The antimicrobial peptide trichogin and its interaction with phospholipid membranes.

The interaction of the antimicrobial peptide trichogin GA IV with phospholipid bilayers has been studied. A series of analogs of trichogin was synthesized in which the nitroxide spin label, 4-amino-4-carboxy-2,2,6,6-tetramethylpiperidino-1-oxyl (TOAC), replaced one of the three alpha-aminoisobutyric acid (Aib) residues in the sequence. These modified peptides were used to assess the location of different residues of the peptide in a phospholipid bilayer composed of egg phosphatidylcholine containing 0.4 mol% of a fluorescently labelled phospholipid. We demonstrate that the substitution of Aib residues with TOAC does not alter the manner in which the peptide affects membrane curvature or induces vesicle leakage. The proximity of the nitroxide group on the peptide to the 4,4-difluoro-4-bora-3a,4a-diaza-S-indacene (BODIPY) fluorophore attached to the phospholipid was estimated from the extent of quenching of the fluorescence. By this criterion it was concluded that the peptide penetrates into the bilayer and that Aib4 is the most deeply inserted of the Aib residues. The results suggest that the helix axis of the peptide is oriented along the plane of the membrane. All of the peptides were shown to raise the bilayer to the hexagonal phase transition temperature of dipalmitoleoylphosphatidylethanolamine, indicating that they promote positive membrane curvature. This is a property observed with peptides that do not penetrate deeply into the bilayer or are oriented along the bilayer normal. We also demonstrate trichogin-promoted leakage of the aqueous contents of liposomes. These results indicate that the peptides cause bilayer destabilization. The extent of leakage induced by trichogin is very sensitive to the peptide to lipid ratio over a narrow range.     

Aggregation and water-membrane partition as major determinants of the activity of the antibiotic peptide trichogin GA IV

Water-membrane partition and aggregation behavior are fundamental aspects of the biological activity of antibiotic peptides, natural compounds causing the death of pathogenic organisms by perturbing the permeability of their membranes. A synthetic fluorescent analog of the natural lipopeptaibol trichogin GA IV was used to study its interaction with model membranes. Time-resolved fluorescence data show that in water, an equilibrium between monomers and small aggregates is present, the two species having different affinity for membranes. Therefore, association curves are strongly dependent on peptide concentration. A similar heterogeneity is present in the membrane phase, which strongly suggests the occurrence of a monomer-aggregate equilibrium in this case, too. The relative population of each species was determined and a strong correlation between the concentration of membrane-bound aggregates and membrane leakage was found, thereby suggesting that liposome perturbation is due to peptide aggregates only. Light-scattering measurements demonstrate that leakage is not due to liposome micellization. Moreover, experiments with markers of different sizes show that molecules with a diameter of approximately 4 nm are released only to a minor extent. Overall, these results suggest that, within the concentration range explored, pore formation by peptide aggregates is the most likely mechanism of action for trichogin in membranes.     

Location and aggregation of the spin-labeled peptide trichogin GA IV in a phospholipid membrane as revealed by pulsed EPR

The lipopeptaibol trichogin GA IV is a 10 amino acid-long residue and alpha-aminoisobutyric acid-rich antibiotic peptide of fungal origin. TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) spin-labeled analogs of this membrane active peptide were investigated in hydrated bilayers of dipalmitoylphosphatidylcholine by electron spin echo envelope modulation (ESEEM) spectroscopy and pulsed electron-electron double resonance (PELDOR). Since, the ESEEM of the spin label appears to be strongly dependent on the presence of water molecules penetrated into the membrane, this phenomenon was used to study the location of this peptide in the membrane. This was achieved by comparing the ESEEM spectra for peptides labeled at different positions along the amino acid sequence with spectra known for lipids with spin labels at different positions along the hydrocarbon chain. To increase the ESEEM amplitude and to distinguish the hydrogen nuclei of water from lipid protons, membranes were hydrated with deuterated water. The PELDOR spectroscopy technique was chosen to study peptide aggregation and to determine the mutual distance distribution of the spin-labeled peptides in the membrane. The location of the peptide in the membrane and its aggregation state were found to be dependent on the peptide concentration. At a low peptide/lipid molar ratio (less than 1:100) the nonaggregated peptide chain of the trichogin molecules lie parallel to the membrane surface, with TOAC at the 4th residue located near the 9th-11th carbon positions of the sn-2 lipid chain. Increasing this ratio up to 1:20 leads to a change in peptide orientation, with the N-terminus of the peptide buried deeper into membrane. Under these conditions peptide aggregates are formed with a mean aggregate number of about N = 2. The aggregates are further characterized by a broad range of intermolecular distances (1.5-4 nm) between the labels at the N-terminal residues. The major population exhibits a distance of approximately 2.5 nm, which is of the same order as the length of the helical peptide. We suggest that the constituting monomers of the dimer are antiparallel oriented.     

