Farnesylated peptides in model membranes: a biophysical investigation

Protein prenylation plays an important role in signal transduction, protein-protein interactions, and the localization and association of proteins with membranes. Using three different techniques, this study physically characterizes the interactions between model dimyristoylphosphatidylcholine membranes and a series of farnesylated peptides. Magic angle spinning nuclear Overhauser enhancement spectroscopy and differential scanning calorimetry reveal that both charged [Ac-Asn-Lys-Asn-Cys-(farnesyl)-OMe and Ac-Asn-Lys-Asn-Cys-(farnesyl)-NH(2)] and uncharged [Ac-Cys-(farnesyl)-OMe and farnesol] species partition into dimyristoylphosphatidylcholine bilayers. Calorimetry and vesicle fluctuation analysis of giant unilamellar vesicles show that the charged peptides modestly decrease the main gel-fluid phase transition and markedly increase the bending rigidity of large unilamellar vesicles. Uncharged species, on the other hand, dramatically decrease the main phase transition and modestly decrease the bending rigidity. No difference with carboxyl methylation is detected.     

Transduction peptides: from technology to physiology

During the past fifteen years, a variety of peptides have been characterized for their ability to translocate into live cells. Most are efficient vectors that can internalize hydrophilic cargoes, and so provide a valuable biological (and potentially therapeutic) tool for targeting proteins into cells. Furthermore, translocation of cell-permeable peptides across the plasma membrane and their subsequent access to the cytosol, even when fused to large hydrophilic proteins, is challenging the perception of the plasma membrane as an impermeable barrier.     

The cooperative behaviour of antimicrobial peptides in model membranes

A systematic analysis of the hypothesis of the antimicrobial peptides' (AMPs) cooperative action is performed by means of full atomistic molecular dynamics simulations accompanied by circular dichroism experiments. Several AMPs from the aurein family (2.5,2.6, 3.1), have a similar sequence in the first ten amino acids, are investigated in different environments including aqueous solution, trifluoroethanol (TFE), palmitoyloleoylphosphatidylethanolamine (POPE), and palmitoyloleoylphosphatidylglycerol (POPG) lipid bilayers. It is found that the cooperative effect is stronger in aqueous solution and weaker in TFE. Moreover, in the presence of membranes, the cooperative effect plays an important role in the peptide/lipid bilayer interaction. The action of AMPs is a competition of the hydrophobic interactions between the side chains of the peptides and the hydrophobic region of lipid molecules, as well as the intra peptide interaction. The aureins 2.5-COOH and 2.6-COOH form a hydrophobic aggregate to minimize the interaction between the hydrophobic group and the water. Once that the peptides reach the water/lipid interface the hydrophobic aggregate becomes smaller and the peptides start to penetrate into the membrane. In contrast, aurein 3.1-COOH forms only a transient aggregate which disintegrates once the peptides reached the membrane, and it shows no cooperativity in membrane penetration.     

Interaction of enkephalin peptides with anionic model membranes

According to the model for passive transport across the membranes, the total flow of permeant molecules is related to the product of the water-membrane partition coefficient and the diffusion coefficient, and to the water-membrane interfacial barrier. The effect of membrane surface charge on the permeability and interaction of analgesic peptide ligands with model membranes was investigated. A mixture of zwitterionic phospholipids with cholesterol was used as a model membrane. The lipid membrane charge density was controlled by the addition of anionic 1-palmitoyl-2-oleoylphosphatidylserine. Two classes of highly potent analgesic peptides were studied, c[D-Pen²,D-Pen⁵]enkephalin (DPDPE) and biphalin, a dimeric analog of enkephalin. The effect of increased surface charge on the permeability of the zwitterionic DPDPE is a relatively modest decrease, that appears to be due to a diminished partition coefficient. On the other hand the binding of the dicationic biphalin ligands to membranes increases proportionally with increased negative surface charge. This effect translates into a significant reduction of biphalin permeability by reducing the diffusion of the peptide across the bilayer. These experiments show the importance of electrostatic effects on the peptide-membrane interactions and suggest that the negative charge naturally present in cell membranes may hamper the membrane transport of some peptide drugs, especially cationic ones, unless there are cationic transporters present.     

