Development of a pathogenesis-based therapy for peeling skin syndrome type 1.

BACKGROUND: Peeling skin syndrome type 1 (PSS1) is a rare and severe autosomal recessive form of congenital ichthyosis. Patients are affected by pronounced erythroderma accompanied by pruritus and superficial generalized peeling of the skin. The disease is caused by nonsense mutations or complete deletion of the CDSN gene encoding for corneodesmosin (CDSN). PSS1 severely impairs quality of life and therapeutic approaches are totally unsatisfactory. OBJECTIVES: The objective of this study was to develop the first steps towards a specific protein replacement therapy for CDSN deficiency. Using this approach, we aimed to restore the lack of CDSN and improve cell-cell cohesion in the transition area of the stratum granulosum (SG) to the stratum corneum. METHODS: Human CDSN was recombinantly expressed in Escherichia coli. A liposome-based carrier system, prepared with a cationic lipopeptide to mediate the transport to the outer membrane of keratinocytes, was developed. This formulation was chosen for CDSN delivery into the skin. The liposomal carrier system was characterized with respect to size, stability and toxicity. Furthermore, the interaction with primary keratinocytes and human epidermal equivalents was investigated. RESULTS: The liposomes showed an accumulation at the membranes of keratinocytes. CDSN-deficient epidermal equivalents that were treated with liposomal encapsulated CDSN demonstrated presence of CDSN in the SG. Finally, the penetration assay and histological examinations revealed an improved epidermal integrity for CDSN-deficient epidermal equivalents, if they were treated with liposomal encapsulated CDSN. CONCLUSIONS: This study presents the first preclinical in vitro experiments for a future specific protein replacement therapy for patients affected by PSS1.

Liquid storage of boar semen: Current and future perspectives on the use of cationic antimicrobial peptides to replace antibiotics in semen extenders.

Antibiotics are of great importance in boar semen extenders to ensure long shelf life of spermatozoa and to reduce transmission of pathogens into the female tract. However, the use of antibiotics carries a risk of developing resistant bacterial strains in artificial insemination laboratories and their spread via artificial insemination. Development of multiresistant bacteria is a major concern if mixtures of antibiotics are used in semen extenders. Minimal contamination prevention techniques and surveillance of critical hygiene control points proved to be efficient in reducing bacterial load and preventing development of antibiotic resistance. Nevertheless, novel antimicrobial concepts are necessary for efficient bacterial control in extended boar semen with a minimum risk of evoking antibiotic resistance. Enhanced efforts have been made in recent years in the design and use of antimicrobial peptides (AMPs) as alternatives to conventional antibiotics. The male genital tract harbors a series of endogenic substances with antimicrobial activity and additional functions relevant to the fertilization process. However, exogenic AMPs often exert dose- and time-dependent toxic effects on mammalian spermatozoa. Therefore, it is important that potential newly designed AMPs have only minor impacts on eukaryotic cells. Recently, synthetic magainin derivatives and cyclic hexapeptides were tested for their application in boar semen preservation. Bacterial selectivity, proteolytic stability, thermodynamic resistance, and potential synergistic interaction with conventional antibiotics propel predominantly cyclic hexapeptides into highly promising, leading candidates for further development in semen preservation. The time scale for the development of resistant pathogens cannot be predicted at this moment.

Challenges and Limits Using Antimicrobial Peptides in Boar Semen Preservation.

Antibiotics are of great importance for the preservation of ejaculates for livestock breading. The use of antibiotics, however, is not an appropriate compensation for a lack of hygiene standards in artificial insemination (AI) centres. Sophisticated hygiene management and the proper identification of hygienic critical control points (HCCPs) at AI centres provide the basis for counteracting the development of antibiotic resistance in contaminant bacteria and their settlement in AI centres. In recent years, efforts have been made to use antimicrobial peptides (AMPs) in the preservation of boar semen. Investigations have included the testing of synthetic magainin derivatives and cyclic hexapeptides. One prerequisite for the application of AMPs is that they have a minor impact on eukaryotic cells. Bacterial selectivity, proteolytic stability, thermodynamic resistance, and mechanisms including synergistic interaction with conventional antibiotics have made cyclic hexapeptides highly promising candidates for potential application as peptide antibiotics for semen preservation.

Interaction of drug-carrier systems with targets--a study using atomic force microscopy.

