Structure of the bovine antimicrobial peptide indolicidin bound to dodecylphosphocholine and sodium dodecyl sulfate micelles

Indolicidin is a cationic, 13-residue antimicrobial peptide (ILPWKWPWWPWRR-NH(2)) which is unusually rich in tryptophan and proline. Its antimicrobial action involves the bacterial cytoplasmic membrane. Fluorescence and circular dichroism spectra demonstrated the structural similarity of indolicidin in complexes with large unilamellar phospolipid vesicles and with detergent micelles. The structure of indolicidin bound to zwitterionic dodecylphosphocholine (DPC) and anionic sodium dodecyl sulfate (SDS) micelles was determined using NMR methods and shown to represent a unique membrane-associated peptide structure. The backbone structure in DPC, well defined between residues 3 and 11, was extended, with two half-turns at residues Lys-5 and Trp-8. The backbone structure in SDS, well defined between residues 5 and 11, was also extended, but lacked the bend in the C-terminal half. Indolicidin in complexes with DPC had a central hydrophobic core composed of proline and tryptophan, which was bracketed by positively charged regions near the peptide termini. The tryptophan side chains, with one exception, folded flat against the peptide backbone, thus giving the molecule a wedge shape. Indolicidin in complexes with SDS had an arrangement of hydrophobic and cationic regions similar to that found in the presence of DPC. The tryptophan side chains were less well defined than for indolicidin in DPC and extended away from the peptide backbone. The preferred location of indolicidin in DPC micelles and lipid bilayers, analyzed using spin-label probes, was at the membrane interface.     

Covalent binding of the natural antimicrobial peptide indolicidin to DNA abasic sites

Indolicidin is a host defense tridecapeptide that inhibits the catalytic activity of HIV-1 integrase in vitro. Here we have elucidated its mechanism of integrase inhibition. Using crosslinking and mass spectrometric footprinting approaches, we found that indolicidin interferes with formation of the catalytic integrase-DNA complex by directly binding DNA. Further characterization revealed that the peptide forms covalent links with abasic sites. Indolicidin crosslinks single- or double-stranded DNAs and various positions of the viral cDNA with comparable efficiency. Using truncated and chemically modified peptides, we show that abasic site crosslinking is independent of the PWWP motif but involves the indolicidin unique lysine residue and the N- and C- terminal NH₂ groups. Because indolicidin can also inhibit topoisomerase I, we believe that multiple actions at the level of DNA might be a common property of antimicrobial peptides.     

Serial Expression and Activity Analysis of LNK-16: A Bovine Antimicrobial Peptide Analogue

Indolicidin is a broad-spectrum antimicrobial peptide (AMP) with great therapeutic potential; however, high manufacturing costs associated with industrial-scale chemical synthesis have limited its delivery. Therefore, the use of recombinant DNA technology to produce this peptide is urgently needed. In this study, a new methodology for the large-scale production of a novel bovine AMP was developed. LNK-16 is an analogue of indolicidin that contains a kallikrein protease site at its C-terminus. The amino acid sequence of LNK-16 was synthesized using Escherichia coli-preferred codons. Three copies of the target gene were assembled in series by overlapping PCR and cloned into pET-30a(+) for the expression of His-(LNK-16)₃ in E. coli BL21 (DE3) cells. The expressed fusion protein His-(LNK-16)₃ was purified by Ni²⁺-chelating chromatography and then cleaved by kallikrein to release LNK-16. The recombinant LNK-16 peptide showed antimicrobial activity similar to that of chemically synthesized LNK-16 and indolicidin. Together, these data indicate that the use of serial expression can improve the large-scale production of AMPs for clinical and research applications.     

Structure and mechanism of action of an indolicidin peptide derivative with improved activity against gram-positive bacteria

Indolicidin, an antimicrobial peptide with a unique amino acid sequence (ILPWKWPWWPWRR-NH(2)) is found in bovine neutrophils. A derivative of indolicidin, CP10A, has alanine residues substituted for proline residues and has improved activity against Gram-positive organisms. Transmission electron microscopy of Staphylococcus aureus and Staphylococcus epidermidis treated with CP10A showed mesosome-like structures in the cytoplasm. The peptide at 2-fold the minimal inhibitory concentration did not show significant killing of S. aureus ISP67 (a histidine, uridine, and thymidine auxotroph) but did show an early effect on histidine and uridine incorporation and, later, an effect on thymidine incorporation. Upon interaction with liposomes, detergents, and lipoteichoic acid, CP10A was shown by circular dichroism spectroscopy to undergo a change in secondary structure. Fluorescence spectroscopy indicated that the tryptophan residues were located at the hydrophobic/hydrophilic interface of liposomes and detergent micelles and were inaccessible to the aqueous quencher KI. The three-dimensional structure of CP10A in the lipid mimetic dodecylphosphocholine was determined using two-dimensional NMR methods and was characterized as a short, amphipathic helical structure, whereas indolicidin was previously shown to have an extended structure. These studies have introduced a cationic peptide with a unique structure and an ability to interact with membranes and to affect intracellular synthesis of proteins, RNA, and DNA.     