Analogs of the antimicrobial peptide trichogin having opposite membrane properties.

Four analogs of the antimicrobial peptide trichogin GA IV were studied. Their sequences are as follows: GT, n-octanoyl-Aib-Gly-Leu-Aib-Gly-Gly-Leu-Aib-Gly-Ile-Leu-OMe; ST, n-octanoyl-Aib-Ser-Leu-Aib-Ser-Ser-Leu-Aib-Ser-Ile-Leu-OMe; BT, n-octanoyl-Aib-Ser(tBu)-Leu-Aib-Ser(tBu)-Ser(tBu)-Leu-Aib-Ser(tBu)-Ile-Leu-OMe; and DT, n-octanoyl-Aib-Ser(tBu)-Leu-Aib-Ser(tBu)-Ser(tBu)-Leu-Aib-Ser(tBu)-Ile-Leu-Aib-Ser(tBu)-Leu-Aib-Ser(tBu)-Ser(tBu)-Leu-Aib-Ser(tBu)-Ile-Leu-OMe. The trichogin GA IV differs from GT only in the nature of the C-terminal residue, being a 1,2 aminoalcohol (leucinol) in the case of the parent peptide. Compared with GT, ST has an increased amphiphilicity. In contrast, BT has little amphiphilicity being composed only of hydrophobic amino acids. DT is an octanoylated head-to-tail dimer of BT. We show that BT and DT lower the bilayer-to-hexagonal phase transition temperature (T(H)) of dipalmitoleoylphosphatidylethanolamine, indicating that the peptides promote negative curvature. These two peptides, composed of only hydrophobic amino acids, have their bulkier groups on one face of the helix, suggesting that they may penetrate membranes at an oblique angle. In contrast, GT and ST, like trichogin itself, increase TH, promoting positive curvature. These peptides have contrasting membrane lytic activities. Whereas DT and BT did not produce leakage of aqueous contents, GT and ST, like trichogin, did cause rapid leakage. The leakage activity with liposomes also correlates with the greater potency of GT and ST, compared with the hydrophobic analogs, in their hemolytic and bacteriostatic action. ST has greater lytic ability than GT in liposomal leakage as well as hemolysis. We also measured the rate of peptide-promoted lipid mixing as an indication of membrane fusion. BT produced lipid mixing only with large unilamellar vesicles enriched with dioleoylphosphatidylethanolamine; ST did not produce lipid mixing, as its apparent reduction of energy transfer proved to be artifactual. Quasi-elastic light scattering of large unilamellar vesicles was also carried out after adding ST and BT. Peptide BT, but not ST, was able to aggregate large unilamellar vesicles. Thus, one of the properties of BT that leads to the induction of lipid mixing is that it is able to aggregate vesicles, placing the bilayers in juxtaposition. Thus, the two pairs of peptides, BT and DT vs GT and ST, exhibit contrasting behaviour with respect to a number of membrane biophysical properties. This occurs despite the fact that the chemical structures of the peptides are rather similar. Such distinct behavior is also reflected in their hemolytic and bacteriostatic actions.     

Fluorescence analysis of tryptophan-containing variants of the LamB signal sequence upon insertion into a lipid bilayer

