the bilayer. In the case of mitochondria, cardiolipin is sequestered on the inner leaflet of the inner mitochondrial membrane. However, during apoptosis this lipid translocates to the outer surface of the outer mitochondrial membrane. This phenomenon must involve an increase in the rate of transbilayer diffusion. The results of the present paper demonstrate that an activated form of Bax can cause this increased rate.

Apoptosis↗
PubMed2003

Peptide-induced formation of cholesterol-rich domains.

The peptide N-acetyl-LWYIK-amide causes the reorganization of bilayers of phosphatidylcholine and cholesterol to produce domains enriched in cholesterol. At a cholesterol mol fraction of 0.5, addition of N-acetyl-LWYIK-amide results in the formation of cholesterol crystallites. Addition of this peptide to mixtures of 1-stearoyl-2-oleoylphosphatidylcholine with lower mol fractions of cholesterol results in an increase in the enthalpy of the chain melting transition of the phospholipid, indicating the depletion of cholesterol from a domain in the membrane. The peptide binds to membranes both with and without cholesterol. However, (1)H magic-angle spinning (MAS) nuclear Overhauser effect spectroscopy (NOESY) indicates that in the presence of cholesterol the peptide has greater penetration into the bilayer. (13)C MAS NMR indicates that the peptide has stronger interactions with the A ring of cholesterol than it does with the interior of the bilayer. These results are in contrast with those of another peptide, N-acetyl-KYWFYR-amide, which does not promote the formation of cholesterol crystallites and does not show preferential interaction with cholesterol by NMR. Therefore, cholesterol can promote the insertion of N-acetyl-LWYIK-amide into a membrane and this peptide will sequester cholesterol into domains. These properties help to explain the observation that this sequence is found to be important in causing the fusion protein of human immunodeficiency virus (HIV) to sequester into raft domains in biological membranes.

Calorimetry, Differential Scanning↗
PubMed2003

Interaction with a membrane surface triggers a reversible conformational change in Bax normally associated with induction of apoptosis.

The Bcl-2 family member Bax is an apoptosis-promoting protein that normally resides in an inactive state within the cytoplasm of healthy cells. Upon induction of apoptosis by diverse stimuli, Bax undergoes a conformational change and translocates to mitochondria, where it oligomerizes and forms pores that allow the release of cytochrome c and other cytotoxic factors. Protein-protein interactions between Bax and other Bcl-2 family members are strongly implicated in Bax activation, but a compelling case has recently been made for the involvement of lipids in this process as well. Here we report that purified Bax undergoes a reversible conformational change upon incubation with lipid vesicles in the absence of other proteins. This Bax-liposome interaction does not depend on a specific lipid composition. Changes in Bax conformation were observed by immunoprecipitation with the conformation-specific antibody 6A7, circular dichroism spectroscopy, and differential scanning calorimetry. Although liposomes induced Bax to become 6A7-reactive (a feature normally associated with the onset of apoptosis), the protein did not insert into membranes, become oligomeric, or form pores, clearly indicating that other triggers are required for Bax to achieve its final pro-apoptotic state. Indeed, the lipid-induced Bax conformational change is shown to be required for tBid-induced Bax oligomerization and pore formation, putting it upstream of tBid activity in this molecular pathway to Bax activation. These data demonstrate that Bax is sensitized to activation by transient interaction with lipid membrane surfaces and provide evidence that Bax activation proceeds in a stepwise fashion, with multiple triggers and potential levels of regulation.

Apoptosis↗
PubMed2003

Quaternary structure of the neuronal protein NAP-22 in aqueous solution.

