Use of amphipathic polymers to deliver a membrane protein to lipid bilayers

Data are presented which suggest that a class of amphiphilic polymers known as 'amphipols' may serve as a vehicle for delivering complex integral membrane proteins into membranes. The integral membrane protein diacylglycerol kinase (DAGK) was maintained in soluble form by either of two different amphipols. Small aliquots of these solutions were added to pre-formed lipid vesicles and the appearance of DAGK catalytic activity was monitored as an indicator of the progress of productive protein insertion into the bilayers. For one of the two amphipols tested, DAGK was observed to productively transfer from its amphipol complex into vesicles with moderate efficiency. Results were not completely clear for the other amphipol.     

Interaction of amphipathic peptides with an immobilised model membrane

The interaction of melittin and a truncated analogue of melittin with an immobilised phosphatidylcholine monolayer has been studied using dynamic elution techniques. The melittin analogue (21Q analogue) had five amino acids omitted from the C-terminal region of melittin. The influence of temperature and methanol concentration on the binding affinity of the two peptides was determined and compared to the binding behaviour of two control molecules N-acetyltryptophanamide and diphenylalanine. Both peptides exhibited non-linear dependence of affinity on % methanol at different temperatures, while N-acetyltryptophanamide and diphenylalanine exhibited linear behaviour. In addition, both melittin and the 21Q analogue exhibited significant band broadening under a range of experimental conditions, which was not evident for N-acetyltryptophanamide and diphenylalanine. As melittin is known to adopt a significant degree of -helical conformation in the presence of lipids, the results suggest that melittin and the 21Q analogue adopt different conformations and orientations upon binding to the immobilised phosphatidylcholine surface. Overall, the results of this study demonstrate that the immobilised lipid monolayer provides a powerful system to rapidly assess the affinity of peptides for different lipid surfaces.     

Interaction of amphipathic peptides mediated by elastic membrane deformations

Amphipathic alpha-helical peptides are perspective antimicrobial drugs. These peptides are partially embedded into the membrane to a shallow depth so that the longitudinal axis of the helix is parallel to the plane of the membrane or deviates from it by a small angle. In the framework of theory of elasticity of liquid crystals, adapted to lipid membranes, we calculated the energy of deformations occurring near the peptides partially embedded into the membrane. The energy of deformations is minimal when two peptides are parallel to each other and stay at a distance of about 5 nm. This configuration is stable with respect to small parallel displacements of the peptides and with respect to small variation of the angle between their axes both in the plane of the membrane and in the perpendicular direction. As a result of deformation the average thickness of the membrane decreases. The distribution of the elastic energy density has a maximum in the middle between the peptides. This region is the most likely place for formation of the through pores in the membrane. Since the equilibrium distance between the peptides is relatively large, it is assumed that the originally appearing pore should be purely lipidic.     

Interactions of basement membrane components

The binding of laminin, type IV collagen, and heparan sulfate proteoglycan to each other was assessed. Laminin binds preferentially to native type IV (basement membrane) collagen over other collagens. A fragment of laminin (M_r 600 000) containing the three short chains (M_r 200 000) but lacking the long chain M_r 400 000) showed the same affinity for type IV collagen as the intact protein. The heparan sulfate proteoglycan binds well to laminin and to type IV collagen. These studies show that laminin, type IV collagen and heparan sulfate proteoglycan interact with each other. Such interactions in situ may determine the structure of basement membranes.     

