Enhanced antitumour activity of 15-residue bovine lactoferricin derivatives containing bulky aromatic amino acids and lipophilic N-terminal modifications.

In a structure-antibacterial activity relationship study of a peptide fragment of bovine lactoferricin consisting of FKCRRWQWRMKKLGA (LFB 17-31), it was revealed that the two Trp residues were important for antibacterial activity. It has further been demonstrated that the size, shape and the aromatic character of the side chains were even more important than the Trp itself. In this study the antitumour effect of a series of LFB 17-31 derivatives are reported, in which the two Trp residues in position 6 and 8 were replaced with the larger non-coded aromatic amino acids Tbt, Tpc, Bip and Dip. The counterproductive Cys in position 3 was also substituted with these larger aromatic residues. In addition, the effect of introducing lipophilic groups of different size and shape in the N-terminal of the LFB 17-31 sequence was addressed. The resulting peptide derivatives were tested for activity against three human tumour cell lines and against normal human umbilical vein endothelial cells and fibroblasts. High antitumour activity by several of the peptides demonstrated that Trp successfully could be substituted by the bulky aromatic residues, and peptides containing the large and rigid Tbt residue in position 6 and/or 8 in LFB 17-31 were the most active candidates. The antitumour effect was even more increased by the Tbt-modified peptides when the three counterproductive amino acids Cys3, Gln7 and Gly14 were replaced by Ala. Enhanced antitumour activity was also obtained by modifying the N-terminal of LFB 17-31 with either long-chained fatty acids or bulky moieties. Thus, our results revealed that the size and shape of the lipophilic groups and their position in the peptide sequence were important for antitumour activity.     

The role of tryptophan in the antibacterial activity of a 15-residue bovine lactoferricin peptide.

Bovine lactoferricin is a 25-residue antibacterial peptide isolated after gastric cleavage of the iron transporting protein lactoferrin. A 15-residue fragment, FKCRRWQWRMKKLGA of this peptide sustains most of the antibacterial activity. In this truncated sequence, the two Trp residues are found to be essential for antibacterial activity. The anchoring properties of Trp, as have been observed in membrane proteins, are believed to be important for the interaction of Trp containing antibacterial peptides with bacterial cell membranes. We have investigated the molecular properties which make Trp important for the antibacterial activity of the 15-residue peptide by replacing Trp with natural and unnatural aromatic amino acids. This series of peptides was tested for antibacterial activity against Echerichia coli and Staphylococcus aureus. We found that neither the hydrogen bonding ability nor the amphipathicity of the indole system are essential properties for the effect of Trp on the antibacterial activity of the peptides. Replacement of Trp with residues containing aromatic hydrocarbon side chains gave the most active peptides. We propose that aromatic hydrocarbon residues are able to position themselves deeper into the bacterial cell membrane, making the peptide more efficient in disrupting the bacterial cell membrane. From our results the size, shape and aromatic character of Trp seem to be the most important features for the activity of this class of Trp containing antibacterial peptides.     

Relative spatial positions of tryptophan and cationic residues in helical membrane-active peptides determine their cytotoxicity

The cytotoxic activity of 10 analogs of the idealized amphipathic helical 21-mer peptide (KAAKKAA)3, where three of the Ala residues at different positions have been replaced with Trp residues, has been investigated. The peptide's cytotoxic activity was found to be markedly dependent upon the position of the Trp residues within the hydrophobic sector of an idealized α-helix. The peptides with Trp residues located opposite the cationic sector displayed no antitumor activity, whereas those peptides with two or three Trp residues located adjacent to the cationic sector exhibited high cytotoxic activity when tested against three different cancer cell lines. Dye release experiments revealed that in contrast to the peptides with Trp residues located opposite the cationic sector, the peptides with Trp residues located adjacent to the cationic sector induced a strong permeabilizing activity from liposomes composed of a mixture of zwitterionic phosphatidylcholine and negatively charged phosphatidylserine (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS)) (2:1) but not from liposomes composed of zwitterionic phosphatidylcholine, POPC. Fluorescence blue shift and quenching experiments revealed that Trp residues inserted deeper into the hydrophobic environment of POPC/POPS liposomes for peptides with high cytotoxic activity. Through circular dichroism studies, a correlation between the cytotoxic activity and the α-helical propensity was established. Structural studies of one inactive and two active peptides in the presence of micelles using NMR spectroscopy showed that only the active peptides adopted highly coiled to helical structures when bound to a membrane surface.     