To learn about the interaction between drug agents and nanoparticular carrier systems, the physical analytical methods of parelectric, electron spin and fluorescence spectroscopy have proven helpful tools to yield descriptive models of such complex systems. For a deeper understanding of drug absorption from body surfaces and drug distribution into the tissues, however, the lack of knowledge about the interaction between such agents and membranes on different levels is a severe drawback. This gap can be closed by the application of atomic force microscopy at normal temperatures and under the admission of liquid surroundings. Moreover, this method allows the inspection of such system-membrane interactions in dependence on time. We studied membrane topography in liquid and gel-phase mixtures, structural changes of membranes during their destruction by aqueous peptide solutions as well as the stability of the membranes exposed to surfactants of increasing concentration and to lipid nanoparticles (solid lipid nanoparticles, nanostructured lipid carriers). For future modelling we can describe the geometry of lipid nanoparticles as well.

Antimicrobial activity of arginine- and tryptophan-rich hexapeptides: the effects of aromatic clusters, D-amino acid substitution and cyclization.

Many antimicrobial peptides bear arginine (R)- and tryptophan (W)-rich sequence motifs. Based on the sequence Ac-RRWWRF-NH2, sets of linear and cyclic peptides were generated by changes in the amino acid sequence, L-D-amino acid exchange and naphthylalanine substituted for tryptophan. Linear RW-peptides displayed moderate activity towards Gram-positive Bacillus subtilis (15 < MIC < 31 microm) and were inactive against Gram-negative Escherichia coli at peptide concentrations < 100 microm. Cyclization induced high antimicrobial activity. The effect of cyclization was most pronounced for peptides with three adjacent aromatic residues. Incorporation of d-amino acid residues had minor influence on the biological activity. The haemolytic activity of all RW-peptides at 100 microm concentration was low (< 7% lysis for linear R/W-rich peptides and < 28% for the cyclic analogues). Introduction of naphthylalanine enhanced the biological activities of both the linear and cyclic peptides. All peptides induced permeabilization of large unilamellar vesicles (LUVs) composed of lipids of the membrane of B. subtilis and erythrocytes, but surprisingly had no effect on LUVs composed of lipids of the E. coli inner membrane. The profiles of peptide activity against B. subtilis and red blood cells correlated with the permeabilizing effects on the corresponding model membranes and were related to hydrophobicity parameters as derived from reversed phase high-performance liquid chromatography (HPLC). The results underlined the importance of amphipathicity as a driving force for cell lytic activity and suggest that conformational constraints and an appropriate position of aromatic residues allowing the formation of hydrophobic clusters are highly favourable for antimicrobial activity and selectivity.

Optimization of the antimicrobial activity of magainin peptides by modification of charge.

Investigation of magainin II amide analogs with cationic charges ranging between +3 and +7 showed that enhancement of the peptide charge up to a threshold value of +5 and conservation of appropriate hydrophobic properties optimized the antimicrobial activity and selectivity. High selectivity was the result of both enhanced antimicrobial and reduced hemolytic activity. Charge increase beyond +5 with retention of other structural motifs led to a dramatic increase of hemolytic activity and loss of antimicrobial selectivity. Selectivity could be restored by reduction of the hydrophobicity of the hydrophobic helix surface (H(hd)), a structural parameter not previously considered to modulate activity. Dye release experiments with lipid vesicles revealed that the potential of peptide charge to modulate membrane activity is limited: on highly negatively charged 1-palmitoyl-2-oleoylphosphatidyl-DL-glycerol bilayers, reinforcement of electrostatic interactions had an activity-reducing effect. On neutral 1-palmitoyl-2-oleoylphosphatidylcholine bilayers, the high activity was determined by H(hd). H(hd) values above a certain threshold led to effective permeabilization of all lipid systems and even compensated for the activity-reducing effect of charge increase on highly negatively charged membranes.

Peptide leucine arginine, a potent immunomodulatory peptide isolated and structurally characterized from the skin of the Northern Leopard frog, Rana pipiens.

On the basis of histamine release from rat peritoneal mast cells, an octadecapeptide was isolated from the skin extract of the Northern Leopard frog (Rana pipiens). This peptide was purified to homogeneity using reversed-phase high performance liquid chromatography and found to have the following primary structure by Edman degradation and pyridylethylation: LVRGCWTKSYPPKPCFVR, in which Cys(5) and Cys(15) are disulfide bridged. The peptide was named peptide leucine-arginine (pLR), reflecting the N- and C-terminal residues. Molecular modeling predicted that pLR possessed a rigid tertiary loop structure with flexible end regions. pLR was synthesized and elicited rapid, noncytolytic histamine release that had a 2-fold greater potency when compared with one of the most active histamine-liberating peptides, namely melittin. pLR was able to permeabilize negatively charged unilamellar lipid vesicles but not neutral vesicles, a finding that was consistent with its nonhemolytic action. pLR inhibited the early development of granulocyte macrophage colonies from bone marrow stem cells but did not induce apoptosis of the end stage granulocytes, i.e. mature neutrophils. pLR therefore displays biological activity with both granulopoietic progenitor cells and mast cells and thus represents a novel bioactive peptide from frog skin.