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.     

Antimicrobial peptides derived from different animals: comparative studies of antimicrobial properties,cytotoxicity and mechanism of action

Animals posses a large variety of antimicrobial peptides (AMPs) that serve as effective components in innate host defenses against microbial infections. These antimicrobial peptides differ in amino acid composition, range of antimicrobial specificities, hemolysis, cytotoxicity and mechanisms of action. This study was designed to evaluate their therapeutic potential of the following six antimicrobial peptides initially found from animals: cecropin P1, indolicidin, LL-37, palustrin-OG1, LFP-20 and LFB-11. Our results indicated that cecropin P1 possessed the most desired biological activity, with fast and potent antimicrobial activity but only slight hemolytic or cytotoxic activity against human cells. Indolicidin was more effective against gram-positive bacteria but with higher hemolytic and cytotoxic activity on human peripheral blood mononuclear cell (PBMCs) (P < 0.05). Although LFP-20 and LFB-11 had moderate activity against tested strains and need 30 min to kill E. coli, they showed almost no hemolytic and cytotoxic activity towards PBMCs (P < 0.01). Indolicidin could form pores of well-defined structure in bacterial membranes whereas lysis of E. coli cells was observed after addition LFB-11 and LL-37 at 1 × MIC for 1 h. LL-37 treatment could lead to the leakage of entire bacterial cytoplasmic contents. The most obvious phenomenon was protuberant structures on the E. coli cell surface after incubation with LFP-20, cecropin P1 and palustrin-OG1. The results presented here illustrate that AMPs derived from different animals exhibited different antimicrobial characteristics. Because of their potent and broad-spectrum antimicrobial activity, low cytotoxicity towards normal cells, and the unique mechanism of action, these peptides may provide the impetus for the development of novel strategies for the prevention of bacterial infections in animals.     

Indolicidin,a 13-residue basic antimicrobial peptide rich in tryptophan and proline,interacts with Ca(2+)-calmodulin

Indolicidin, ILPWKWPWWPWRR-NH(2), a short 13-residue antimicrobial and cytolytic peptide characterized from bovine neutrophils, has the calmodulin-recognition 1-5-10 hydrophobic pattern (indicated by amino acids in bold), is cationic, and thereby fulfills the requirements to interact with calmodulin. Hence, we have investigated the calmodulin-binding properties of indolicidin. Indolicidin interacted with calmodulin with fairly high affinity in a Ca(2+)-dependent manner. However, when bound, the peptide did not adopt helical conformation. Indolicidin also inhibited calmodulin-stimulated phosphodiesterase activity with IC(50) values in the nanomolar range. Replacement of either the proline residues of indolicidin with alanines or tryptophan residues with phenylalanines did not affect binding to calmodulin. However, these replacements had distinctive effects on the conformations of the bound peptides. While the alanine analog of indolicidin adopted predominantly alpha-helical conformation, the phenylalanine analog remained largely unordered. Differences in the ability of these analogs to inhibit the calmodulin-stimulated phosphodiesterase activity were observed. While the alanine analog was capable of inhibiting the activity with IC(50) values comparable to that of indolicidin, the phenylalanine analog did not inhibit the activity. Our results indicate that ability to adopt amphiphilic alpha-helical structure is not a prerequisite for binding to calmodulin and also binding does not necessarily result in inhibition of calmodulin-stimulated enzyme activities.     