To investigate the interaction of the LamB signal sequence with lipid bilayers, we have synthesized three tryptophan-containing analogues of the wild-type signal peptide. The tryptophan residues were used as intrinsic fluorescent probes of the N-terminal (position 5), central (position 18), and C-terminal (position 24) regions of the 25-residue peptide. The tryptophan substitutions did not significantly alter the physical properties of the wild-type signal peptide. In the presence of lipid vesicles which mimic the composition of the Escherichia coli inner membrane, the peptides adopt alpha-helical structure, and the tryptophan fluorescence emission maximum is shifted to shorter wavelength, indicating that the peptides insert into the acyl chain region of the lipid bilayer. Fluorescence quenching by soluble, aqueous-phase (I-), and membrane-resident (nitroxide-labeled lipids) quenchers was used to locate the tryptophans in each peptide within the bilayer. The C-terminus was interfacial while the central region of the signal sequence was deeply buried within the acyl chain region of the bilayer. The tryptophan at position 5 was buried but less deeply than the tryptophan at position 18. This topology is consistent with either a looped or a transmembrane orientation of signal peptide. However, either structure must accommodate the high helical content of the peptides in vesicles. These results indicate that the LamB signal sequence spontaneously inserts into the acyl chain region of lipid membranes in the absence of any of the proteins involved in protein secretion.     

Self-assembling and membrane modifying properties of a lipopeptaibol studied by CW-ESR and PELDOR spectroscopies.

Trichogin GA IV is a short lipopeptaibol antibiotic that is capable of enhancing the transport of small cations through the phospholipid double layer of the membrane. The antibiotic activity of the undecapeptide is thought to be based on either its self-assembling or membrane-modifying property. The chemical equilibrium between self-aggregated and non-aggregated molecular states was studied by CW-ESR spectroscopy using solutions of TOAC nitroxide spin-labelled trichogin analogues in an apolar solvent to mimic the membrane bound state. At room temperature the two different sets of signals observed in the spectrum were attributed to the presence of both monomers and aggregates in the sample. The ESR spectra of the monomeric and aggregated forms were separated and the dependence of the fraction of monomeric peptide molecules on concentration was obtained over the range 5 x 10(-6) to 7 x 10(-4) M. A two-step aggregation mechanism is proposed: dimerization of peptide molecules followed by aggregation of dimers to assemblies of four peptide molecules per aggregate. The equilibrium constants were estimated for both steps. In addition, the lower lifetime limit was determined for dimers and tetramers. It is shown that when the peptide concentration exceeds 10(-5) M. the major part of the peptide molecules in solution has the form of tetrameric aggregates. Independently, the PELDOR technique was used to investigate the concentration dependence of the parameters of the dipole-dipole interaction between spin labels in frozen (77 K] glassy solutions of aggregates of mono-labelled TOAC analogues. The number of molecules in aggregates as well as the frequency and amplitude of PELDOR signal oscillations were found to be concentration independent in the range 5 x 10(-4) to 8 x 10(-3) M. In the frozen glassy solution state, the number of peptide molecules per aggregate was determined to be close to four, which is in agreement with the value obtained for spin-labelled trichogin at room temperature. The present data provide experimental evidence in favour of a self-assembling rather than a membrane-modifying ion conduction mechanism.     

Peptide-membrane binding is not enough to explain bioactivity: A case study

Membrane-active peptides are a promising class of antimicrobial and anticancer therapeutics. For this reason, their molecular mechanisms of action are currently actively investigated. By exploiting Electron Paramagnetic Resonance, we study the membrane interaction of two spin-labeled analogs of the antimicrobial and cytotoxic peptide trichogin GA IV (Tri), with opposite bioactivity: Tri(Api⁸), able to selectively kill cancer cells, and Tri(Leu⁴), which is completely nontoxic. In our attempt to determine the molecular basis of their different biological activity, we investigate peptide impact on the lateral organization of lipid membranes, peptide localization and oligomerization, in the zwitter-ionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) model membrane We show that, despite their divergent bioactivity, both peptide analogs (i) are membrane-bound, (ii) display a weak tendency to oligomerization, and (iii) do not induce significant lipid rearrangement. Conversely, literature data show that the parent peptide trichogin, which is cytotoxic without any selectivity, is strongly prone to dimerization and affects the reorganization of POPC membranes. Its dimers are involved in the rotation around the peptide helix, as observed at cryogenic temperatures in the millisecond timescale. Since this latter behavior is not observed for the inactive Tri(Leu⁴), we propose that for short-length peptides as trichogin oligomerization and molecular motions are crucial for bioactivity, and membrane binding alone is not enough to predict or explain it. We envisage that small changes in the peptide sequence that affect only their ability to oligomerize, or their molecular motions inside the membrane, can tune the peptide activity on membranes of different compositions.     