Continuous measurement of rapid transbilayer movement of a pyrene-labeled phospholipid analogue.

The excimer forming capacity of the fluorescent moiety pyrene is employed to measure continuously the transbilayer (re)distribution of a pyrene-labeled phosphatidylcholine analogue (pyPC) in liposomal membranes. pyPC with a lauroyl residue (sn-1 position) and a short (butyroyl) fatty acid chain (sn-2 position) bearing the pyrene moiety incorporates rapidly into the outer leaflet of liposomes. The fluorescence intensities of excimers (I(E)) and of monomers (I(M)) of pyPC depend on the concentration of the analogue in a membrane leaflet. Therefore, the redistribution of pyPC from the outer to the inner leaflet can be followed by changes of the ratio I(E)/I(M). The transbilayer movement of pyPC in pure phospholipid vesicles is very slow indicated by a constant I(E)/I(M). However, addition of membrane active peptides (melittin, magainin 2 amide or a mutant of magainin 2 amide) induced a rapid translocation of pyPC from the outer to the inner leaflet. An approach is presented which allows estimating the transbilayer distribution of pyPC from the measured ratio I(E)/I(M).

A role for a helical connector between two receptor binding sites of a long-chain peptide hormone.

The conformational freedom of single-chain peptide hormones, such as the 41-amino acid hormone corticotropin releasing factor (CRF), is a major obstacle to the determination of their biologically relevant conformation, and thus hampers insights into the mechanism of ligand-receptor interaction. Since N- and C-terminal truncations of CRF lead to loss of biological activity, it has been thought that almost the entire peptide is essential for receptor activation. Here we show the existence of two segregated receptor binding sites at the N and C termini of CRF, connection of which is essential for receptor binding and activation. Connection of the two binding sites by highly flexible epsilon-aminocaproic acid residues resulted in CRF analogues that remained full, although weak agonists (EC(50): 100-300 nM) independent of linker length. Connection of the two sites by an appropriate helical peptide led to a very potent analogue, which adopted, in contrast to CRF itself, a stable, monomer conformation in aqueous solution. Analogues in which the two sites were connected by helical linkers of different lengths were potent agonists; their significantly different biopotencies (EC(50): 0.6-50 nM), however, suggest the relative orientation between the two binding sites rather than the maintenance of a distinct distance between them to be essential for a high potency.

Structural features of helical antimicrobial peptides: their potential to modulate activity on model membranes and biological cells.

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.

Noncovalent immobilized artificial membrane chromatography, an improved method for describing peptide-lipid bilayer interactions.

A promising approach in assessing hydrophobic peptide-membrane interactions is the use of reversed-phase high-performance liquid chromatography. The present study describes the preparation and properties of a noncovalent immobilized artificial membrane (noncovalent IAM) stationary phase. The noncovalent IAM phase was prepared by coating the C18 chains of a reversed-phase HPLC column with the phospholipid ditetradecanoyl-sn-glycero-3-phosphocholine. Lipid coating was achieved by pumping a lipid solution in water-2-propanol through the column. The formation of a bilayer-like structure on the chromatographic surface was confirmed by calculating the phospholipid surface density of the stationary phase. The surface density was determined to be approximately 1.95 mumol m-2, which is close to that of lipid vesicles. The coating was found to be stable in chromatographic elution systems containing less than 35% of acetonitrile. Employing this new technique, we determined interaction parameters of a set of helical antibacterial magainin-2-amide peptides with pairwise substitutions of adjacent amino acids by their D-enatiomers. The results demonstrate that the chromatographic retention behavior of peptides on noncovalent IAM stationary phase shows an excellent correlation with lipid affinities to phospholipid vesicles.

Structural requirements for cellular uptake of alpha-helical amphipathic peptides.

