Antibacterial activity in bovine lactoferrin-derived peptides.

Science.gov (United States)

Hoek, K S; Milne, J M; Grieve, P A; Dionysius, D A; Smith, R

1997-01-01

Several peptides sharing high sequence homology with lactoferricin B (Lf-cin B) were generated from bovine lactoferrin (Lf) with recombinant chymosin. Two peptides were copurified, one identical to Lf-cin B and another differing from Lf-cin B by the inclusion of a C-terminal alanine (lactoferricin). Two other peptides were copurified from chymosin-hydrolyzed Lf, one differing from Lf-cin B by the inclusion of C-terminal alanyl-leucine and the other being a heterodimer linked by a disulfide bond. These peptides were isolated in a single step from chymosin-hydrolyzed Lf by membrane ion-exchange chromatography and were purified by reverse-phase high-pressure liquid chromatography (HPLC). They were characterized by N-terminal Edman sequencing, mass spectrometry, and antibacterial activity determination. Pure lactoferricin, prepared from pepsin-hydrolyzed Lf, was purified by standard chromatography techniques. This peptide was analyzed against a number of gram-positive and gram-negative bacteria before and after reduction of its disulfide bond or cleavage after its single methionine residue and was found to inhibit the growth of all the test bacteria at a concentration of 8 microM or less. Subfragments of lactoferricin were isolated from reduced and cleaved peptide by reverse-phase HPLC. Subfragment 1 (residues 1 to 10) was active against most of the test microorganisms at concentrations of 10 to 50 microM. Subfragment 2 (residues 11 to 26) was active against only a few microorganisms at concentrations up to 100 microM. These antibacterial studies indicate that the activity of lactoferricin is mainly, but not wholly, due to its N-terminal region. PMID:8980754

Biodegradable copolymers carrying cell-adhesion peptide sequences.

Science.gov (United States)

Proks, Vladimír; Machová, Lud'ka; Popelka, Stepán; Rypácek, Frantisek

2003-01-01

Amphiphilic block copolymers are used to create bioactive surfaces on biodegradable polymer scaffolds for tissue engineering. Cell-selective biomaterials can be prepared using copolymers containing peptide sequences derived from extracellular-matrix proteins (ECM). Here we discuss alternative ways for preparation of amphiphilic block copolymers composed of hydrophobic polylactide (PLA) and hydrophilic poly(ethylene oxide) (PEO) blocks with cell-adhesion peptide sequences. Copolymers PLA-b-PEO were prepared by a living polymerisation of lactide in dioxane with tin(II)2-ethylhexanoate as a catalyst. The following approaches for incorporation of peptides into copolymers were elaborated. (a) First, a side-chain protected Gly-Arg-Gly-Asp-Ser-Gly (GRGDSG) peptide was prepared by solid-phase peptide synthesis (SPPS) and then coupled with delta-hydroxy-Z-amino-PEO in solution. In the second step, the PLA block was grafted to it via a controlled polymerisation of lactide initiated by the hydroxy end-groups of PEO in the side-chain-protected GRGDSG-PEO. Deprotection of the peptide yielded a GRGDSG-b-PEO-b-PLA copolymer, with the peptide attached through its C-end. (b) A protected GRGDSG peptide was built up on a polymer resin and coupled with Z-carboxy-PEO using a solid-phase approach. After cleavage of the delta-hydroxy-PEO-GRGDSG copolymer from the resin, polymerisation of lactide followed by deprotection of the peptide yielded a PLA-b-PEO-b-GRGDSG block copolymer, in which the peptide is linked through its N-terminus.

Library Design-Facilitated High-Throughput Sequencing of Synthetic Peptide Libraries.

Science.gov (United States)

Vinogradov, Alexander A; Gates, Zachary P; Zhang, Chi; Quartararo, Anthony J; Halloran, Kathryn H; Pentelute, Bradley L

2017-11-13

A methodology to achieve high-throughput de novo sequencing of synthetic peptide mixtures is reported. The approach leverages shotgun nanoliquid chromatography coupled with tandem mass spectrometry-based de novo sequencing of library mixtures (up to 2000 peptides) as well as automated data analysis protocols to filter away incorrect assignments, noise, and synthetic side-products. For increasing the confidence in the sequencing results, mass spectrometry-friendly library designs were developed that enabled unambiguous decoding of up to 600 peptide sequences per hour while maintaining greater than 85% sequence identification rates in most cases. The reliability of the reported decoding strategy was additionally confirmed by matching fragmentation spectra for select authentic peptides identified from library sequencing samples. The methods reported here are directly applicable to screening techniques that yield mixtures of active compounds, including particle sorting of one-bead one-compound libraries and affinity enrichment of synthetic library mixtures performed in solution.

The fsr Quorum-Sensing System and Cognate Gelatinase Orchestrate the Expression and Processing of Proprotein EF_1097 into the Mature Antimicrobial Peptide Enterocin O16

OpenAIRE

Dundar, Halil; Brede, Dag A.; La Rosa, Sabina Leanti; El-Gendy, Ahmed Osama; Diep, Dzung B.; Nes, Ingolf F.

2015-01-01

A novel antimicrobial peptide designated enterocin O16 was purified from Enterococcus faecalis. Mass spectrometry showed a monoisotopic mass of 7,231 Da, and N-terminal Edman degradation identified a 29-amino-acid sequence corresponding to residues 90 to 119 of the EF_1097 protein. Bioinformatic analysis showed that enterocin O16 is composed of the 68 most C-terminal residues of the EF_1097 protein. Introduction of an in-frame isogenic deletion in the ef1097 gene abolished the production of e...

BIOPEP database and other programs for processing bioactive peptide sequences.

Science.gov (United States)

Minkiewicz, Piotr; Dziuba, Jerzy; Iwaniak, Anna; Dziuba, Marta; Darewicz, Małgorzata

2008-01-01

This review presents the potential for application of computational tools in peptide science based on a sample BIOPEP database and program as well as other programs and databases available via the World Wide Web. The BIOPEP application contains a database of biologically active peptide sequences and a program enabling construction of profiles of the potential biological activity of protein fragments, calculation of quantitative descriptors as measures of the value of proteins as potential precursors of bioactive peptides, and prediction of bonds susceptible to hydrolysis by endopeptidases in a protein chain. Other bioactive and allergenic peptide sequence databases are also presented. Programs enabling the construction of binary and multiple alignments between peptide sequences, the construction of sequence motifs attributed to a given type of bioactivity, searching for potential precursors of bioactive peptides, and the prediction of sites susceptible to proteolytic cleavage in protein chains are available via the Internet as are other approaches concerning secondary structure prediction and calculation of physicochemical features based on amino acid sequence. Programs for prediction of allergenic and toxic properties have also been developed. This review explores the possibilities of cooperation between various programs.

Novel antimicrobial peptides isolated from the venom of wild bees

Czech Academy of Sciences Publication Activity Database

Čeřovský, Václav; Monincová, Lenka; Slaninová, Jiřina; Fučík, Vladimír; Borovičková, Lenka; Hovorka, Oldřich; Voburka, Zdeněk; Cvačka, Josef; Bednárová, Lucie; Buděšínský, Miloš; Straka, J.

2009-01-01

Roč. 276, Suppl. 1 (2009), s. 106-106 ISSN 1742-464X. [FEBS Congress /34/. 04.07.2009-09.07.2009, Praha] Institutional research plan: CEZ:AV0Z40550506 Keywords : linear cationic alpha-helical antimicrobial peptides * Edman degradation * mass spectrometry Subject RIV: CC - Organic Chemistry

Polymeric peptide pigments with sequence-encoded properties

Energy Technology Data Exchange (ETDEWEB)

Lampel, Ayala; McPhee, Scott A.; Park, Hang-Ah; Scott, Gary G.; Humagain, Sunita; Hekstra, Doeke R.; Yoo, Barney; Frederix, Pim W. J. M.; Li, Tai-De; Abzalimov, Rinat R.; Greenbaum, Steven G.; Tuttle, Tell; Hu, Chunhua; Bettinger, Christopher J.; Ulijn, Rein V.

2017-06-08

Melanins are a family of heterogeneous polymeric pigments that provide ultraviolet (UV) light protection, structural support, coloration, and free radical scavenging. Formed by oxidative oligomerization of catecholic small molecules, the physical properties of melanins are influenced by covalent and noncovalent disorder. We report the use of tyrosine-containing tripeptides as tunable precursors for polymeric pigments. In these structures, phenols are presented in a (supra-)molecular context dictated by the positions of the amino acids in the peptide sequence. Oxidative polymerization can be tuned in a sequence-dependent manner, resulting in peptide sequence–encoded properties such as UV absorbance, morphology, coloration, and electrochemical properties over a considerable range. Short peptides have low barriers to application and can be easily scaled, suggesting near-term applications in cosmetics and biomedicine.

Peptide Pattern Recognition for high-throughput protein sequence analysis and clustering

DEFF Research Database (Denmark)

Busk, Peter Kamp

2017-01-01

Large collections of protein sequences with divergent sequences are tedious to analyze for understanding their phylogenetic or structure-function relation. Peptide Pattern Recognition is an algorithm that was developed to facilitate this task but the previous version does only allow a limited...... number of sequences as input. I implemented Peptide Pattern Recognition as a multithread software designed to handle large numbers of sequences and perform analysis in a reasonable time frame. Benchmarking showed that the new implementation of Peptide Pattern Recognition is twenty times faster than...... the previous implementation on a small protein collection with 673 MAP kinase sequences. In addition, the new implementation could analyze a large protein collection with 48,570 Glycosyl Transferase family 20 sequences without reaching its upper limit on a desktop computer. Peptide Pattern Recognition...

Structural characterization of peptides derived from prosomatostatins I and II isolated from the pancreatic islets of two species of teleostean fish: the daddy sculpin and the flounder.

Science.gov (United States)

Conlon, J M; Davis, M S; Falkmer, S; Thim, L

1987-11-02

The primary structures of three peptides from extracts from the pancreatic islets of the daddy sculpin (Cottus scorpius) and three analogous peptides from the islets of the flounder (Platichthys flesus), two species of teleostean fish, have been determined by automated Edman degradation. The structures of the flounder peptides were confirmed by fast-atom bombardment mass spectrometry. The peptides show strong homology to residues (49-60), (63-96) and (98-125) of the predicted sequence of preprosomatostatin II from the anglerfish (Lophius americanus). The amino acid sequences of the peptides suggest that, in the sculpin, prosomatostatin II is cleaved at a dibasic amino acid residue processing site (corresponding to Lys61-Arg62 in anglerfish preprosomatostatin II). The resulting fragments are further cleaved at monobasic residue processing sites (corresponding to Arg48 and Arg97 in anglerfish preprosomatostatin II). In the flounder the same dibasic residue processing site is utilised but cleavage at different monobasic sites takes place (corresponding to Arg50 and Arg97 in anglerfish preprosomatostatin II). A peptide identical to mammalian somatostatin-14 was also isolated from the islets of both species and is presumed to represent a cleavage product of prosomatostatin I.

Effect of sequence and stereochemistry reversal on p53 peptide mimicry.

Directory of Open Access Journals (Sweden)

Alessio Atzori

Full Text Available Peptidomimetics effective in modulating protein-protein interactions and resistant to proteolysis have potential in therapeutic applications. An appealing yet underperforming peptidomimetic strategy is to employ D-amino acids and reversed sequences to mimic a lead peptide conformation, either separately or as the combined retro-inverso peptide. In this work, we examine the conformations of inverse, reverse and retro-inverso peptides of p53(15-29 using implicit solvent molecular dynamics simulation and circular dichroism spectroscopy. In order to obtain converged ensembles for the peptides, we find enhanced sampling is required via the replica exchange molecular dynamics method. From these replica exchange simulations, the D-peptide analogues of p53(15-29 result in a predominantly left-handed helical conformation. When the parent sequence is reversed sequence as either the L-peptide and D-peptide, these peptides display a greater helical propensity, feature reflected by NMR and CD studies in TFE/water solvent. The simulations also indicate that, while approximately similar orientations of the side-chains are possible by the peptide analogues, their ability to mimic the parent peptide is severely compromised by backbone orientation (for D-amino acids and side-chain orientation (for reversed sequences. A retro-inverso peptide is disadvantaged as a mimic in both aspects, and further chemical modification is required to enable this concept to be used fruitfully in peptidomimetic design. The replica exchange molecular simulation approach adopted here, with its ability to provide detailed conformational insights into modified peptides, has potential as a tool to guide structure-based design of new improved peptidomimetics.