Primary amphipathic cell-penetrating peptides: structural requirements and interactions with model membranes

To identify rules for the design of efficient cell-penetrating peptides that deliver therapeutic agents into subcellular compartments, we compared the properties of two closely related primary amphipathic peptides that mainly differ by their conformational state. On the basis of a peptide Pbeta that is nonstructured in water and that promotes efficient cellular uptake of nucleic acids through noncovalent association, we have designed a peptide [Palpha] that is predicted to adopt a helical conformation. We show that [Pbeta] undergoes a lipid-induced conformational transition into a sheet structure, while [Palpha] remains helical. Penetration experiments show that both peptides can spontaneously insert into phospholipid membranes. Analysis of compression isotherms indicates that both peptides interact with phospholipids in the liquid expanded and liquid condensed states. AFM observations reveal that the peptides strongly disrupt the lipid organization of the monolayers and that the conformational state can influence the uptake by model membranes.     

Lipid-packing perturbation of model membranes by pH-responsive antimicrobial peptides

The indiscriminate use of conventional antibiotics is leading to an increase in the number of resistant bacterial strains, motivating the search for new compounds to overcome this challenging problem. Antimicrobial peptides, acting only in the lipid phase of membranes without requiring specific membrane receptors as do conventional antibiotics, have shown great potential as possible substituents of these drugs. These peptides are in general rich in basic and hydrophobic residues forming an amphipathic structure when in contact with membranes. The outer leaflet of the prokaryotic cell membrane is rich in anionic lipids, while the surface of the eukaryotic cell is zwitterionic. Due to their positive net charge, many of these peptides are selective to the prokaryotic membrane. Notwithstanding this preference for anionic membranes, some of them can also act on neutral ones, hampering their therapeutic use. In addition to the electrostatic interaction driving peptide adsorption by the membrane, the ability of the peptide to perturb lipid packing is of paramount importance in their capacity to induce cell lysis, which is strongly dependent on electrostatic and hydrophobic interactions. In the present research, we revised the adsorption of antimicrobial peptides by model membranes as well as the perturbation that they induce in lipid packing. In particular, we focused on some peptides that have simultaneously acidic and basic residues. The net charges of these peptides are modulated by pH changes and the lipid composition of model membranes. We discuss the experimental approaches used to explore these aspects of lipid membranes using lipid vesicles and lipid monolayer as model membranes.     

Interaction of enkephalin peptides with anionic model membranes.

According to the model for passive transport across the membranes, the total flow of permeant molecules is related to the product of the water-membrane partition coefficient and the diffusion coefficient, and to the water-membrane interfacial barrier. The effect of membrane surface charge on the permeability and interaction of analgesic peptide ligands with model membranes was investigated. A mixture of zwitterionic phospholipids with cholesterol was used as a model membrane. The lipid membrane charge density was controlled by the addition of anionic 1-palmitoyl-2-oleoylphosphatidylserine. Two classes of highly potent analgesic peptides were studied, c[D-Pen(2),D-Pen(5)]enkephalin (DPDPE) and biphalin, a dimeric analog of enkephalin. The effect of increased surface charge on the permeability of the zwitterionic DPDPE is a relatively modest decrease, that appears to be due to a diminished partition coefficient. On the other hand the binding of the dicationic biphalin ligands to membranes increases proportionally with increased negative surface charge. This effect translates into a significant reduction of biphalin permeability by reducing the diffusion of the peptide across the bilayer. These experiments show the importance of electrostatic effects on the peptide-membrane interactions and suggest that the negative charge naturally present in cell membranes may hamper the membrane transport of some peptide drugs, especially cationic ones, unless there are cationic transporters present.     