The structure of the cell-permeable alpha-helical amphipathic model peptide FLUOS-KLALKLALKALKAALKLA-NH2 (I) was modified stepwise with respect to its helix parameters hydrophobicity, hydrophobic moment and hydrophilic face as well as molecular size and charge. Cellular uptake and membrane destabilizing activity of the resulting peptides were studied using aortic endothelial cells and HPLC combined with CLSM. With the exceptions that a reduction of molecule size below 16 amino acid residues and the introduction of a negative net charge abolished uptake, none of the investigated structural parameters proved to be essential for the passage of these peptides across the plasma membrane. Membrane toxicity also showed no correlation to any of the parameters investigated and could be detected only at concentrations higher than 2 microM. These results implicate helical amphipathicity as the only essential structural requirement for the entry of such peptides into the cell interior, in accord with earlier studies. The pivotal role of helical amphipathicity was confirmed by uptake results obtained with two further pairs of amphipathic/non-amphipathic 18-mer peptides with different primary structure, net charge and helix parameters from I. The amphipathic counterparts were internalized into the cells to a comparable extent as I, whereas no cellular uptake could be detected for the non-amphipathic analogues. The mode of uptake remains unclear and involves both temperature-sensitive and -insensitive processes, indicating non-endocytic contributions.

Secondary structure of bacteriorhodopsin fragments. External sequence constraints specify the conformation of transmembrane helices.

The secondary structure of bacteriorhodopsin polypeptides comprising two (AB, CD, DE, FG), three (AC, CE, EG), four (AD, DG), or five (AE, CG) of the seven transmembrane segments has been analyzed by circular dichroism spectroscopy. A comparison of the alpha-helix content with that predicted from the high resolution structure of the native protein revealed that the N-terminal AB, AC, AD, and AE fragments and the C-terminal CG fragment are completely refolded in the presence of mixed phospholipid micelles. In contrast, the DG, EG, FG, CD, CE, and DE fragments did not form alpha-helices of the expected lengths at pH 6. Each of the latter fragments displayed, however, an increased helicity upon lowering the pH to 4. Fluorescence measurements with the CD and FG fragments suggest that this helix formation occurs within transmembrane segments C and G, respectively, and thus is likely to originate from the protonation of carboxyl residues that participate in proton translocation. The partial misfolding at neutral pH observed for the shorter fragments from the central and C-terminal part of bacteriorhodopsin indicates that the conformation of some transmembrane segments is specified by interactions with neighboring helices in the assembled structure. Moreover, the data demonstrate that two stable helices at the N terminus of a multihelical membrane protein are sufficient as a folding template to induce a native conformation to the following transmembrane domains.

Functional modifications of alamethicin ion channels by substitution of glutamine 7, glycine 11 and proline 14.

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.

Proline at position 14 of alamethicin is essential for hemolytic activity, catecholamine secretion from chromaffin cells and enhanced metabolic activity in endothelial cells.

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.

Modulation of membrane activity of amphipathic, antibacterial peptides by slight modifications of the hydrophobic moment.

Starting from the sequences of magainin 2 analogs, peptides with slightly increased hydrophobic moment (mu) but retained other structural parameters were designed. Circular dichroism investigations revealed that all peptides adopt an alpha-helical conformation when bound to phospholipid vesicles. Analogs with increased mu were considerably more active in permeabilizing vesicles mainly composed of zwitterionic lipid. In addition, the antibacterial and hemolytic activities of these analogs were enhanced. Correlation of permeabilization and binding indicated that the activity increase is predominantly caused by an increased membrane affinity of the peptides due to strengthened hydrophobic interactions.

Influence of the angle subtended by the positively charged helix face on the membrane activity of amphipathic, antibacterial peptides.

To investigate the influence of the angle subtended by the positively charged helix face on membrane activity, six amphipathic alpha-helical peptides with angles between 80 degrees and 180 degrees, but with retained hydrophobicity, hydrophobic moment, and positive overall charge, were designed starting from the sequence of the antibacterial peptide magainin 2. CD investigations revealed that all analogs are in an alpha-helical conformation in vesicle suspension. The ability of the peptides to induce dye release from negatively charged phosphatidylglycerol (PG) vesicles decreased with increasing angle. However, peptides with a large angle of positively charged residues (140-180 degrees) exhibited a considerably higher permeabilizing activity at zwitterionic phosphatidylcholine (PC) and mixed PC/PG (3:1) vesicles than analogs with a small angle (80-120 degrees). In addition, analogs with large angles were more active in antibacterial and hemolytic assays. The antibacterial specificity of these analogs was decreased. Binding investigations showed that peptide binding is favored by a large angle and a high content of negatively charged phospholipid. In contrast, a small angle and a low negative membrane charge enhanced the membrane-permeabilizing efficiency of the bound peptide fraction. All analogs stabilized the bilayer phase of phosphatidylethanolamine over the inverted hexagonal phase. Therefore, a class L mechanism of permeabilization can be excluded. Furthermore, the analogs do not act by the induction of positive curvature strain or by a "carpet-like" mechanism. Our results are in accordance with a pore mechanism: The membrane-permeabilizing efficiency of analogs with enhanced angle of positively charged residues is reduced due to electrostatic repulsion between adjacent helices within the pore, thus resulting in a decreased pore-forming probability and/or pore destabilization.

