Tryptophan fluorescence study on the interaction of the signal peptide of the Escherichia coli outer membrane protein PhoE with model membranes

The interaction of the signal peptide of the Escherichia coli outer membrane protein PhoE with different phospholipid vesicles was investigated by fluorescence techniques, using a synthetic mutant signal peptide in which valine at position -8 in the hydrophobic sequence was replaced by tryptophan. First it was established that this mutation in the signal sequence of prePhoE does not affect in vivo and in vitro translocation efficiency and that the biophysical properties of the synthetic mutant signal peptide are similar to those of the wild-type signal peptide. Next, fluorescence experiments were performed which showed an increase in quantum yield and a blue shift of the emission wavelength maximum upon interaction of the signal peptide with lipid vesicles, indicating that the tryptophan moiety enters a more hydrophobic environment. These changes in intrinsic fluorescence were found to be more pronounced in the presence of phosphatidylglycerol (PG) or cardiolipin (CL) than with phosphatidylcholine (PC). In addition, quenching experiments demonstrated a shielding of the tryptophan fluorescence from quenching by the aqueous quenchers iodide and acrylamide upon interaction of the signal peptide with lipid vesicles, a shielding in the case of acrylamide that was more pronounced in the presence of negatively charged lipids. Finally it was found that acyl chain brominated lipids incorporated into phospholipid bilayers were able to quench the tryptophan fluorescence of the signal peptide, with the quenching efficiency in CL vesicles being much higher than in PC vesicles. The results clearly demonstrate that the PhoE signal peptide interacts strongly with different lipid vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of a membrane potential on the interaction of mastoparan X, a mitochondrial presequence, and several regulatory peptides with phospholipid vesicles.

Recently the pH gradient evoked by a K+ diffusion potential was shown to translocate a synthetic monobasic amphipathic hexapeptide across the bilayer of lipid vesicles (De Kroon, A.I.P.M., Vogt, B., Van 't Hof, R., De Kruijff, B. and De Gier, J. (1991) Biophys. J. 60, in press). Here this observation is extended by studying the effect of a membrane potential on a set of bioactive peptides. The panel of peptides comprises the toxin mastoparan X, a tryptophan-containing analogue of the presequence of the mitochondrial protein cytochrome oxidase subunit IV (preCoxIV(1-25)W18), and the regulatory peptides ACTH(1-24), alpha-MSH, ACTH(1-10), dynorphin A, bombesin, and LHRH. The interaction of these peptides with phospholipid vesicles has been measured using the intrinsic tryptophan residue as fluorescent probe. In the absence of a K+ diffusion potential only mastoparan X and the presequence show considerable binding to vesicles consisting of phosphatidylcholine (PC). In contrast, under these conditions all peptides display affinity for vesicles consisting of the acidic phospholipid cardiolipin (CL), the extent of which depends on the net positive charge of the peptide. Application of a K+ diffusion potential to large unilamellar vesicles (LUV) consisting of PC results in a time dependent tryptophan fluorescence increase for mastoparan X, which is accelerated upon incorporating increasing amounts of CL into the LUV. A similar fluorescence increase in response to a K+ diffusion potential was observed for the above model peptide. Yet the mechanism resulting in the fluorescence increase of mastoparan X is completely different from that of the hexapeptide. Binding experiments indicate that a membrane potential-induced enhanced binding of the peptide to the outer surface of the vesicles contributes to the fluorescence increase. PreCoxIV(1-25)W18, dynorphin A, and ACTH(1-24) show fluorescence responses upon applying a membrane potential that are consistent with that of mastoparan X, whereas the other peptides tested do not respond up to a LUV CL content of 50%. The results tentatively suggest that the membrane potential only affects a peptide when it has the ability to adopt a stable membrane bound conformation.     

Ion gradient-induced membrane translocation of model peptides.