Fluctuations and the Rate-Limiting Step of Peptide-Induced Membrane Leakage

Peptide-induced vesicle leakage is a common experimental test for the membrane-perturbing activity of antimicrobial peptides. The leakage kinetics is usually very slow, requiring minutes to hours for complete release of vesicle contents, and exhibits a biphasic behavior. We report here that, in the case of the peptaibol trichogin GA IV, all processes involved in peptide-membrane interaction, such as peptide-membrane association, peptide aggregation, and peptide translocation, take place on a timescale much shorter than the leakage kinetics. On the basis of these findings, we propose a stochastic model in which the leakage kinetics is determined by the discrete nature of a vesicle suspension: peptides are continuously exchanging among vesicles, producing significant fluctuations over time in the number of peptide molecules bound to each vesicle, and in the formation of pores. According to this model, the fast initial leakage is caused by vesicles that contain at least one pore after the peptides are randomly distributed among the liposomes, whereas the slower release is associated with the time needed to occasionally reach in an intact vesicle the critical number of bound peptides necessary for pore formation. Fluctuations due to peptide exchange among vesicles therefore represent the rate-limiting step of such a slow mechanism.     

Conformation and membrane activity of an analogue of the peptaibol antibiotic trichogin GA IV with a lipophilic amino acid at the N-terminus

We have synthesized by solution-phase methods two analogues of the 11-residue lipopeptaibol antibiotic trichogin GA IV in which the N-terminal n-octanoyl group is replaced either by an N-acetylated 2-amino-2-methyl-l -undecanoic acid or by an N-acetylated α-aminoisobutyric acid. CD, FTIR absorption, and NMR analyses unequivocally show that the main structural features of trichogin GA IV are preserved in these analogues. Since only the peptide containing the lipophilic chain exhibits membrane-modifying properties, these results strongly support the view that moving the long acyl moiety from the N^α-blocking group to the side chain of the N-terminal extra-residue does not affect the conformational properties or the membrane activity of trichogin GA IV. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Ion transport across a phospholipid membrane mediated by the peptide trichogin GA IV

Trichogin GA IV is a special member of a class of peptaibols that are linear peptide antibiotics of fungal origin, characterised by the presence of a variable number of alpha-aminoisobutyric acid residues, an acyl group at the N-terminus and a 1,2-amino alcohol at the C-terminus. Most of the peptaibols display ion-channel-forming or at least membrane-modifying properties. The 11-residue-long trichogin GA IV is not only one of shortest peptaibols, but it is also unique for its n-octanoyl group instead of the more common found acetyl group at the N-terminus. For the first time we have found that this lipopeptaibol is able to enhance conduction of monovalent cations through membranes of large unilamellar vesicles (LUVs). The influence of the [Leu-OMe]trichogin GA IV analogue (TRI) on ion permeation was studied under a variety of conditions (lipid composition, lipid-to-peptide ratio and a transmembrane potential). Parallel experiments were performed with the 16-residue long, channel-forming peptaibol, zervamicin (ZER). For the two peptides, the permeability between K(+) and Na(+) was found to be different. In addition, the ion diffusion rate dependencies on the peptide concentration are observed to be different. This might indicate that a different number of aggregated molecules are involved in the rate-limiting step, i.e. 3-4 (TRI) and 4-7 (ZER). In the presence of TRI, dissipation of the transmembrane potential, Delta psi, was observed with a rate to be dependent on the magnitude of both initial Delta psi and peptide concentration. Both peptides were activated by a cis-positive but not by cis-negative Delta psi. Under identical conditions the ion-conducting efficiency of zervamicin was 100-200 times higher than that of trichogin. Our results show that, unlike for zervamicin, the membrane-modifying activity of trichogin is not associated with a channel mechanism.     

Peptide antibiotic trichogin in model membranes: Self-association and capture of fatty acids

The antimicrobial action of peptides in bacterial membranes is commonly related to their mode of self-assembling which results in pore formation. To optimize peptide antibiotic use for therapeutic purposes, a study on the concentration dependence of self-assembling process is thus desirable. In this work, we investigate this dependence for peptaibol trichogin GA IV (Tric) in the 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) model membrane in the range of peptide concentrations between 0.5 and 3.3?mol%. Pulsed double electron-electron resonance (PELDOR) applied on spin-labeled peptide analogs highlights the onset of peptide dimerization above a critical peptide concentration value, namely ~ 2?mol%. Electron spin echo (ESE) envelope modulation (ESEEM) for D₂O-hydrated bilayers shows that dimerization is accompanied by peptide re-orientation towards a trans-membrane disposition. For spin-labeled stearic acids (5-DSA) in POPC bilayers, the study of ESE decays and ESEEM in the presence of a deuterated peptide analog indicates that above the critical peptide concentration the 5-DSA molecules are attracted by peptide molecules, forming nanoclusters. As the 5-DSA molecules represent a model for the behavior of fatty acids participating in bacterial membrane homeostasis, such capturing action by Tric may represent an additional mechanism of its antibiotic activity.     