ApoA-I mimetic peptides with differing ability to inhibit atherosclerosis also exhibit differences in their interactions with membrane bilayers

Two homologous apoA-I mimetic peptides, 3F-2 and 3F(14), differ in their in vitro antiatherogenic properties (Epand, R. M., Epand, R. F., Sayer, B. G., Datta, G., Chaddha, M., and Anantharamaiah, G. M. (2004) J. Biol. Chem. 279, 51404-51414). In the present work, we demonstrate that the peptide 3F-2, which has more potent anti-inflammatory activity in vitro when administered intraperitoneally to female apoE null mice (20 microg/mouse/day) for 6 weeks, inhibits atherosclerosis (lesion area 15,800 +/- 1000 microm(2), n = 29), whereas 3F(14) does not (lesion area 20,400 +/- 1000 microm(2), n = 26) compared with control saline administered (19,900 +/- 1400 microm(2), n = 22). Plasma distribution of the peptides differs in that 3F-2 preferentially associates with high density lipoprotein, whereas 3F(14) preferentially associates with apoB-containing particles. After intraperitoneal injection of (14)C-labeled peptides, 3F(14) reaches a higher maximal concentration and has a longer half-time of elimination than 3F-2. A study of the effect of these peptides on the motional and organizational properties of phospholipid bilayers, using several NMR methods, demonstrates that the two peptides insert to different extents into membranes. 3F-2 with aromatic residues at the center of the nonpolar face partitions closer to the phospholipid head group compared with 3F(14). In contrast, only 3F(14) affects the terminal methyl group of the acyl chain, decreasing the (2)H order parameter and at the same time also decreasing the molecular motion of this methyl group. This dual effect of 3F(14) can be explained in terms of the cross-sectional shape of the amphipathic helix. These results support the proposal that the molecular basis for the difference in the biological activities of the two peptides lies with their different interactions with membranes.     

Interactions of basement membrane components

The binding of laminin, type IV collagen, and heparan sulfate proteoglycan to each other was assessed. Laminin binds preferentially to native type IV (basement membrane) collagen over other collagens. A fragment of laminin (Mr 600 000) containing the three short chains (Mr 200 000) but lacking the long chain (Mr 400 000) showed the same affinity for type IV collagen as the intact protein. The heparan sulfate proteoglycan binds well to laminin and to type IV collagen. These studies show that laminin, type IV collagen and heparan sulfate proteoglycan interact with each other. Such interactions in situ may determine the structure of basement membranes.     

Interactions of amphipathic CPPs with model membranes

We have investigated the interactions between two carrier peptides and model membrane systems as well as the conformational consequences of these interactions. Studies performed with lipid monolayers at the air-water interface have enabled identification of the nature of the lipid-peptide interactions and characterization of the influence of phospholipids on the ability of these peptides to penetrate into lipidic media. Penetration experiments reveal that both peptides interact strongly with phospholipids. Conformational investigations indicate that the lipid-peptide interaction govern the conformational state of the peptides. Based on the ability of both peptides to promote ion permeabilization of both natural and artificial membranes, we propose a model illustrating the translocation process. For MPG, it is based on the formation of a β-barrel pore-like structure, while for Pep-1, it is based on association of helices.     

Interactions of amphipathic CPPs with model membranes

We have investigated the interactions between two carrier peptides and model membrane systems as well as the conformational consequences of these interactions. Studies performed with lipid monolayers at the air-water interface have enabled identification of the nature of the lipid-peptide interactions and characterization of the influence of phospholipids on the ability of these peptides to penetrate into lipidic media. Penetration experiments reveal that both peptides interact strongly with phospholipids. Conformational investigations indicate that the lipid-peptide interaction govern the conformational state of the peptides. Based on the ability of both peptides to promote ion permeabilization of both natural and artificial membranes, we propose a model illustrating the translocation process. For MPG, it is based on the formation of a beta-barrel pore-like structure, while for Pep-1, it is based on association of helices.     

Rapidly transported organelles containing membrane and cytoskeletal components: their relation to axonal growth