Antibacterial activity of 15-residue lactoferricin derivatives.

Lactoferricins are a class of antibacterial peptides isolated after gastric-pepsin digest of the mammalian iron-chelating-protein lactoferrin. For investigation of antibacterial activity, we prepared short synthetic derivatives of bovine, human, caprine, murine and porcine lactoferricins with 15-amino-acid residues of high sequence homology. The peptides corresponded to amino-acid residues 17-31 of the mature bovine lactoferrin. Only the bovine and caprine derivatives displayed measurable antibacterial activity, with the bovine one having a minimal inhibitory concentration of 24 microM and being 10 times more active than the caprine one against Escherichia coli. An alanine-scan of the bovine lactoferricin derivative was performed to identify specific amino acids that were important for the antibacterial activity. We found that neither of the two tryptophan residues (Trp 6 and Trp 8) present in the bovine lactoferricin derivative could be replaced by alanine without a major loss of antibacterial activity. The other lactoferricin derivatives tested contained only one tryptophan residue (Trp 6). Modified human, caprine and porcine lactoferricin derivatives containing two tryptophan residues (Trp 6 and Trp 8) displayed minimal inhibitory concentrations of 74, 174 and 219 microM, respectively, which represented up to a six-fold increase in antibacterial activity. The alanine-scan also revealed that the antibacterial activity was increased when acetamidomethyl-protected cysteine and unprotected glutamine (Cys 3 and Gln 7) were replaced with alanine. Only the bovine lactoferricin derivative and a few of its alanine-modified derivatives displayed measurable activity against Staphylococcus aureus.     

New indolicidin analogues with potent antibacterial activity.

Indolicidin is a 13-residue antimicrobial peptide amide, ILPWKWPWWPWRR-NH2, isolated from the cytoplasmic granules of bovine neutrophils. Indolicidin is active against a wide range of microorganisms and has also been shown to be haemolytic and cytotoxic towards erythrocytes and human T lymphocytes. The aim of the present paper is two-fold. First, we examine the importance of tryptophan in the antibacterial activity of indolicidin. We prepared five peptide analogues with the format ILPXKXPXXPXRR-NH2 in which Trp-residues 4,6,8,9,11 were replaced in all positions with X = a single non-natural building block; N-substituted glycine residue or nonproteinogenic amino acid. The analogues were tested for antibacterial activity against both Staphylococcus aureus American type culture collection (ATCC) 25923 and Escherichia coli ATCC 25922. We found that tryptophan is not essential in the antibacterial activity of indolicidin, and even more active analogues were obtained by replacing tryptophan with non-natural aromatic amino acids. Using this knowledge, we then investigated a new principle for improving the antibacterial activity of small peptides. Our approach involves changing the hydrophobicity of the peptide by modifying the N-terminus with a hydrophobic non-natural building block. We prepared 22 analogues of indolicidin and [Phe(4,6,8,9,11)] indolicidin, 11 of each, carrying a hydrophobic non-natural building block attached to the N-terminus. Several active antibacterial analogues were identified. Finally, the cytotoxicity of the analogues against sheep erythrocytes was assessed in a haemolytic activity assay. The results presented here suggest that modified analogues of antibacterial peptides, containing non-natural building blocks, are promising lead structures for developing future therapeutics.     

The effects of charge and lipophilicity on the antibacterial activity of undecapeptides derived from bovine lactoferricin.

