Antimicrobial activity of short arginine- and tryptophan-rich peptides.

Highly antimicrobial active arginine- and tryptophan-rich peptides were synthesized ranging in size from 11 to five amino acid residues in order to elucidate the main structural requirement for such short antimicrobial peptides. The amino acid sequences of the peptides were based on previous studies of longer bovine and murine lactoferricin derivatives. Most of the peptides showed strong inhibitory action against the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, and the Gram-positive bacterium Staphylococcus aureus. For the most active derivatives, the minimal inhibitory concentration values observed for the Gram-negative bacteria were 5 microg/ml (3.5 microM), whereas it was 2.5 microg/ml (1.5 microM) for the Gram-positive bacterium. It was essential for the antimicrobial activity that the peptides contained a minimum of three tryptophan and three arginine residues, and carried a free N-terminal amino group and an amidated C-terminal end. Furthermore, a minimum sequence size of seven amino acid residues was required for a high antimicrobial activity against Pseudomonas aeruginosa. The insertion of additional arginine and tryptophan residues into the peptides resulted only in small variations in the antimicrobial activity, whereas replacement of a tryptophan residue with tyrosine in the hepta- and hexapeptides resulted in reduced antimicrobial activity, especially against the Gram-negative bacteria. The peptides were non-haemolytic, making them highly potent as prospective antibiotic agents.     

Anti‐biofilm and sporicidal activity of peptides based on wheat puroindoline and barley hordoindoline proteins

The broad‐spectrum activity of antimicrobial peptides (AMPs) and low probability of development of host resistance make them excellent candidates as novel bio‐control agents. A number of AMPs are found to be cationic, and a small proportion of these are tryptophan‐rich. The puroindolines (PIN) are small, basic proteins found in wheat grains with proposed roles in biotic defence of seeds and seedlings. Synthetic peptides based on their unique tryptophan‐rich domain (TRD) display antimicrobial properties. Bacterial endospores and biofilms are highly resistant cells, with significant implications in both medical and food industries. In this study, the cationic PIN TRD‐based peptides PuroA (FPVTWRWWKWWKG‐NH₂) and Pina‐M (FSVTWRWWKWWKG‐NH₂) and the related barley hordoindoline (HIN) based Hina (FPVTWRWWTWWKG‐NH₂) were tested for effects on planktonic cells and biofilms of the common human pathogens including Pseudomonas aeruginosa, Listeria monocytogenes and the non‐pathogenic Listeria innocua. All peptides showed significant bactericidal activity. Further, PuroA and Pina‐M at 2 × MIC prevented initial biomass attachment by 85–90% and inhibited >90% of 6‐h preformed biofilms of all three organisms. However Hina, with a substitution of Lys‐9 with uncharged Thr, particularly inhibited Listeria biofilms. The PIN based peptides were also tested against vegetative cells and endospores of Bacillus subtilis. The results provided evidence that these tryptophan‐rich peptides could kill B. subtilis even in sporulated state, reducing the number of viable spores by 4 log units. The treated spores appeared withered under scanning electron microscopy. The results establish the potential of these tryptophan‐rich peptides in controlling persistent pathogens of relevance to food industries and human health. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Development of indole chemistry to label tryptophan residues in protein for determination of tryptophan surface accessibility

Solvent accessibility can be used to evaluate protein structural models, identify binding sites, and characterize protein conformational changes. The differential modification of amino acids at specific sites enables the accessible surface residues to be identified by mass spectrometry. Tryptophan residues within proteins can be differentially labeled with halocompounds by a photochemical reaction. In this study, tryptophan residues of carbonic anhydrase are reacted with chloroform, 2,2,2-trichloroethanol (TCE), 2,2,2-trichloroacetate (TCA), or 3-bromo-1-propanol (BP) under UV irradiation at 280 nm. The light-driven reactions with chloroform, TCE, TCA, and BP attach a formyl, hydroxyethanone, carboxylic acid, and propanol group, respectively, onto the indole ring of tryptophan. Trypsin and chymotrypsin digests of the modified carbonic anhydrase are used to map accessible tryptophan residues using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Tryptophan reactivity is determined by identifying peptides with tryptophan residues modified with the appropriate label. The reactivity is calculated from the frequency that the modification is identified and a semiquantitative measure of the amount of products formed. Both of these measures of tryptophan reactivity correlate significantly with the accessible surface area of tryptophan residues in carbonic anhydrase determined from the X-ray crystal structure. Therefore the photochemical reaction of halocompounds with tryptophan residues in carbonic anhydrase indicates the degree of solvent accessibility of these residues.     

