Interaction of amphipathic peptides with an immobilised model membrane

Summary 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 moleculesN-acetyltryptophanamide and diphenylalanine. Both peptides exhibited non-linear dependence of affinity on % methanol at different temperatures, whileN-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 forN-acetyltryptophanamide and diphenylalanine. As melittin is known to adopt a significant degree of α-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.     

Effect of end group blockage on the properties of a class A amphipathic helical peptide

In a recent classification of biologically active amphipathic α-helixes, the lipid-associating domains in exchangeable plasma apolipoproteins have been classified as class A amphipathic helixes (Segrest, J. P., De Loof, H., Dohlman, J. G., Brouillette, C. G., Anantharamaiah, G. M. Proteins 8:103–117, 1990). A model peptide analog with the sequence, Asp Trp Leu Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Leu Lys Glu Ala Phe (18A), possesses the characteristics of a class A amphipathic helix. The addition of an acetyl group at the α-amino terminus and an amide at the α-carboxyl terminus, to obtain Ac-18A-NH₂, produces large increases in helicity for the peptide both in solution and when associated with lipid (for 18A vs Ac-18A-NH₂, from 6 to 38% helix in buffer and from 49 to 92% helix when bound to dimyristoyl phosphatidylcholine in discoidal complexes). Blocking of the end-groups of 18A stabilizes the α-helix in the presence of lipid by approximately 1.3 kcal/mol. There is also an increase in the self-association of the blocked peptide in aqueous solution. The free energy of binding to the PC–water interface is increased only by about 3% (from ?8.0 kcal/mol for 18A to ?8.3 kcal/mol for Ac-18A-NH₂). The Ac-18A-NH₂ has a much greater potency in raising the bilayer to hexagonal phase transition temperature of dipalmitoleoyl phosphatidylethanolamine than does 18A. In this regard Ac-18A-NH₂ more closely resembles the behavior of the apolipoprotein A-I, which is the major protein component of high-density lipoprotein and a potent inhibitor of lipid hexagonal phase formation. The activation of the plasma enzyme lecithin: cholesterol acyltransferase by the Ac-18A-NH₂ peptide is greater than the 18A analog and comparable to that observed with the apo A-I. In the case of Ac-18A-NH₂, the higher activating potency may be due, at least in part, to the ability of the peptide to micellize egg PC vesicles. © 1993 Wiley-Liss, Inc.     

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.     

Sub‐angstrom modeling of complexes between flexible peptides and globular proteins

A wide range of regulatory processes in the cell are mediated by flexible peptides that fold upon binding to globular proteins. Computational efforts to model these interactions are hindered by the large number of rotatable bonds in flexible peptides relative to typical ligand molecules, and the fact that different peptides assume different backbone conformations within the same binding site. In this study, we present Rosetta FlexPepDock, a novel tool for refining coarse peptide–protein models that allows significant changes in both peptide backbone and side chains. We obtain high resolution models, often of sub‐angstrom backbone quality, over an extensive and general benchmark that is based on a large nonredundant dataset of 89 peptide–protein interactions. Importantly, side chains of known binding motifs are modeled particularly well, typically with atomic accuracy. In addition, our protocol has improved modeling quality for the important application of cross docking to PDZ domains. We anticipate that the ability to create high resolution models for a wide range of peptide–protein complexes will have significant impact on structure‐based functional characterization, controlled manipulation of peptide interactions, and on peptide‐based drug design. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Effects antifreeze peptides on the thermotropic properties of a model membrane

In this paper, we report on the effect of short segments of type I antifreeze protein (AFP I) on the thermotropic properties of a model membrane. Two different types of dimyristoylphosphatidylcholine model membranes were used, multilamellar vesicles and small unilamellar vesicles. The membrane properties were studied by differential scanning calorimetry (DSC) and fluorescence anisotropy. With the incorporation of AFP I and its short segments, the order of the model membrane increased both in the gel state and in the liquid crystalline state. The interaction of AFPs with the model membrane caused a shift in the phase transition to lower temperatures, which is accompanied by a broadening of the DSC thermogram. This preferential stabilization to a more ordered phase by the AFPs could be due to ordering the hydrophobic membrane core and separation into domains. Overall, this approach of employing short segments of AFP I simplifies the correlation between antifreeze protein characteristics and the effect of these parameters on the interaction mechanism of AFP with cell membranes. The success of this approach can lead to the identification of short peptides with high antifreeze activity.     