We have investigated the effects of charge and lipophilicity on the antibacterial activity of an undecapeptide (FKCRRWQWRMK) derived from the sequence of bovine lactoferricin. We prepared ten analogues that were modified by the incorporation of Ala, Tyr, Trp, Met and Arg residues, which are amino acids known to be important for the antibacterial activity of longer derivatives of lactoferricins. All undecapeptides contained the native Trp residues in positions 6 and 8, and the Arg residues in positions 5 and 9. Generally, the Gram-positive bacterium Staphylococcus aureus was more susceptible to these undecapeptides than the Gram-negative bacteria, and a higher antibacterial activity was observed against Escherichia coli than against Pseudomonas aeruginosa. The only exception was the peptide Undeca 9 (RRWYRWAWRMR-NH2), which was almost equally active against all three test strains, displaying minimal inhibitory concentrations of 10 microg/ml (5.8 microM), 7.5 microg/ml (4.4 microM) and 5 microg/ml (2.9 microM) against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus, respectively. The peptides Undeca 6 (YRAWRWAWRWR-NH2) and Undeca 7 (YRMWRWAWRWR-NH2) were the two most active undecapeptides against Staphylococcus aureus, both displaying a minimal inhibitory concentration of 2.5 microg/ml (1.5 microM). The study showed that a level was reached in which undecapeptides having a net charge above +4 and containing three or four Trp residues all displayed a high antibacterial activity. All undecapeptides prepared were essentially non-haemolytic, but undecapeptides containing more than three Trp residues displayed 50% haemolysis of human red blood cells at concentrations above 400 microg/ml (>230 microM).     

Tryptophan- and arginine-rich antimicrobial peptides: Structures and mechanisms of action

Antimicrobial peptides encompass a number of different classes, including those that are rich in a particular amino acid. An important subset are peptides rich in Arg and Trp residues, such as indolicidin and tritrpticin, that have broad and potent antimicrobial activity. The importance of these two amino acids for antimicrobial activity was highlighted through the screening of a complete combinatorial library of hexapeptides. These residues possess some crucial chemical properties that make them suitable components of antimicrobial peptides. Trp has a distinct preference for the interfacial region of lipid bilayers, while Arg residues endow the peptides with cationic charges and hydrogen bonding properties necessary for interaction with the abundant anionic components of bacterial membranes. In combination, these two residues are capable of participating in cation-π interactions, thereby facilitating enhanced peptide-membrane interactions. Trp sidechains are also implicated in peptide and protein folding in aqueous solution, where they contribute by maintaining native and nonnative hydrophobic contacts. This has been observed for the antimicrobial peptide from human lactoferrin, possibly restraining the peptide structure in a suitable conformation to interact with the bacterial membrane. These unique properties make the Arg- and Trp-rich antimicrobial peptides highly active even at very short peptide lengths. Moreover, they lead to structures for membrane-mimetic bound peptides that go far beyond regular α-helices and β-sheet structures. In this review, the structures of a number of different Trp- and Arg-rich antimicrobial peptides are examined and some of the major mechanistic studies are presented.     

Tryptophan- and arginine-rich antimicrobial peptides: structures and mechanisms of action

Antimicrobial peptides encompass a number of different classes, including those that are rich in a particular amino acid. An important subset are peptides rich in Arg and Trp residues, such as indolicidin and tritrpticin, that have broad and potent antimicrobial activity. The importance of these two amino acids for antimicrobial activity was highlighted through the screening of a complete combinatorial library of hexapeptides. These residues possess some crucial chemical properties that make them suitable components of antimicrobial peptides. Trp has a distinct preference for the interfacial region of lipid bilayers, while Arg residues endow the peptides with cationic charges and hydrogen bonding properties necessary for interaction with the abundant anionic components of bacterial membranes. In combination, these two residues are capable of participating in cation-pi interactions, thereby facilitating enhanced peptide-membrane interactions. Trp sidechains are also implicated in peptide and protein folding in aqueous solution, where they contribute by maintaining native and nonnative hydrophobic contacts. This has been observed for the antimicrobial peptide from human lactoferrin, possibly restraining the peptide structure in a suitable conformation to interact with the bacterial membrane. These unique properties make the Arg- and Trp-rich antimicrobial peptides highly active even at very short peptide lengths. Moreover, they lead to structures for membrane-mimetic bound peptides that go far beyond regular alpha-helices and beta-sheet structures. In this review, the structures of a number of different Trp- and Arg-rich antimicrobial peptides are examined and some of the major mechanistic studies are presented.     