Determining the occurrence of a 3(10)-helix and an alpha-helix in two different segments of a lipopeptaibol antibiotic using TOAC, a nitroxide spin-labeled C(alpha)-tetrasubstituted alpha-aminoacid

Trichogin GA IV is a 11-residue lipopeptaibol antibiotic exhibiting membrane modifying properties. We synthesized step-by-step by solution methods three trichogin analogues, each with a double Aib (alpha-aminoisobutyric acid)-->TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) replacement. The strict similarity in the conformational propensities of Aib and TOAC allowed us to exploit these analogues in a detailed investigation of the conformation of this lipopeptaibol in different organic solvents and in a membrane-mimetic environment using in particular the double spin labeling ESR technique. We conclude that the secondary structure in solution remains essentially unchanged if compared to that previously found in the crystal state for trichogin. More specifically, the N-terminal region of the peptide folds in a 3(10)-helix, while the central and C-terminal regions are mainly alpha-helical. An additional, significant proof for the modest plasticity of the trichogin structure was obtained by an X-ray diffraction analysis of the nOct-[TOAC4,8, Leu-OMe11] analogue. For the three analogues permeability measurements revealed membrane-modifying properties comparable to those of natural trichogin.     

Orientation and immersion depth of a helical lipopeptaibol in membranes using TOAC as an ESR probe

Trichogin GA IV is a lipopeptaibol antibiotic characterized by the sequence nOct–Aib¹–Gly–Leu–Aib⁴–Gly–Gly–Leu–Aib⁸–Gly–Ile–Lol (nOct: n‐octanoyl; Aib: α‐aminoisobutyric acid; Lol, leucinol), which exhibits membrane‐modifying properties. We synthesized step‐by‐step by solution methods three trichogin analogues, each with a single Aib → 2,2,6,6‐tetramethylpiperidin‐1‐oxyl‐4‐amino‐4‐carboxylic acid (TOAC) substitution. The similarity in the conformational propensities of the C^α‐tetrasubstituted α‐amino acids Aib and TOAC allowed us to exploit these analogues to investigate the orientation and therefore the mechanism of action of trichogin in the membranes by the electron spin resonance (ESR) technique. A conformational analysis by Fourier transform ir absorption and CD in different organic solvents and in a membrane‐mimetic environment indicated that the conformation of the natural lipopeptaibol remains almost unchanged in the three analogues. Moreover, for all of the analogues permeability measurements revealed membrane‐modifying properties comparable to those of trichogin. Our ESR investigation demonstrated that, in liposomes based on phosphatidylcholine, trichogin lays parallel to the membrane surface with its hydrophobic face oriented toward the membrane interior. These results suggest that trichogin might modify membrane permeability via a carpet‐like mechanism, at least in liposomes and in the absence of a transmembrane potential. © 1999 John Wiley & Sons, Inc. Biopoly 50: 239–253, 1999     

Total synthesis of the natural,medium-length,peptaibol pentadecaibin and study of the chemical features responsible for its membrane activity

Peptaibols are naturally occurring, antimicrobial peptides endowed with well-defined helical conformations and resistance to proteolysis. Both features stem from the presence in their sequence of several, C^α-tetrasubstituted, α-aminoisobutyric acid (Aib) residues. Peptaibols interact with biological membranes, usually causing their leakage. All of the peptaibol–membrane interaction mechanisms proposed so far begin with peptide aggregation or accumulation. The long-length alamethicin, the most studied peptaibol, acts by forming pores in the membranes. Conversely, the carpet mechanism has been claimed for short-length peptaibols, such as trichogin. The mechanism of medium-length peptaibols is far less studied, and this is partly due to the difficulties of their synthesis. They are believed to perturb membrane permeability in different ways, depending on the membrane properties. The present work focuses on pentadecaibin, a recently discovered, medium-length peptaibol. In contrast to the majority of its family members, its sequence does not comprise hydroxyprolines or prolines, and its helix is not kinked. A reliable and effective synthesis procedure is described that allowed us to produce also two shorter analogs. By a combination of techniques, we were able to establish a 3D-structure–activity relationship. In particular, the membrane activity of pentadecaibin heavily depends on the presence of three consecutive Aib residues that are responsible for the clear, albeit modest, amphiphilic character of its helix. The shortest analog, devoid of two of these three Aib residues, preserves a well-defined helical conformation, but not its amphipathicity, and loses almost completely the ability to cause membrane leakage. We conclude that pentadecaibin amphiphilicity is probably needed for the peptide ability to perturb model membranes.     