Synthesis of peptides containing 5‐hydroxytryptophan,oxindolylalanine, N‐formylkynurenine and kynurenine

ROS, continuously produced in cells, can reversibly or irreversibly oxidize proteins, lipids, and DNA. At the protein level, cysteine, methionine, tryptophan, and tyrosine residues are particularly prone to oxidation. Here, we describe the solid phase synthesis of peptides containing four different oxidation products of tryptophan residues that can be formed by oxidation in proteins in vitro and in vivo: 5‐HTP, Oia, Kyn, and NFK. First, we synthesized Oia and NFK by selective oxidation of tryptophan and then protected the ${\bf \alpha}$ ‐amino group of both amino acids, and the commercially available 5‐HTP, with Fmoc‐succinimide. High yields of Fmoc‐Kyn were obtained by acid hydrolysis of Fmoc‐NFK. All four Fmoc derivatives were successfully incorporated, at high yields, into three different peptide sequences from skeletal muscle actin, creatin kinase (M‐type), and ${\bf \beta}$ ‐enolase. The correct structure of all modified peptides was confirmed by tandem mass spectrometry. Interestingly, isobaric peptides containing 5‐HTP and Oia were always well separated in an acetonitrile gradient with TFA as the ion‐pair reagent on a C₁₈‐phase. Such synthetic peptides should prove useful in future studies to distinguish isobaric oxidation products of tryptophan. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Helical structure and packing orientation of the S2 segment in the Shaker K+ channel

Six transmembrane segments, S1-S6, cluster around the central pore-forming region in voltage-gated K+ channels. To investigate the structural characteristics of the S2 segment in the Shaker K+ channel, we replaced each residue in S2 singly with tryptophan (or with alanine for the native tryptophan). All but one of the 23 Trp mutants expressed voltage-dependent K+ currents in Xenopus oocytes. The effects of the mutations were classified as being of low or high impact on channel gating properties. The periodicity evident in the effects of these mutations supports an alpha-helical structure for the S2 segment. The high- and low-impact residues cluster onto opposite faces of a helical wheel projection of the S2 segment. The low-impact face is also tolerant of single mutations to asparagine. All results are consistent with the idea that the low-impact face projects toward membrane lipids and that changes in S2 packing occur upon channel opening. We conclude that the S2 segment is a transmembrane alpha helix and that the high-impact face packs against other transmembrane segments in the functional channel.     

Tryptophan-130 is the most reactive tryptophan residue in rabbit skeletal myosin subfragment-1

Rabbit skeletal muscle myosin subfragment-1 (S-1) was reacted with dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide (DHNBS) resulting in modification of 0.8 tryptophan residues per S-1. In order to assign the most reactive tryptophan of the 5 S-1 tryptophans, antibodies were raised in rabbits against bovine serum albumin modified with DHNBS. The antibodies reacted with the 27 kDa tryptic fragment of DHNBS-treated S-1, indicating that the reactive tryptophan resides on this domain. The 27 kDa fragment was isolated from DHNBS-treated S-1 and was further cleaved at a single cysteine residue by 2-nitro-5-thiocyanobenzoic acid. This cleavage resulted in two peptides, each of them containing one tryptophan. The antibodies reacted with the smaller peptide consisting of residues 122-204. The only tryptophan residing on this peptide is Trp130, and this is therefore the most reactive tryptophan of S-1.     

