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Various models have been proposed for the sequence of events occurring after binding of specific antimicrobial peptides to lipid membranes. The lipid clustering model arose by the finding that antimicrobial peptides can induce a segregation of certain negatively charged lipids in lipid model membranes. Anionic lipid segregation by cationic peptides is initially an effect of charge interaction where the ratio of peptide and lipid charges is thought to be the decisive parameter in the peptide induced lipid demixing. However, the sequence of events following this initial lipid clustering is more complex and can lead to deactivation of membrane proteins involved in cell division or perturbation of lipid reorganization essential for cell division. In this study we used DSC and ITC techniques to investigate the effect of binding different cyclic hexapeptides with varying antimicrobial efficacy, to phosphatidylglycerol (PG)/phosphatidylethanolamine (PE) lipid membranes and their ability to induce lipid segregation in these mixtures. We found that these cyclic hexapeptides consisting of three charged and three aromatic amino acids showed indeed different abilities to induce lipid demixing depending on their amino acid composition and their sequence. The results clearly showed that the cationic amino acids are essential for electrostatic binding but that the three hydrophobic amino acids in the peptides and their position in the sequence also contribute to binding affinity and to the extent of induction of lipid clustering. The efficacy of these different hexapeptides to induce PG clusters in PG/PE membranes was found to be correlated with their antimicrobial activity., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Antimicrobial peptides attract a lot of interest as potential candidates to overcome bacterial resistance. So far, nearly all the proposed scenarios for their mechanism of action are associated with perforating and breaking down bacterial membranes after a binding process. In this study we obtained additional information on peptide induced demixing of bacterial membranes as a possible mechanism of specificity of antimicrobial peptides. We used DSC and FT-IR to study the influence of a linear and cyclic arginine- and tryptophan-rich antimicrobial peptide having the same sequence (RRWWRF) on the thermotropic phase transitions of lipid membranes. The cyclization of the peptide was found to enhance its antimicrobial activity and selectivity ( Dathe, M. Nikolenko, H. Klose, J. Bienert, M. Biochemistry 43 (2004) 9140-9150). A particular preference of the binding of the peptides to DPPG headgroups compared to other headgroups of negatively charged phospholipids, namely DMPA, DPPS and cardiolipin was observed. The main transition temperature of DPPG bilayers was considerably decreased by the bound peptides. The peptides caused a substantial down-shift of the transition of DPPG/DMPC. In contrast, they induced a demixing in DPPG/DPPE bilayers and led to the appearance of two peaks in the DSC curves indicating a DPPG-peptide-enriched domain and a DPPE-enriched domain. These results could be confirmed by FT-IR-spectroscopic measurements. We therefore propose that the observed peptide-induced lipid demixing in PG/PE-membranes could be a further specific effect of the antimicrobial peptides operating only on bacterial membranes, which contain appreciable amounts of PE and PG, and which could in principle also occur in liquid-crystalline membranes.

Supramolecular structures, particularly micelles and liposomes equipped with uptake-mediating address compounds, have attracted much attention as pharmaceutical formulations. Their development requires an understanding of the mechanism by which the carrier systems interact with and translocate into the target cells. We developed an apolipoprotein E-derived peptide, called A2, that efficiently translocates across cell membranes. Upon coupling of two palmitoyl chains (P2), the highly cationic sequence acquires detergent-like properties such as a strong tendency to self-associate and the ability to integrate into lipid bilayers. Confocal laser scanning microscopy and fluorescence activated cell sorting were used to compare the internalization of the fluorescence-labeled monomeric A2 with the uptake of the colloidal P2A2 micelles and P2A2-tagged liposomes into endothelial cells of blood vessels. Specific inhibitors of endocytosis were used to identify the underlying mechanisms. b.End3 and BAEC cells as example of endothelial cells of small capillaries and large vascular vessels, respectively, were examined. The uptake of monomeric A2 was characterized by poor cellular selectivity. A2 was efficiently internalized into both cell lines via at least two different mechanisms. Besides an endocytotic uptake route, a second passive pathway exists, that leads to a rapid distribution of A2 within the cytoplasm. Also liposomes tagged with P2A2 were non-selectively internalized into both b.End3 and BAEC cells. Their nonselective uptake was mediated by clathrin- and caveolin-independent endocytosis. In contrast, micellar P2A2 entered b.End3 cells via clathrin-mediated endocytosis, while no uptake of P2A2 into BAEC cells was observed. In conclusion, the specific clathrin-mediated uptake mode of P2A2 micelles might provide the basis for a blood brain barrier-specific targeting.