Biotinylated lipid bilayer disks as model membranes for biosensor analyses

The aim of this study was to investigate the potential of polyethylene glycol (PEG)-stabilized lipid bilayer disks as model membranes for surface plasmon resonance (SPR)-based biosensor analyses. Nanosized bilayer disks that included 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)₂₀₀₀] (DSPE-PEG₂₀₀₀-biotin) were prepared and structurally characterized by cryo-transmission electron microscopy (cryo-TEM) imaging. The biotinylated disks were immobilized via streptavidin to three different types of sensor chips (CM3, CM4, and CM5) varying in their degree of carboxymethylation and thickness of the dextran matrix. The bilayer disks were found to interact with and bind stably to the streptavidin-coated sensor surfaces. As a first step toward the use of these bilayer disks as model membranes in SPR-based studies of membrane proteins, initial investigations were carried out with cyclooxygenases 1 and 2 (COX 1 and COX 2). Bilayer disks were preincubated with the respective protein and thereafter allowed to interact with the sensor surface. The signal resulting from the interaction was, in both cases, significantly enhanced as compared with the signal obtained when disks alone were injected over the surface. The results of the study suggest that bilayer disks constitute a new and promising type of model membranes for SPR-based biosensor studies.     

Interaction of hagfish cathelicidin antimicrobial peptides with model lipid membranes

Hagfish intestinal antimicrobial peptides (HFIAPs) are a family of polycationic peptides exhibiting potent, broad-spectrum bactericidal activity. In an attempt to unravel the mechanism of action of HFIAPs, we have studied their interaction with model membranes. Synthetic HFIAPs selectively bound to liposomes mimicking bacterial membranes, and caused the release of vesicle-encapsulated fluorescent markers in a size-dependent manner. In planar lipid bilayer membranes, HFIAPs induced erratic current fluctuations and reduced membrane line tension according to a general theory for lipidic pores, suggesting that HFIAP pores contain lipid molecules. Consistent with this notion, lipid transbilayer redistribution accompanied HFIAP pore formation, and membrane monolayer curvature regulated HFIAP pore formation. Based on these studies, we propose that HFIAPs kill target cells, at least in part, by interacting with their plasma membrane to induce formation of lipid-containing pores. Such a membrane-permeabilizing function appears to be an evolutionarily conserved host-defense mechanism of antimicrobial peptides.     

Changes in the structural-functional state of erythrocyte membranes treated with anion transport inhibitors

Effect of 4,4-dyisotiocyanostilben-2,2-disulfonate (DIDS) and 1-ftor-2,4-dinitrobenzol (NDFB) on the rate of phosphate ion transport in erythrocytes, filtrability and thermal stability of erythrocytes and on the structural state of the erythrocyte membrane estimated by UV-fluorescence, PAAG--electrophoresis and measuring of the activity of membrane-bound acetylcholinesterase (AChE) has been studied. Unpenetrating anion transport inhibitor DIDS is shown to induce structural modifications of bands 3 of protein and AChE, while DNFB penetrating the membrane causes a significant reorganization of many membrane proteins (including spectrin) resulting in changes of transport and mechanical properties of erythrocytes.     

A thermodynamic approach to the mechanism of cell-penetrating peptides in model membranes

We report a first test of the hypothesis that the mechanism of antimicrobial, cytolytic, and amphipathic cell-penetrating peptides in model membranes is determined by the thermodynamics of insertion of the peptide into the lipid bilayer from the surface-associated state. Three peptides were designed with minimal mutations relative to the sequence of TP10W, the Y3W variant of transportan 10, which is a helical, amphipathic cell-penetrating peptide previously studied. Binding to 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) membranes and release of dye from those vesicles were assessed by stopped-flow fluorescence, and the secondary structure of the peptides on the membrane was determined by circular dichroism. The Gibbs energy of binding determined experimentally was in excellent agreement with that calculated using the Wimley-White interfacial hydrophobicity scale, taking into account the helical content of the membrane-associated peptide. Release of dye from POPC vesicles remained graded, as predicted by the hypothesis. More significantly, as the Gibbs energy of insertion into the bilayer became more unfavorable, which was estimated using the Wimley-White octanol hydrophobicity scale, dye release became slower, in quantitative agreement with the prediction.     