We have examined the movements, composition, and cellular origin of phase-dense varicosities in cultures of chick sympathetic and sensory neurons. These organelles are variable in diameter (typically between 0.2 and 2 microns) and undergo saltatory movements both towards and away from the neuronal cell body. Their mean velocities vary inversely with the size of the organelle and are greater in the retrograde than the anterograde direction. Organelles stain with the lipophilic dye 1, 1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine and with antibodies to cytoskeletal components. In cultures double-stained with antibodies to alpha-tubulin and 70-kD neurofilament protein (NF-L), approximately 40% of the organelles stain for tubulin, 30% stain for NF- L, 10% stain for both tubulin and NF-L, and 40% show no staining with either antibody. The association of cytoskeletal proteins with the organelles shows that these proteins are able to move by a form of rapid axonal transport. Under most culture conditions the predominant direction of movement is towards the cell body, suggesting that the organelles are produced at or near the growth cone. Retrograde movements continue in culture medium lacking protein or high molecular mass components and increase under conditions in which the advance of the growth cone is arrested. There is a fourfold increase in the number of organelles moving retrogradely in neurites that encounter a substratum-associated barrier to elongation; retrograde movements increase similarly in cultures exposed to cytochalasin at levels known to block growth cone advance. No previously described organelle shows behavior coordinated with axonal growth in this way. We propose that the organelles contain membrane and cytoskeletal components that have been delivered to the growth cone, by slow or fast anterograde transport, in excess of the amounts required to synthesize more axon. In view of their rapid mobility and variable contents, we suggest that they be called "neuronal parcels."     

Mechanisms for the modulation of membrane bilayer properties by amphipathic helical peptides

The amphipathic helix, in which hydrophobia and hydrophilic residues are grouped on opposing faces, is a structural mot if found in many peptides and proteins that bind to membranes. One of the physical properties of membranes that can be altered by the binding of amphipathic helices is membrane monolayer curvature strain. Class A amphipathic helices, which are present in exchangeable plasma lipoproteins, can stabilize membranes by reducing negative monolayer curvature strain; proline-punctuated class A amphipathic helical segments are particularly effective in this regard. This property is suggested to be associated with some of the beneficial biological effects of this protein. On the other hand, lytic amphipathic helical peptides can act by increasing negative curvature strain or by forming pores composed of helical clusters. Thus, different amphipathic helical peptides can be membrane stabilizing or be lytic to membranes, depending on the structural motif of the helix, which in turn determines the nature of its association with membranes. Features of these peptides that are responsible for their specific properties are discussed. © 1994 John Wiley & Sons, Inc.     

Action mechanism of amphipathic peptides gramicidin S and melittin on erythrocyte membrane

Amphipathic peptides gramicidin S and melittin caused a characteristic colloid-osmotic hemolysis on human erythrocytes; that is, the peptides produced initially a small membrane lesion in erythrocyte membrane, followed by the release of hemoglobin. The size of membrane lesion increased with an increase in the concentration of peptide. Under the conditions causing membrane lesion, we observed the release of membrane fragments containing phospholipids. The present results show that both the peptides have the ability to stimulate the release of membrane fragments out of the cells and this brings about the perforation of molecules of small size, leading to a colloid-osmotic hemolysis.     

Orientations of amphipathic helical peptides in membrane bilayers determined by solid-state NMR spectroscopy

Summary Solid-state NMR spectroscopy was used to determine the orientations of two amphipathic helical peptides associated with lipid bilayers. A single spectral parameter provides sufficient orientational information for these peptides, which are known, from other methods, to be helical. The orientations of the peptides were determined using the¹⁵N chemical shift observed for specifically labeled peptide sites. Magainin, an antibiotic peptide from frog skin, was found to lie in the plane of the bilayer. M2, a helical segment of the nicotinic acetylcholine receptor, was found to span the membrane, perpendicular to the plane of the bilayer. These findings have important implications for the mechanisms of biological functions of these peptides.     

Peptide-membrane interactions and mechanisms of membrane destruction by amphipathic alpha-helical antimicrobial peptides

Antimicrobial peptides (AMPs) have received considerable interest as a source of new antibiotics with the potential for treatment of multiple-drug resistant infections. An important class of AMPs is composed of linear, cationic peptides that form amphipathic alpha-helices. Among the most potent of these are the cecropins and synthetic peptides that are hybrids of cecropin and the bee venom peptide, mellitin. Both cecropins and cecropin-mellitin hybrids exist in solution as unstructured monomers, folding into predominantly alpha-helical structures upon membrane binding with their long helical axis parallel to the bilayer surface. Studies using model membranes have shown that these peptides intercalate into the lipid bilayer just below the level of the phospholipid glycerol backbone in a location that requires expansion of the outer leaflet of the bilayer, and evidence from a variety of experimental approaches indicates that expansion and thinning of the bilayer are common characteristics during the early stages of antimicrobial peptide-membrane interactions. Subsequent disruption of the membrane permeability barrier may occur by a variety of mechanisms, leading ultimately to loss of cytoplasmic membrane integrity and cell death.     