Identification and functional analysis of a novel bradykinin inhibitory peptide in the venoms of New World Crotalinae pit vipers

International Nuclear Information System (INIS)

James Graham, Robert Leslie; Graham, Ciaren; McClean, Stephen; Chen, Tianbao; O'Rourke, Martin; Hirst, David; Theakston, David; Shaw, Chris

2005-01-01

A novel undecapeptide has been isolated and structurally characterized from the venoms of three species of New World pit vipers from the subfamily, Crotalinae. These include the Mexican moccasin (Agkistrodon bilineatus), the prairie rattlesnake (Crotalus viridis viridis), and the South American bushmaster (Lachesis muta). The peptide was purified from all three venoms using a combination of gel permeation chromatography and reverse-phase HPLC. Automated Edman degradation sequencing and MALDI-TOF mass spectrometry established its peptide primary structure as: Thr-Pro-Pro-Ala-Gly-Pro-Asp-Val-Gly-Pro-Arg-OH, with a non-protonated molecular mass of 1063.18 Da. A synthetic replicate of the peptide was found to be an antagonist of bradykinin action at the rat vascular B2 receptor. This is the first bradykinin inhibitory peptide isolated from snake venom. Database searching revealed the peptide to be highly structurally related (10/11 residues) with a domain residing between the bradykinin-potentiating peptide and C-type natriuretic peptide domains of a recently cloned precursor from tropical rattlesnake (Crotalus durissus terrificus) venom gland. BIP thus represents a novel biological entity from snake venom

A theoretical justification for single molecule peptide sequencing.

Directory of Open Access Journals (Sweden)

Jagannath Swaminathan

2015-02-01

Full Text Available The proteomes of cells, tissues, and organisms reflect active cellular processes and change continuously in response to intracellular and extracellular cues. Deep, quantitative profiling of the proteome, especially if combined with mRNA and metabolite measurements, should provide an unprecedented view of cell state, better revealing functions and interactions of cell components. Molecular diagnostics and biomarker discovery should benefit particularly from the accurate quantification of proteomes, since complex diseases like cancer change protein abundances and modifications. Currently, shotgun mass spectrometry is the primary technology for high-throughput protein identification and quantification; while powerful, it lacks high sensitivity and coverage. We draw parallels with next-generation DNA sequencing and propose a strategy, termed fluorosequencing, for sequencing peptides in a complex protein sample at the level of single molecules. In the proposed approach, millions of individual fluorescently labeled peptides are visualized in parallel, monitoring changing patterns of fluorescence intensity as N-terminal amino acids are sequentially removed, and using the resulting fluorescence signatures (fluorosequences to uniquely identify individual peptides. We introduce a theoretical foundation for fluorosequencing and, by using Monte Carlo computer simulations, we explore its feasibility, anticipate the most likely experimental errors, quantify their potential impact, and discuss the broad potential utility offered by a high-throughput peptide sequencing technology.

Peptomics, identification of novel cationic Arabidopsis peptides with conserved sequence motifs

DEFF Research Database (Denmark)

Olsen, Addie Nina; Mundy, John; Skriver, Karen

2002-01-01

Arabidopsis family of 34 genes. The predicted peptides are characterized by a conserved C-terminal sequence motif and additional primary structure conservation in a core region. The majority of these genes had not previously been annotated. A subset of the predicted peptides show high overall sequence...... similarity to Rapid Alkalinization Factor (RALF), a peptide isolated from tobacco. We therefore refer to this peptide family as RALFL for RALF-Like. RT-PCR analysis confirmed that several of the Arabidopsis genes are expressed and that their expression patterns vary. The identification of a large gene family...

Mapping membrane activity in undiscovered peptide sequence space using machine learning.

Science.gov (United States)

Lee, Ernest Y; Fulan, Benjamin M; Wong, Gerard C L; Ferguson, Andrew L

2016-11-29

There are some ∼1,100 known antimicrobial peptides (AMPs), which permeabilize microbial membranes but have diverse sequences. Here, we develop a support vector machine (SVM)-based classifier to investigate ⍺-helical AMPs and the interrelated nature of their functional commonality and sequence homology. SVM is used to search the undiscovered peptide sequence space and identify Pareto-optimal candidates that simultaneously maximize the distance σ from the SVM hyperplane (thus maximize its "antimicrobialness") and its ⍺-helicity, but minimize mutational distance to known AMPs. By calibrating SVM machine learning results with killing assays and small-angle X-ray scattering (SAXS), we find that the SVM metric σ correlates not with a peptide's minimum inhibitory concentration (MIC), but rather its ability to generate negative Gaussian membrane curvature. This surprising result provides a topological basis for membrane activity common to AMPs. Moreover, we highlight an important distinction between the maximal recognizability of a sequence to a trained AMP classifier (its ability to generate membrane curvature) and its maximal antimicrobial efficacy. As mutational distances are increased from known AMPs, we find AMP-like sequences that are increasingly difficult for nature to discover via simple mutation. Using the sequence map as a discovery tool, we find a unexpectedly diverse taxonomy of sequences that are just as membrane-active as known AMPs, but with a broad range of primary functions distinct from AMP functions, including endogenous neuropeptides, viral fusion proteins, topogenic peptides, and amyloids. The SVM classifier is useful as a general detector of membrane activity in peptide sequences.

Peptides derivatized with bicyclic quaternary ammonium ionization tags. Sequencing via tandem mass spectrometry.

Science.gov (United States)

Setner, Bartosz; Rudowska, Magdalena; Klem, Ewelina; Cebrat, Marek; Szewczuk, Zbigniew

2014-10-01

Improving the sensitivity of detection and fragmentation of peptides to provide reliable sequencing of peptides is an important goal of mass spectrometric analysis. Peptides derivatized by bicyclic quaternary ammonium ionization tags: 1-azabicyclo[2.2.2]octane (ABCO) or 1,4-diazabicyclo[2.2.2]octane (DABCO), are characterized by an increased detection sensitivity in electrospray ionization mass spectrometry (ESI-MS) and longer retention times on the reverse-phase (RP) chromatography columns. The improvement of the detection limit was observed even for peptides dissolved in 10 mM NaCl. Collision-induced dissociation tandem mass spectrometry of quaternary ammonium salts derivatives of peptides showed dominant a- and b-type ions, allowing facile sequencing of peptides. The bicyclic ionization tags are stable in collision-induced dissociation experiments, and the resulted fragmentation pattern is not significantly influenced by either acidic or basic amino acid residues in the peptide sequence. Obtained results indicate the general usefulness of the bicyclic quaternary ammonium ionization tags for ESI-MS/MS sequencing of peptides. Copyright © 2014 John Wiley & Sons, Ltd.

LESSONS IN DE NOVO PEPTIDE SEQUENCING BY TANDEM MASS SPECTROMETRY

Science.gov (United States)

Medzihradszky, Katalin F.; Chalkley, Robert J.

2015-01-01

Mass spectrometry has become the method of choice for the qualitative and quantitative characterization of protein mixtures isolated from all kinds of living organisms. The raw data in these studies are MS/MS spectra, usually of peptides produced by proteolytic digestion of a protein. These spectra are “translated” into peptide sequences, normally with the help of various search engines. Data acquisition and interpretation have both been automated, and most researchers look only at the summary of the identifications without ever viewing the underlying raw data used for assignments. Automated analysis of data is essential due to the volume produced. However, being familiar with the finer intricacies of peptide fragmentation processes, and experiencing the difficulties of manual data interpretation allow a researcher to be able to more critically evaluate key results, particularly because there are many known rules of peptide fragmentation that are not incorporated into search engine scoring. Since the most commonly used MS/MS activation method is collision-induced dissociation (CID), in this article we present a brief review of the history of peptide CID analysis. Next, we provide a detailed tutorial on how to determine peptide sequences from CID data. Although the focus of the tutorial is de novo sequencing, the lessons learned and resources supplied are useful for data interpretation in general. PMID:25667941

Sequencing Lys-N Proteolytic Peptides by ESI and MALDI Tandem Mass Spectrometry

Science.gov (United States)

Dupré, Mathieu; Cantel, Sonia; Verdié, Pascal; Martinez, Jean; Enjalbal, Christine

2011-02-01

In this study, we explored the MS/MS behavior of various synthetic peptides that possess a lysine residue at the N-terminal position. These peptides were designed to mimic peptides produced upon proteolysis by the Lys-N enzyme, a metalloendopeptidase issued from a Japanese fungus Grifola frondosa that was recently investigated in proteomic studies as an alternative to trypsin digestion, as a specific cleavage at the amide X-Lys chain is obtained that provides N-terminal lysine peptide fragments. In contrast to tryptic peptides exhibiting a lysine or arginine residue solely at the C-terminal position, and are thus devoid of such basic amino acids within the sequence, these Lys-N proteolytic peptides can contain the highly basic arginine residue anywhere within the peptide chain. The fragmentation patterns of such sequences with the ESI-QqTOF and MALDI-TOF/TOF mass spectrometers commonly used in proteomic bottom-up experiments were investigated.

Purification and amino acid sequence of a bacteriocins produced by Lactobacillus salivarius K7 isolated from chicken intestine

Directory of Open Access Journals (Sweden)

Kenji Sonomoto

2006-03-01

Full Text Available A bacteriocin-producing strain, Lactobacillus K7, was isolated from a chicken intestine. The inhibitory activity was determined by spot-on-lawn technique. Identification of the strain was performed by morphological, biochemical (API 50 CH kit and molecular genetic (16S rDNA basis. Bacteriocin purification processes were carried out by amberlite adsorption, cation exchange and reverse-phase high perform- ance liquid chromatography. N-terminal amino acid sequences were performed by Edman degradation. Molecular mass was determined by electrospray-ionization (ESI mass spectrometry (MS. Lactobacillus K7 showed inhibitory activity against Lactobacillus sakei subsp. sakei JCM 1157T, Leuconostoc mesenteroides subsp. mesenteroides JCM 6124T and Bacillus coagulans JCM 2257T. This strain was identified as Lb. salivarius. The antimicrobial substance was destroyed by proteolytic enzymes, indicating its proteinaceous structure designated as a bacteriocin type. The purification of bacteriocin by amberlite adsorption, cation exchange, and reverse-phase chromatography resulted in only one single active peak, which was designated FK22. Molecular weight of this fraction was 4331.70 Da. By amino acid sequence, this peptide was homology to Abp 118 beta produced by Lb. salivarius UCC118. In addition, Lb. salivarius UCC118 produced 2-peptide bacteriocin, which was Abp 118 alpha and beta. Based on the partial amino acid sequences of Abp 118 beta, specific primers were designed from nucleotide sequences according to data from GenBank. The result showed that the deduced peptide was high homology to 2-peptide bacteriocin, Abp 118 alpha and beta.

PhcrTx2, a New Crab-Paralyzing Peptide Toxin from the Sea Anemone Phymanthus crucifer

Science.gov (United States)

Garateix, Anoland; Salceda, Emilio; Zaharenko, André Junqueira; Pons, Tirso; Santos, Yúlica; Arreguín, Roberto; Ständker, Ludger; Forssmann, Wolf-Georg; Tytgat, Jan; Vega, Rosario

2018-01-01

Sea anemones produce proteinaceous toxins for predation and defense, including peptide toxins that act on a large variety of ion channels of pharmacological and biomedical interest. Phymanthus crucifer is commonly found in the Caribbean Sea; however, the chemical structure and biological activity of its toxins remain unknown, with the exception of PhcrTx1, an acid-sensing ion channel (ASIC) inhibitor. Therefore, in the present work, we focused on the isolation and characterization of new P. crucifer toxins by chromatographic fractionation, followed by a toxicity screening on crabs, an evaluation of ion channels, and sequence analysis. Five groups of toxic chromatographic fractions were found, and a new paralyzing toxin was purified and named PhcrTx2. The toxin inhibited glutamate-gated currents in snail neurons (maximum inhibition of 35%, IC50 4.7 µM), and displayed little or no influence on voltage-sensitive sodium/potassium channels in snail and rat dorsal root ganglion (DRG) neurons, nor on a variety of cloned voltage-gated ion channels. The toxin sequence was fully elucidated by Edman degradation. PhcrTx2 is a new β-defensin-fold peptide that shares a sequence similarity to type 3 potassium channels toxins. However, its low activity on the evaluated ion channels suggests that its molecular target remains unknown. PhcrTx2 is the first known paralyzing toxin in the family Phymanthidae. PMID:29414882

PhcrTx2, a New Crab-Paralyzing Peptide Toxin from the Sea Anemone Phymanthus crucifer

Directory of Open Access Journals (Sweden)

Armando Alexei Rodríguez

2018-02-01

Full Text Available Sea anemones produce proteinaceous toxins for predation and defense, including peptide toxins that act on a large variety of ion channels of pharmacological and biomedical interest. Phymanthus crucifer is commonly found in the Caribbean Sea; however, the chemical structure and biological activity of its toxins remain unknown, with the exception of PhcrTx1, an acid-sensing ion channel (ASIC inhibitor. Therefore, in the present work, we focused on the isolation and characterization of new P. crucifer toxins by chromatographic fractionation, followed by a toxicity screening on crabs, an evaluation of ion channels, and sequence analysis. Five groups of toxic chromatographic fractions were found, and a new paralyzing toxin was purified and named PhcrTx2. The toxin inhibited glutamate-gated currents in snail neurons (maximum inhibition of 35%, IC50 4.7 µM, and displayed little or no influence on voltage-sensitive sodium/potassium channels in snail and rat dorsal root ganglion (DRG neurons, nor on a variety of cloned voltage-gated ion channels. The toxin sequence was fully elucidated by Edman degradation. PhcrTx2 is a new β-defensin-fold peptide that shares a sequence similarity to type 3 potassium channels toxins. However, its low activity on the evaluated ion channels suggests that its molecular target remains unknown. PhcrTx2 is the first known paralyzing toxin in the family Phymanthidae.