The K+ diffusion potential-induced association of synthetic model peptides carrying a single positive charge originating from the NH2-terminal amino function with large unilamellar vesicles (LUV) consisting of phosphatidylcholine (PC) has been reported previously (de Kroon, A. I. P. M., J. de Gier, and B. de Kruijff. 1989. Biochim. Biophys. Acta. 981:371-373). To determine the vesicle localization of the associated peptides, fluorescence measurements utilizing the peptides' tryptophan residue as intrinsic fluorescent probe were performed. The application in these measurements, of vesicles that exhibit an asymmetric transbilayer distribution of brominated PC which is a quencher of tryptophan fluorescence, unequivocally demonstrated that the peptide H3N(+)-AIMLWA-Ome (AIXme+) is accumulated in the interface of the inner leaflet of the vesicle membrane in response to the valinomycin-induced K+ diffusion potential (negative inside). The relative contributions of the membrane potential (delta psi) and the pH gradient (delta pH, acidic inside) induced by the K+ diffusion potential, to the process have been assessed. An analysis of the pH and delta pH dependencies of the process demonstrated that the K+ diffusion potential-induced peptide accumulation is largely determined by a redistribution of peptide according to the transbilayer pH gradient, in agreement with a translocation across the vesicle membrane of the neutral, deprotonated form of the peptide. The general validity of the mechanism proposed for the vesicle-uptake of AIXme+ has been examined by extending the experiments to peptide analogues with a single negative charge and to peptides with two positive charges, and by investigating the effect of incorporating the acidic phospholipid cardiolipin (CL) into the LUV. The incorporation of CL appeared not to affect the K+ diffusion, potential-induced vesicle uptake of AIXme+. The peptide H3N(+)-RMLWA-Ome (RXme2+) showed a small delta pH independent fluorescence response to the delta psi upon raising the CL content of the vesicles to 25%.     

The effect of acidic residues and amphipathicity on the lytic activities of mastoparan peptides studied by fluorescence and CD spectroscopy

Some mastoparan peptides extracted from social wasps display antimicrobial activity and some are hemolytic and cytotoxic. Although the cell specificity of these peptides is complex and poorly understood, it is believed that their net charges and their hydrophobicity contribute to modulate their biological activities. We report a study, using fluorescence and circular dichroism spectroscopies, evaluating the influence of these two parameters on the lytic activities of five mastoparans in zwitterionic and anionic phospholipid vesicles. Four of these peptides, extracted from the venom of the social wasp Polybia paulista, present both acidic and basic residues with net charges ranging from +1 to +3 which were compared to Mastoparan-X with three basic residues and net charge +4. Previous studies revealed that these peptides have moderate-to-strong antibacterial activity against Gram-positive and Gram-negative microorganisms and some of them are hemolytic. Their affinity and lytic activity in zwitterionic vesicles decrease with the net electrical charges and the dose response curves are more cooperative for the less charged peptides. Higher charged peptides display higher affinity and lytic activity in anionic vesicles. The present study shows that the acidic residues play an important role in modulating the peptides’ lytic and biological activities and influence differently when the peptide is hydrophobic or when the acidic residue is in a hydrophilic peptide.     

Preferential binding of apolipoprotein E derived peptides with oxidized phospholipid

The physiological function of apolipoprotein E (apoE) includes transport and metabolism of lipids and its C-terminal domain harbors high affinity lipid-binding sites. Although the binding of apoE with non-oxidized phospholipid containing membranes has been characterized earlier, the interaction of apoE or its fragments with oxidized phospholipid containing membrane has never been studied. In this study we have compared the interaction of amphipathic helical peptide sequences derived from the C-terminal domain of apoE with membrane vesicles containing oxidized phospholipid, 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC), with membrane vesicles without PazePC. The interaction was studied by monitoring (a) fluorescence emission maxima of the peptides, (b) acrylamide quenching of the peptides tryptophan residues and (c) by measuring the equilibrium binding constants by resonance energy transfer (RET) analysis. Our result shows that peptide sequence 202-223, 245-266 and 268-289 of apoE has higher affinity towards membrane containing PazePC, compared to membrane without PazePC. Presence of 1mM divalent cation or 50 mM NaCl in the buffer decreased the binding of peptides to PazePC containing membrane vesicles suggesting possible involvement of the electrostatic interaction in the binding. These observations suggest that the preferential binding of apoE to oxidized phospholipid containing membrane may play a role in the anti-oxidative properties of apoE.     