Effect of amphipathic peptides with different alpha-helical contents on liposome-fusion.

The peptide-induced fusion of neutral and acidic liposomes was studied in relation to the amphiphilicities evaluated by alpha-helical contents of peptides by means of a carboxyfluorescein leakage assay, light scattering, a membrane intermixing assay and electron microscopy. An amphipathic mother peptide, Ac-(Leu-Ala-Arg-Leu)3-NHCH3 (4(3], and its derivatives, [Pro6]4(3) (1), [Pro2,6]4(3) (2), and [Pro2,6,10]4(3) (3), which have very similar hydrophobic moments, caused a leakage of contents from small unilamellar vesicles composed of egg yolk phosphatidylcholine and egg yolk phosphatidic acid (3:1). The abilities of the peptides to induce the fusion of the acidic liposomes increased with increasing alpha-helical content: in acidic liposomes the helical contents were in the order of 4(3) greater than 1 greater than 2 greater than 3 (Lee et al. (1989) Chem. Lett., 599-602). Electron microscopic data showed that 1 caused a transformation of the small unilamellar vesicles (20-50 nm in diameter) to large ones (100-300 nm). Based on the fact that these peptides have very similar hydrophobic moments despite of decreasing in the mean residue hydrophobicities to some extent, it was concluded that the abilities of the peptides to induce the fusion of liposomes depend on the extent of amphiphilic conformation evaluated by alpha-helical contents of the peptides in the presence of liposomes. For neutral liposomes of egg yolk phosphatidylcholine, all the proline-containing peptides showed no fusogenic ability but weak leakage abilities, suggesting that the charge interaction between the basic peptides and acidic phospholipid is an important factor to induce the perturbation and fusion of the bilayer.     

Influence of lipophilic groups in cationic alpha-helical peptides on their abilities to bind with DNA and deliver genes into cells.

For the purpose of achieving gene transfer into cells mediated by peptides with a short chain length, we employed two kinds of amphiphilic alpha-helix peptides, mastoparan (INLK-ALAA-LAKK-IL-NH2) obtained from wasp venom and an alpha-helix model peptide (LARL-LARL-LARL-NH2). Furthermore, to strengthen the hydrophobicity of the peptide required for the formation of the aggregates with the DNA, we modified these peptides using several lipophilic groups, i.e. acyl groups with a single chain, a dialkylcarbamoyl group and a cholesteryloxycarbonyl group. We examined the ability of the peptides and their derivatives to bind and aggregate with plasmid DNA, the structural change in the peptides caused by binding with the DNA and the in vitro gene transfer abilities into COS-7 cells. As a result, mastoparan was found to acquire the DNA binding ability by introduction of the lipophilic group. The conformational change in the peptides depended on the hydrophobicity of the introduced acyl group. The DNA complex of most lipophilic mastoparan derivatives could be incorporated into the cells via the endocytosis pathway. In the case of the helix model peptide, the acyl group with a moderate chain length was required for the formation of the aggregate which is competent for incorporation into the cells. In this study, we succeeded in giving such short peptides sufficient gene transfer ability by modifying them with some lipophilic groups. However, the influence of the modification by the lipophilic groups on the formation of aggregates with DNA and the gene transfer ability depended on the structure of the peptide portion. These results indicate that consideration of total hydrophobicity balance is needed for the design of an efficient gene carrier peptide.     