Peptide hydrophobicity controls the activity and selectivity of magainin 2 amide in interaction with membranes.

The magainins are antibacterial peptides from the skin of Xenopus laevis. They show a broad range of activity against prokaryotic cells but lyse eukaryotic cells poorly. To elucidate the influence of peptide hydrophobicity on membrane activity and selectivity, we designed and synthesized analogs of magainin 2 amide with slightly varying hydrophobicities but retained hydrophobic moment, peptide charge, and angle subtended by the hydrophilic helix region. Circular dichroism investigations of the peptides revealed that all peptides investigated adopt an alpha-helical conformation when bound to phospholipid vesicles. Dye-releasing experiments from vesicles of phosphatidylglycerol (PG) showed that the membrane-permeabilizing activity of the analogs is not influenced by peptide hydrophobicity. In contrast, the permeability-enhancing activity on vesicles bearing high amounts of phosphatidylcholine (PC) increases drastically with enhanced peptide hydrophobicity, resulting in a reduced selectivity of more hydrophobic analogs for negatively charged membranes. Likewise, the peptide affinity to PC-rich membranes increases in the order of hydrophobicity. Correlation of peptide binding and membrane permeabilization of PC/PG (3:1) vesicles revealed that the observed differences in peptide activity on membranes of low negative surface charge are mainly caused by the different binding affinities. The antibacterial and hemolytic activity of the peptides increases with enhanced hydrophobicity. A strong correlation was found between the hemolytic effect and the bilayer-permeabilizing activity against PC-rich vesicles. Whereas the antibacterial specificity of the more hydrophobic analogs is retained for Escherichia coli, the specificity for Pseudomonas aeruginosa decreases with increasing hydrophobicity.

Influence of proline position upon the ion channel activity of alamethicin.

Alamethicin, a 20-residue peptaibol, induces voltage-dependent ion channels in lipid bilayers according to the barrel-stave model. To study relationships between the proline-14-induced kink region and the channel-forming behavior of the peptide, a set of alamethicin analogs with proline incorporated at positions 11, 12, 13, 14, 15, 16, and 17, respectively, as well as an analog with alanine instead of proline at position 14 were synthesized. Macroscopic conductance experiments show that the voltage dependence of the peptides is conserved although slightly influenced, but the apparent mean number of monomers forming the channels is significantly reduced when proline is not located at position 14. This is confirmed in single-channel experiments. The analogs with proline next to position 14 (i.e., 13, 15, 16) show stable conductance levels, but of reduced number, which follows the order Alam-P14 > Alam-P15 > Alam-P16 > Alam-P13. This reduction in the number of levels is connected with changes in the lifetime of the channels. Analogs with proline at position 11, 12, or 17 produce erratic, extremely short-lived current events that could not be resolved. The changes in functional properties are related to structural properties as probed by circular dichroism. The results indicate that proline at position 14 results in optimal channel activity, whereas channels formed by the analogs bearing proline at different positions are considerably less stable.

The tendency of magainin to associate upon binding to phospholipid bilayers.

Fluorescence energy transfer (FET) from [Trp16]-magainin-2-amide (Trp-Mag) and [D-Ala15,D-Trp16]magainin-2-amide (DD-Trp-Mag) to N(alpha)-dansyl-magainin-2-amide (DNS-Mag) was used to study the association of magainin 2 analogs bound to phosphatidylglycerol vesicles. As shown by circular dichroism and fluorescence spectroscopy, the all-L-analogs exist in a helical conformation and are completely bound to the lipid membrane. The observed FET between Trp-Mag and DNS-Mag is rather small and increases with the DNS-Mag surface concentration. The experimentally determined transfer efficiency is lower than predicted for monomeric magainin analogs randomly distributed exclusively at the outer leaflet of lipid vesicles. These observations can be explained by two different models of spatial distribution for the monomeric magainin analogs. The first model takes into account translocation of magainin which might result in a uniform distribution of magainin at the inner and outer vesicle leaflets. The second model assumes that at least one shell of lipids exists between two magainin molecules, thus reducing the probability of direct contact. Both models explain the measured FET without any contribution of stable associates of magainin analogs. Furthermore, for Trp-Mag and DD-Trp-Mag, an identical energy transfer efficiency was observed, although the nonhelical double-D substituted analog should have a significantly reduced association tendency resulting in decreased FET. Our conclusion that the observed FET is not the result of magainin association is confirmed by the equivalence of the measured energy transfer efficiencies.