Antagonistic Activities of Novel Peptides from Bacillus amyloliquefaciens PT14 against Fusarium solani and Fusarium oxysporum.

Science.gov (United States)

Kim, Young Gwon; Kang, Hee Kyoung; Kwon, Kee-Deok; Seo, Chang Ho; Lee, Hyang Burm; Park, Yoonkyung

2015-12-09

Bacillus species have recently drawn attention due to their potential use in the biological control of fungal diseases. This paper reports on the antifungal activity of novel peptides isolated from Bacillus amyloliquefaciens PT14. Reverse-phase high-performance liquid chromatography revealed that B. amyloliquefaciens PT14 produces five peptides (PT14-1, -2, -3, -4a, and -4b) that exhibit antifungal activity but are inactive against bacterial strains. In particular, PT14-3 and PT14-4a showed broad-spectrum antifungal activity against Fusarium solani and Fusarium oxysporum. The PT14-4a N-terminal amino acid sequence was identified through Edman degradation, and a BLAST homology analysis showed it not to be identical to any other protein or peptide. PT14-4a displayed strong fungicidal activity with minimal inhibitory concentrations of 3.12 mg/L (F. solani) and 6.25 mg/L (F. oxysporum), inducing severe morphological deformation in the conidia and hyphae. On the other hand, PT14-4a had no detectable hemolytic activity. This suggests PT14-4a has the potential to serve as an antifungal agent in clinical therapeutic and crop-protection applications.

Proteomics shows Hsp70 does not bind peptide sequences indiscriminately in vivo

International Nuclear Information System (INIS)

Grossmann, Michael E.; Madden, Benjamin J.; Gao, Fan; Pang, Yuan-Ping; Carpenter, John E.; McCormick, Daniel; Young, Charles Y.F.

2004-01-01

Heat shock protein 70 (Hsp70) binds peptide and has several functions that include protein folding, protein trafficking, and involvement with immune function. However, endogenous Hsp70-binding peptides had not previously been identified. Therefore, we eluted and identified several hundred endogenously bound peptides from Hsp70 using liquid chromatography ion trap mass spectrophotometry (LC-ITMS). Our work shows that the peptides are capable of binding Hsp70 as previously described. They are generally 8-26 amino acids in length and correspond to specific regions of many proteins. Through computationally assisted analysis of peptides eluted from Hsp70 we determined variable amino acid sequences, including a 5 amino acid core sequence that Hsp70 favorably binds. We also developed a computer algorithm that predicts Hsp70 binding within proteins. This work helps to define what peptides are bound by Hsp70 in vivo and suggests that Hsp70 facilitates peptide selection by aiding a funneling mechanism that is flexible but allows only a limited number of peptides to be processed

Unraveling the sequence and structure of the protein osteocalcin from a 42 ka fossil horse

Science.gov (United States)

Ostrom, Peggy H.; Gandhi, Hasand; Strahler, John R.; Walker, Angela K.; Andrews, Philip C.; Leykam, Joseph; Stafford, Thomas W.; Kelly, Robert L.; Walker, Danny N.; Buckley, Mike; Humpula, James

2006-04-01

We report the first complete amino acid sequence and evidence of secondary structure for osteocalcin from a temperate fossil. The osteocalcin derives from a 42 ka equid bone excavated from Juniper Cave, Wyoming. Results were determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-MS) and Edman sequencing with independent confirmation of the sequence in two laboratories. The ancient sequence was compared to that of three modern taxa: horse ( Equus caballus), zebra ( Equus grevyi), and donkey ( Equus asinus). Although there was no difference in sequence among modern taxa, MALDI-MS and Edman sequencing show that residues 48 and 49 of our modern horse are Thr, Ala rather than Pro, Val as previously reported (Carstanjen B., Wattiez, R., Armory, H., Lepage, O.M., Remy, B., 2002. Isolation and characterization of equine osteocalcin. Ann. Med. Vet.146(1), 31-38). MALDI-MS and Edman sequencing data indicate that the osteocalcin sequence of the 42 ka fossil is similar to that of modern horse. Previously inaccessible structural attributes for ancient osteocalcin were observed. Glu 39 rather than Gln 39 is consistent with deamidation, a process known to occur during fossilization and aging. Two post-translational modifications were documented: Hyp 9 and a disulfide bridge. The latter suggests at least partial retention of secondary structure. As has been done for ancient DNA research, we recommend standards for preparation and criteria for authenticating results of ancient protein sequencing.

Structural and biological characterization of three novel mastoparan peptides from the venom of the neotropical social wasp Protopolybia exigua (Saussure).

Science.gov (United States)

Mendes, Maria Anita; de Souza, Bibiana Monson; Palma, Mario Sergio

2005-01-01

The venom of the Neotropical social wasp Protopolybia exigua(Saussure) was fractionated by RP-HPLC resulting in the elution of 20 fractions. The homogeneity of the preparations were checked out by using ESI-MS analysis and the fractions 15, 17 and 19 (eluted at the most hydrophobic conditions) were enough pure to be sequenced by Edman degradation chemistry, resulting in the following sequences: Protopolybia MPI I-N-W-L-K-L-G-K-K-V-S-A-I-L-NH2 Protopolybia-MP II I-N-W-K-A-I-I-E-A-A-K-Q-A-L-NH2 Protopolybia-MP III I-N-W-L-K-L-G-K-A-V-I-D-A-L-NH2 All the peptides were manually synthesized on-solid phase and functionally characterized. Protopolybia-MP I is a hemolytic mastoparan, probably acting on mast cells by assembling in plasma membrane, resulting in pore formation; meanwhile, the peptides Protopolybia-MP II and -MP III were characterized as a non-hemolytic mast cell degranulator toxins, which apparently act by virtue of their binding to G-protein receptor, activating the mast cell degranulation.

Isolation of biologically active peptides from the venom of Japanese carpenter bee, Xylocopa appendiculata.

Science.gov (United States)

Kawakami, Hiroko; Goto, Shin G; Murata, Kazuya; Matsuda, Hideaki; Shigeri, Yasushi; Imura, Tomohiro; Inagaki, Hidetoshi; Shinada, Tetsuro

2017-01-01

Mass spectrometry-guided venom peptide profiling is a powerful tool to explore novel substances from venomous animals in a highly sensitive manner. In this study, this peptide profiling approach is successfully applied to explore the venom peptides of a Japanese solitary carpenter bee, Xylocopa appendiculata (Hymenoptera: Apoidea: Apidae: Anthophila: Xylocopinae: Xylocopini). Although interesting biological effects of the crude venom of carpenter bees have been reported, the structure and biological function of the venom peptides have not been elucidated yet. The venom peptide profiling of the crude venom of X. appendiculata was performed by matrix-assisted laser desorption/ionization-time of flight mass spectroscopy. The venom was purified by a reverse-phase HPLC. The purified peptides were subjected to the Edman degradation, MS/MS analysis, and/or molecular cloning methods for peptide sequencing. Biological and functional characterization was performed by circular dichroism analysis, liposome leakage assay, and antimicrobial, histamine releasing and hemolytic activity tests. Three novel peptides with m / z 16508, 1939.3, and 1900.3 were isolated from the venom of X. appendiculata . The peptide with m / z 16508 was characterized as a secretory phospholipase A 2 (PLA 2 ) homolog in which the characteristic cysteine residues as well as the active site residues found in bee PLA 2 s are highly conserved. Two novel peptides with m/z 1939.3 and m/z 1900.3 were named as Xac-1 and Xac-2, respectively. These peptides are found to be amphiphilic and displayed antimicrobial and hemolytic activities. The potency was almost the same as that of mastoparan isolated from the wasp venom. We found three novel biologically active peptides in the venom of X. appendiculata and analyzed their molecular functions, and compared their sequential homology to discuss their molecular diversity. Highly sensitive mass analysis plays an important role in this study.

Can Natural Proteins Designed with ‘Inverted’ Peptide Sequences Adopt Native-Like Protein Folds?

Science.gov (United States)

Sridhar, Settu; Guruprasad, Kunchur

2014-01-01

We have carried out a systematic computational analysis on a representative dataset of proteins of known three-dimensional structure, in order to evaluate whether it would possible to ‘swap’ certain short peptide sequences in naturally occurring proteins with their corresponding ‘inverted’ peptides and generate ‘artificial’ proteins that are predicted to retain native-like protein fold. The analysis of 3,967 representative proteins from the Protein Data Bank revealed 102,677 unique identical inverted peptide sequence pairs that vary in sequence length between 5–12 and 18 amino acid residues. Our analysis illustrates with examples that such ‘artificial’ proteins may be generated by identifying peptides with ‘similar structural environment’ and by using comparative protein modeling and validation studies. Our analysis suggests that natural proteins may be tolerant to accommodating such peptides. PMID:25210740

A fossil protein chimera; difficulties in discriminating dinosaur peptide sequences from modern cross-contamination

Science.gov (United States)

Warwood, Stacey; van Dongen, Bart; Kitchener, Andrew C.; Manning, Phillip L.

2017-01-01

A decade ago, reports that organic-rich soft tissue survived from dinosaur fossils were apparently supported by proteomics-derived sequence information of exceptionally well-preserved bone. This initial claim to the sequencing of endogenous collagen peptides from an approximately 68 Myr Tyrannosaurus rex fossil was highly controversial, largely on the grounds of potential contamination from either bacterial biofilms or from laboratory practice. In a subsequent study, collagen peptide sequences from an approximately 78 Myr Brachylophosaurus canadensis fossil were reported that have remained largely unchallenged. However, the endogeneity of these sequences relies heavily on a single peptide sequence, apparently unique to both dinosaurs. Given the potential for cross-contamination from modern bone analysed by the same team, here we extract collagen from bone samples of three individuals of ostrich, Struthio camelus. The resulting LC–MS/MS data were found to match all of the proposed sequences for both the original Tyrannosaurus and Brachylophosaurus studies. Regardless of the true nature of the dinosaur peptides, our finding highlights the difficulty of differentiating such sequences with confidence. Our results not only imply that cross-contamination cannot be ruled out, but that appropriate measures to test for endogeneity should be further evaluated. PMID:28566488

Sequence diversity and evolution of antimicrobial peptides in invertebrates.

Science.gov (United States)

Tassanakajon, Anchalee; Somboonwiwat, Kunlaya; Amparyup, Piti

2015-02-01

Antimicrobial peptides (AMPs) are evolutionarily ancient molecules that act as the key components in the invertebrate innate immunity against invading pathogens. Several AMPs have been identified and characterized in invertebrates, and found to display considerable diversity in their amino acid sequence, structure and biological activity. AMP genes appear to have rapidly evolved, which might have arisen from the co-evolutionary arms race between host and pathogens, and enabled organisms to survive in different microbial environments. Here, the sequence diversity of invertebrate AMPs (defensins, cecropins, crustins and anti-lipopolysaccharide factors) are presented to provide a better understanding of the evolution pattern of these peptides that play a major role in host defense mechanisms. Copyright © 2014 Elsevier Ltd. All rights reserved.

Primary structure of a 14 kDa basic structural protein (Lm-76) from the cuticle of the migratory locust, Locusta migratoria

DEFF Research Database (Denmark)

Andersen, Jens S.; Andersen, S O; Højrup, P

1993-01-01

The complete amino acid sequence of a 14 kDa structural protein (LM-76) isolated from pharate cuticle of the locust, Locusta migratoria, was determined by Edman degradation of the intact protein and enzymatically derived peptides. Plasma desorption and electrospray mass spectrometry was used as a...

Negative Ion In-Source Decay Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry for Sequencing Acidic Peptides

Science.gov (United States)

McMillen, Chelsea L.; Wright, Patience M.; Cassady, Carolyn J.

2016-05-01

Matrix-assisted laser desorption/ionization (MALDI) in-source decay was studied in the negative ion mode on deprotonated peptides to determine its usefulness for obtaining extensive sequence information for acidic peptides. Eight biological acidic peptides, ranging in size from 11 to 33 residues, were studied by negative ion mode ISD (nISD). The matrices 2,5-dihydroxybenzoic acid, 2-aminobenzoic acid, 2-aminobenzamide, 1,5-diaminonaphthalene, 5-amino-1-naphthol, 3-aminoquinoline, and 9-aminoacridine were used with each peptide. Optimal fragmentation was produced with 1,5-diaminonphthalene (DAN), and extensive sequence informative fragmentation was observed for every peptide except hirudin(54-65). Cleavage at the N-Cα bond of the peptide backbone, producing c' and z' ions, was dominant for all peptides. Cleavage of the N-Cα bond N-terminal to proline residues was not observed. The formation of c and z ions is also found in electron transfer dissociation (ETD), electron capture dissociation (ECD), and positive ion mode ISD, which are considered to be radical-driven techniques. Oxidized insulin chain A, which has four highly acidic oxidized cysteine residues, had less extensive fragmentation. This peptide also exhibited the only charged localized fragmentation, with more pronounced product ion formation adjacent to the highly acidic residues. In addition, spectra were obtained by positive ion mode ISD for each protonated peptide; more sequence informative fragmentation was observed via nISD for all peptides. Three of the peptides studied had no product ion formation in ISD, but extensive sequence informative fragmentation was found in their nISD spectra. The results of this study indicate that nISD can be used to readily obtain sequence information for acidic peptides.