NAP-22, a myristoylated, anionic protein, is a major protein component of the detergent-insoluble fraction of neurons. After extraction from the membrane, it is readily soluble in water. NAP-22 will partition only into membranes with specific lipid compositions. The lipid specificity is not expected for a monomeric myristoylated protein. We have studied the self-association of NAP-22 in solution. Sedimentation velocity experiments indicated that the protein is largely associated. The low concentration limiting s value is approximately 1.3 S, indicating a highly asymmetric monomer. In contrast, a nonmyristoylated form of the protein shows no evidence of oligomerization by velocity sedimentation and has an s value corresponding to the smallest component of NAP-22, but without the presence of higher oligomers. Sedimentation equilibrium runs indicate that there is a rapidly reversible equilibrium between monomeric and oligomeric forms of the protein followed by a slower, more irreversible association into larger aggregates. In situ atomic force microscopy of the protein deposited on mica from freshly prepared dilute solution revealed dimers on the mica surface. The values of the association constants obtained from the sedimentation equilibrium data suggest that the weight concentration of the monomer exceeds that of the dimer below a total protein concentration of 0.04 mg/ml. Since the concentration of NAP-22 in the neurons of the developing brain is approximately 0.6 mg/ml, if the protein were in solution, it would be in oligomeric form and bind specifically to cholesterol-rich domains. We demonstrate, using fluorescence resonance energy transfer, that at low concentrations, NAP-22 labeled with Texas Red binds equally well to liposomes of phosphatidylcholine either with or without the addition of 40 mol% cholesterol. Thus, oligomerization of NAP-22 contributes to its lipid selectivity during membrane binding.

Animals↗
PubMed2003

Membrane fusion activity of vesicular stomatitis virus glycoprotein G is induced by low pH but not by heat or denaturant.

The fusogenic envelope glycoprotein G of the rhabdovirus vesicular stomatitis virus (VSV) induces membrane fusion at acidic pH. At acidic pH the G protein undergoes a major structural reorganization leading to the fusogenic conformation. However, unlike other viral fusion proteins, the low-pH-induced conformational change of VSV G is completely reversible. As well, the presence of an alpha-helical coiled-coil motif required for fusion by a number of viral and cellular fusion proteins was not predicted in VSV G protein by using a number of algorithms. Results of pH dependence of the thermal stability of G protein as determined by intrinsic Trp fluorescence and circular dichroism (CD) spectroscopy show that the G protein is equally stable at neutral or acidic pH. Destabilization of G structure at neutral pH with either heat or urea did not induce membrane fusion or conformational change(s) leading to membrane fusion. Taken together, these data suggest that the mechanism of VSV G-induced fusion is distinct from the fusion mechanism of fusion proteins that involve a coiled-coil motif.

Animals↗
PubMed2003

Irreversible unfolding of the neutral pH form of influenza hemagglutinin demonstrates that it is not in a metastable state.

The thermal denaturation of the proteins of influenza virus has been measured by differential scanning calorimetry in the presence and absence of lipids as a function of scan rate. We have applied theories of irreversible thermodynamics to obtain the activation energy. In the presence of liposomes of dioleoylphosphatidylcholine with the ganglioside, GD(1a), the denaturation temperature of the hemagglutinin protein is lowered. This lowering of thermal stability is also reflected in the temperature dependence of the circular dichroism spectra. Quasi-elastic light scattering confirms that liposomes containing GD(1a) interact with the virus and inhibit the growth in the size of the particle as a function of temperature. Although the virus can fuse with the liposomes at higher temperatures, the enthalpy change for this process is not detectable. Our results also demonstrate that the compact folded structure of the influenza hemagglutinin protein is not a kinetically trapped metastable high-energy form.

Calorimetry, Differential Scanning↗
PubMed2003

On the interaction between gp41 and membranes: the immunodominant loop stabilizes gp41 helical hairpin conformation.

gp41 is the protein responsible for the process of membrane fusion that allows primate lentiviruses (HIV and SIV) to enter into their host cells. gp41 ectodomain contains an N-terminal and a C-terminal heptad repeat region (NHR and CHR) connected by an immunodominant loop. In the absence of membranes, the NHR and CHR segments fold into a protease-resistant core with a trimeric helical hairpin structure. However, when the immunodominant loop is not present (either in a complex formed by HIV-1 gp41-derived NHR and CHR peptides or by mild treatment with protease of recombinant constructs of HIV-1 gp41 ectodomain, which also lack the N-terminal fusion peptide and the C-terminal Trp-rich region) membrane binding induces a conformational change in the gp41 core structure. Here, we further investigated whether covalently linking the NHR and CHR segments by the immunodominant loop affects this conformational change. Specifically, we analyzed a construct corresponding to a fragment of SIVmac239 gp41ectodomain (residues 27-149, named e-gp41) by means of surface plasmon resonance, Trp and rhodamine fluorescence, ATR-FTIR spectroscopy, and differential scanning calorimetry. Our results suggest that the presence of the loop stabilizes the trimeric helical hairpin both when e-gp41 is in aqueous solution and when it is bound to the membrane surface. Bearing in mind possible differences between HIV-1 and SIV gp41, and considering that the gp41 ectodomain constructs analyzed to date lack the N-terminal fusion peptide and the C-terminal Trp-rich region, we discuss our observations in relation to the mechanism of virus-induced membrane fusion.