Effects of Hydrophobic Amino Acid Substitutions on Antimicrobial Peptide Behavior

Antimicrobial peptides (AMPs) are naturally occurring components of the immune system that act against bacteria in a variety of organisms throughout the evolutionary hierarchy. There have been many studies focused on the activity of AMPs using biophysical and microbiological techniques; however, a clear and predictive mechanism toward determining if a peptide will exhibit antimicrobial activity is still elusive, in addition to the fact that the mechanism of action of AMPs has been shown to vary between peptides, targets, and experimental conditions. Nonetheless, the majority of AMPs contain hydrophobic amino acids to facilitate partitioning into bacterial membranes and a net cationic charge to promote selective binding to the anionic surfaces of bacteria over the zwitterionic host cell surfaces. This study explores the role of hydrophobic amino acids using the peptide C18G as a model system. These changes were evaluated for the effects on antimicrobial activity, peptide-lipid interactions using Trp fluorescence spectroscopy, peptide secondary structure formation, and bacterial membrane permeabilization. The results show that while secondary structure formation was not significantly impacted by the substitutions, antibacterial activity and binding to model lipid membranes were well correlated. The variants containing Leu or Phe as the sole hydrophobic groups bound bilayers with highest affinity and were most effective at inhibiting bacterial growth. Peptides with Ile exhibited intermediate behavior while those with Val or α-aminoisobutyric acid (Aib) showed poor binding and activity. The Leu, Phe, and Ile peptides demonstrated a clear preference for anionic bilayers, exhibiting significant emission spectrum shifts upon binding. Similarly, the Leu, Phe, and Ile peptides demonstrated greater ability to disrupt lipid vesicles and bacterial membranes. In total, the data indicate that hydrophobic moieties in the AMP sequence play a significant role in the binding and ability of the peptide to exhibit antibacterial activity.     

On resin amino acid side chain attachment strategy for the head to tail synthesis of new glutamine containing gramicidin-S analogs and their antimicrobial activity

The alarming increase in infections caused by multiple drug resistant bacteria including methicillin-resistant Staphylococcus aureus has prompted a desperate search for new antimicrobials. Augmenting the discoveries of completely new scaffolds with antimicrobial activity are efforts aimed at modifying existing molecules to optimize activity or reduce toxicity. We report herein the parallel solid-phase synthesis of analogues of the cationic antimicrobial peptide gramicidin S (GS) using amino acid side chain attachment strategy. The ornithine (Orn) residues were replaced by glutamine (Gln) and the aromatic d-phenylalanine (Phe) were replaced by different aromatic d-amino acids. Additional Gln containing GS analogues with all the possible combinations of the hydrophobic amino acids valine and leucine were also synthesized. In this work we also report the antibacterial activity of these analogs against several clinically-important drug-resistant Gram-positive and Gram-negative pathogens.     