Formation and characterization of a single Trp-Trp cross-link in indolicidin that confers protease stability without altering antimicrobial activity

Indolicidin is a 13-residue cationic, antimicrobial peptide-amide isolated from the cytoplasmic granules of bovine neutrophils. The unique composition of indolicidin distinguishes it from alpha-helical and beta-structured cationic peptides, because five of indolicidin's 13 residues are tryptophans: H-Ile-Leu-Pro-Trp-Lys-Trp-Pro-Trp-Trp-Pro-Trp-Arg-Arg-NH(2). Solid phase synthesis of indolicidin gave rise to a minor byproduct that possessed unusual fluorescence and UV absorbance properties compared with authentic indolicidin. The byproduct was purified by combined ion exchange and reversed phase high pressure liquid chromatography steps and was shown be identical to authentic indolicidin in its microbicidal activity against Staphylococcus aureus, Escherichia coli, Candida albicans, and Cryptococcus neoformans. Mass analysis of the byproduct revealed a 2-atomic mass unit reduction compared with indolicidin, suggesting the deprotonation of two indole side chains to form an intrachain delta(1),delta(1)'-ditryptophan derivative. We confirmed the nature of the cross-linked byproduct, termed X-indolicidin, by absorbance and fluorescence spectroscopy, peptide mapping, and sequence analysis. Edman degradation revealed that Trp-6 and Trp-9 were covalently cross-linked. Compared with indolicidin, X-indolicidin was partially resistant to digestion with trypsin and chymotrypsin, suggesting that the ditryptophan stabilizes a subset of molecular conformations that are protease resistant but that are absent in the native structure.     

Structure-function relationships in the tryptophan-rich, antimicrobial peptide indolicidin.

Indolicidin is a cationic 13 amino acid peptide amide produced in the granules of bovine neutrophils with the sequence H-ILPWKWPWWPWRR-NH2. Indolicidin is both antimicrobial and, to a lesser extent, haemolytic. In order to systematically investigate structure-function relationships, the solid-phase synthesis of indolicidin and 48 distinct analogues are reported, as well as the characterization of their respective biological properties. Peptides synthesized and characterized include analogues with modified terminal functions, truncations from either terminus, an alanine scan to determine the role of each individual amino acid, specific amino acid exchanges of aromatic, charged and structural residues and several retro-, inverso- and retroinverso-analogues. Together, characterization of these analogues identifies specific residues involved in antimicrobial or haemolytic activity and suggests a core structure that may form a scaffold for the further development of peptidomimetic analogues of indolicidin.     

Comparison of the membrane association of two antimicrobial peptides, magainin 2 and indolicidin

Interactions of two antimicrobial peptides, magainin 2 and indolicidin, with three different model biomembranes, namely, monolayers, large unilamellar vesicles (LUVs), and giant liposomes, were studied. Insertion of both peptides into lipid monolayers was progressively enhanced when the content of an acidic phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) in a film of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) was increased. Indolicidin and magainin 2 penetrated also into lipid monolayers containing cholesterol (mole fraction, X = 0.1). Membrane association of magainin 2 attenuated lipid lateral diffusion in POPG-containing LUVs as revealed by the decrease in the excimer/monomer fluorescence ratio I(e)/I(m) for the pyrene fatty-acid-containing phospholipid derivative 1-palmitoyl-2-[10-(pyren-1-yl) decanoyl]-sn-glycero-3-phospho-rac-glycerol (PPDPG). Likewise, an increase in steady-state fluorescence anisotropy of the membrane-incorporated diphenylhexatriene (DPH) was observed, revealing magainin 2 to increase acyl chain order and induce segregation of acidic phospholipids. Similar effects were observed for indolicidin. The topological effects of magainin 2 and indolicidin on phospholipid membranes were investigated using optical microscopy of giant vesicles. Magainin 2 had essentially no influence on either SOPC or SOPC:cholesterol (X = 0.1) giant liposomes. However, effective vesiculation was observed when acidic phospholipid (X(PG) = 0.1) was included in the giant vesicles. Indolicidin caused only a minor shrinkage of giant SOPC vesicles whereas the formation of endocytotic vesicles was observed when the giant liposome contained POPG (X(PG) = 0.1). Interestingly, for indolicidin, vesiculation was also observed for giant vesicles composed of SOPC/cholesterol (X(chol) = 0.1). Possible mechanisms of membrane transformation induced by these two peptides are discussed.     