Activity of a novel‐designed antimicrobial peptide and its interaction with lipids

A new antimicrobial peptide l‐RW containing double amphipathic binding sequences was designed, and its biological activities were investigated in the present study. L‐RW showed antibacterial activity against several bacterial strains but low cytotoxicity to mammalian cells and low hemolytic activity to red blood cells, which makes it a potential and promising peptide for further development. Microscale thermophoresis (MST), a new technique, was applied to study the antimicrobial peptide–lipid interaction for the first time, which examined the binding affinities of this new antimicrobial peptide to various lipids, including different phospholipids, mixture lipids and bacterial lipid extracts. The results demonstrated that l‐RW bound preferentially to negatively charged lipids over neutral lipids, which was consistent with the biological activities, revealing the important role of electrostatic interaction in the binding process. L‐RW also showed higher binding affinity for lipid extract from Staphyloccocus aureus compared with Pseudomonas aeruginosa and Escherichia coli, which were in good agreement with the higher antibacterial activity against S. aureus than P. aeruginosa and E. coli, suggesting that the binding affinity is capable to predict the antibacterial activity to some extent. Additionally, the binding of l‐RW to phospholipids was also performed in fetal bovine serum solution by MST, which revealed that the components in biological solution may have interference with the binding event. The results proved that MST is a useful and potent tool in antimicrobial peptide–lipid interaction investigation. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Immunogobulin-lipid interaction: A model membrane study

We recently presented evidence (Vandenbranden, M., De Coen, J.L., Jeener, R., Kanarek, L. and Ruysschaert, J.M. (1981) Mol. Immunol. 8, 621–631) for the existence of two conformational rabbit serum IgG immunoglobulin isomers. In the present report, their capacity to interact with lipid is investigated in model membranes. (1) One isomer, IgG(H), behaves like several intrinsic membrane proteins: it induces a large surface pressure increase when injected under a lipid monolayer in the close packed state and increases by 20-fold the conductance of a planar bilayer. The other isomer, IgG(S) doesn't interact significantly with the lipids. (2) IgG(H) marked a preference for monolayers made of lipids with a negatively charged polar headgroup and bearing unsaturations in their acyl chains. Penetration is stronger with lipid monolayer in the fluid state than in the rigid state. (3) As previously shown (Vandenbranden, M., De Coen, J.L., Jeener, R., Kanarek, L. and Ruysschaert, J.M. (1981) Mol. Immunol. 18, 621–631), circular dichroïsm spectra and antigen precipitation tests don't allow to detect any difference in the overall secondary conformation and antigen recognition properties of the two isomers. (4) Papaïn cleavage of the hinge region suppresses the hydrophobic properties of IgG towards lipid monolayers. (5) The hypothesis of a binding of the hinge region with the lipid bilayer is discussed.     

Interaction of 18-residue peptides derived from amphipathic helical segments of globular proteins with model membranes

We investigated the interaction of six 18-residue peptides derived from amphipathic helical segments of globular proteins with model membranes. The net charge of the peptides at neutral pH varies from −1 to +6. Circular dichroism spectra indicate that peptides with a high net positive charge tend to fold into a helical conformation in the presence of negatively charged lipid vesicles. In helical conformation, their average hydrophobic moment and hydrophobicity would render them surface-active. The composition of amino acids on the polar face of the helix in the peptides is considerably different. The peptides show variations in their ability to permeabilise zwitterionic and anionic lipid vesicles. Whereas increased net positive charge favours greater permeabilisation, the distribution of charged residues in the polar face also plays a role in determining membrane activity. The distribution of amino acids in the polar face of the helix in the peptides that were investigated do not fall into the canonical classes described. Amphipathic helices, which are part of proteins, with a pattern of amino acid distribution different from those observed in class L, A and others, could help in providing newer insights into peptide-membrane interactions.     