Conformational Flexibility Determines Selectivity and Antibacterial, Antiplasmodial, and Anticancer Potency of Cationic ��-Helical Peptides

We used a combination of fluorescence, circular dichroism (CD), and NMR spectroscopies in conjunction with size exclusion chromatography to help rationalize the relative antibacterial, antiplasmodial, and cytotoxic activities of a series of proline-free and proline-containing model antimicrobial peptides (AMPs) in terms of their structural properties. When compared with proline-free analogs, proline-containing peptides had greater activity against Gram-negative bacteria, two mammalian cancer cell lines, and intraerythrocytic Plasmodium falciparum, which they were capable of killing without causing hemolysis. In contrast, incorporation of proline did not have a consistent effect on peptide activity against Mycobacterium tuberculosis. In membrane-mimicking environments, structures with high α-helix content were adopted by both proline-free and proline-containing peptides. In solution, AMPs generally adopted disordered structures unless their sequences comprised more hydrophobic amino acids or until coordinating phosphate ions were added. Proline-containing peptides resisted ordering induced by either method. The roles of the angle subtended by positively charged amino acids and the positioning of the proline residues were also investigated. Careful positioning of proline residues in AMP sequences is required to enable the peptide to resist ordering and maintain optimal antibacterial activity, whereas varying the angle subtended by positively charged amino acids can attenuate hemolytic potential albeit with a modest reduction in potency. Maintaining conformational flexibility improves AMP potency and selectivity toward bacterial, plasmodial, and cancerous cells while enabling the targeting of intracellular pathogens.     

Interaction studies of novel cell selective antimicrobial peptides with model membranes and E. coli ATCC 11775

Cationic antimicrobial peptides (CAMPs) are novel candidates for drug development. Here we describe design of six short and potent CAMPs (SA-1 to SA-6) based on a minimalist template of 12 residues H+HHG+HH+HH+NH2 (where H: hydrophobic amino acid and +: charged hydrophilic amino acid). Designed peptides exhibit good antibacterial activity in micro molar concentration range (1-32 μg/ml) and rapid clearance of Gram-positive and Gram-negative bacterial strains at concentrations higher than MIC. For elucidating mode of action of designed peptides various biophysical studies including CD and Trp fluorescence were performed using model membranes. Further based on activity, selectivity and membrane bound structure; modes of action of Trp rich peptide SA-3 and template based peptide SA-4 were compared. Calcein dye leakage and transmission electron microscopic studies with model membranes exhibited selective membrane active mode of action for peptide SA-3 and SA-4. Extending our work from model membranes to intact E. coli ATCC 11775 in scanning electron micrographs we could visualize different patterns of surface perturbation caused by peptide SA-3 and SA-4. Further at low concentration rapid translocation of FITC-tagged peptide SA-3 into the cytoplasm of E. coli cells without concomitant membrane perturbation indicates involvement of intracellular targeting mechanism as an alternate mode of action as was also evidenced in DNA retardation assay. For peptide SA-4 concentration dependent translocation into the bacterial cytoplasm along with membrane perturbation was observed. Establishment of a non specific membrane lytic mode of action of these peptides makes them suitable candidates for drug development.     

Tuning β-sheet peptide self-assembly and hydrogelation behavior by modification of sequence hydrophobicity and aromaticity