What determines the activity of antimicrobial and cytolytic peptides in model membranes

We previously proposed three hypotheses relating the mechanism of antimicrobial and cytolytic peptides in model membranes to the Gibbs free energies of binding and insertion into the membrane [Almeida, P. F., and Pokorny, A. (2009) Biochemistry 48, 8083-8093]. Two sets of peptides were designed to test those hypotheses, by mutating of the sequences of δ-lysin, cecropin A, and magainin 2. Peptide binding and activity were measured on phosphatidylcholine membranes. In the first set, the peptide charge was changed by mutating basic to acidic residues or vice versa, but the amino acid sequence was not altered much otherwise. The type of dye release changed from graded to all-or-none according to prediction. However, location of charged residues in the sequence with the correct spacing to form salt bridges failed to improve binding. In the second set, the charged and other key residues were kept in the same positions, whereas most of the sequence was significantly but conservatively simplified, maintaining the same hydrophobicity and amphipathicity. This set behaved completely different from predicted. The type of release, which was expected to be maintained, changed dramatically from all-or-none to graded in the mutants of cecropin and magainin. Finally, contrary to the hypotheses, the results indicate that the Gibbs energy of binding to the membrane, not the Gibbs energy of insertion, is the primary determinant of peptide activity.     

Transduction of biochemical signals across cell membranes

Biological cells need to be responsive to various stimuli, primarily chemical ligands from their environments. Specific receptor molecules embedded in the plasma membrane detect the different biochemical signals that impact the cell, and these receptors are the conduits for transmission of this information to the cell interior for action. There are several classes of signal transduction receptors and many specific receptors within each of the major classes. This review emphasizes the structural biology of three major classes of transmembrane receptors - tyrosine kinase receptors, histidine kinase sensors, and G-protein coupled receptors. Biophysical principles that govern the processes of signal transduction across cell membranes are also discussed.     

Interactions of mast cell degranulating peptides with model membranes: A comparative biophysical study

In the last decade, there has been renewed interest in biologically active peptides in fields like allergy, autoimmune diseases and antibiotic therapy. Mast cell degranulating peptides mimic G-protein receptors, showing different activity levels even among homologous peptides. Another important feature is their ability to interact directly with membrane phospholipids, in a fast and concentration-dependent way. The mechanism of action of peptide HR1 on model membranes was investigated comparatively to other mast cell degranulating peptides (Mastoparan, Eumenitin and Anoplin) to evidence the features that modulate their selectivity. Using vesicle leakage, single-channel recordings and zeta-potential measurements, we demonstrated that HR1 preferentially binds to anionic bilayers, accumulates, folds, and at very low concentrations, is able to insert and create membrane spanning ion-selective pores. We discuss the ion selectivity character of the pores based on the neutralization or screening of the peptides charges by the bilayer head group charges or dipoles.     

Melittin peptides exhibit different activity on different cells and model membranes

Melittin (MLT) is a lytic peptide with a broad spectrum of activity against both eukaryotic and prokaryotic cells. To understand the role of proline and the thiol group of cysteine in the cytolytic activity of MLT, native MLT and cysteine-containing analogs were prepared using solid phase peptide synthesis. The antimicrobial and cytolytic activities of the monomeric and dimeric MLT peptides against different cells and model membranes were investigated. The results indicated that the proline residue was necessary for antimicrobial activity and cytotoxicity and its absence significantly reduced lysis of model membranes and hemolysis. Although lytic activity against model membranes decreased for the MLT dimer, hemolytic activity was increased. The native peptide and the MLT-P14C monomer were mainly unstructured in buffer while the dimer adopted a helical conformation. In the presence of neutral and negatively charged vesicles, the helical content of the three peptides was significantly increased. The lytic activity, therefore, is not correlated to the secondary structure of the peptides and, more particularly, on the propensity to adopt helical conformation.     

Transduction of peptides and proteins into live cells by cell penetrating peptides