Conformational states of the cell-penetrating peptide penetratin when interacting with phospholipid vesicles: effects of surface charge and peptide concentration

The most commonly studied of the cell-penetrating peptides (CPP) is "penetratin" (pAntp), which functions as a carrier (vector), even for large hydrophilic (cargo) molecules. pAntp originates from the third helix of the Antennapedia homeodomain protein. The peptide is known to interact with negatively charged phospholipid vesicles, which leads to induction of secondary structure. In the present study, circular dichroism (CD) spectroscopy has been used to characterize the different secondary structures induced upon interaction with small unilamellar vesicles (SUVs) from mixtures of zwitterionic 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and negatively charged 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG). The interaction was monitored using an electron paramagnetic resonance (EPR) spin probe attached to the peptide, and the intrinsic fluorophore (tryptophan). We measured the secondary structure as a function of surface charge density, total lipid-to-peptide (L/P) molar ratio, and salt concentration, for completely bound peptide. With vesicles from POPG/POPC in a molar ratio below 30:70, at a high L/P, the peptide adopts a mainly helical conformation. Increasing the charge density, at the same L/P, promotes a higher degree of beta-structure. At a fixed charge density, reducing the L/P also results in an alpha-->beta structure conversion. Hence, low membrane surface charge density and low pAntp concentration both favor a mainly helical conformation, while high charge density and pAntp concentration promote a dominating beta-structure. We conclude that pAntp, when residing at the surface of a membrane, is chameleon-like in terms of its induced structure.     

Thermodynamics of the interactions of tryptophan-rich cathelicidin antimicrobial peptides with model and natural membranes

Tritrpticin and indolicidin are short 13-residue tryptophan-rich antimicrobial peptides that hold potential as future alternatives for antibiotics. Isothermal titration calorimetry (ITC) has been applied as the main tool in this study to investigate the thermodynamics of the interaction of these two cathelicidin peptides as well as five tritrpticin analogs with large unilamellar vesicles (LUVs), representing model and natural anionic membranes. The anionic LUVs were composed of (a) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPE/POPG) (7:3) and (b) natural E. coli polar lipid extract. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was used to make model zwitterionic membranes. Binding isotherms were obtained to characterize the antimicrobial peptide binding to the LUVs, which then allowed for calculation of the thermodynamic parameters of the interaction. All peptides exhibited substantially stronger binding to anionic POPE/POPG and E. coli membrane systems than to the zwitterionic POPC system due to strong electrostatic attractions between the highly positively charged peptides and the negatively charged membrane surface, and results with tritrpticin derivatives further revealed the effects of various amino acid substitutions on membrane binding. No significant improvement was observed upon increasing the Tritrp peptide charge from + 4 to + 5. Replacement of Arg residues with Lys did not substantially change peptide binding to anionic vesicles but moderately decreased the binding to zwitterionic LUVs. Pro to Ala substitutions in tritrpticin, allowing the peptide to adopt an α-helical structure, resulted in a significant increase of the binding to both anionic and zwitterionic vesicles and therefore reduced the selectivity for bacterial and mammalian membranes. In contrast, substitution of Trp with other aromatic amino acids significantly decreased the peptide's ability to bind to anionic LUVs and essentially eliminated binding to zwitterionic LUVs. The ITC results were consistent with the outcome of fluorescence spectroscopy membrane binding and perturbation studies. Overall, our work showed that a natural E. coli polar lipid extract as a bacterial membrane model was advantageous compared to the simpler and more widely used POPE/POPG lipid system.     