Peptide self-assembly leading to cross-β amyloid structures is a widely studied phenomenon because of its role in amyloid pathology and the exploitation of amyloid as a functional biomaterial. The self-assembly process is governed by hydrogen bonding, hydrophobic, aromatic π-π, and electrostatic Coulombic interactions. A role for aromatic π-π interactions in peptide self-assembly leading to amyloid has been proposed, but the relative contributions of π-π versus general hydrophobic interactions in these processes are poorly understood. The Ac-(XKXK)(2)-NH(2) peptide was used to study the contributions of aromatic and hydrophobic interactions to peptide self-assembly. Position X was globally replaced by valine (Val), isoleucine (Ile), phenylalanine (Phe), pentafluorophenylalanine (F(5)-Phe), and cyclohexylalanine (Cha). At low pH, these peptides remain monomeric because of repulsion of charged lysine (Lys) residues. Increasing the solvent ionic strength to shield repulsive charge-charge interactions between protonated Lys residues facilitated cross-β fibril formation. It was generally found that as peptide hydrophobicity increased, the required ionic strength to induce self-assembly decreased. At [NaCl] ranging from 0 to 1000 mM, the Val sequence failed to assemble. Assembly of the Phe sequence commenced at 700 mM NaCl and at 300 mM NaCl for the less hydrophobic Ile variant, even though it displayed a mixture of random coil and β-sheet secondary structures over all NaCl concentrations. β-Sheet formation for F(5)-Phe and Cha sequences was observed at only 20 and 60 mM NaCl, respectively. Whereas self-assembly propensity generally correlated to peptide hydrophobicity and not aromatic character the presence of aromatic amino acids imparted unique properties to fibrils derived from these peptides. Nonaromatic peptides formed fibrils of 3-15 nm in diameter, whereas aromatic peptides formed nanotape or nanoribbon architectures of 3-7 nm widths. In addition, all peptides formed fibrillar hydrogels at sufficient peptide concentrations, but nonaromatic peptides formed weak gels, whereas aromatic peptides formed rigid gels. These findings clarify the influence of aromatic amino acids on peptide self-assembly processes and illuminate design principles for the inclusion of aromatic amino acids in amyloid-derived biomaterials.     

De novo design and structure-activity relationships of peptide emulsifiers and foaming agents

A series of eight amphipathic peptides (8, 11, 15, 2 x 18, 22, 26, 29 amino acids in length) were designed to investigate the effects of amino acid composition, peptide length and secondary structure on surface activity assessed as emulsification and foaming activity. The potential for alpha-helix formation at the hydrophobic/hydrophilic interface was maximized through the use of helix-forming amino acids, a relatively large hydrophobic surface of 200 degrees of arc and ion pairs between basic and acidic amino acids on the hydrophilic surface. Emulsification activity increased rapidly between 11 and 22 residues as alpha-helicity in aqueous solution increased. Despite their small size, the peptides produced exceptionally stable emulsions, compared with proteins. Foaming activity was enhanced by the presence of aromatic amino acids and the activity of the best peptide examined was superior to that of bovine serum albumin and beta-lactoglobulin.     

Tyrosine replacing tryptophan as an anchor in GWALP peptides

Synthetic model peptides have proven useful for examining fundamental peptide-lipid interactions. A frequently employed peptide design consists of a hydrophobic core of Leu-Ala residues with polar or aromatic amino acids flanking each side at the interfacial positions, which serve to "anchor" a specific transmembrane orientation. For example, WALP family peptides (acetyl-GWW(LA)(n)LWWA-[ethanol]amide), anchored by four Trp residues, have received particular attention in both experimental and theoretical studies. A recent modification proved successful in reducing the number of Trp anchors to only one near each end of the peptide. The resulting GWALP23 (acetyl-GGALW(5)(LA)(6)LW(19)LAGA-[ethanol]amide) displays reduced dynamics and greater sensitivity to lipid-peptide hydrophobic mismatch than traditional WALP peptides. We have further modified GWALP23 to incorporate a single tyrosine, replacing W(5) with Y(5). The resulting peptide, Y(5)GWALP23 (acetyl-GGALY(5)(LA)(6)LW(19)LAGA-amide), has a single Trp residue that is sensitive to fluorescence experiments. By incorporating specific (2)H and (15)N labels in the core sequence of Y(5)GWALP23, we were able to use solid-state NMR spectroscopy to examine the peptide orientation in hydrated lipid bilayer membranes. The peptide orients well in membranes and gives well-defined (2)H quadrupolar splittings and (15)N/(1)H dipolar couplings throughout the core helical sequence between the aromatic residues. The substitution of Y(5) for W(5) has remarkably little influence on the tilt or dynamics of GWALP23 in bilayer membranes of the phospholipids DOPC, DMPC, or DLPC. A second analogue of the peptide with one Trp and two Tyr anchors, Y(4,5)GWALP23, is generally less responsive to the bilayer thickness and exhibits lower apparent tilt angles with evidence of more extensive dynamics. In general, the peptide behavior with multiple Tyr anchors appears to be quite similar to the situation when multiple Trp anchors are present, as in the original WALP series of model peptides.     