Lipid shape is a key factor for membrane interactions of amphipathic helical peptides

The membrane alignment of the amphiphilic α-helical model peptide MSI-103 (sequence [KIAGKIA](3)-NH(2)) was examined by solid state (2)H-NMR in different lipid systems by systematically varying the acyl chain length and degree of saturation, the lipid head group type, and the peptide-to-lipid molar ratio. In liquid crystalline phosphatidylcholine (PC) lipids with saturated chains, the amphiphilic helix changes its orientation from a surface-bound S-state to a tilted T-state with increasing peptide concentration. In PC lipids with unsaturated chains, on the other hand, the S-state is found throughout all concentrations. Using phosphatidylethanolamine lipids with a small head group or by addition of lyso-lipids with only one acyl chain, the spontaneous curvature of the bilayer was purposefully changed. In the first case with a negative curvature only the S-state was found, whereas in systems with a positive curvature the peptide preferred the obliquely immersed T-state at high concentration. The orientation of MSI-103 thus correlates very well with the shape of the lipid molecules constituting the membrane. Lipid charge, on the other hand, was found to affect only the initial electrostatic attraction to the membrane surface but not the alignment preferences. In bilayers that are sealed with 20% cholesterol, MSI-103 cannot bind in a well-oriented manner and forms immobilized aggregates instead. We conclude that the curvature properties of a membrane are a key factor in the interactions of amphiphilic helical peptides in general, whose re-alignment and immersion preferences may thus be inferred in a straightforward manner from the lipid-shape concept.     

Mechanism of membrane damage induced by the amphipathic peptides gramicidin S and melittin

The action of gramicidin S and melittin on human erythrocytes, Staphylococcus aureus and Escherichia coli was studied as an extension of the previous study (Katsu, T., Ninomiya, C., Kuroko, M., Kobayashi, H., Hirota, T. and Fujita, Y. (1988) Biochim. Biophys. Acta 939, 57-63). These amphipathic peptides stimulated the release of membrane phospholipids outside cells in a concentration range causing permeability change. The shape change of erythrocytes from normal discoid to spiculate form was observed just prior to the release of membrane components. We have proposed the following action mechanism of gramicidin S and melittin. The peptide molecules were predominantly accumulated in the outer half of the bilayer, deforming the erythrocyte cell into crenature. A large accumulation made the membrane structure unstable, resulting in the release of membrane fragments and the simultaneous enhancement of permeability. The action mechanism of these peptides was compared with that of simple surfactants.     

Osmotically induced membrane tension modulates membrane permeabilization by class L amphipathic helical peptides: nucleation model of defect formation.

The mechanism of action of lytic peptides on membranes is widely studied and is important in view of potential medical applications. Previously (I. V. Polozov, A. I. Polozova, E. M. Tytler, G. M. Anantharamaiah, J. P. Segrest, G. A. Woolley, and R. M., Biochemistry, 36:9237--9245) we analyzed the mechanism of membrane permeabilization by 18L, the archetype lytic peptide featuring the class L amphipathic alpha-helix, according to the classification of Segrest et al. (J. P. Segrest, G. de Loof, J. G. Dohlman, C. G. Brouillette, and G. M. Anantharamaiah, 1990, Proteins, 8:103--117). We concluded that the 18L peptide destabilizes membranes, leading to a transient formation of large defects that result in contents leakage and, in the presence of bilayer-bilayer contact, could lead to vesicle fusion. Here we report that this defect formation is strongly enhanced by the membrane tension induced by osmotic swelling of vesicles. Even below standard leakage-inducing peptide/lipid ratios, membrane resistance to osmotic tension drops from hundreds to tens of milliosmoles. The actual decrease is dependent on the peptide/lipid ratio and on the type of lipid. We propose that under membrane tension a peptidic pore serves as a nucleation site for the transient formation of a lipidic pore. The tension is released upon pore expansion with inclusion of more peptides and lipids into the pore lining. This tension modulation of leakage was observed for other class L peptides (mastoparan, K18L) and thus may be of general applicability for the action of membrane active lytic peptides.