Thermodynamics of the interactions of tryptophan-rich cathelicidin antimicrobial peptides with model and natural membranes

Tritrpticin and indolicidin are short 13-residue tryptophan-rich antimicrobial peptides that hold potential as future alternatives for antibiotics. Isothermal titration calorimetry (ITC) has been applied as the main tool in this study to investigate the thermodynamics of the interaction of these two cathelicidin peptides as well as five tritrpticin analogs with large unilamellar vesicles (LUVs), representing model and natural anionic membranes. The anionic LUVs were composed of (a) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPE/POPG) (7:3) and (b) natural E. coli polar lipid extract. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was used to make model zwitterionic membranes. Binding isotherms were obtained to characterize the antimicrobial peptide binding to the LUVs, which then allowed for calculation of the thermodynamic parameters of the interaction. All peptides exhibited substantially stronger binding to anionic POPE/POPG and E. coli membrane systems than to the zwitterionic POPC system due to strong electrostatic attractions between the highly positively charged peptides and the negatively charged membrane surface, and results with tritrpticin derivatives further revealed the effects of various amino acid substitutions on membrane binding. No significant improvement was observed upon increasing the Tritrp peptide charge from +4 to +5. Replacement of Arg residues with Lys did not substantially change peptide binding to anionic vesicles but moderately decreased the binding to zwitterionic LUVs. Pro to Ala substitutions in tritrpticin, allowing the peptide to adopt an alpha-helical structure, resulted in a significant increase of the binding to both anionic and zwitterionic vesicles and therefore reduced the selectivity for bacterial and mammalian membranes. In contrast, substitution of Trp with other aromatic amino acids significantly decreased the peptide's ability to bind to anionic LUVs and essentially eliminated binding to zwitterionic LUVs. The ITC results were consistent with the outcome of fluorescence spectroscopy membrane binding and perturbation studies. Overall, our work showed that a natural E. coli polar lipid extract as a bacterial membrane model was advantageous compared to the simpler and more widely used POPE/POPG lipid system.     

Composition effect on peptide interaction with lipids and bacteria: variants of C3a peptide CNY21

The effect of peptide hydrophobicity and charge on peptide interaction with model lipid bilayers was investigated for the C3a-derived peptide CNY21 by fluorescence spectroscopy, circular dichroism, ellipsometry, z-potential, and photon correlation spectroscopy measurements. For both zwitterionic and anionic liposomes, the membrane-disruptive potency for CNY21 variants increased with increasing net positive charge and mean hydrophobicity and was completely lost on elimination of all peptide positive charges. Analogous effects of elimination of the peptide positive net charge in particular were found regarding bacteria killing for both Pseudomonas aeruginosa and Bacillus subtilis. The peptides, characterized by moderate helix content both in buffer and when attached to the liposomes, displayed high adsorption for the net positively charged peptide variants, whereas adsorption was non-measurable for the uncharged peptide. That electrostatically driven adsorption represents the main driving force for membrane disruption in lipid systems was also demonstrated by a drastic reduction in both liposome leakage and peptide adsorption with increasing ionic strength, and this salt inactivation can be partly avoided by increasing the peptide hydrophobicity. This increased electrolyte resistance translates also to a higher antibacterial effect for the hydrophobically modified variant at high salt concentration. Overall, our findings demonstrate the importance of the peptide adsorption and resulting peptide interfacial density for membrane-disruptive effects of these peptides.     

Role of peptide structure in lipid-peptide interactions: a fluorescence study of the binding of pentagastrin-related pentapeptides to phospholipid vesicles