Target of rapamycin FATC domain as a general membrane anchor: The FKBP‐12 like domain of FKBP38 as a case study

Increased efforts have been undertaken to better understand the formation of signaling complexes at cellular membranes. Since the preparation of proteins containing a transmembrane domain or a prenylation motif is generally challenging an alternative membrane anchoring unit that is easy to attach, water‐soluble and binds to different membrane mimetics would find broad application. The 33‐residue long FATC domain of yeast TOR1 (y1fatc) fulfills these criteria and binds to neutral and negatively charged micelles, bicelles, and liposomes. As a case study, we fused it to the FKBP506‐binding region of the protein FKBP38 (FKBP38‐BD) and used ¹H–¹⁵N NMR spectroscopy to characterize localization of the chimeric protein to micelles, bicelles, and liposomes. Based on these and published data for y1fatc, its use as a C‐terminally attachable membrane anchor for other proteins is compatible with a wide range of buffer conditions (pH circa 6–8.5, NaCl 0 to >150 mM, presence of reducing agents, different salts such as MgCl₂ and CaCl₂). The high water‐solubility of y1fatc enables its use for titration experiments against a membrane‐localized interaction partner of the fused target protein. Results from studies with peptides corresponding to the C‐terminal 17–11 residues of the 33‐residue long domain by 1D ¹H NMR and CD spectroscopy indicate that they still can interact with membrane mimetics. Thus, they may be used as membrane anchors if the full y1fatc sequence is disturbing or if a chemically synthesized y1fatc peptide shall be attached by native chemical ligation, for example, unlabeled peptide to ¹⁵N‐labeled target protein for NMR studies.     

A fast and simple method for probing the interaction of peptides and proteins with lipids and membrane-mimetics using GB1 fusion proteins and NMR spectroscopy

The expression of peptides and proteins as fusions to the B1 domain of streptococcal protein G (GB1) is very popular since GB1 often improves the solubility of the target protein and because the first purification step using IgG affinity chromatography is simple and efficient. However, the following protease digest is not always complete or can result in a digest of the target protein. In addition, a further purification step such as RP-HPLC has to be used to get rid of the GB1 tag and undigested fusion protein. Because the protease digest and the following purification step are not only time-consuming but generally also expensive, we tested if GB1 fusion proteins can directly be used for NMR interaction studies using lipids or membrane-mimetics. Based on NMR binding studies using only the GB1 part, this fusion tag does not significantly interact with different membrane-mimetics such as micelles, bicelles, or liposomes. Thus spectral changes observed using GB1-fusion proteins indicate lipid- and membrane interactions of the target protein. The method was initially established to probe membrane interactions of a large number of mutants of the FATC domain of the ser/thr kinase TOR. To demonstrate the usefulness of the approach, we show NMR binding data for the wild type protein and a leucine to alanine mutant.     

Interaction of long-chain nicotinates with dipalmitoylphosphatidylcholine

The interaction of four long-chain nicotinates, compounds that are of interest as potential chemopreventive agents, with dipalmitoylphosphatidylcholine (DPPC) was investigated in monolayers at the air-water interface and in fully hydrated bilayers. For the monolayer studies, the compression isotherms of mixtures of the respective nicotinate with DPPC were recorded at various compositions on a hydrochloric acid subphase (pH 1.9-2.1, 37 +/- 2 degrees C). The headgroup of the nicotinates (24-29 A2/molecule) is larger than that of the hydrophobic tail (20 A2/molecule). The pure nicotinates exhibit a temperature- and chain length-dependent transition from an expanded to a condensed phase. Analysis of the concentration dependence of the average molecular area at constant film pressure and the concentration dependence of the breakpoint of the phase transition from the expanded to the condensed state suggests that all four DPPC-nicotinate mixtures are partially miscible at the air-water interface. Although a complex phase behavior with several phase transitions was observed, differential scanning calorimetry studies of the four mixtures are also indicative of the partial miscibility of DPPC and the respective nicotinate. Overall, the complex phase behavior most likely results from the head-tail mismatch of the nicotinates and the geometric packing constraints in the two-component lipid bilayer.     