A fossil protein chimera; difficulties in discriminating dinosaur peptide sequences from modern cross-contamination.

Science.gov (United States)

Buckley, Michael; Warwood, Stacey; van Dongen, Bart; Kitchener, Andrew C; Manning, Phillip L

2017-05-31

A decade ago, reports that organic-rich soft tissue survived from dinosaur fossils were apparently supported by proteomics-derived sequence information of exceptionally well-preserved bone. This initial claim to the sequencing of endogenous collagen peptides from an approximately 68 Myr Tyrannosaurus rex fossil was highly controversial, largely on the grounds of potential contamination from either bacterial biofilms or from laboratory practice. In a subsequent study, collagen peptide sequences from an approximately 78 Myr Brachylophosaurus canadensis fossil were reported that have remained largely unchallenged. However, the endogeneity of these sequences relies heavily on a single peptide sequence, apparently unique to both dinosaurs. Given the potential for cross-contamination from modern bone analysed by the same team, here we extract collagen from bone samples of three individuals of ostrich, Struthio camelus The resulting LC-MS/MS data were found to match all of the proposed sequences for both the original Tyrannosaurus and Brachylophosaurus studies. Regardless of the true nature of the dinosaur peptides, our finding highlights the difficulty of differentiating such sequences with confidence. Our results not only imply that cross-contamination cannot be ruled out, but that appropriate measures to test for endogeneity should be further evaluated. © 2017 The Authors.

Purification of the labeled cyanogen bromide peptides of the α polypeptide from sodium and potassium ion-activated adenosinetriphosphatase modified with N-[3H]ethylmaleimide

International Nuclear Information System (INIS)

Le, D.T.

1985-01-01

Sodium and potassium ion-activated adenosinetriphosphatase, isolated from canine kidney, was reacted with N-[ 3 H]ethylmaleimide under three different conditions, defined by particular concentrations of ligands for the enzyme, such that after the same amount of time the remaining activity of then enzyme varied from 90% to 30%. The conformation of the enzyme also differed among the three conditions. In all cases, the α-polypeptide was purified and subjected to cyanogen bromide digestion. Two distinct, radioactive peptides were separated by gel filtration of the cyanogen bromide digest on a column of Sephadex LH-60 equilibrated with 95% ethanol: 88% formic acid:4:1. One of the radioactive peptides was shown to contain the sulfhydryl residue whose reaction with N-ethylmaleimide inactivates the enzyme. The other radioactive peptide contained a sulfhydryl residue that seems to react with N-ethylmaleimide only when the binding site for ATP is not occupied. Alkylation of this residue, however, does not result in inactivation of enzyme. Both peptides were purified further by high-pressure liquid chromatography, and their amino-terminal sequences were determined by the manual dansyl-Edman or solid-phase techniques. The peptide containing the sulfhydryl protected by ATP has, as its amino terminus, the lysine that reacts exclusively with fluorescein-5'-isothiocyanate

Prediction of peptide drift time in ion mobility mass spectrometry from sequence-based features

KAUST Repository

Wang, Bing; Zhang, Jun; Chen, Peng; Ji, Zhiwei; Deng, Shuping; Li, Chi

2013-01-01

Background: Ion mobility-mass spectrometry (IMMS), an analytical technique which combines the features of ion mobility spectrometry (IMS) and mass spectrometry (MS), can rapidly separates ions on a millisecond time-scale. IMMS becomes a powerful tool to analyzing complex mixtures, especially for the analysis of peptides in proteomics. The high-throughput nature of this technique provides a challenge for the identification of peptides in complex biological samples. As an important parameter, peptide drift time can be used for enhancing downstream data analysis in IMMS-based proteomics.Results: In this paper, a model is presented based on least square support vectors regression (LS-SVR) method to predict peptide ion drift time in IMMS from the sequence-based features of peptide. Four descriptors were extracted from peptide sequence to represent peptide ions by a 34-component vector. The parameters of LS-SVR were selected by a grid searching strategy, and a 10-fold cross-validation approach was employed for the model training and testing. Our proposed method was tested on three datasets with different charge states. The high prediction performance achieve demonstrate the effectiveness and efficiency of the prediction model.Conclusions: Our proposed LS-SVR model can predict peptide drift time from sequence information in relative high prediction accuracy by a test on a dataset of 595 peptides. This work can enhance the confidence of protein identification by combining with current protein searching techniques. 2013 Wang et al.; licensee BioMed Central Ltd.

Prediction of peptide drift time in ion mobility mass spectrometry from sequence-based features

KAUST Repository

Wang, Bing

2013-05-09

Background: Ion mobility-mass spectrometry (IMMS), an analytical technique which combines the features of ion mobility spectrometry (IMS) and mass spectrometry (MS), can rapidly separates ions on a millisecond time-scale. IMMS becomes a powerful tool to analyzing complex mixtures, especially for the analysis of peptides in proteomics. The high-throughput nature of this technique provides a challenge for the identification of peptides in complex biological samples. As an important parameter, peptide drift time can be used for enhancing downstream data analysis in IMMS-based proteomics.Results: In this paper, a model is presented based on least square support vectors regression (LS-SVR) method to predict peptide ion drift time in IMMS from the sequence-based features of peptide. Four descriptors were extracted from peptide sequence to represent peptide ions by a 34-component vector. The parameters of LS-SVR were selected by a grid searching strategy, and a 10-fold cross-validation approach was employed for the model training and testing. Our proposed method was tested on three datasets with different charge states. The high prediction performance achieve demonstrate the effectiveness and efficiency of the prediction model.Conclusions: Our proposed LS-SVR model can predict peptide drift time from sequence information in relative high prediction accuracy by a test on a dataset of 595 peptides. This work can enhance the confidence of protein identification by combining with current protein searching techniques. 2013 Wang et al.; licensee BioMed Central Ltd.

Identification of a novel Plasmopara halstedii elicitor protein combining de novo peptide sequencing algorithms and RACE-PCR

Directory of Open Access Journals (Sweden)

Madlung Johannes

2010-05-01

Full Text Available Abstract Background Often high-quality MS/MS spectra of tryptic peptides do not match to any database entry because of only partially sequenced genomes and therefore, protein identification requires de novo peptide sequencing. To achieve protein identification of the economically important but still unsequenced plant pathogenic oomycete Plasmopara halstedii, we first evaluated the performance of three different de novo peptide sequencing algorithms applied to a protein digests of standard proteins using a quadrupole TOF (QStar Pulsar i. Results The performance order of the algorithms was PEAKS online > PepNovo > CompNovo. In summary, PEAKS online correctly predicted 45% of measured peptides for a protein test data set. All three de novo peptide sequencing algorithms were used to identify MS/MS spectra of tryptic peptides of an unknown 57 kDa protein of P. halstedii. We found ten de novo sequenced peptides that showed homology to a Phytophthora infestans protein, a closely related organism of P. halstedii. Employing a second complementary approach, verification of peptide prediction and protein identification was performed by creation of degenerate primers for RACE-PCR and led to an ORF of 1,589 bp for a hypothetical phosphoenolpyruvate carboxykinase. Conclusions Our study demonstrated that identification of proteins within minute amounts of sample material improved significantly by combining sensitive LC-MS methods with different de novo peptide sequencing algorithms. In addition, this is the first study that verified protein prediction from MS data by also employing a second complementary approach, in which RACE-PCR led to identification of a novel elicitor protein in P. halstedii.

Catalytic center of lecithin:cholesterol acyltransferase: isolation and sequence of diisopropyl fluorophosphate-labeled peptides

Energy Technology Data Exchange (ETDEWEB)

Park, Y.B.; Yueksel, U.G.; Gracy, R.W.; Lacko, A.G.

1987-02-27

Lecithin:cholesterol acyltransferase (LCAT) was purified from hog plasma and subsequently reacted with (/sup 3/H)-Diisopropyl fluorophosphate (DFP). The labeled enzyme was digested with pepsin and the peptides separated by high performance liquid chromatography (HPLC). Two radioactive peptides were isolated, subjected to automated amino acid sequencing and yielded the following data: A) Ile-Ser-Leu-Gly-Ala-Pro-Trp-Gly-Gly-Ser, and B) Tyr-Ile-Phe-Asp-x-Gly-Phe-Pro-Tyr-x-Asp-Pro-Val. Both of these sequences represent very highly conserved regions of the enzyme when compared to the sequence of human LCAT. Peptide (A) is considered to represent the catalytic center of LCAT based on comparisons with data reported in the literature.

Purification of the labeled cyanogen bromide peptides of the. cap alpha. polypeptide from sodium and potassium ion-activated adenosinetriphosphatase modified with N-(/sup 3/H)ethylmaleimide

Energy Technology Data Exchange (ETDEWEB)

Le, D.T.

1985-01-01

Sodium and potassium ion-activated adenosinetriphosphatase, isolated from canine kidney, was reacted with N-(/sup 3/H)ethylmaleimide under three different conditions, defined by particular concentrations of ligands for the enzyme, such that after the same amount of time the remaining activity of then enzyme varied from 90% to 30%. The conformation of the enzyme also differed among the three conditions. In all cases, the ..cap alpha..-polypeptide was purified and subjected to cyanogen bromide digestion. Two distinct, radioactive peptides were separated by gel filtration of the cyanogen bromide digest on a column of Sephadex LH-60 equilibrated with 95% ethanol: 88% formic acid:4:1. One of the radioactive peptides was shown to contain the sulfhydryl residue whose reaction with N-ethylmaleimide inactivates the enzyme. The other radioactive peptide contained a sulfhydryl residue that seems to react with N-ethylmaleimide only when the binding site for ATP is not occupied. Alkylation of this residue, however, does not result in inactivation of enzyme. Both peptides were purified further by high-pressure liquid chromatography, and their amino-terminal sequences were determined by the manual dansyl-Edman or solid-phase techniques. The peptide containing the sulfhydryl protected by ATP has, as its amino terminus, the lysine that reacts exclusively with fluorescein-5'-isothiocyanate.

Multifunctional hybrid networks based on self assembling peptide sequences

Science.gov (United States)

Sathaye, Sameer

The overall aim of this dissertation is to achieve a comprehensive correlation between the molecular level changes in primary amino acid sequences of amphiphilic beta-hairpin peptides and their consequent solution-assembly properties and bulk network hydrogel behavior. This has been accomplished using two broad approaches. In the first approach, amino acid substitutions were made to peptide sequence MAX1 such that the hydrophobic surfaces of the folded beta-hairpins from the peptides demonstrate shape specificity in hydrophobic interactions with other beta-hairpins during the assembly process, thereby causing changes to the peptide nanostructure and bulk rheological properties of hydrogels formed from the peptides. Steric lock and key complementary hydrophobic interactions were designed to occur between two beta-hairpin molecules of a single molecule, LNK1 during beta-sheet fibrillar assembly of LNK1. Experimental results from circular dichroism, transmission electron microscopy and oscillatory rheology collectively indicate that the molecular design of the LNK1 peptide can be assigned the cause of the drastically different behavior of the networks relative to MAX1. The results indicate elimination or significant reduction of fibrillar branching due to steric complementarity in LNK1 that does not exist in MAX1, thus supporting the original hypothesis. As an extension of the designed steric lock and key complementarity between two beta-hairpin molecules of the same peptide molecule. LNK1, three new pairs of peptide molecules LP1-KP1, LP2-KP2 and LP3-KP3 that resemble complementary 'wedge' and 'trough' shapes when folded into beta-hairpins were designed and studied. All six peptides individually and when blended with their corresponding shape complement formed fibrillar nanostructures with non-uniform thickness values. Loose packing in the assembled structures was observed in all the new peptides as compared to the uniform tight packing in MAX1 by SANS analysis. This

Angiotensin I-Converting-Enzyme-Inhibitory and Antibacterial Peptides from Lactobacillus helveticus PR4 Proteinase-Hydrolyzed Caseins of Milk from Six Species

Science.gov (United States)

Minervini, F.; Algaron, F.; Rizzello, C. G.; Fox, P. F.; Monnet, V.; Gobbetti, M.