De novo designed cyclic cationic peptides as inhibitors of plant pathogenic bacteria

Head-to-tail cyclic peptides of 4-10 residues consisting of alternating hydrophilic (Lys) and hydrophobic (Leu and Phe) amino acids were synthesized and tested against the economically important plant pathogenic bacteria Erwinia amylovora, Xanthomonas vesicatoria and Pseudomonas syringae. The antibacterial activity, evaluated as the minimal inhibitory concentration (MIC), the cytotoxicity against human red blood cells and stability towards protease degradation were determined. The influence of cyclization, ring size, and replacement of l-Phe with d-Phe on antibacterial and hemolytic activities was studied and correlated with the degree of structuring and hydrophobicity. Our results showed that linear peptides were inactive against the three bacteria tested. Cyclic peptides were active only toward X. vesicatoria and P. syringae, being c(KLKLKFKLKQ) (BPC10L) the most active peptide with MIC values of 6.25 and 12.5 microM, respectively. The improved antibacterial activity of cyclic peptides compared to their linear counterparts was associated to an increase of the hydrophobicity, represented as RP-HPLC retention time (t(R)), and secondary structure content which are related to an enhanced amphipathicity. A decrease of antibacterial and hemolytic activities was observed when a d-Phe was introduced into the cyclic sequences, which was attributed to their low amphipathicity as shown by their low secondary structure content and low t(R). The small size, simple structure, bactericidal effect, and stability to protease degradation of the best peptides make them potential candidates for the development of effective antibacterial agents for use in plant protection.     

Novel lactoferrampin antimicrobial peptides derived from human lactoferrin

Human lactoferrampin is a novel antimicrobial peptide found in the cationic N-terminal lobe of the iron-binding human lactoferrin protein. The amino acid sequence that directly corresponds to the previously characterized bovine lactoferrin-derived lactoferrampin peptide is inactive on its own (WNLLRQAQEKFGKDKSP, residues 269-285). However, by increasing the net positive charge near the C-terminal end of human lactoferrampin, a significant increase in its antibacterial and Candidacidal activity was obtained. Conversely, the addition of an N-terminal helix cap (sequence DAI) did not have any appreciable effect on the antibacterial or antifungal activity of human lactoferrampin peptides, even though it markedly influenced that of bovine lactoferrampin. The solution structure of five human lactoferrampin variants was determined in SDS micelles and all of the structures display a well-defined amphipathic N-terminal helix and a flexible cationic C-terminus. Differential scanning calorimetry studies indicate that this peptide is capable of inserting into the hydrophobic core of a membrane, while fluorescence spectroscopy results suggest that a hydrophobic patch encompassing the single Trp and Phe residues as well as Leu, Ile and Ala side chains mediates the interaction between the peptide and the hydrophobic core of a phospholipid bilayer.     

Peptide inhibitors of melittin action

The sequence of peptides necessary to inhibit melittin-induced lysis was studied using 13 peptide analogues of the inhibitor Ac-IVIFDC-NH₂. Although this inhibitor is a disulfide-linked dimer, inhibition was equally effective if the thiol SH was blocked or replaced by methionine or lysine. The substitution of phenylalanine with other aromatic residues preserved activity, as did the replacement of aspartic acid by asparagine. The results suggest that the cytolytic activity of melittin can be inhibited by a short peptide of four hydrophobic residues followed by two other nonspecific residues. Fluorescence studies showed that the inhibitor caused a blue shift in the Trp emission spectrum. A spin label attached to the N-terminus of the inhibitor significantly quenched the fluorescence. These data confirmed the involvement of Trp 19 with the inhibitor, also predicted by molecular modeling of the probable binding site. Density gradient studies with large unilamellar vesicles indicated that the inhibitor prevented melittin from reacting with the lipid bilayer.