Interactions of tryptophan-rich cathelicidin antimicrobial peptides with model membranes studied by differential scanning calorimetry

The 13-residue cathelicidins indolicidin and tritrpticin are part of a group of relatively short tryptophan-rich antimicrobial peptides that hold potential as future substitutes for antibiotics. Differential scanning calorimetry (DSC) has been applied here to study the effect of indolicidin and tritrpticin as well as five tritrpticin analogs on the phase transition behaviour of model membranes made up of zwitterionic dimyristoylphosphatidylcholine (DMPC, DMPC/cholesterol) and anionic dimyristoylphosphatidyl glycerol (DMPG) phospholipids. Most of the peptides studied significantly modified the phase transition profile, suggesting the importance of hydrophobic forces for the peptide interactions with the lipid bilayers and their insertion into the bilayer. Indolicidin and tritrpticin are both known to be flexible in aqueous solution, but they adopt turn-turn structures when they bind to and insert in a membrane surface. Pro-to-Ala substitutions in tritrpticin, which result in the formation of a stable α-helix in this peptide, lead to a substantial increase in the peptide interactions with both zwitterionic and anionic phospholipid vesicles. In contrast, the substitution of the three Trp residues by Tyr or Phe resulted in a significant decrease of the peptide's interaction with anionic vesicles and virtually eliminated binding of these peptides to the zwitterionic vesicles. An increase of the cationic charge of the peptide induced much smaller changes to the peptide interaction with all lipid systems than substitution of particular amino acids or modification of the peptide conformation. The presence of multiple lipid domains with a non-uniform peptide distribution was noticed. Slow equilibration of the lipid-peptide systems due to peptide redistribution was observed in some cases. Generally good agreement between the present DSC data and peptide antimicrobial activity data was obtained.     

Interactions of tryptophan-rich cathelicidin antimicrobial peptides with model membranes studied by differential scanning calorimetry

The 13-residue cathelicidins indolicidin and tritrpticin are part of a group of relatively short tryptophan-rich antimicrobial peptides that hold potential as future substitutes for antibiotics. Differential scanning calorimetry (DSC) has been applied here to study the effect of indolicidin and tritrpticin as well as five tritrpticin analogs on the phase transition behaviour of model membranes made up of zwitterionic dimyristoylphosphatidylcholine (DMPC, DMPC/cholesterol) and anionic dimyristoylphosphatidyl glycerol (DMPG) phospholipids. Most of the peptides studied significantly modified the phase transition profile, suggesting the importance of hydrophobic forces for the peptide interactions with the lipid bilayers and their insertion into the bilayer. Indolicidin and tritrpticin are both known to be flexible in aqueous solution, but they adopt turn-turn structures when they bind to and insert in a membrane surface. Pro-to-Ala substitutions in tritrpticin, which result in the formation of a stable alpha-helix in this peptide, lead to a substantial increase in the peptide interactions with both zwitterionic and anionic phospholipid vesicles. In contrast, the substitution of the three Trp residues by Tyr or Phe resulted in a significant decrease of the peptide's interaction with anionic vesicles and virtually eliminated binding of these peptides to the zwitterionic vesicles. An increase of the cationic charge of the peptide induced much smaller changes to the peptide interaction with all lipid systems than substitution of particular amino acids or modification of the peptide conformation. The presence of multiple lipid domains with a non-uniform peptide distribution was noticed. Slow equilibration of the lipid-peptide systems due to peptide redistribution was observed in some cases. Generally good agreement between the present DSC data and peptide antimicrobial activity data was obtained.     

An optimized Boc synthesis of indolicidin

Summary Indolicidin, an antimicrobial peptide from bovine neutrophils containing five tryptophan out of a total of 13 residues, has the highest molar proportion of tryptophan of any known peptide sequence and is thus considered a difficult synthetic target. Conventional Boc chemistry can be applied to the synthesis of indolicidin with an appropriate choice of scavenger mixtures, reaction times and temperatures at the crucial acidolytic cleavage and deprotection step. In particular, treatment with HF/p-cresol/p-thiocresol (90:7:3) for 40 min at −8°C results in a crude product containing ca. 90% indolicidin, from which the target compound can be isolated in satisfactory yields and purity after reverse-phase purification. The main byproducts arising during the synthesis and cleavage steps have been identified by HPLC with on-line electrospray mass spectrometric detection.     