2003-01-01

Sodium caseinates prepared from bovine, sheep, goat, pig, buffalo or human milk were hydrolyzed by a partially purified proteinase of Lactobacillus helveticus PR4. Peptides in each hydrolysate were fractionated by reversed-phase fast-protein liquid chromatography. The fractions which showed the highest angiotensin I-converting-enzyme (ACE)-inhibitory or antibacterial activity were sequenced by mass spectrum and Edman degradation analyses. Various ACE-inhibitory peptides were found in the hydrolysates: the bovine αS1-casein (αS1-CN) 24-47 fragment (f24-47), f169-193, and β-CN f58-76; ovine αS1-CN f1-6 and αS2-CN f182-185 and f186-188; caprine β-CN f58-65 and αS2-CN f182-187; buffalo β-CN f58-66; and a mixture of three tripeptides originating from human β-CN. A mixture of peptides with a C-terminal sequence, Pro-Gly-Pro, was found in the most active fraction of the pig sodium caseinate hydrolysate. The highest ACE-inhibitory activity of some peptides corresponded to the concentration of the ACE inhibitor (S)-N-(1-[ethoxycarbonyl]-3-phenylpropyl)-ala-pro maleate (enalapril) of 49.253 μg/ml (100 μmol/liter). Several of the above sequences had features in common with other ACE-inhibitory peptides reported in the literature. The 50% inhibitory concentration (IC50) of some of the crude peptide fractions was very low (16 to 100 μg/ml). Some identified peptides were chemically synthesized, and the ACE-inhibitory activity and IC50s were confirmed. An antibacterial peptide corresponding to β-CN f184-210 was identified in human sodium caseinate hydrolysate. It showed a very large spectrum of inhibition against gram-positive and -negative bacteria, including species of potential clinical interest, such as Enterococcus faecium, Bacillus megaterium, Escherichia coli, Listeria innocua, Salmonella spp., Yersinia enterocolitica, and Staphylococcus aureus. The MIC for E. coli F19 was ca. 50 μg/ml. Once generated, the bioactive peptides were resistant to further

MRUniNovo: an efficient tool for de novo peptide sequencing utilizing the hadoop distributed computing framework.

Science.gov (United States)

Li, Chuang; Chen, Tao; He, Qiang; Zhu, Yunping; Li, Kenli

2017-03-15

Tandem mass spectrometry-based de novo peptide sequencing is a complex and time-consuming process. The current algorithms for de novo peptide sequencing cannot rapidly and thoroughly process large mass spectrometry datasets. In this paper, we propose MRUniNovo, a novel tool for parallel de novo peptide sequencing. MRUniNovo parallelizes UniNovo based on the Hadoop compute platform. Our experimental results demonstrate that MRUniNovo significantly reduces the computation time of de novo peptide sequencing without sacrificing the correctness and accuracy of the results, and thus can process very large datasets that UniNovo cannot. MRUniNovo is an open source software tool implemented in java. The source code and the parameter settings are available at http://bioinfo.hupo.org.cn/MRUniNovo/index.php. s131020002@hnu.edu.cn ; taochen1019@163.com. Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Structural studies of polypeptides: Mechanism of immunoglobin catalysis and helix propagation in hybrid sequence, disulfide containing peptides

Energy Technology Data Exchange (ETDEWEB)

Storrs, Richard Wood [Univ. of California, Berkeley, CA (United States)

1992-08-01

Catalytic immunoglobin fragments were studied Nuclear Magnetic Resonance spectroscopy to identify amino acid residues responsible for the catalytic activity. Small, hybrid sequence peptides were analyzed for helix propagation following covalent initiation and for activity related to the protein from which the helical sequence was derived. Hydrolysis of p-nitrophenyl carbonates and esters by specific immunoglobins is thought to involve charge complementarity. The pK of the transition state analog P-nitrophenyl phosphate bound to the immunoglobin fragment was determined by ³¹P-NMR to verify the juxtaposition of a positively charged amino acid to the binding/catalytic site. Optical studies of immunoglobin mediated photoreversal of cis, syn cyclobutane thymine dimers implicated tryptophan as the photosensitizing chromophore. Research shows the chemical environment of a single tryptophan residue is altered upon binding of the thymine dimer. This tryptophan residue was localized to within 20 Å of the binding site through the use of a nitroxide paramagnetic species covalently attached to the thymine dimer. A hybrid sequence peptide was synthesized based on the bee venom peptide apamin in which the helical residues of apamin were replaced with those from the recognition helix of the bacteriophage 434 repressor protein. Oxidation of the disufide bonds occured uniformly in the proper 1-11, 3-15 orientation, stabilizing the 434 sequence in an α-helix. The glycine residue stopped helix propagation. Helix propagation in 2,2,2-trifluoroethanol mixtures was investigated in a second hybrid sequence peptide using the apamin-derived disulfide scaffold and the S-peptide sequence. The helix-stop signal previously observed was not observed in the NMR NOESY spectrum. Helical connectivities were seen throughout the S-peptide sequence. The apamin/S-peptide hybrid binded to the S-protein (residues 21-166 of ribonuclease A) and reconstituted enzymatic activity.

Structural studies of polypeptides: Mechanism of immunoglobin catalysis and helix propagation in hybrid sequence, disulfide containing peptides

Energy Technology Data Exchange (ETDEWEB)

Storrs, R.W.

1992-08-01

Catalytic immunoglobin fragments were studied Nuclear Magnetic Resonance spectroscopy to identify amino acid residues responsible for the catalytic activity. Small, hybrid sequence peptides were analyzed for helix propagation following covalent initiation and for activity related to the protein from which the helical sequence was derived. Hydrolysis of p-nitrophenyl carbonates and esters by specific immunoglobins is thought to involve charge complementarity. The pK of the transition state analog P-nitrophenyl phosphate bound to the immunoglobin fragment was determined by [sup 31]P-NMR to verify the juxtaposition of a positively charged amino acid to the binding/catalytic site. Optical studies of immunoglobin mediated photoreversal of cis, syn cyclobutane thymine dimers implicated tryptophan as the photosensitizing chromophore. Research shows the chemical environment of a single tryptophan residue is altered upon binding of the thymine dimer. This tryptophan residue was localized to within 20 [Angstrom] of the binding site through the use of a nitroxide paramagnetic species covalently attached to the thymine dimer. A hybrid sequence peptide was synthesized based on the bee venom peptide apamin in which the helical residues of apamin were replaced with those from the recognition helix of the bacteriophage 434 repressor protein. Oxidation of the disufide bonds occured uniformly in the proper 1-11, 3-15 orientation, stabilizing the 434 sequence in an [alpha]-helix. The glycine residue stopped helix propagation. Helix propagation in 2,2,2-trifluoroethanol mixtures was investigated in a second hybrid sequence peptide using the apamin-derived disulfide scaffold and the S-peptide sequence. The helix-stop signal previously observed was not observed in the NMR NOESY spectrum. Helical connectivities were seen throughout the S-peptide sequence. The apamin/S-peptide hybrid binded to the S-protein (residues 21-166 of ribonuclease A) and reconstituted enzymatic activity.

Novel haemoglobin-derived antimicrobial peptides from chicken (Gallus gallus) blood: purification, structural aspects and biological activity.

Science.gov (United States)

Vasilchenko, A S; Rogozhin, E A; Vasilchenko, A V; Kartashova, O L; Sycheva, M V

2016-12-01

To purify and characterize antimicrobial peptides derived from the acid extract of Gallus gallus blood cells. Two polypeptides (i.e. CHb-1 and CHb-2) with antibacterial activity were detected in the acidic extract of blood cells from chicken (G. gallus). The isolated peptides that possessed a potent antibacterial activity were purified using a two-step chromatography procedure that involved solid-phase extraction of a total protein/peptide extract followed by thin fractionation by reversed-phase high performance liquid chromatography (RP-HPLC). The molecular masses of the purified peptides were similar and were 4824·4 and 4825·2 Da, which have been measured by matrix-assisted laser desorption/ionization mass spectrometry (MALDI TOF MS). Their amino acid sequences were determined by Edman degradation and showed that the peptides were fully identical to the two fragments of G. gallus α-haemoglobin localized into different subunits (A and D respectively). The peptides were active in micromolar concentrations against Gram-negative Escherichia coli K12 TG1. Using the 1-N-phenylnaphthylamine, the FITC-dextran labelled probes and the live/dead staining allowed to show the hemocidin mode of action and estimate the pore size. In this study, for the first time, α-haemoglobin from chicken (G. gallus) has been investigated as a donor of the two high homologous native peptide fragments that possess potent antibacterial activity in vitro. These are membrane-active peptides and their mechanism of action against E. coli involves a toroidal pore formation. The obtained results expand the perception of the role of haemoglobin in a living system, describing it as a source of multifunction substances. Additionally, the data presented in this paper may contribute to the development of new, cost-effective, antimicrobial agents. © 2016 The Society for Applied Microbiology.

Purification of labeled cyanogen bromide peptides of the alpha polypeptide from sodium ion and potassium ion activated adenosinetriphosphatase modified with N-[3H]ethylmaleimide

International Nuclear Information System (INIS)

Le, D.T.

1986-01-01

Sodium ion and potassium ion activated adenosinetriphosphatase, isolated from canine kidney, was reacted with N-[ 3 H]ethylmaleimide while it was poised in three different conformations, ostensibly E2-P, E2, and E1, respectively. These assignments were made from a consideration of the particular concentrations of ligands in the respective alkylation mixtures. After a 30-min reaction, the remaining enzymatic activity was found to vary among these three different samples from 90 to 30% of that of unalkylated controls. In all cases, the alpha polypeptide was purified and subjected to digestion with cyanogen bromide, and in each digest the same two distinct radioactive peptides were identified and purified by gel filtration on a column of Sephadex LH-60. The incorporation of N-[ 3 H]ethylmaleimide into one of these two peptides correlated closely with enzymatic inactivation, while the incorporation into the other was most extensive when the portion of the active site to which ATP binds was unoccupied. Alkylation of the residue within the latter peptide, however, does not result in inactivation of the enzyme. Both peptides were further purified by high-pressure liquid chromatography, and their amino-terminal sequences were determined by manual dansyl Edman or solid-phase techniques. The peptide containing the sulfhydryl protected by ATP has, as its amino terminus, the lysine that reacts exclusively with fluoresceinyl 5'-isothiocyanate

Sequence-Specific Model for Peptide Retention Time Prediction in Strong Cation Exchange Chromatography.

Science.gov (United States)

Gussakovsky, Daniel; Neustaeter, Haley; Spicer, Victor; Krokhin, Oleg V

2017-11-07

The development of a peptide retention prediction model for strong cation exchange (SCX) separation on a Polysulfoethyl A column is reported. Off-line 2D LC-MS/MS analysis (SCX-RPLC) of S. cerevisiae whole cell lysate was used to generate a retention dataset of ∼30 000 peptides, sufficient for identifying the major sequence-specific features of peptide retention mechanisms in SCX. In contrast to RPLC/hydrophilic interaction liquid chromatography (HILIC) separation modes, where retention is driven by hydrophobic/hydrophilic contributions of all individual residues, SCX interactions depend mainly on peptide charge (number of basic residues at acidic pH) and size. An additive model (incorporating the contributions of all 20 residues into the peptide retention) combined with a peptide length correction produces a 0.976 R 2 value prediction accuracy, significantly higher than the additive models for either HILIC or RPLC. Position-dependent effects on peptide retention for different residues were driven by the spatial orientation of tryptic peptides upon interaction with the negatively charged surface functional groups. The positively charged N-termini serve as a primary point of interaction. For example, basic residues (Arg, His, Lys) increase peptide retention when located closer to the N-terminus. We also found that hydrophobic interactions, which could lead to a mixed-mode separation mechanism, are largely suppressed at 20-30% of acetonitrile in the eluent. The accuracy of the final Sequence-Specific Retention Calculator (SSRCalc) SCX model (∼0.99 R 2 value) exceeds all previously reported predictors for peptide LC separations. This also provides a solid platform for method development in 2D LC-MS protocols in proteomics and peptide retention prediction filtering of false positive identifications.

Purification of labeled cyanogen bromide peptides of the alpha polypeptide from sodium ion and potassium ion activated adenosinetriphosphatase modified with N-(/sup 3/H)ethylmaleimide

Energy Technology Data Exchange (ETDEWEB)

Le, D.T.

1986-05-06

Sodium ion and potassium ion activated adenosinetriphosphatase, isolated from canine kidney, was reacted with N-(/sup 3/H)ethylmaleimide while it was poised in three different conformations, ostensibly E2-P, E2, and E1, respectively. These assignments were made from a consideration of the particular concentrations of ligands in the respective alkylation mixtures. After a 30-min reaction, the remaining enzymatic activity was found to vary among these three different samples from 90 to 30% of that of unalkylated controls. In all cases, the alpha polypeptide was purified and subjected to digestion with cyanogen bromide, and in each digest the same two distinct radioactive peptides were identified and purified by gel filtration on a column of Sephadex LH-60. The incorporation of N-(/sup 3/H)ethylmaleimide into one of these two peptides correlated closely with enzymatic inactivation, while the incorporation into the other was most extensive when the portion of the active site to which ATP binds was unoccupied. Alkylation of the residue within the latter peptide, however, does not result in inactivation of the enzyme. Both peptides were further purified by high-pressure liquid chromatography, and their amino-terminal sequences were determined by manual dansyl Edman or solid-phase techniques. The peptide containing the sulfhydryl protected by ATP has, as its amino terminus, the lysine that reacts exclusively with fluoresceinyl 5'-isothiocyanate.

Sequence-selective targeting of duplex DNA by peptide nucleic acids

DEFF Research Database (Denmark)

Nielsen, Peter E

2010-01-01

Sequence-selective gene targeting constitutes an attractive drug-discovery approach for genetic therapy, with the aim of reducing or enhancing the activity of specific genes at the transcriptional level, or as part of a methodology for targeted gene repair. The pseudopeptide DNA mimic peptide...

Practical multipeptide synthesis: dedicated software for the definition of multiple, overlapping peptides covering polypeptide sequences.