Calorimetry, Differential Scanning↗
PubMed2003

Direct comparison of membrane interactions of model peptides composed of only Leu and Lys residues.

We compared the properties of two peptides of identical size and amino acid composition, Ac-(LKKL)(5)-NHEt and Ac-(KL)(10)-NHEt. Both are amphipathic, but only Ac-(LKKL)(5)-NHEt is a potent promoter of negative curvature. CD studies performed in the presence of lipids confirmed that under these conditions Ac-(LKKL)(5)-NHEt forms an alpha-helix, and Ac-(KL)(10)-NHEt adopts a beta structure. We studied their binding affinity by centrifugation and isothermal titration calorimetry techniques. The Ac-(LKKL)(5)-NHEt bound to zwitterionic and anionic liposomes, while Ac-(KL)(10)-NHEt interacted mainly with anionic liposomes. Ac-(LKKL)(5)-NHEt was more lytic than Ac-(KL)(10)-NHEt for zwitterionic palmitoyloleoylphosphatidylcholine (POPC) liposomes, and for liposomes composed of lipids extracted from either sheep or human erythrocytes (RBC). Both peptides had similar lytic and lipid mixing activities for liposomes containing anionic lipids. Both peptides were highly hemolytic, with Ac-(LKKL)(5)-NHEt active against sheep RBC and Ac-(KL)(10)-NHEt more active against human RBC. From their respective minimal effective concentrations (MECs) as antimicrobial agents, we judged Ac-(KL)(10)-NHEt to be 2 to 5-fold more potent than Ac-(LKKL)(5)-NHEt in media that contained physiological concentrations of NaCl. Notwithstanding, both peptides had MECs <1 microg/mL for Escherichia coli and Pseudomonas aeruginosa and <4 microg/mL for Staphylococcus aureus and methicillin-resistant Staphylococcus aureus. Although selectivity of antimicrobial peptides for bacterial membranes may result, in part, from the preferential display of anionic residues in these membranes, inability to interact with or bind to zwitterionic phospholipids offers no guarantee that the peptide will lack appreciable cytotoxicity for host cells.

Anti-Infective Agents↗
PubMed2003

The arrangement of cholesterol in membranes and binding of NAP-22.

Cholesterol forms crystals when the mol fraction of sterol in a membrane bilayer exceeds a certain value. The solubility limit of cholesterol is very dependent on the nature of the phospholipid with which it is mixed. NMR methods have proven useful in quantifying the amount of cholesterol monohydrate crystals present in mixtures with phospholipids. A protein, NAP-22, present in high abundance in the synaptic cell membrane and synaptic vesicle, promotes the formation of cholesterol crystallites in lipid mixtures in which cholesterol would be completely dissolved in the membrane in the absence of protein. This finding, along with effects of the protein on the phase transitions of mixtures of phosphatidylcholine (PC) and cholesterol indicate that NAP-22 facilitates the formation of cholesterol-rich domains. This protein will bind only to membranes of PC that contain either cholesterol or phosphatidylethanolamine (PE). The process requires the presence of a myristoyl group on the N-terminus of NAP-22. The phenomenon also does not occur with a 19 amino acid myristoylated peptide corresponding to the amino terminal segment of NAP-22. The basis of the selectivity of NAP-22 for interacting with membranes of specific composition is suggested to be due to the accessibility of the myristoyl group.

Calmodulin-Binding Proteins↗