Methionine-containing zipper peptides

Using circular dichroism, we have examined the effect of single and multiple methionine mutations on the dimerization function of a previously reported engineered leucine zipper peptide. Our results show that the methionine-containing zipper peptides self-associate to form coiled coils that are less stable than that of the reference leucine zipper. The circular dichroism data also indicate that leucine at position d is more tolerant of methionine substitution than isoleucine at position a.     

Functional Domains within Fusion Proteins: Prospectives for Development of Peptide Inhibitors of Viral Cell Fusion

The entry of enveloped viruses into host cells is accomplished by fusion ofthe viral envelope and target plasma membrane and is mediated by fusionproteins. Recently, several functional domains within fusion proteins fromdifferent viral families were identified. Some are directly involved inconformational changes after receptor binding, as suggested by the recentrelease of crystallographically determined structures of a highly stablecore structure of the fusion proteins in the absence of membranes. However,in the presence of membranes, this core binds strongly to the membrane'ssurface and dissociates therein. Other regions, besides the N-terminal fusionpeptide, which include the core region and an internal fusion peptide inparamyxoviruses, are directly involved in the actual membrane fusion event,suggesting an umbrella like model for the membrane inducedconformational change of fusion proteins. Peptides resembling these regionshave been shown to have specific antiviral activity, presumably because theyinterfere with the corresponding domains within the viruses. Overall, thesestudies shed light into the molecular mechanism of membrane fusion induced byenvelope glycoproteins and suggest that fusion proteins from different viralfamilies share common structural and functional motifs.     

Anthrylvinyl-labeled phospholipids as fluorescent membrane probes. The action of melittin on mulitlipid systems

The interaction of melittin with multicomponent lipid mixtures composed of phosphatidylcholine, sphingomyelin and phosphatidylserine or phosphatidylglycerol was investigated by measuring the intrinsic fluorescence of the peptide, steady state fluorescence anisotropy of, and Trp-fluorescence energy transfer to fluorescent analogs of the same phospholipids bearing the anthrylvinyl fluorophore in one of the aliphatic chains at various distances from the polar head group. Based on the finding that at high lipid/peptide ratio the peptide induces unequal changes in the fluorescence parameters of phospholipid probes differing structurally only in their polar head groups, it is concluded that melittin induces lipid demixing in its nearest environment. Comparison of the fluorescence energy transfer from Trp to different lipid probes indicates that the depth of penetration of melittin into the bilayer depends on the polar head group composition of the phospholipid matrix and that certain segments of the melittin chain display a specific affinity for a given lipid head group.     

A molecular model for membrane fusion based on solution studies of an amphiphilic peptide from HIV gp41.

The mechanism of protein-mediated membrane fusion and lysis has been investigated by solution-state studies of the effects of peptides on liposomes. A peptide (SI) corresponding to a highly amphiphilic C-terminal segment from the envelope protein (gp41) of the human immunodeficiency virus (HIV) was synthesized and tested for its ability to cause lipid membranes to fuse together (fusion) or to break open (lysis). These effects were compared to those produced by the lytic and fusogenic peptide from bee venom, melittin. Other properties studied included the changes in visible absorbance and mean particle size, and the secondary structure of peptides as judged by CD spectroscopy. Taken together, the observations suggest that protein-mediated membrane fusion is dependent not only on hydrophobic and electrostatic forces but also on the spatial arrangement of the amino acid residues to form an amphiphilic structure that promotes the mixing of the lipids between membranes. A speculative molecular model is proposed for membrane fusion by alpha-helical peptides, and its relationship to the forces involved in protein-membrane interactions is discussed.     

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.     