The binding of pentagastrin and three other structurally related pentapeptides to phospholipid vesicles has been studied by fluorescence spectroscopy. The fluorescence of the tryptophan residues of these peptides exhibits an increased quantum yield upon binding to phospholipid vesicles. This is accompanied by a blue shift of the maximum emission, indicative of the incorporation of the tryptophan residue into a more hydrophobic environment. The affinity of the peptides for a zwitterionic phospholipid, dimyristoylphosphatidylcholine (DMPC), increases in the following order: N-t-Boc-beta-Ala-Trp-Met-Gly-Phe-NH2 greater than N-t-Boc-beta-Ala-Trp-Met-Arg-Phe-NH2 greater than N-t-Boc-beta-Ala-Trp-Met-Asp-Phe-NH2 greater than N-t-Boc-beta-Ala-Trp-Met-Phe-Asp-NH2. Comparison of the interaction of these various peptides with this phospholipid indicates that although the interaction is largely of hydrophobic nature, the structure of the polar amino acids and their electrostatic charge have significant influence on the nature of the bindings. In addition, the sequence of polar and apolar amino acids appears to be of importance. The higher affinity for DMPC of N-t-Boc-beta-Ala-Trp-Met-Asp-Phe-NH2 as compared to its "reversed" analogue N-t-Boc-beta-Ala-Trp-Met-Phe-Asp-NH2 suggests that the ability of the peptides to fold into amphiphatic structures can enhance their lipid binding affinity. For all peptides the interaction with DMPC is greater at 8 degrees C, i.e., below the lipid phase transition temperature, than at 40 degrees C, i.e., above the lipid phase transition temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Autonomous transmembrane segment S4 of the voltage sensor domain partitions into the lipid membrane

The S4 transmembrane segment in voltage-gated ion channels, a highly basic α helix, responds to changes in membrane potential and induces channel opening. Earlier work by others indicates that the S4 segment interacts with lipids in plasma membrane, but its mechanism is unclear. Working with synthetic tryptophan-labeled S4 peptides, we characterized binding of autonomous S4 to lipid membranes. The binding free energy (5.2±0.2kcal/mol) of the peptide-lipid interaction was estimated from the apparent dissociation constants, determined from the changes in anisotropy of tryptophan fluorescence induced by addition of lipid vesicles with 30mol% phosphatidylglycerol. The results are in good agreement with the prediction based on the Wimley-White hydrophobicity scale for interfacial (IF) binding of an alpha-helical peptide to the lipid bilayer (6.98kcal/mol). High salt inhibited the interaction, thus indicating that the peptide/membrane interaction has both electrostatic and non-electrostatic components. Furthermore, the synthetic S4 corresponding to the Shaker potassium channel was found to spontaneously penetrate into the negatively charged lipid membrane to a depth of about 9?. Our results revealed important biophysical parameters that influence the interaction of S4 with the membrane: they include fluidity, surface charge, and surface pressure of the membrane, and the α helicity and regular spacing of basic amino-acid residues in the S4 sequence.     

Both acidic and basic amino acids in an amphitropic enzyme,CTP:phosphocholine cytidylyltransferase,dictate its selectivity for anionic membranes

Amphitropic proteins are regulated by reversible membrane interaction. Anionic phospholipids generally promote membrane binding of such proteins via electrostatics between the negatively charged lipid headgroups and clusters of basic groups on the proteins. In this study of one amphitropic protein, a cytidylyltransferase (CT) that regulates phosphatidylcholine synthesis, we found that substitution of lysines to glutamine along both interfacial strips of the membrane-binding amphipathic helix eliminated electrostatic binding. Unexpectedly, three glutamates also participate in the selectivity for anionic membrane surfaces. These glutamates become protonated in the low pH milieu at the surface of anionic, but not zwitterionic membranes, increasing protein positive charge and hydrophobicity. The binding and insertion into lipid vesicles of a synthetic peptide containing the three glutamates was pH-dependent with an apparent pK(a) that varied with anionic lipid content. Glutamate to glutamine substitution eliminated the pH dependence of the membrane interaction, and reduced anionic membrane selectivity of both the peptide and the whole CT enzyme examined in cells. Thus anionic lipids, working via surface-localized pH effects, can promote membrane binding by modifying protein charge and hydrophobicity, and this novel mechanism contributes to the membrane selectivity of CT in vivo.     

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.     