Antimicrobial lipopeptaibol trichogin GA IV: role of the three Aib residues on conformation and bioactivity

The lipopeptaibol trichogin GA IV is a natural, non-ribosomally synthesized, antimicrobial peptide remarkably resistant to the action of hydrolytic enzymes. This feature may be connected to the multiple presence in its sequence of the non-coded residue α-aminoisobutyric acid (Aib), which is known to be responsible for the adoption of particularly stable helical structures already at the level of short peptides. To investigate the role of Aib residues on the 3D-structure and bioactivity of trichogin GA IV, we synthesized and fully characterized four analogs where one or two Aib residues are replaced by L-Leu. Our extensive conformational studies (including an X-ray diffraction analysis) and biological assays performed on these analogs showed that the Aib to L-Leu replacements do not affect the resistance to proteolysis, but modulate the bioactivity of trichogin GA IV in a 3D-structure related manner.     

Some regularities of dynamic accessibility of buried fluorescent residues to external quenchers in proteins

The temperature and viscosity dependences of quenching of buried tryptophan residues of several proteins by external ionic (iodide) and neutral (acrylamide) quenchers have been studied. The effective quenching rate constant is shown to be proportional to the diffusion coefficient of the free solvent (water or 20 vol% glycerol). This fact supports the idea that the accessibility of buried chromophores of proteins to quenchers is based on the dynamic perturbations in protein structure (the dynamic accessibility). These structural perturbations are assumed to be governed by some diffusion-limited processes in the solvent surrounding the protein molecule.     

Trichogin: a paradigm for lipopeptaibols.

Lipopeptaibols are members of a novel family of naturally occurring, short peptides with antimicrobial activity, characterized by a lipophilic acyl chain at the N-terminus, a high content of turn/helix inducing alpha-aminoisobutyric acid and a 1,2-amino alcohol at the C-terminus. Using solution methods, the prototypical lipopeptaibol trichogin GA IV and a large series of appropriately designed analogues were synthesized, which allow: (i) determination of the minimal lipid chain and peptide main-chain lengths for the onset of membrane activity, and (ii) exploitation of a number of physico-chemical techniques aimed at assessing the trichogin preferred conformation under a variety of conditions and at investigating its mechanism of interaction with the phospholipid membranes.     

Role of peptide structure in lipid-peptide interactions: high-sensitivity differential scanning calorimetry and electron spin resonance studies of the structural properties of dimyristoylphosphatidylcholine membranes interacting with pentagastrin-related pentapeptides

The effects of amino acid substitutions in the pentapeptide pentagastrin on the nature of its interactions with dimyristoylphosphatidylcholine (DMPC) are assessed by differential scanning calorimetry and electron spin resonance. In two peptide analogues, the Asp at position 4 in pentagastrin (N-t-Boc-beta-Ala-Trp-Met-Asp-Phe-NH2) is replaced by Gly or Phe. These uncharged, more hydrophobic peptides have little effect on the transition temperature of DMPC, but they broaden the transition and lower the transition enthalpy as do integral membrane proteins. These peptides also mimic the behavior of integral membrane proteins in decreasing the order of a 5-doxylstearic acid spin probe below the transition temperature and in exhibiting a second immobilized lipid component using a 16-doxylstearic acid spin probe in DMPC. Three charged peptides were studied: pentagastrin, an analogue with positions 4 and 5 reversed (i.e., ending in Phe-Asp-NH2), and one with Asp replaced by Arg at position 4. All three of these charged peptides altered the phase transition behavior of DMPC to give two components, one above and one below the transition temperature of the pure lipid. With increasing peptide concentration, the higher melting transition became more prominent. The arginine-containing peptide produced the largest shifts in melting temperature followed by pentagastrin and then the "reversed" peptide. The arginine-containing peptide also increased the enthalpy of the transition. These peptides also increased the ordering of DMPC below the phase transition as measured with both 5- and 16-doxylstearic acid. The ordering effect was most pronounced with the arginine-containing peptide using the 5-doxylstearic acid probe. The results demonstrate that even the zwitterionic DMPC can interact more strongly with positively charged peptides than with negatively charged ones. In addition, peptide sequence as well as composition is important in determining the nature of peptide-lipid interactions. The markedly different effects of these pentagastrin peptides on the phase transition and motional properties of DMPC occur despite the similar depth of burial of these peptides with DMPC.     