Antimicrobial activity of the indolicidin‐derived novel synthetic peptide In‐58

Natural peptides with antimicrobial activity are extremely diverse, and peptide synthesis technologies make it possible to significantly improve their properties for specific tasks. Here, we investigate the biological properties of the natural peptide indolicidin and the indolicidin‐derived novel synthetic peptide In‐58. In‐58 was generated by replacing all tryptophan residues on phenylalanine in D‐configuration; the α‐amino group in the main chain also was modified by unsaturated fatty acid. Compared with indolicidin, In‐58 is more bactericidal, more resistant to proteinase K, and less toxic to mammalian cells. Using molecular physics approaches, we characterized the action of In‐58 on bacterial cells at the cellular level. Also, we have found that studied peptides damage bacterial membranes. Using the Escherichia coli luminescent biosensor strain MG1655 (pcolD’::lux), we investigated the action of indolicidin and In‐58 at the subcellular level. At subinhibitory concentrations, indolicidin and In‐58 induced an SOS response. Our data suggest that indolicidin damages the DNA, but bacterial membrane perturbation is its principal mode of action. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

An optimized Boc synthesis of indolicidin

Indolicidin, an antimicrobial peptide from bovine neutrophils containing five tryptophan out of a total of 13 residues, has the highest molar proportion of tryptophan of any known peptide sequence and is thus considered a difficult synthetic target. Conventional Boc chemistry can be applied to the synthesis of indolicidin with an appropriate choice of scavenger mixtures, reaction times and temperatures at the crucial acidolytic cleavage and deprotection step. In particular, treatment with HF/p-cresol/p-thiocresol (90:7:3) for 40 min at –8 °C results in a crude product containing ca. 90% indolicidin, from which the target compound can be isolated in satisfactory yields and purity after reverse-phase purification. The main byproducts arising during the synthesis and cleavage steps have been identified by HPLC with on-line electrospray mass spectrometric detection.     

Increased pathogen resistance and yield in transgenic plants expressing combinations of the modified antimicrobial peptides based on indolicidin and magainin

Reverse peptide of indolicidin (Rev4), a 13-residue peptide based on the sequence of indolicidin, has been shown to possess both strong antimicrobial and protease inhibitory activities in vitro. To evaluate its efficacy in vivo, we produced and evaluated transgenic tobacco (Nicotiana tabacum L.) and Arabidopsis thaliana [(L.) Heynh.] plants expressing Rev4 with different signal peptide sequences for pathogen resistance. All transgenic plants showed normal growth and development, an indication of no or low cytotoxicity of the peptide. Furthermore, the transgenic plants exhibited elevated resistance to three bacterial and two oomycete pathogens. Interestingly, tobacco plants expressing Rev4 displayed enhanced yield compared to the control as indicated by an increased biomass production by as much as 34% in two field trials. When Rev4 was coexpressed with another antimicrobial peptide, Myp30, the disease resistance levels in the transgenic Arabidopsis were enhanced. These findings suggest the potential of using these peptides to protect plants from microbial pathogens and to enhance yield.     

Bacterial selectivity and plausible mode of antibacterial action of designed Pro-rich short model antimicrobial peptides.

To develop novel Pro-rich model AMPs with shorter length and higher bacterial selectivity/therapeutic index (TI) than natural AMP, indolicidin, we synthesized a series of undodecapeptides derived from the sequence XXPXXPWXPXX-NH2 (X indicates Leu or Lys) with different ratios of Lys and Leu residues. Several Pro-rich model peptides (K7 WP3, K6 WL1 P3, K5 WL2 P3-1, K5 WL2 P3-2, and K4 WL3 P3) had approximate 8- to 11-fold higher bacterial selectivity/TI compared to indolicidin. These peptides selectively bind to negatively charged liposomes (EYPG/EYPG; 7:3, w/w) mimicking bacterial membranes. Their high selectivity to negatively charged phospholipids corresponds well with their high bacterial selectivity. Indolicidin showed almost complete depolarization of the cytoplasmic membrane of Staphylococcus aureus and dye-leakage from negatively charged liposomes at 10 microM, whereas all of Pro-rich model peptides had very little activity in these assays even at 80 microM, as observed in buforin 2. These results suggest that the ultimate target of our designed Pro-rich model peptides is probably the intracellular components (e.g. protein, DNA or RNA) rather than the cytoplasmic membranes. Collectively, our designed Pro-rich short model peptides appear to be excellent candidates for future development as a novel antimicrobial agent.     