Science.gov (United States)

Heegaard, P M; Holm, A; Hagerup, M

1993-01-01

A personal computer program for the conversion of linear amino acid sequences to multiple, small, overlapping peptide sequences has been developed. Peptide lengths and "jumps" (the distance between two consecutive overlapping peptides) are defined by the user. To facilitate the use of the program for parallel solid-phase chemical peptide syntheses for the synchronous production of multiple peptides, amino acids at each acylation step are laid out by the program in a convenient standard multi-well setup. Also, the total number of equivalents, as well as the derived amount in milligrams (depend-ending on user-defined equivalent weights and molar surplus), of each amino acid are given. The program facilitates the implementation of multipeptide synthesis, e.g., for the elucidation of polypeptide structure-function relationships, and greatly reduces the risk of introducing mistakes at the planning step. It is written in Pascal and runs on any DOS-based personal computer. No special graphic display is needed.

Precursors of vertebrate peptide antibiotics dermaseptin b and adenoregulin have extensive sequence identities with precursors of opioid peptides dermorphin, dermenkephalin, and deltorphins.

Science.gov (United States)

Amiche, M; Ducancel, F; Mor, A; Boulain, J C; Menez, A; Nicolas, P

1994-07-08

The dermaseptins are a family of broad spectrum antimicrobial peptides, 27-34 amino acids long, involved in the defense of the naked skin of frogs against microbial invasion. They are the first vertebrate peptides to show lethal effects against the filamentous fungi responsible for severe opportunistic infections accompanying immunodeficiency syndrome and the use of immunosuppressive agents. A cDNA library was constructed from skin poly(A+) RNA of the arboreal frog Phyllomedusa bicolor and screened with an oligonucleotide probe complementary to the COOH terminus of dermaseptin b. Several clones contained a full-length DNA copy of a 443-nucleotide mRNA that encoded a 78-residue dermaseptin b precursor protein. The deduced precursor contained a putative signal sequence at the NH2 terminus, a 20-residue spacer sequence extremely rich (60%) in glutamic and aspartic acids, and a single copy of a dermaseptin b progenitor sequence at the COOH terminus. One clone contained a complete copy of adenoregulin, a 33-residue peptide reported to enhance the binding of agonists to the A1 adenosine receptor. The mRNAs encoding adenoregulin and dermaseptin b were very similar: 70 and 75% nucleotide identities between the 5'- and 3'-untranslated regions, respectively; 91% amino acid identity between the signal peptides; 82% identity between the acidic spacer sequences; and 38% identity between adenoregulin and dermaseptin b. Because adenoregulin and dermaseptin b have similar precursor designs and antimicrobial spectra, adenoregulin should be considered as a new member of the dermaseptin family and alternatively named dermaseptin b II. Preprodermaseptin b and preproadenoregulin have considerable sequence identities to the precursors encoding the opioid heptapeptides dermorphin, dermenkephalin, and deltorphins. This similarity extended into the 5'-untranslated regions of the mRNAs. These findings suggest that the genes encoding the four preproproteins are all members of the same family

De novo sequencing of two novel peptides homologous to calcitonin-like peptides, from skin secretion of the Chinese Frog, Odorrana schmackeri

NARCIS (Netherlands)

Evaristo, Geisa P C; Pinkse, Martijn W H; Chen, Tianbao; Wang, Lei; Mohammed, Shabaz; Heck, Albert J R; Mathes, Isabella; Lottspeich, Friedrich; Shaw, Chris; Albar, Juan Pablo; Verhaert, Peter D E M

2015-01-01

An MS/MS based analytical strategy was followed to solve the complete sequence of two new peptides from frog (Odorrana schmackeri) skin secretion. This involved reduction and alkylation with two different alkylating agents followed by high resolution tandem mass spectrometry. De novo sequencing was

Human acid β-glucosidase: isolation and amino acid sequence of a peptide containing the catalytic site

International Nuclear Information System (INIS)

Dinur, T.; Osiecki, K.M.; Legler, G.; Gatt, S.; Desnick, R.J.; Grabowski, G.A.

1986-01-01

Human acid β-glucosidase (D-glucosyl-N-acylsphingosine glucohydrolase, EC 3.2.1.45) cleaves the glucosidic bonds of glucosylceramide and synthetic β-glucosides. The deficient activity of this hydrolase is the enzymatic defect in the subtypes and variants of Gaucher disease, the most prevalent lysosomal storage disease. To isolate and characterize the catalytic site of the normal enzyme, brominated 3 H-labeled conduritol B epoxide ( 3 H-Br-CBE), which inhibits the enzyme by binding covalently to this site, was used as an affinity label. Under optimal conditions 1 mol of 3 H-Br-CBE bound to 1 mol of pure enzyme protein, indicating the presence of a single catalytic site per enzyme subunit. After V 8 protease digestion of the 3 H-Br-CBE-labeled homogeneous enzyme, three radiolabeled peptides, designated peptide A, B, or C, were resolved by reverse-phase HPLC. The partial amino acid sequence (37 residues) of peptide A (M/sub r/, 5000) was determined. The sequence of this peptide, which contained the catalytic site, had exact homology to the sequence near the carboxyl terminus of the protein, as predicted from the nucleotide sequence of the full-length cDNA encoding acid β-glucosidase

Cloning and characterization of 2S albumin, Car i 1, a major allergen in pecan.

Science.gov (United States)

Sharma, Girdhari M; Irsigler, Andre; Dhanarajan, Pushparani; Ayuso, Rosalia; Bardina, Luda; Sampson, Hugh A; Roux, Kenneth H; Sathe, Shridhar K

2011-04-27

Although pecans are associated with IgE-mediated food allergies, the allergens responsible remain to be identified and characterized. The 2S albumin gene was amplified from the pecan cDNA library. Dot-blots were used to screen the recombinant protein with pecan allergic patients' serum. The affinity purified native protein was analyzed by Edman sequencing and mass spectrometry/mass spectrometry (MS/MS) analysis. Cross-reactivity with walnut was determined by inhibition enzyme-linked immunosorbent assay (ELISA). Sequential epitopes were determined by probing the overlapping peptides with three different patients' serum pool. The 3-dimensional homology model was generated, and the locations of the pecan epitopes were compared with those of known sequential epitopes on other allergenic tree nut homologues. Of 28 patients tested by dot-blot, 22 (79%) bound to 2S albumin, designated as Car i 1. Edman sequencing and the MS/MS sequencing of native 2S albumin confirmed the identity of recombinant (r) Car i 1. Both pecan and walnut protein extracts inhibited the IgE-binding to rCar i 1. Sequential epitope mapping indicated weak, moderate, and strong reactivity against 12, 7, and 5 peptides, respectively. Of the 11 peptides recognized by all serum pools, 5 peptides were strongly reactive and located in 3 discrete regions of the Car i 1 (amino acids 43-57, 67-78, and 106-120). Three-dimensional modeling revealed IgE-reactive epitopes to be solvent accessible and share significant homology with other tree nuts providing a possible basis for previously observed cross-reactivity.

Error Analysis of Deep Sequencing of Phage Libraries: Peptides Censored in Sequencing

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Wadim L. Matochko

2013-01-01

Full Text Available Next-generation sequencing techniques empower selection of ligands from phage-display libraries because they can detect low abundant clones and quantify changes in the copy numbers of clones without excessive selection rounds. Identification of errors in deep sequencing data is the most critical step in this process because these techniques have error rates >1%. Mechanisms that yield errors in Illumina and other techniques have been proposed, but no reports to date describe error analysis in phage libraries. Our paper focuses on error analysis of 7-mer peptide libraries sequenced by Illumina method. Low theoretical complexity of this phage library, as compared to complexity of long genetic reads and genomes, allowed us to describe this library using convenient linear vector and operator framework. We describe a phage library as N×1 frequency vector n=ni, where ni is the copy number of the ith sequence and N is the theoretical diversity, that is, the total number of all possible sequences. Any manipulation to the library is an operator acting on n. Selection, amplification, or sequencing could be described as a product of a N×N matrix and a stochastic sampling operator (Sa. The latter is a random diagonal matrix that describes sampling of a library. In this paper, we focus on the properties of Sa and use them to define the sequencing operator (Seq. Sequencing without any bias and errors is Seq=Sa IN, where IN is a N×N unity matrix. Any bias in sequencing changes IN to a nonunity matrix. We identified a diagonal censorship matrix (CEN, which describes elimination or statistically significant downsampling, of specific reads during the sequencing process.

Crystal structure of importin-{alpha} complexed with a classic nuclear localization sequence obtained by oriented peptide library screening

Energy Technology Data Exchange (ETDEWEB)

Takeda, A.A.S.; Fontes, M.R.M. [UNESP, Universidade Estadual Paulista, Botucatu, SP (Brazil); Yang, S.N.Y. [University of Melbourne, Melbourne (Australia); Harris, J.M. [Queensland University of Technology, Brisbane (Australia); Jans, D.A. [Monash University, Clayton (Australia); Kobe, B. [University of Queensland, Brisbane, QU (Australia)

2012-07-01

Full text: Importin-{alpha} (Imp{alpha}) plays a role in the classical nuclear import pathway, binding to cargo proteins with activities in the nucleus. Different Imp{alpha} paralogs responsible for specific cargos can be found in a single organism. The cargos contain nuclear localization sequences (NLSs), which are characterized by one or two clusters of basic amino acids (monopartite and bipartite NLSs, respectively). In this work we present the crystal structure of Imp{alpha} from M. musculus (residues 70-529, lacking the auto inhibitory domain) bound to a NLS peptide (pepTM). The peptide corresponds to the optimal sequence obtained by an oriented peptide library experiment designed to probe the specificity of the major NLS binding site. The peptide library used five degenerate positions and identified the sequence KKKRR as the optimal sequence for binding to this site for mouse Imp{alpha} (70-529). The protein was obtained using an E. coli expression system and purified by affinity chromatography followed by an ion exchange chromatography. A single crystal of Imp{alpha} -pepTM complex was grown by the hanging drop method. The data were collected using the Synchrotron Radiation Source LNLS, Brazil and processed to 2.3. Molecular replacement techniques were used to determine the crystal structure. Electron density corresponding to the peptide was present in both major and minor binding sites The peptide is bound to Imp{alpha} similar as the simian virus 40 (SV40) large tumour (T)-antigen NLS. Binding assays confirmed that the peptide bound to Imp{alpha} with low nM affinities. This is the first time that structural information has been linked to an oriented peptide library screening approach for importin-{alpha}; the results will contribute to understanding of the sequence determinants of classical NLSs, and may help identify as yet unidentified classical NLSs in novel proteins. (author)

Complete sequences of glucagon-like peptide-1 from human and pig small intestine

DEFF Research Database (Denmark)

Orskov, C; Bersani, M; Johnsen, A H

1989-01-01

intestine of the proglucagon precursor were determined by pairs of basic amino acid residues flanking the two peptides. Earlier studies have shown that synthetic glucagon-like peptide-1 (GLP-1) synthesized according to the proposed structure (proglucagon 71-108 or because residue 108 is Gly, 72-107 amide......) had no physiological effects, whereas a truncated from of GLP-1, corresponding to proglucagon 78-107 amide, strongly stimulated insulin secretion and depressed glucagon secretion. To determine the amino acid sequence of the naturally occurring peptide we isolated GLP-1 from human small intestine...

Partial sequence determination of metabolically labeled radioactive proteins and peptides

International Nuclear Information System (INIS)

Anderson, C.W.

1982-01-01

The author has used the sequence analysis of radioactive proteins and peptides to approach several problems during the past few years. They, in collaboration with others, have mapped precisely several adenovirus proteins with respect to the nucleotide sequence of the adenovirus genome; identified hitherto missed proteins encoded by bacteriophage MS2 and by simian virus 40; analyzed the aminoterminal maturation of several virus proteins; determined the cleavage sites for processing of the poliovirus polyprotein; and analyzed the mechanism of frameshifting by excess normal tRNAs during cell-free protein synthesis. This chapter is designed to aid those without prior experience at protein sequence determinations. It is based primarily on the experience gained in the studies cited above, which made use of the Beckman 890 series automated protein sequencers

UniNovo: a universal tool for de novo peptide sequencing.

Science.gov (United States)

Jeong, Kyowon; Kim, Sangtae; Pevzner, Pavel A

2013-08-15

Mass spectrometry (MS) instruments and experimental protocols are rapidly advancing, but de novo peptide sequencing algorithms to analyze tandem mass (MS/MS) spectra are lagging behind. Although existing de novo sequencing tools perform well on certain types of spectra [e.g. Collision Induced Dissociation (CID) spectra of tryptic peptides], their performance often deteriorates on other types of spectra, such as Electron Transfer Dissociation (ETD), Higher-energy Collisional Dissociation (HCD) spectra or spectra of non-tryptic digests. Thus, rather than developing a new algorithm for each type of spectra, we develop a universal de novo sequencing algorithm called UniNovo that works well for all types of spectra or even for spectral pairs (e.g. CID/ETD spectral pairs). UniNovo uses an improved scoring function that captures the dependences between different ion types, where such dependencies are learned automatically using a modified offset frequency function. The performance of UniNovo is compared with PepNovo+, PEAKS and pNovo using various types of spectra. The results show that the performance of UniNovo is superior to other tools for ETD spectra and superior or comparable with others for CID and HCD spectra. UniNovo also estimates the probability that each reported reconstruction is correct, using simple statistics that are readily obtained from a small training dataset. We demonstrate that the estimation is accurate for all tested types of spectra (including CID, HCD, ETD, CID/ETD and HCD/ETD spectra of trypsin, LysC or AspN digested peptides). UniNovo is implemented in JAVA and tested on Windows, Ubuntu and OS X machines. UniNovo is available at http://proteomics.ucsd.edu/Software/UniNovo.html along with the manual.