Interaction of a synthetic antimicrobial peptide with model membrane by fluorescence spectroscopy

Static and time-resolved fluorescence of tryptophan and ortho-aminobenzoic acid was used to investigate the interaction of the synthetic antimicrobial peptide L1A (IDGLKAIWKKVADLLKNT-NH₂) with POPC and POPC:POPG. N-acetylated (Ac-L1A) and N-terminus covalently bonded ortho-aminobenzoic acid (Abz-L1A-W8V) were also used. Static fluorescence and quenching by acrylamide showed that the peptides adsorption to the lipid bilayers was accompanied by spectral blue shift and by a decrease in fluorescence quenching, indicating that the peptides moved to a less polar environment probably buried in the lipidic phase of the vesicles. These results also suggest that the loss of the N-terminus charge allowed deeper fluorophore insertion in the bilayer. Despite the local character of spectroscopic information, conclusions can be drawn about the peptides as a whole. The dynamic behaviors of the peptides are such that the mean intensity lifetimes, the long correlation time and the residual anisotropy at long times increased when the peptides adsorb in lipid vesicles, being larger in anionic vesicles. From the steady-state increase in fluorescence intensity and anisotropy, we observed that the partition coefficient of peptides L1A and its Abz analog in both types of vesicles are higher than the acetylated analog; moreover, the affinity to the anionic vesicle is higher than to the zwitterionic.     

Membrane binding and translocation of cell-penetrating peptides

Cell-penetrating peptides (CPPs) have been extensively studied during the past decade, because of their ability to promote the cellular uptake of various cargo molecules, e.g., oligonucleotides and proteins. In a recent study of the uptake of several analogues of penetratin, Tat(48-60) and oligoarginine in live (unfixed) cells [Thorén et al. (2003) Biochem. Biophys. Res. Commun. 307, 100-107], it was found that both endocytotic and nonendocytotic uptake pathways are involved in the internalization of these CPPs. In the present study, the membrane interactions of some of these novel peptides, all containing a tryptophan residue to facilitate spectroscopic studies, are investigated. The peptides exhibit a strong affinity for large unilamellar vesicles (LUVs) containing zwitterionic and anionic lipids, with binding constants decreasing in the order penetratin > R(7)W > TatP59W > TatLysP59W. Quenching studies using the aqueous quencher acrylamide and brominated lipids indicate that the tryptophan residues of the peptides are buried to a similar extent into the membrane, with an average insertion depth of approximately 10-11 A from the bilayer center. The membrane topology of the peptides was investigated using an assay based on resonance energy transfer between tryptophan and a fluorescently labeled lysophospholipid, lysoMC, distributed asymmetrically in the membranes of LUVs. By determination of the energy transfer efficiency when peptide was added to vesicles with lysoMC present exclusively in the inner leaflet, it was shown that none of the peptides investigated is able to translocate across the lipid membranes of LUVs. By contrast, confocal laser scanning microscopy studies on carboxyfluorescein-labeled peptides showed that all of the peptides rapidly traverse the membranes of giant unilamellar vesicles (GUVs). The choice of model system is thus crucial for the conclusions about the ability of CPPs to translocate across lipid membranes. Under the conditions used in the present study, peptide-lipid interactions alone cannot explain the different cellular uptake characteristics exhibited by these peptides.     

Structures and mode of membrane interaction of a short alpha helical lytic peptide and its diastereomer determined by NMR, FTIR, and fluorescence spectroscopy.

The interaction of many lytic cationic antimicrobial peptides with their target cells involves electrostatic interactions, hydrophobic effects, and the formation of amphipathic secondary structures, such as alpha helices or beta sheets. We have shown in previous studies that incorporating approximately 30%d-amino acids into a short alpha helical lytic peptide composed of leucine and lysine preserved the antimicrobial activity of the parent peptide, while the hemolytic activity was abolished. However, the mechanisms underlying the unique structural features induced by incorporating d-amino acids that enable short diastereomeric antimicrobial peptides to preserve membrane binding and lytic capabilities remain unknown. In this study, we analyze in detail the structures of a model amphipathic alpha helical cytolytic peptide KLLLKWLL KLLK-NH2 and its diastereomeric analog and their interactions with zwitterionic and negatively charged membranes. Calculations based on high-resolution NMR experiments in dodecylphosphocholine (DPCho) and sodium dodecyl sulfate (SDS) micelles yield three-dimensional structures of both peptides. Structural analysis reveals that the peptides have an amphipathic organization within both membranes. Specifically, the alpha helical structure of the L-type peptide causes orientation of the hydrophobic and polar amino acids onto separate surfaces, allowing interactions with both the hydrophobic core of the membrane and the polar head group region. Significantly, despite the absence of helical structures, the diastereomer peptide analog exhibits similar segregation between the polar and hydrophobic surfaces. Further insight into the membrane-binding properties of the peptides and their depth of penetration into the lipid bilayer has been obtained through tryptophan quenching experiments using brominated phospholipids and the recently developed lipid/polydiacetylene (PDA) colorimetric assay. The combined NMR, FTIR, fluorescence, and colorimetric studies shed light on the importance of segregation between the positive charges and the hydrophobic moieties on opposite surfaces within the peptides for facilitating membrane binding and disruption, compared to the formation of alpha helical or beta sheet structures.     