Poly-l-lysines and poly-l-arginines induce leakage of negatively charged phospholipid vesicles and translocate through the lipid bilayer upon electrostatic binding to the membrane

Poly-l-lysines (PLL) and poly-l-arginines (PLA) of different polymer chain lengths interact strongly with negatively charged phospholipid vesicles mainly due to their different electrical charges. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) and their mixtures (1/1 mol/mol) with the respective phosphatidylcholines of equivalent chain length were chosen as model membrane systems that form at room temperature either the fluid L_α or the gel phase L_β lipid bilayer membranes, respectively. Leakage experiments revealed that the fluid POPG membranes are more perturbed compared to the gel phase DPPG membranes upon peptide binding. Furthermore, it was found that pure PG membranes are more prone to release the vesicle contents as a result of pore formation than the lipid mixtures POPG/POPC and DPPG/DPPC. For the longer polymers (≥ 44 amino acids) maximal dye-release was observed when the molar ratio of the concentrations of amino acid residues to charged lipid molecules reached a value of R_P = 0.5, i.e. when the outer membrane layer was theoretically entirely covered by the polymer. At ratios lower or higher than 0.5 leakage dropped significantly. Furthermore, PLL and PLA insertions and/or translocations through lipid membranes were analyzed by using FITC-labeled polymers by monitoring their fluorescence intensity upon membrane binding. Short PLL molecules and PLA molecules of all lengths seemed to translocate through both fluid and gel phase lipid bilayers. Comparison of the PLL and PLA fluorescence assay results showed that PLA interacts stronger with phospholipid membranes compared to PLL. Isothermal titration calorimetry (ITC) measurements were performed to give further insight into these mechanisms and to support the findings obtained by fluorescence assays. Cryo-transmission electron microscopy (cryo-TEM) was used to visualize changes in the vesicles' morphology after addition of the polypeptides.     

Penetration into membrane of amino‐terminal region of SecA when associated with SecYEG in active complexes

The general secretory (Sec) system of Escherichia coli translocates both periplasmic and outer membrane proteins through the cytoplasmic membrane. The pathway through the membrane is provided by a highly conserved translocon, which in E. coli comprises two heterotrimeric integral membrane complexes, SecY, SecE, and SecG (SecYEG), and SecD, SecF, and YajC (SecDF/YajC). SecA is an associated ATPase that is essential to the function of the Sec system. SecA plays two roles, it targets precursors to the translocon with the help of SecB and it provides energy via hydrolysis of ATP. SecA exists both free in the cytoplasm and integrally membrane associated. Here we describe details of association of the amino‐terminal region of SecA with membrane. We use site‐directed spin labelling and electron paramagnetic resonance spectroscopy to show that when SecA is co‐assembled into lipids with SecYEG to yield highly active translocons, the N‐terminal region of SecA penetrates the membrane and lies at the interface between the polar and the hydrophobic regions, parallel to the plane of the membrane at a depth of approximately 5 Å. When SecA is bound to SecYEG, preassembled into proteoliposomes, or nonspecifically bound to lipids in the absence of SecYEG, the N‐terminal region penetrates more deeply (8 Å). Implications of partitioning of the SecA N‐terminal region into lipids on the complex between SecB carrying a precursor and SecA are discussed.     

Structural studies and model membrane interactions of two peptides derived from bovine lactoferricin.

The powerful antimicrobial properties of bovine lactoferricin (LfcinB) make it attractive for the development of new antimicrobial agents. An 11-residue linear peptide portion of LfcinB has been reported to have similar antimicrobial activity to lactoferricin itself, but with lower hemolytic activity. The membrane-binding and membrane-perturbing properties of this peptide were studied together with an amidated synthetic version with an added disulfide bond, which was designed to confer increased stability and possibly activity. The antimicrobial and cytotoxic properties of the peptides were measured against Staphylococcus aureus and Escherichia coli and by hemolysis assays. The peptides were also tested in an anti-cancer assay against neuroblastoma cell lines. Vesicle disruption caused by these LfcinB derivatives was studied using the fluorescent reporter molecule calcein. The extent of burial of the two Trp residues in membrane mimetic environments were quantitated by fluorescence. Finally, the solution NMR structures of the peptides bound to SDS micelles were determined to provide insight into their membrane bound state. The cyclic peptide was found to have greater antimicrobial potency than its linear counterpart. Consistent with this property, the two Trp residues of the modified peptide were suggested to be embedded deeper into the membrane. Although both peptides adopt an amphipathic structure without any regular alpha-helical or beta-sheet conformation, the 3D-structures revealed a clearer partitioning of the cationic and hydrophobic faces for the cyclic peptide.