A procedure for easy removal of acrylamide gel rods from the casting glass tubes

A new method specific for the determination of subpicomole quantities of tryptophan has been developed by elaboration of the Pictet-Spengler reaction. It permitted reproducible quantitation of tryptophan in less than 1 μl of plasma ultrafiltrate or 1 mg of brain tissue. Samples deproteinized by trichloroacetic acid were boiled for 15 min with formaldehyde and potassium ferricyanide at controlled acidity, where tryptophan was converted to a single new product identified as 9-hydroxymethyl-β-carboline. It was quantitated by either direct spectrofluorometry or a reversed-phase HPLC system developed for β-carbolines. Under our conditions, peptides containing N-terminal tryptophan such as Trp-Leu and delta sleep-inducing peptide gave N-(9-hydroxymethyl-β-carboline-3-carbonyl) peptides which retained all amino acid residues except tryptophan.     

Effect of modification on physico-chemical and biological properties of haptoglobin. Reaction with N-bromusuccinimide and 2-hydroxy-5-nitrobenzyl bromide.

N-Bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide have been used for modification of tryptophan residues in human haptoglobin (Hp) type 2-1. Modification of three exposed tryptophan residues reduced considerably both the Hp-haemoglobin interaction and binding of the antibody against the native protein. Modification of the remaining 7-8 tryptophan residues resulted in a complete loss of those properties. Antisera directed against Hp with the modified tryptophan residues appeared to be highly specific in immunological reactions.     

Identification of amino acids involved in the Flo11p-mediated adhesion of Saccharomyces cerevisiae to a polystyrene surface using phage display with competitive elution

AIMS: To identify the main amino acids involved in the Flo11p-mediated adhesion of Saccharomyces cerevisiae to the polystyrene surface PolySorp. METHODS AND RESULTS: Using a combination of phage display and competitive elution revealed that 12-mer peptides of phages from competitive panning with S. cerevisiae FLO11 wild-type (TBR1) cells had a higher consensus than those from competitive panning with S. cerevisiae flo11Delta mutant (TBR5) cells, suggesting that the wild-type cells interact with the plastic surface in a stronger and more similar way than the mutant cells. Tryptophan and proline were more abundant in the peptides of phages from competitive elution with FLO11 cells than in those from competitive elution with flo11Delta cells. Furthermore, two phages with hydrophobic peptides containing 1 or 2 tryptophan, and 3 or 5 proline, residues inhibited the adhesion of FLO11 cells to PolySorp more than a phage with a hydrophobic peptide containing no tryptophan and only two proline residues. CONCLUSIONS: Our results suggest a key role of tryptophan and proline in the hydrophobic interactions between Flo11p on the S. cerevisiae cell surface and the PolySorp surface. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study may contribute to the development of novel strategies to limit yeast infections in hospitals and other medical environments.     

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.     

Inactivation of Lysozyme By Alkylperoxyl Radicals

Thermolysis of 2, 2'-azo-bis-(2-amidinopropane) under air in the presence of lysozyme leads to extensive inactivation of the enzyme. The number of inactivated enzyme molecules per radical produced increases with the enzyme concentration up to values considerably larger than one. Enzyme inacitivation is accompanied by extensive tryptophan modification. Over the enzyme concentration range considered (1.7 to 130μM) nearly 4 tryptophan groups are modified per enzyme molecule inactivated. Both the inactivation and tryptophan modification are prevented by micromolar concentrations of propyl gallate. The results are interpreted in terms of an efficient inactivation of the enzyme by the alkylperoxyl radicals generated by thermolysis of the azocompound.     

Geranyl modification on the tryptophan residue of ComXRO-E-2 pheromone by a cell-free system

ComX pheromone is an isoprenoidal oligopeptide containing a modified tryptophan residue, which stimulates natural genetic competence in the gram-positive bacterium Bacillus. Since posttranslational prenylation on the tryptophan residue has not been reported except in ComX pheromone, the universality of this modification has not yet been elucidated. In this paper, we established a cell-free system, whereby the tryptophan residue in peptides is modified with a geranyl group by modifying enzyme ComQ. In addition, we investigated enzymatic reaction conditions using an in vitro enzyme reaction system. This is the first report of in vitro geranylation on the tryptophan residue. This system is potentially a useful tool for elucidating the universality of prenylation on the tryptophan residue.     