Interaction of indolicidin, a 13-residue peptide rich in tryptophan and proline and its analogues with model membranes

Indolicidin is a 13-residue broad-spectrum antibacterial peptide isolated from bovine neutrophils. The primary structure of the peptide ILPWKWPWWPWRR-amide (IL) reveals an unusually high percentage of tryptophan residues. IL and its analogues where proline residues have been replaced by alanine (ILA) and trp replaced by phe (ILF) show comparable antibacterial activitieso While IL and ILA are haemolytic, ILF does not have this property. Since aromatic residues would strongly favour partitioning of the peptide into the lipid bilayer interface, the biological activities of IL and its analogues could conceivably arise due perturbation of the lipid bilayer of membranes. We have therefore investigated the interaction of IL and its analogues with lipid vesicles. Peptides IL and ILA bind to lipid vesicles composed of phosphatidylcholine and phosphatidylethanol amine: phosphatidyl glycerol: cardiolipin. The position of λ_max and I^- quenching experiments suggest that the trp residues are localized at the membrane interface and not associated with the hydrophobic core of the lipid bilayer in both the peptides. Hence, membrane permeabilization is likely to occur due to deformation of the membrane surface rather than formation of transmembrane channels by indolicidin and its analogues. Peptides ILA, IL and ILF cause the release of entrapped carboxyfluorescein from phosphatidyl choline vesicles. The peptide-lipid ratios indicate that ILF is less effective than IL and ILA in permeabilizing lipid vesicles, correlating with their haemolytic activities. An erratum to this article is available at .     

CD spectra of indolicidin antimicrobial peptides suggest turns, not polyproline helix.

Indolicidin is a 13-residue antimicrobial peptide-amide isolated from the cytoplasmic granules of bovine neutrophils that contains five Trp and three Pro residues. Falla et al. [(1996) J. Biol. Chem. 271, 19298] suggested that indolicidin forms a poly-L-proline II helix based upon the circular dichroism (CD) spectra of a closely related peptide (indolicidin methyl ester). In contrast, we found no evidence of poly-L-proline II helix formation in the CD spectra of native indolicidin in various solvents or when bound to micelles and membranes [Ladokhin et al. (1997) Biophys. J. 72, 794]. We interpreted the spectra as arising from unordered and/or beta-turn structures, but noted a sharp negative band at 227 nm arising from the tryptophan residues that would mask spectral features characteristic of poly-L-proline II helix. We have reexamined this issue by means of CD measurements of native indolicidin and several of its analogues. None of the features characteristic of a poly-L-proline helix (or alpha- or 3(10)-helix) were observed for any of the peptides studied. To eliminate artifacts associated with tryptophan, we synthesized indolicidin-L and indolicidin-F in which all five tryptophans were replaced with leucines or phenylalanines, respectively. The changes in CD spectra of these Trp-free peptides upon transfer into membrane-like environments were found to be consistent with the formation of beta-turns. For the native indolicidin in SDS micelles, temperature increases resulted in a coupled diminution of two sharp bands, a negative one at 227 nm and a positive one at 217 nm. This phenomenon, which is absent in indolicidin-L variants with single Leu-->Trp substitutions, is consistent with exciton splitting produced by the stacking of indole rings. Type VI turns in model peptides in aqueous solution are known to be promoted by stacking interactions between cis-proline and neighboring aromatic residues [Yao et al. (1994) J. Mol. Biol. 243, 754]. Molecular modeling of indolicidin with a -Trp(6)-cis-Pro(7)-Trp(8)- type VIa turn demonstrated the feasibility of this turn conformation and revealed the possibility of an accompanying amphipathic structure. We therefore suggest that turn conformations are the principal structural motif of indolicidin and that these turns greatly enhance membrane activity.     

Combinatorial microscopy for the study of protein–membrane interactions in supported lipid bilayers: Order parameter measurements by combined polarized TIRFM/AFM

Understanding the mechanisms of peptide-induced membrane disorder is critical to the design of novel antimicrobial and cell-penetrating peptides. One means of quantifying local structure and order/disorder is through the orientational order parameter, typically obtained using various spectroscopic approaches. We report here on the use of an image-based means of tracking the order parameter in supported lipid bilayers during peptide-induced disordering. By coupling polarized total internal reflection fluorescence microscopy with in situ atomic force microscopy, it is now possible to track changes in order parameter associated with peptide binding and insertion, as well as lipid headgroup and acyl chain reordering, while simultaneously resolving molecular-scale topographical changes. Interactions between the model antimicrobial peptide, indolicidin, and its fluorescent analog, TAMRA-indolicidin, with model eukaryotic (DOPC:DSPC:cholesterol) and prokaryotic (DOPE/DOPG) membranes were tracked using the fluorescent lipid reporters, DiI-C₂₀ and BODIPY-PC. Changes in the order parameter upon membrane binding and insertion provided insights into the orientation of the peptide and the role of membrane chemistry and composition on insertion dynamics and membrane restructuring.