Proteomics of the chloroplast: systematic identification and targeting analysis of lumenal and peripheral thylakoid proteins

DEFF Research Database (Denmark)

Peltier, J B; Friso, G; Kalume, D E

2000-01-01

The soluble and peripheral proteins in the thylakoids of pea were systematically analyzed by using two-dimensional electrophoresis, mass spectrometry, and N-terminal Edman sequencing, followed by database searching. After correcting to eliminate possible isoforms and post-translational modificati......The soluble and peripheral proteins in the thylakoids of pea were systematically analyzed by using two-dimensional electrophoresis, mass spectrometry, and N-terminal Edman sequencing, followed by database searching. After correcting to eliminate possible isoforms and post......-translational modifications, we estimated that there are at least 200 to 230 different lumenal and peripheral proteins. Sixty-one proteins were identified; for 33 of these proteins, a clear function or functional domain could be identified, whereas for 10 proteins, no function could be assigned. For 18 proteins, no expressed...... sequence tag or full-length gene could be identified in the databases, despite experimental determination of a significant amount of amino acid sequence. Nine previously unidentified proteins with lumenal transit peptides are presented along with their full-length genes; seven of these proteins possess...

Antimicrobial Activity of Truncated and Polyvalent Peptides Derived from the FKCRRQWQWRMKKGLA Sequence against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923

Directory of Open Access Journals (Sweden)

Nataly de Jesús Huertas

2017-06-01

Full Text Available Peptides derived from LfcinB were designed and synthesized, and their antibacterial activity was tested against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923. Specifically, a peptide library was constructed by systemically removing the flanking residues (N or C-terminal of Lfcin 17–31 (17FKCRRWQWRMKKLGA31, maintaining in all peptides the 20RRWQWR25 sequence that corresponds to the minimal antimicrobial motif. For this research, also included were (i a peptide containing an Ala instead of Cys ([Ala19]-LfcinB 17–31 and (ii polyvalent peptides containing the RRWQWR sequence and a non-natural amino acid (aminocaproic acid. We established that the lineal peptides LfcinB 17–25 and LfcinB 17–26 exhibited the greatest activity against E. coli ATCC 25922 and S. aureus ATCC 25923, respectively. On the other hand, polyvalent peptides, a dimer and a tetramer, exhibited the greatest antibacterial activity, indicating that multiple copies of the sequence increase the activity. Our results suggest that the dimeric and tetrameric sequence forms potentiate the antibacterial activity of lineal sequences that have exhibited moderate antibacterial activity.

A Combinational Strategy upon RNA Sequencing and Peptidomics Unravels a Set of Novel Toxin Peptides in Scorpion Mesobuthus martensii

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Ning Luan

2016-10-01

Full Text Available Scorpion venom is deemed to contain many toxic peptides as an important source of natural compounds. Out of the two hundred proteins identified in Mesobuthus martensii (M. martensii, only a few peptide toxins have been found so far. Herein, a combinational approach based upon RNA sequencing and Liquid chromatography-mass spectrometry/mass spectrometry (LC MS/MS was employed to explore the venom peptides in M. martensii. A total of 153 proteins were identified from the scorpion venom, 26 previously known and 127 newly identified. Of the novel toxins, 97 proteins exhibited sequence similarities to known toxins, and 30 were never reported. Combining peptidomic and transcriptomic analyses, the peptide sequence of BmKKx1 was reannotated and four disulfide bridges were confirmed within it. In light of the comparison of conservation and variety of toxin amino acid sequences, highly conserved and variable regions were perceived in 24 toxins that were parts of two sodium channel and two potassium channel toxins families. Taking all of this evidences together, the peptidomic analysis on M. martensii indeed identified numerous novel scorpion peptides, expanded our knowledge towards the venom diversity, and afforded a set of pharmaceutical candidates.

Enhancement of the Enterocin CRL35 Activity by a Synthetic Peptide Derived from the NH2-Terminal Sequence

Science.gov (United States)

Saavedra, Lucila; Minahk, Carlos; de Ruiz Holgado, Aída P.; Sesma, Fernando

2004-01-01

The enterocin CRL35 biosynthetic gene cluster was cloned and sequenced. The sequence was revealed to be highly identical to that of the mundticin KS gene cluster (S. Kawamoto, J. Shima, R. Sato, T. Eguchi, S. Ohmomo, J. Shibato, N. Horikoshi, K. Takeshita, and T. Sameshima, Appl. Environ. Microbiol. 68:3830-3840, 2002). Short synthetic peptides were designed based on the bacteriocin sequence and were evaluated in antimicrobial competitive assays. The peptide KYYGNGVSCNKKGCS produced an enhancement of enterocin CRL35 antimicrobial activity in a buffer system. PMID:15215149

Identification of Alternative Splice Variants Using Unique Tryptic Peptide Sequences for Database Searches.

Science.gov (United States)

Tran, Trung T; Bollineni, Ravi C; Strozynski, Margarita; Koehler, Christian J; Thiede, Bernd

2017-07-07

Alternative splicing is a mechanism in eukaryotes by which different forms of mRNAs are generated from the same gene. Identification of alternative splice variants requires the identification of peptides specific for alternative splice forms. For this purpose, we generated a human database that contains only unique tryptic peptides specific for alternative splice forms from Swiss-Prot entries. Using this database allows an easy access to splice variant-specific peptide sequences that match to MS data. Furthermore, we combined this database without alternative splice variant-1-specific peptides with human Swiss-Prot. This combined database can be used as a general database for searching of LC-MS data. LC-MS data derived from in-solution digests of two different cell lines (LNCaP, HeLa) and phosphoproteomics studies were analyzed using these two databases. Several nonalternative splice variant-1-specific peptides were found in both cell lines, and some of them seemed to be cell-line-specific. Control and apoptotic phosphoproteomes from Jurkat T cells revealed several nonalternative splice variant-1-specific peptides, and some of them showed clear quantitative differences between the two states.

Enterocin T, a novel class IIa bacteriocin produced by Enterococcus sp. 812.

Science.gov (United States)

Chen, Yi-Sheng; Yu, Chi-Rong; Ji, Si-Hua; Liou, Min-Shiuan; Leong, Kun-Hon; Pan, Shwu-Fen; Wu, Hui-Chung; Lin, Yu-Hsuan; Yu, Bi; Yanagida, Fujitoshi

2013-09-01

Enterococcus sp. 812, isolated from fresh broccoli, was previously found to produce a bacteriocin active against a number of Gram-positive bacteria, including Listeria monocytogenes. Bacteriocin activity decreased slightly after autoclaving (121 °C for 15 min), but was inactivated by protease K. Mass spectrometry analysis revealed the bacteriocin mass to be approximately 4,521.34 Da. N-terminal amino acid sequencing yielded a partial sequence, NH2-ATYYGNGVYXDKKKXWVEWGQA, by Edman degradation, which contained the consensus class IIa bacteriocin motif YGNGV in the N-terminal region. The obtained partial sequence showed high homology with some enterococcal bacteriocins; however, no identical peptide or protein was found. This peptide was therefore considered to be a novel bacteriocin produced by Enterococcus sp. 812 and was termed enterocin T.

Sequence of the radioactive tryptic peptide obtained after inactivating the F1-ATPase of the thermophilic bacterium PS3 with 5'-p-fluorosulfonylbenzoyl[3H]adenosine at 65 degrees C

International Nuclear Information System (INIS)

Bullough, D.A.; Yoshida, M.; Allison, W.S.

1986-01-01

Following a lag of about 30 min, the F1-ATPase from the thermophilic bacterium, PS3 (TF1), was inactivated slowly by 0.8 mM 5'-p-fluorosulfonylbenzoyladenosine (FSBA) at 23 degrees C and pH 7.0. When the enzyme was treated with 0.2 mM FSBA at pH 7.0 and 23 degrees C for 15 min and gel-filtered, no enzyme activity was lost. However, the lag in inactivation was abolished when the enzyme was subsequently incubated with 2.0 mM FSBA at 23 degrees C in the pH range from 6.8 to 10.0. The pH-inactivation profile obtained under these conditions revealed a pK alpha of about 9.3 which was associated with the inactivation. When pretreated TF1 was inactivated at 23 degrees C with [3H]FSBA by about 90%, greater than 20 mol of [3H]SBA was incorporated per mole of enzyme. TF1 was inactivated rapidly by 0.8 mM FSBA at pH 6.4 and 65 degrees C, and no lag was observed. Following inactivation of TF1 with 0.8 mM [3H]FSBA at 65 degrees C and pH 6.4, about 10 mol of [3H]SBA was incorporated per mole of enzyme. When a tryptic digest of the labeled enzyme was fractionated by reversed-phase high-performance liquid chromatography, a single major radioactive peptide was isolated. When subjected to automatic Edman degradation, this peptide was shown to have the amino acid sequence: A-L-A-P-E-I-V-G-E-E-H-X-Q-V-A-R, where X indicates that a phenylthiohydantoin derivative was not detected in cycle 12. However, from the DNA sequence of the gene encoding the subunit of TF1 (Y. Kagawa, M. Ishizuka, T. Saishu, and S. Nakao (1985)), this position has been shown to be occupied by tyrosine. This tyrosine is homologous with beta-Tyr-368 of the bovine mitochondrial F1-ATPase (MF1) the modification of which is responsible for the inactivation MF1 by FSBA

The two capsid proteins of maize rayado fino virus contain common peptide sequences.

Science.gov (United States)

Falk, B W; Tsai, J H

1986-01-01

Virions of maize rayado fino virus (MRFV) were purified and two major capsid proteins (ca. Mr 29,000 and 22,000) were resolved by SDS-PAGE. When the two major capsid proteins were isolated from gels and compared by one-dimensional peptide mapping after digestion with Staphylococcus aureus V-8 protease, indistinguishable peptide maps were obtained, suggesting that these two proteins contain common peptide sequences. Some preparations also showed minor protein components that were intermediate between the Mr 22,000 and Mr 29,000 capsid proteins. One of the minor proteins, ca. Mr 27,000, gave a peptide map indistinguishable from the major capsid proteins. In vitro ageing of partially purified preparations or virion treatment with proteolytic enzymes failed to show conversion of the Mr 29,000 protein to a Mr 22,000. Protease inhibitors added to the buffers used for virion purification did not affect the apparent 1:3 ratio of 29,000 to 22,000 proteins in the purified preparations.

Modeling of the Ebola Virus Delta Peptide Reveals a Potential Lytic Sequence Motif

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William R. Gallaher

2015-01-01

Full Text Available Filoviruses, such as Ebola and Marburg viruses, cause severe outbreaks of human infection, including the extensive epidemic of Ebola virus disease (EVD in West Africa in 2014. In the course of examining mutations in the glycoprotein gene associated with 2014 Ebola virus (EBOV sequences, a differential level of conservation was noted between the soluble form of glycoprotein (sGP and the full length glycoprotein (GP, which are both encoded by the GP gene via RNA editing. In the region of the proteins encoded after the RNA editing site sGP was more conserved than the overlapping region of GP when compared to a distant outlier species, Tai Forest ebolavirus. Half of the amino acids comprising the “delta peptide”, a 40 amino acid carboxy-terminal fragment of sGP, were identical between otherwise widely divergent species. A lysine-rich amphipathic peptide motif was noted at the carboxyl terminus of delta peptide with high structural relatedness to the cytolytic peptide of the non-structural protein 4 (NSP4 of rotavirus. EBOV delta peptide is a candidate viroporin, a cationic pore-forming peptide, and may contribute to EBOV pathogenesis.

Prediction of Xaa-Pro peptide bond conformation from sequence and chemical shifts

Energy Technology Data Exchange (ETDEWEB)

Shen Yang; Bax, Ad, E-mail: bax@nih.go [National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Laboratory of Chemical Physics (United States)

2010-03-15

We present a program, named Promega, to predict the Xaa-Pro peptide bond conformation on the basis of backbone chemical shifts and the amino acid sequence. Using a chemical shift database of proteins of known structure together with the PDB-extracted amino acid preference of cis Xaa-Pro peptide bonds, a cis/trans probability score is calculated from the backbone and {sup 13}C{sup {beta}} chemical shifts of the proline and its neighboring residues. For an arbitrary number of input chemical shifts, which may include Pro-{sup 13}C{sup {gamma}}, Promega calculates the statistical probability that a Xaa-Pro peptide bond is cis. Besides its potential as a validation tool, Promega is particularly useful for studies of larger proteins where Pro-{sup 13}C{sup {gamma}} assignments can be challenging, and for on-going efforts to determine protein structures exclusively on the basis of backbone and {sup 13}C{sup {beta}} chemical shifts.