Liposomes and micellar carriers equipped with targeting and cellular uptake mediating peptides have attracted attention for numerous applications. The optimization of the carrier requires an understanding of how their properties influence target cell recognition and uptake. We developed a dipalmitoylated apolipoprotein E-derived peptide, named P2A2 as promising vector to mediate cellular uptake of potential micellar and liposomal carriers. Confocal laser scanning microscopy (CLSM) and fluorescence-activated cell sorting (FACS) were used to get insight into the internalization mediated by carboxyfluoresceine-labeled P2fA2 and the all-D amino acid analogue P2fa2 into brain capillary endothelial cells. Both peptide micelles and liposomes entered cells via endocytosis. Cell surface heparan sulfate proteoglycans (HSPGs) were involved in the internalization process of peptide-bearing liposomes characterized by a diameter of 100 nm, a low surface density of 100 peptide molecules per vesicle and a helical conformation of the vector. In contrast, peptide micelles characterized by a diameter of about 10 nm, a high peptide density caused by 19 associated molecules and a high conformational flexibility of the vector sequence did not address HSPG. Unspecific interactions between the carriers and membrane constituents predominate the two uptake processes but stereospecific components seem to be involved. Both routes differ with respect to transport efficiency. The results provide a prospective basis to optimize liposomes and micelles as drug delivery systems.

Liposomes equipped with cellular uptake-mediating peptidic vector compounds have attracted much attention as target-specific drug delivery systems. Aside from the development of the target recognition motif itself, vector coupling to liposomes while conserving the active conformation constitutes an important element in carrier development. To elucidate the most efficient way for adsorptive peptide binding to liposomes, we synthesized and characterized two-domain peptides comprising a cationic sequence derived from the binding domain of apolipoprotein E (apoE) for the low-density lipoprotein receptor and different lipid-binding motifs, that is, an amphipathic helix, a transmembrane helix, single fatty acids or two palmitoyl chains. Peptide properties considered relevant for peptide-liposome complexes to initiate an endocytotic cellular uptake such as lipid binding, helicity, stability of anchorage, bilayer-disturbing activity, and toxicity showed that the dipalmitoyl derivative was the most suitable to associate the apoE peptide to the surface of liposomes. The peptide showed pronounced lipid affinity and was stably anchored within the lipid bilayer on a time scale of at least 30 min. The helicity of about 40% in the lipid-bound state and the location of the amphipathic helix on the liposomal surface provided the prerequisites for interaction of the complex with the cell surface-located receptor. The concentration of the dipalmitoylated peptide to permeabilize neutral lipid bilayers (lipid concentration 25 microM) was 0.06 microM and a 2 microM concentration reduced cell viability to about 80%. Efficient internalization of liposomes bearing about 180 peptide derivatives on the surface into brain capillary endothelial cells was monitored by confocal laser scanning microscopy. The concept of complexation using dipalmitoylated peptides may offer an efficient substitute to covalent vector coupling and a prospective way to optimize the capacity of liposomes as drug delivery systems also for different targets.

Model compounds of modified hydrophobicity (Eta), hydrophobic moment (mu) and angle subtended by charged residues (Phi) were synthesized to define the general roles of structural motifs of cationic helical peptides for membrane activity and selectivity. The peptide sets were based on a highly hydrophobic, non-selective KLA model peptide with high antimicrobial and hemolytic activity. Variation of the investigated parameters was found to be a suitable method for modifying peptide selectivity towards either neutral or highly negatively charged lipid bilayers. Eta and mu influenced selectivity preferentially via modification of activity on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) bilayers, while the size of the polar/hydrophobic angle affected the activity against 1-palmitoyl-2-oleoylphosphatidyl-DL-glycerol (POPG). The influence of the parameters on the activity determining step was modest in both lipid systems and the activity profiles were the result of the parameters' influence on the second less pronounced permeabilization step. Thus, the activity towards POPC vesicles was determined by the high permeabilizing efficiency, however, changes in the structural parameters preferentially influenced the relatively moderate affinity. In contrast, intensive peptide accumulation via electrostatic interactions was sufficient for the destabilization of highly negatively charged POPG lipid membranes, but changes in the activity profile, as revealed by the modification of Phi, seem to be preferentially caused by variation of the low permeabilizing efficiency. The parameters proved very effective also in modifying antimicrobial and hemolytic activity. However, their influence on cell selectivity was limited. A threshold value of hydrophobicity seems to exist which restricted the activity modifying potential of mu and Phi on both lipid bilayers and cell membranes.