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.     

Comparison of the membrane interaction and permeabilization by the designed peptide Ac-MB21-NH2 and truncated dermaseptin S3

Ac-MB21-NH(2) (Ac-FASLLGKALKALAKQ-NH(2)) and dermaseptin S3(1-16)-NH(2) (ALWKNMLKGIGKLAGK-NH(2)) are cationic amphipathic peptides with antimicrobial activity against a broad spectrum of microorganisms including various fungi. The interaction of the peptides with liposomes was studied by exploiting the tryptophan fluorescence of F1W-Ac-MB21-NH(2) and dermaseptin S3(1-16)-NH(2). Spectral analysis and the use of quenchers indicate that the tryptophans of both peptides insert more deeply in anionic than in zwitterionic liposomes. Membrane insertion correlates with the formation of an alpha-helical peptide structure. Both peptides permeabilize liposomes composed of anionic, cylindric phospholipids more efficiently than liposomes formed of zwitterionic, conic (phospho)lipids.     

Excitation energy transfer from tryptophan residues of peptides and intrinsic proteins to diphenylhexatriene in phospholipid vesicles and biological membranes

An efficient excitation energy transfer from tryptophan residues of intrinsic membrane proteins to an extrinsic fluorescent probe (diphenylhexatriene) has been demonstrated in rat erythrocyte ghosts. To correlate this transfer with the localization of the probe, a model system has been investigated. It consists of peptides containing lysine and tryptophan residues bound to negatively charged phosphatidylserine vesicles. Absorption and fluorescence spectroscopies were used to follow peptide binding and diphenylhexatriene incorporation. Peptide binding is accompanied by a blue shift of the tryptophan fluorescence together with an increase of the quantum yield and of the fluorescence decay time. An experimental Föster critical distance value of 4.0 nm was found for energy transfer from tryptophan residues of peptides to diphenylhexatriene which approaches the range of calculated values (3.1–3.7 nm) using a two-dimensional model. These results demonstrate that efficient energy transfer can occur from tryptophan residues of intrinsic proteins to diphenylhexatriene without any interaction between diphenylhexatriene and proteins in biological membranes.     

Comparison of the interaction,positioning, structure induction and membrane perturbation of cell-penetrating peptides and non-translocating variants with phospholipid vesicles

Cell-penetrating peptides (CPPs) are able to translocate and carry cargo molecules across cell membranes. Using fluorescence techniques (polarization and quenching) and CD spectroscopy we studied the interaction, conformation and topology of two such peptides, transportan and 'penetratin' (pAntp), and two variants of differing translocating abilities, with small phospholipid vesicles of varying charge density. The induced structure of transportan is always helical independent of vesicle surface charge. pAntp and its two variants interact significantly only with negatively charged vesicles. The induced secondary structure depends on membrane charge and lipid/peptide ratio. The degree of membrane perturbation, evidenced by fluorescence polarization, of pAntp and its variants is related to their secondary structure. In the helical state, the peptides have little effect on the membrane. Under conditions where pAntp and its variants are converted into beta-structures, they cause membrane perturbation. Oriented CD suggests that the two CPPs (pAntp and transportan) in their helical state lie along the vesicle surface, while the two pAntp variants appear to penetrate deeper into the membrane.