Internalization of peptides and proteins into live cells is an essential prerequisite for studies on intracellular signal pathways, for treatment of certain microbial diseases and for signal transduction therapy, especially for cancer treatment. Cell penetrating peptides (CPPs) facilitate the transport of cargo-proteins through the cell membrane into live cells. CPPs which allow formation of non-covalent complexes with the cargo are used primarily in this study due to the relatively easy handling procedure. Efficiency of the protein uptake is estimated qualitatively by fluorescence microscopy and quantitatively by SDS-PAGE. Using the CPP cocktail JBS-Proteoducin, the intracellular concentrations of a secondary antibody and bovine serum albumin can reach the micromolar range. Internalization of antibodies allows mediation of intracellular pathways including knock down of signal transduction. The high specificity and affinity of antibodies makes them potentially more powerful than siRNA. Thus, CPPs represent a significant new possibility to study signal transduction processes in competition or in comparison to the commonly used other techniques. To estimate the highest attainable intracellular concentrations of cargo proteins, the CPPs are tested for cytotoxicity. Cell viability and membrane integrity relative to concentration of CPPs are investigated. Viability as estimated by the reductive activity of mitochondria (MTT-test) is more sensitive to higher concentrations of CPPs versus membrane integrity, as measured by the release of dead cell protease. Distinct differences in uptake efficiency and cytotoxic effects are found using six different CPPs and six different adhesion and suspension cell lines.     

Differential effects of five types of antipathogenic plant peptides on model membranes

The effects of five antipathogenic plant peptides, wheat α-thionin, potato PTH1 defensin, barley LTP2 lipid transfer protein, and potato tuber DL1 and DL2 defensins, have been tested against phospholipid vesicles (liposomes). Wheat thionin very actively induces aggregation and leakage of negatively charged vesicles. LTP2 displays the same activities, although to a limited extent. Under certain conditions PTH1 and DL2 induce vesicle aggregation, but not leakage. Potato defensin DL1 failed to show any effect on liposomes. The same peptides have been assayed against a plant pathogenic bacterium, both the membrane-active and -inactive compounds having efficient antibacterial action.     

Phospholipid flop induced by transmembrane peptides in model membranes is modulated by lipid composition

Since phospholipid synthesis is generally confined to one leaflet of a membrane, membrane growth requires phospholipid translocation (flip-flop). It is generally assumed that this process is protein-mediated; however, the mechanism of flip-flop remains elusive. Previously, we have demonstrated flop of 2-[6-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]caproyl] (C6NBD) phospholipids, induced by the presence of membrane-spanning peptides in vesicles composed of an Escherichia coli phospholipid extract, supporting the hypothesis that the presence of transmembrane stretches of proteins in the bilayer is sufficient to allow phospholipid flip-flop in the inner membrane of E. coli [Kol et al. (2001) Biochemistry 40, 10500]. Here, we investigated whether the specific phospholipid composition of E. coli is a prerequisite for transmembrane helix-induced flop of phospholipids. This was tested by determining the amount of C6NBD-phospholipid that was translocated from the inner leaflet to the outer leaflet of a model membrane in time, using a dithionite reduction assay. The transmembrane peptides GWWL(AL)8WWA (WALP23) and GKKL(AL)8KKA (KALP23) induced phospholipid flop in model membranes composed of various lipid mixtures. The rate of peptide-induced flop was found to decrease with increasing dioleoylphosphatidylethanolamine (DOPE) content of vesicles composed of DOPE and dioleoylphosphatidylcholine (DOPC), and the rate of KALP23-induced flop was shown to be stimulated by higher dioleoylphosphatidylglycerol (DOPG) content in model membranes composed of DOPG and DOPC. Furthermore, the incorporation of cholesterol had an inhibitory effect on peptide-induced flop. Finally, flop efficiency was strongly dependent on the phospholipid headgroup of the NBD-phospholipid analogue. Possible implications for transmembrane helix-induced flop in biomembranes in general are discussed.     

Membrane insertion and lateral mobility of synthetic amphiphilic signal peptides in lipid model membranes

Amphiphilic signal sequences with the potential to form alpha-helices with a polar, charged face and an apolar face are common in proteins which are imported into mitochondria, in the PTS permeases of bacteria, and in bacterial rhodopsins. Synthetic peptides of such sequences partition into the surface region of lipid membranes where they can adopt different secondary structures. A finely controlled balance of electrostatic and hydrophobic interactions determines the 'affinity' of amphiphilic signal peptides for lipid membranes, as well as the structure, orientation and depth of penetration of these peptides in lipid bilayer membranes. The ability of an individual peptide to associate with lipid bilayer membranes in several different modes is, most likely, a general feature of amphiphilic signal peptides and is reflected in several common physical properties of their amino acid sequences.