Showing papers by "Richard M. Epand published in 2003"

Trichogin: a paradigm for lipopeptaibols.

Abstract: Lipopeptaibols are members of a novel family of naturally occurring, short peptides with antimicrobial activity, characterized by a lipophilic acyl chain at the N-terminus, a high content of turn/helix inducing α-aminoisobutyric acid and a 1,2-amino alcohol at the C-terminus. Using solution methods, the prototypical lipopeptaibol trichogin GA IV and a large series of appropriately designed analogues were synthesized, which allow: (i) determination of the minimal lipid chain and peptide main-chain lengths for the onset of membrane activity, and (ii) exploitation of a number of physico-chemical techniques aimed at assessing the trichogin preferred conformation under a variety of conditions and at investigating its mechanism of interaction with the phospholipid membranes. Copyright © 2003 European Peptide Society and John Wiley & Sons, Ltd.

Interactions of the antimicrobial β‐peptide β‐17 with phospholipid vesicles differ from membrane interactions of magainins

Abstract: We have studied the interaction of β-17, a potent synthetic antimicrobial β-peptide, with phospholipids. We find that unlike other antimicrobial peptides such as magainin II, β-17 facilitates the formation of nonbilayer phases, indicating that the peptide promotes negative curvature. Studies of liposomal leakage also indicate a different mode of membrane interaction relative to magainin II, but both leakage and membrane binding show that β-17, like magainin II, has strong affinity for membranes containing anionic lipids. This is likely to be an important factor contributing to the antimicrobial specificity of the β-peptide.

Transbilayer lipid diffusion promoted by Bax: implications for apoptosis.

Abstract: 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 BaxΔC 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

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

Abstract: 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.

Binding of NAP-22, a calmodulin-binding neuronal protein, to raft-like domains in model membranes.

Abstract: The cholesterol-binding protein NAP-22 is a major component of the detergent-insoluble low-density fraction of rat brain. In this study, we found, using fluorescence microscopy, that native NAP-22, but not a demyristoylated form, binds to cholesterol-rich raft-like domains in planar-supported monolayers and remains bound after nonionic detergent extraction. NAP-22 also protects the cholesterol-rich domains during extraction by methyl-β-cyclodextrin. The lateral mobility of this protein is much lower than that of other raft components in model membranes, suggesting that both cholesterol binding and inter-NAP-22 interactions markedly reduce its lateral diffusion. This study suggests that NAP-22 binding may be employed to image cholesterol-rich regions, such as caveolae/rafts, on the plasma membrane of cells, and preliminary efforts in that direction are presented.

Liposomes as models for antimicrobial peptides.

Abstract: Publisher Summary Liposomes are useful tools to identify the mechanism of action of antimicrobial peptides, and they provide a simple system to complement cell studies for optimization of the potency and specificity of these agents. The chapter provides several methods that can be used to study the binding of peptides to membranes. Centrifugation is a simple and direct method for studying the binding of peptides to membranes, and it has general applicability. The basis of the method is simple. It requires the physical separation of lipid-bound peptide from free peptide. Fluorescence is a particularly convenient method to use for peptides that contain tryptophan (Trp). The method is sensitive to low concentrations of peptide and precautions can be taken to diminish light-scattering artifacts. Many studies have applied isothermal titration calorimetry to the study of the binding of antimicrobial peptides to membranes. A variety of methods can be used to assess the conformational properties of an antimicrobial peptide, as well as the manner in which it inserts into membranes. One aspect of the interaction of the peptide with the membrane is its orientation with respect to the plane of the bilayer.

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

Abstract: 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, GD1a, 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 GD1a 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.

Specificity of membrane binding of the neuronal protein NAP-22.

Abstract: NAP-22, a major protein of neuronal rafts is known to preferentially bind to membranes containing cholesterol. In this work we establish the requirements for membrane binding of NAP-22. We find that other sterols can replace cholesterol to promote binding. In addition, bilayers containing phosphatidylethanolamine bind NAP-22 in the absence of cholesterol. Thus, there is not a specific interaction of NAP-22 with cholesterol that determines its binding to membranes. Addition of a mol fraction of phosphatidylserine of 0.05 to membranes of phosphatidylcholine and cholesterol enhances the membrane binding of NAP-22. The dependence of binding on the mol fraction of phosphatidylserine indicates that NAP-22 binds to membranes with its amino-terminal segment closer to the membrane than the remainder of the protein. We have also determined which segments of NAP-22 are required for membrane binding. A non-myristoylated form binds only weakly to membranes. Truncating the protein from 226 amino acids to the myristoylated amino-terminal 60 amino acids does not prevent binding to membranes in a cholesterol-dependent manner, but this binding is of weaker affinity. However, myristoylation is not sufficient to promote binding to cholesterol-rich domains. An N-terminal 19-amino-acid, myristoylated peptide binds to membranes but without requiring specific lipids. Thus, the remainder of the protein contributes to the lipid specificity of the membrane binding of NAP-22.

Design and function of a conformationally restricted analog of the influenza virus fusion peptide.

Abstract: A conformationally restricted analog of the N-terminal 12-residue peptide segment of the HA2 subunit of the PPV/34, PR/8/34, and Jap/57 strains of influenza virus hemagglutinin was synthesized containing three residues of Calpha-methyl-valine. This peptide has a higher content of helical structure in the presence of 50% trifluoroethanol or when interacting with liposomes of egg phosphatidylcholine compared with the conformationally more flexible control peptide with the native sequence. The control and analog peptides had opposite effects on membrane curvature as measured by shifts in the bilayer-to-hexagonal phase transition temperature of a synthetic phosphatidylethanolamine derivative. The control peptide promoted more negative curvature, particularly at acidic pH and was also more potent than the analog in promoting lipid mixing. The results indicate that the ability of the peptide to sample a broader range of conformations is required for the influenza fusion peptide to destabilize membranes and that a rigid helical structure is less fusogenic. The difference between the two peptides and between pH 7.4 and pH 5.0 show a correlation between the ability to promote negative curvature and to accelerate lipid mixing.