The role of charge and hydrophobicity in peptide-lipid interaction: a comparative study based on tryptophan fluorescence measurements combined with the use of aqueous and hydrophobic quenchers

The interaction of interrelated model peptides with model membranes has been studied by techniques based on tryptophan fluorescence. The peptides used are derivatives of the sequence H-Ala-Met-Leu-Trp-Ala-OH, which was designed for this purpose. Several modifications yielded a set of 13 penta- and hexapeptides varying in net charge, hydrophobicity, charge distribution, and the intramolecular position of the tryptophan residue with respect to the charge(s). The affinity of these peptides for small unilamellar vesicles (SUV) consisting of zwitterionic egg phosphatidylcholine (eggPC) and negatively charged beef heart cardiolipin (bhCL) has been investigated in a comparative way. The criteria for affinity comprise (1) intrinsic fluorescence changes upon titration of the peptides with the lipid vesicles, (2) reduced accessibility of the peptides to aqueous quenchers of tryptophan fluorescence (I- and acrylamide) in the presence of lipid, and (3) exposure to membrane-incorporated fluorescence quenchers, brominated phosphatidylcholines (BrPC). Application of BrPC brominated at different positions along the acyl chains provided information on the membrane topology of the peptides. With respect to the extent of affinity for zwitterionic membranes, the overall hydrophobicity of the peptides is the main determinant. A comparison of the affinity for PC of equally hydrophobic peptides carrying either a single positive or negative charge reveals preferential interaction of the cationic peptide. Both hydrophobic and electrostatic interactions determine the affinity of positively charged mono- and divalent peptides for CL vesicles. The distribution of the charged moieties in divalent positively charged peptides, either both at one end of the molecule or one at each end, has little influence on the affinity of these peptides for CL but does affect the extent of exposure to BrPC. Upon decreasing the surface charge density of the vesicles by diluting CL with increasing amounts of PC, both types of peptides show different behavior. The position of the tryptophan relative to the charged moiety in the peptide molecule is shown to affect the fluorescent properties upon interaction with vesicles. Concerning the membrane topology, all peptides adopt a localization near the membrane surface, with the neutral peptides inserting slightly deeper into the bilayer than the charged peptides. The results allow a comparative analysis of the factors determining the extents and modes of lipid-model peptide interaction; in addition, the validity of the methods applied is discussed.     

Partial [alphaMe]Aun scan of [l-Leu11-OMe]-trichogin GA IV, a membrane active synthetic precursor of the natural lipopeptaibol.

We synthesized using solution-phase methods three analogs of [l-Leu11-OMe] trichogin GA IV, a membrane active synthetic precursor of the lipopeptaibol antibiotic in which the N-terminal n-octanoyl group and each of the three Aib residues in positions 1, 4 and 8 are replaced by an acetyl group and the lipophilic Calpha,alpha-disubstituted glycine l-(alphaMe)Aun, respectively [partial (alphaMe)Aun scan]. FT-IR absorption and CD analyses unequivocally show that the main three-dimensional structural features of [l-Leu11-OMe] trichogin GA IV are preserved in the analogs. Also, [l-Leu11-OMe] trichogin GA IV and the three Nalpha-acetylated l-(alphaMe)Aun analogs exhibit strictly comparable membrane-modifying properties. Taken together, these results strongly favor the conclusion that a shift of the long hydrocarbon moiety from the Nalpha-blocking group to the side-chain of the 1, 4 or 8 residue does not have any significant effect on the conformational properties or the membrane activity of [l-Leu11-OMe] trichogin GA IV and, by extension, of the natural lipopeptaibol.