Antibacterial, membrane-lytic peptides belong to the innate immune system and host defense mechanism of a multitude of animals and plants. The largest group of peptide antibiotics comprises peptides which fold into an amphipathic alpha-helical conformation when interacting with the target. The activity of these peptides is thought to be determined by global structural parameters rather than by the specific amino acid sequence. This review is concerned with the influence of structural parameters, such as peptide helicity, hydrophobicity, hydrophobic moment, peptide charge and the size of the hydrophobic/hydrophilic domain, on membrane activity and selectivity. The potential of these parameters to increase the antibacterial activity and to improve the prokaryotic selectivity of natural and model peptides is assessed. Furthermore, biophysical studies are summarized which elucidated the molecular basis for activity and selectivity modulations on the level of model membranes. Finally, the knowledge about the role of peptide structural parameters is applied to understand the different activity spectra of natural membrane-lytic peptides.

Alamethicin is a 20 amino acid, potentially helical peptaibol which forms voltage-dependent ion channels in bilayer systems. Two aspects of alamethicin structure have been suggested to be of particular functional significance for stabilization of alamethicin channels. (i) Proline 14 inducing a helix kink is together with glycine at position 11 responsible for an appropriate orientation of the molecules in the conducting associates. (ii) Glutamine 7 lining the channel interior is assumed to stabilize the channel structure by forming inter-helix hydrogen bonds. The functional importance of these residues was probed in macroscopic and single-channel experiments with alamethicin analogs containing polar, side chain bearing residues at position 11 (glutamine, asparagine) or at position 14 (glutamine). In order to investigate the crucial role of glutamine 7 for the stabilization of channel aggregates, this residue was substituted by alanine. The conformation of the lipid bound peptides was determined by circular dichroism spectroscopy. The results show that glutamine 7, glycine 11 and proline 14 are not essential for channel formation but substitution of any residue reduced the number of conductance levels and significantly reduced their lifetimes. Channel stabilization by the introduction of residues with potential hydrogen bonding capacity at positions 11 and 14 was not observed. Differences in the conformation of the lipid bound peptides, their orientation in the bilayer and their affinity for the lipid membrane appear thus to contribute to the modulation of functional properties.

Alamethicin is known to lyse different biological cells and to induce voltage dependent ion channels in lipid bilayers. A set of analogs with proline shifted from position 14 in the native peptide towards the N- and C-terminus was used to investigate the role of proline in: (i) alamethicin induced hemolysis of human red blood cells, (ii) stimulation of catecholamine secretion from bovine adrenal chromaffin cells and (iii) induction of metabolic activity in bovine aortic endothelial cells. Half maximal hemolytic activity was found at 30 microM alamethicin concentration, complete lysis occurred at 100 microM. The stimulation of catecholamine secretion in the presence of extracellular Ca2+ was concentration dependent up to 50 microM alamethicin. At this high concentration mild secretion was also found in the absence of Ca2+ indicating cell membrane damage. Alamethicin transiently stimulated the metabolic rate of endothelial cells in a concentration dependent mode up to 20 microM while the inhibition of metabolism at higher concentrations pointed to a toxic effect. The alamethicin analogs were completely inactive in all the biological assays. The effects correlated with a loss of dye release inducing activities on phosphatidylcholine vesicles and reduction of channel forming properties in lipid bilayers and were associated with modifications of membrane affinity rather than conformational changes of the peptides. The results indicate that proline at position 14 of the native peptide is essential for the interaction with different membrane systems., (Copyright 1998 Elsevier Science B.V.)