The lysine binding sites of human plasminogen. Evidence for a critical tryptophan in the binding site of kringle 4

Chemical modification of human degraded form of plasminogen with NH2-terminal lysine (Lys-plasminogen) and the elastase fragments kringle 1 + 2 + 3 and kringle 4 with the tryptophan reagent [14C]dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide results in the incorporation of label and the parallel loss of lysine binding ability. In the case of kringle 4, only one-half of the lysine binding sites could be inactivated, but the modified and unmodified forms could be separated by affinity chromatography. The modified form contained 1 mol of 2-hydroxy-5-nitrobenzyl groups/mol of kringle 4 and did not bind to lysine-Sepharose. Lysine analogs such as 6-aminohexanoic acid protected kringle 4 against modification. Peptide-mapping studies on this form showed that essentially all of the label was in two chymotryptic peptides containing a tryptophan corresponding to Trp426 in the plasminogen sequence. Competition experiments with anti-kringle 4 antibodies having an affinity for the lysine binding site showed that the binding of 2-hydroxy-5-nitrobenzyl-kringle 4 to antibodies was about 10 times weaker than for unmodified kringle 4. These results indicate that the integrity of specific tryptophan residue is critical to the binding of lysine and related amino acids to kringle 4of human plasminogen.     

Synthesis and characterization of β‐peptide helices as transmembrane domains in lipid model membranes

Aggregation, orientation and dynamics of transmembrane helices are of relevance for protein function and transmembrane signaling. To explore the interactions of transmembrane helices and the interdependence of peptide structure and lipid composition of the membranes, β‐peptides were explored as model transmembrane domains. Various hydrophobic β‐peptide sequences were synthesized by solid phase peptide synthesis. Conformational analyses of β‐peptide helices were performed in organic solvents (methanol and 2,2,2‐trifluoroethanol) and in large unilamellar liposomes (dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine and dioleoylphosphatidylcholine) indicating 12‐ and 14‐helix conformations, depending on β³‐amino acid sequences. The intrinsic tryptophan fluorescence of β³‐homotryptophan units inserted in the center or near the end of the sequence was used to verify the membrane insertion of the β‐peptides. A characteristic blue shift with peripheral β³‐homotryptophan compared with β‐peptides with central tryptophan served as indication for a transmembrane orientation of the β‐peptides within the lipid bilayer. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Towards structural determination of the ComX pheromone: synthetic studies on peptides containing geranyltryptophan

Bacteria produce and respond to signal molecules depending on their cell density. This process is called "quorum sensing". The ComX pheromone, controlled by quorum sensing, activates natural genetic competence in Bacillus subtilis. ComX is an oligopeptide with a posttranslational modification. It has been suggested that ComX pheromone is modified with an isoprenoid at its tryptophan residue, but the complete chemical structure is unknown. We first determined the molecular formula of ComX(RO-E-2), a competence factor for B. subtilis strain RO-E-2. Then we synthesized putative pheromones with 1-, 2-, 4-, 5-, 6-, or 7-geranyl substituted tryptophan residues. The regio- and stereo-selective synthesis of the geranyl tryptophans was successful, and we prepared the six peptides with modified tryptophan residues. These peptides had the same molecular formula and showed similar hydrophobicity to the natural ComX(RO-E-2) in LC-MS analysis. But, none of them showed the same retention time as the natural pheromone and none exhibited its biological activity. These results suggest that the isoprenoid modification pattern of the tryptophan residue is more complex than postulated.     

Ultraviolet illumination-induced reduction of alpha-lactalbumin disulfide bridges

Prolonged exposure of Ca(2+)-loaded or Ca(2+)-depleted human alpha-lactalbumin to ultraviolet light (270-290 nm, 1 mW/cm(2), for 2 to 4 h) results in a 10-nm red shift of its tryptophan fluorescence spectrum. Gel chromatography of the UV-illuminated samples reveals two non-native protein forms: (1) a component with a red-shifted tryptophan fluorescence spectrum; and (2) a component with kynurenine-like fluorescent properties. The first component has from 0.6 to 0.9 free DTNB-reactive SH groups per protein molecule, which are absent in the native protein and is characterized by slightly lowered Ca(2+)-affinity (2 x 10(8) M(-1) versus 8 x 10(8) M(-1) for the native protein) and absence of observable thermal transition. The second component corresponds to the protein with photochemically modified tryptophan residues. It is assumed that the UV excitation of tryptophan residue(s) in alpha-lactalbumin is followed by a transfer of electrons to the Sbond;S bonds, resulting in their reduction. Mass spectrometry data obtained for trypsin-fragmented UV-illuminated alpha-lactalbumin with acrylodan-modified free thiol groups reveal the reduction of the 61-77 and 73-91 disulfide bridges. The effect observed has to be taken into account in any UV-region spectral studies of alpha-lactalbumin.     

Analysis of tryptophan fluorescence lifetimes in a series of human serum albumin mutants with substitutions in subdomain 2 A

Tryptophan 214, the only tryptophan residue in human serum albumin, is located in the physiologically important subdomain 2A ligand binding site. In the present study the fluorescence lifetime of tryptophan 214 in the following human serum albumin (HSA) mutants with substitutions in subdomain 2A were determined: K195M, K199M, F211V, R218M, R218H, R218A, R222M, H242V, and R257M. An HSA mutant in which tryptophan was moved from subdomain 2A to subdomain 3A (W214L/Y411W) was also examined. Additionally, the fluorescence lifetime of tryptophan 214 in an HSA fragment consisting of subdomains 1A, 1B, and 2A (1A-1B-2A HSA) was determined. For those species expected to have the most dramatic changes in tryptophan microenvironment, W214L/Y411W and 1A-1B-2A HSA, clear changes in tryptophan lifetimes were observed. Significant changes were also seen for those species with mutations at position 218, which is next to tryptophan in the X-ray structure of HSA. However, significant changes were also observed for H242V and R257M, which contain substitutions at positions not immediately adjacent to tryptophan 214, highlighting the conformational flexibility of subdomain 2A.     

Specificity in substrate binding by protein folding catalysts: tyrosine and tryptophan residues are the recognition motifs for the binding of peptides to the pancreas-specific protein disulfide isomerase PDIp

Using a cross-linking approach, we recently demonstrated that radiolabeled peptides or misfolded proteins specifically interact in vitro with two luminal proteins in crude extracts from pancreas microsomes. The proteins were the folding catalysts protein disulfide isomerase (PDI) and PDIp, a glycosylated, PDI-related protein, expressed exclusively in the pancreas. In this study, we explore the specificity of these proteins in binding peptides and related ligands and show that tyrosine and tryptophan residues in peptides are the recognition motifs for their binding by PDIp. This peptide-binding specificity may reflect the selectivity of PDIp in binding regions of unfolded polypeptide during catalysis of protein folding.     

Nitrated and oxidized products of a single tryptophan residue in human Cu,Zn-superoxide dismutase treated with either peroxynitrite-carbon dioxide or myeloperoxidase-hydrogen peroxide-nitrite

We reported previously that a single tryptophan residue, Trp32, in human Cu,Zn-superoxide dismutase is specifically modified by peroxynitrite-CO2 [Yamakura et al. (2001) Biochim. Biophys. Acta 1548, 38-46]. In this study, we modified Cu,Zn-superoxide dismutase by using a combination of myeloperoxidase, hydrogen peroxide, and nitrite. The modified enzyme showed no loss of copper and zinc, and 15% less enzymatic activity. Trp32 was the only significant amino acid lost. After trypsin digestion of the modified SOD with peroxynitrite-CO2 and the myeloperoxidase system, six newly appearing peptides containing tryptophan derivatives were observed on microLC-ESI-Q-TOF mass analyses and HPLC with a photodiode-array detector. The derivatives of the tryptophan residue exhibiting mass increases of 4, 16 (2 peaks), 32, 45 (major), and 45 Da (minor) were identified as kynurenine, oxindole-3-alanine and its derivatives, dihydroxytryptophan, 6-nitrotryptophan and 5-nitrotryptophan, respectively. We further identified 6-nitrotryptophan from the 1H-NMR spectrum for the pronase-digested product and calculated the yield of 6-nitrotryptophan as being about 30% for each of the modification methods. The tryptophan residue in the modified human Cu,Zn-superoxide dismutase gave the same spectra for the products including 6-nitrotryptophan as the major nitrated product with the two different modification systems.     

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.