The Cysteine S-Alkylation Reaction as a Synthetic Method to Covalently Modify Peptide Sequences.

Science.gov (United States)

Calce, Enrica; De Luca, Stefania

2017-01-05

Synthetic methodologies to chemically modify peptide molecules have long been investigated for their impact in the field of chemical biology. They allow the introduction of biochemical probes useful for studying protein functions, for manipulating peptides with therapeutic potential, and for structure-activity relationship investigations. The commonly used approach was the derivatization of an amino acid side chain. In this regard, the cysteine, for its unique reactivity, has been widely employed as the substrate for such modifications. Herein, we report on methodologies developed to modify the cysteine thiol group through the S-alkylation reaction. Some procedures perform the alkylation of cysteine derivatives, in order to prepare building blocks to be used during the peptide synthesis, whilst some others selectively modify peptide sequences containing a cysteine residue with a free thiol group, both in solution and in the solid phase. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Large-scale analysis of peptide sequence variants: the case for high-field asymmetric waveform ion mobility spectrometry.

Science.gov (United States)

Creese, Andrew J; Smart, Jade; Cooper, Helen J

2013-05-21

Large scale analysis of proteins by mass spectrometry is becoming increasingly routine; however, the presence of peptide isomers remains a significant challenge for both identification and quantitation in proteomics. Classes of isomers include sequence inversions, structural isomers, and localization variants. In many cases, liquid chromatography is inadequate for separation of peptide isomers. The resulting tandem mass spectra are composite, containing fragments from multiple precursor ions. The benefits of high-field asymmetric waveform ion mobility spectrometry (FAIMS) for proteomics have been demonstrated by a number of groups, but previously work has focused on extending proteome coverage generally. Here, we present a systematic study of the benefits of FAIMS for a key challenge in proteomics, that of peptide isomers. We have applied FAIMS to the analysis of a phosphopeptide library comprising the sequences GPSGXVpSXAQLX(K/R) and SXPFKXpSPLXFG(K/R), where X = ADEFGLSTVY. The library has defined limits enabling us to make valid conclusions regarding FAIMS performance. The library contains numerous sequence inversions and structural isomers. In addition, there are large numbers of theoretical localization variants, allowing false localization rates to be determined. The FAIMS approach is compared with reversed-phase liquid chromatography and strong cation exchange chromatography. The FAIMS approach identified 35% of the peptide library, whereas LC-MS/MS alone identified 8% and LC-MS/MS with strong cation exchange chromatography prefractionation identified 17.3% of the library.

Use of eluted peptide sequence data to identify the binding characteristics of peptides to the insulin-dependent diabetes susceptibility allele HLA-DQ8 (DQ 3.2).

Science.gov (United States)

Godkin, A; Friede, T; Davenport, M; Stevanovic, S; Willis, A; Jewell, D; Hill, A; Rammensee, H G

1997-06-01

HLA-DQ8 (A1*0301, B1*0302) and -DQ2 (A1*0501, B1*0201) are both associated with diseases such as insulin-dependent diabetes mellitus and coeliac disease. We used the technique of pool sequencing to look at the requirements of peptides binding to HLA-DQ8, and combined these data with naturally sequenced ligands and in vitro binding assays to describe a novel motif for HLA-DQ8. The motif, which has the same basic format as many HLA-DR molecules, consists of four or five anchor regions, in the positions from the N-terminus of the binding core of n, n + 3, n + 5/6 and n + 8, i.e. P1, P4, P6/7 and P9. P1 and P9 require negative or polar residues, with mainly aliphatic residues at P4 and P6/7. The features of the HLA-DQ8 motif were then compared to a pool sequence of peptides eluted from HLA-DQ2. A consensus motif for the binding of a common peptide which may be involved in disease pathogenesis is described. Neither of the disease-associated alleles HLA-DQ2 and -DQ8 have Asp at position 57 of the beta-chain. This Asp, if present, may form a salt bridge with an Arg at position 79 of the alpha-chain and so alter the binding specificity of P9. HLA-DQ2 and -DQ8 both appear to prefer negatively charged amino acids at P9. In contrast, HLA-DQ7 (A1*0301, B1*0301), which is not associated with diabetes, has Asp at beta 57, allowing positively charged amino acids at P9. This analysis of the sequence features of DQ-binding peptides suggests molecular characteristics which may be useful to predict epitopes involved in disease pathogenesis.

Peptide array-based interaction assay of solid-bound peptides and anchorage-dependant cells and its effectiveness in cell-adhesive peptide design.

Science.gov (United States)

Kato, Ryuji; Kaga, Chiaki; Kunimatsu, Mitoshi; Kobayashi, Takeshi; Honda, Hiroyuki

2006-06-01

Peptide array, the designable peptide library covalently synthesized on cellulose support, was applied to assay peptide-cell interaction, between solid-bound peptides and anchorage-dependant cells, to study objective peptide design. As a model case, cell-adhesive peptides that could enhance cell growth as tissue engineering scaffold material, was studied. On the peptide array, the relative cell-adhesion ratio of NIH/3T3 cells was 2.5-fold higher on the RGDS (Arg-Gly-Asp-Ser) peptide spot as compared to the spot with no peptide, thus indicating integrin-mediated peptide-cell interaction. Such strong cell adhesion mediated by the RGDS peptide was easily disrupted by single residue substitution on the peptide array, thus indicating that the sequence recognition accuracy of cells was strictly conserved in our optimized scheme. The observed cellular morphological extension with active actin stress-fiber on the RGD motif-containing peptide supported our strategy that peptide array-based interaction assay of solid-bound peptide and anchorage-dependant cells (PIASPAC) could provide quantitative data on biological peptide-cell interaction. The analysis of 180 peptides obtained from fibronectin type III domain (no. 1447-1629) yielded 18 novel cell-adhesive peptides without the RGD motif. Taken together with the novel candidates, representative rules of ineffective amino acid usage were obtained from non-effective candidate sequences for the effective designing of cell-adhesive peptides. On comparing the amino acid usage of the top 20 and last 20 peptides from the 180 peptides, the following four brief design rules were indicated: (i) Arg or Lys of positively charged amino acids (except His) could enhance cell adhesion, (ii) small hydrophilic amino acids are favored in cell-adhesion peptides, (iii) negatively charged amino acids and small amino acids (except Gly) could reduce cell adhesion, and (iv) Cys and Met could be excluded from the sequence combination since they have

A workflow for peptide-based proteomics in a poorly sequenced plant: A case study on the plasma membrane proteome of banana

DEFF Research Database (Denmark)

Vertommen, A.; Laurell Blom Møller, Anders; Cordewener, J. H. G.

2011-01-01

for membrane proteomics. However, their application in non-model plants demands special precautions to prevent false positive identification of proteins.In the current paper, a workflow for membrane proteomics in banana, a poorly sequenced plant, is proposed. The main steps of this workflow are (i......) optimization of the peptide separation, (ii) performing de novo sequencing to allow a sequence homology search and (iii) visualization of identified peptide–protein associations using Cytoscape to remove redundancy and wrongly assigned peptides, based on species-specific information. By applying this workflow...

Primary structure of Lep d I, the main Lepidoglyphus destructor allergen.

Science.gov (United States)

Varela, J; Ventas, P; Carreira, J; Barbas, J A; Gimenez-Gallego, G; Polo, F

1994-10-01

The most relevant allergen of the storage mite Lepidoglyphus destructor (Lep d I) has been characterized. Lep d I is a monomer protein of 13273 Da. The primary structure of Lep d I was determined by N-terminal Edman degradation and partially confirmed by cDNA sequencing. Sequence polymorphism was observed at six positions, with non-conservative substitutions in three of them. No potential N-glycosylation site was revealed by peptide sequencing. The 125-residue sequence of Lep d I shows approximately 40% identity (including the six cysteines) with the overlapping regions of group II allergens from the genus Dermatophagoides, which, however, do not share common allergenic epitopes with Lep d I.

PH dependent adhesive peptides

Science.gov (United States)

Tomich, John; Iwamoto, Takeo; Shen, Xinchun; Sun, Xiuzhi Susan

2010-06-29

A novel peptide adhesive motif is described that requires no receptor or cross-links to achieve maximal adhesive strength. Several peptides with different degrees of adhesive strength have been designed and synthesized using solid phase chemistries. All peptides contain a common hydrophobic core sequence flanked by positively or negatively charged amino acids sequences.

Expanding the peptide beta-turn in alphagamma hybrid sequences: 12 atom hydrogen bonded helical and hairpin turns.

Science.gov (United States)

Chatterjee, Sunanda; Vasudev, Prema G; Raghothama, Srinivasarao; Ramakrishnan, Chandrasekharan; Shamala, Narayanaswamy; Balaram, Padmanabhan

2009-04-29

Hybrid peptide segments containing contiguous alpha and gamma amino acid residues can form C(12) hydrogen bonded turns which may be considered as backbone expanded analogues of C(10) (beta-turns) found in alphaalpha segments. Exploration of the regular hydrogen bonded conformations accessible for hybrid alphagamma sequences is facilitated by the use of a stereochemically constrained gamma amino acid residue gabapentin (1-aminomethylcyclohexaneacetic acid, Gpn), in which the two torsion angles about C(gamma)-C(beta) (theta(1)) and C(beta)-C(alpha) (theta(2)) are predominantly restricted to gauche conformations. The crystal structures of the octapeptides Boc-Gpn-Aib-Gpn-Aib-Gpn-Aib-Gpn-Aib-OMe (1) and Boc-Leu-Phe-Val-Aib-Gpn-Leu-Phe-Val-OMe (2) reveal two distinct conformations for the Aib-Gpn segment. Peptide 1 forms a continuous helix over the Aib(2)-Aib(6) segment, while the peptide 2 forms a beta-hairpin structure stabilized by four cross-strand hydrogen bonds with the Aib-Gpn segment forming a nonhelical C(12) turn. The robustness of the helix in peptide 1 in solution is demonstrated by NMR methods. Peptide 2 is conformationally fragile in solution with evidence of beta-hairpin conformations being obtained in methanol. Theoretical calculations permit delineation of the various C(12) hydrogen bonded structures which are energetically feasible in alphagamma and gammaalpha sequences.

Multi-species sequence comparison reveals conservation of ghrelin gene-derived splice variants encoding a truncated ghrelin peptide.

Science.gov (United States)

Seim, Inge; Jeffery, Penny L; Thomas, Patrick B; Walpole, Carina M; Maugham, Michelle; Fung, Jenny N T; Yap, Pei-Yi; O'Keeffe, Angela J; Lai, John; Whiteside, Eliza J; Herington, Adrian C; Chopin, Lisa K

2016-06-01

The peptide hormone ghrelin is a potent orexigen produced predominantly in the stomach. It has a number of other biological actions, including roles in appetite stimulation, energy balance, the stimulation of growth hormone release and the regulation of cell proliferation. Recently, several ghrelin gene splice variants have been described. Here, we attempted to identify conserved alternative splicing of the ghrelin gene by cross-species sequence comparisons. We identified a novel human exon 2-deleted variant and provide preliminary evidence that this splice variant and in1-ghrelin encode a C-terminally truncated form of the ghrelin peptide, termed minighrelin. These variants are expressed in humans and mice, demonstrating conservation of alternative splicing spanning 90 million years. Minighrelin appears to have similar actions to full-length ghrelin, as treatment with exogenous minighrelin peptide stimulates appetite and feeding in mice. Forced expression of the exon 2-deleted preproghrelin variant mirrors the effect of the canonical preproghrelin, stimulating cell proliferation and migration in the PC3 prostate cancer cell line. This is the first study to characterise an exon 2-deleted preproghrelin variant and to demonstrate sequence conservation of ghrelin gene-derived splice variants that encode a truncated ghrelin peptide. This adds further impetus for studies into the alternative splicing of the ghrelin gene and the function of novel ghrelin peptides in vertebrates.

The primary structure of rat liver ribosomal protein L37. Homology with yeast and bacterial ribosomal proteins.

Science.gov (United States)

Lin, A; McNally, J; Wool, I G

1983-09-10

The covalent structure of the rat liver 60 S ribosomal subunit protein L37 was determined. Twenty-four tryptic peptides were purified and the sequence of each was established; they accounted for all 111 residues of L37. The sequence of the first 30 residues of L37, obtained previously by automated Edman degradation of the intact protein, provided the alignment of the first 9 tryptic peptides. Three peptides (CN1, CN2, and CN3) were produced by cleavage of protein L37 with cyanogen bromide. The sequence of CN1 (65 residues) was established from the sequence of secondary peptides resulting from cleavage with trypsin and chymotrypsin. The sequence of CN1 in turn served to order tryptic peptides 1 through 14. The sequence of CN2 (15 residues) was determined entirely by a micromanual procedure and allowed the alignment of tryptic peptides 14 through 18. The sequence of the NH2-terminal 28 amino acids of CN3 (31 residues) was determined; in addition the complete sequences of the secondary tryptic and chymotryptic peptides were done. The sequence of CN3 provided the order of tryptic peptides 18 through 24. Thus the sequence of the three cyanogen bromide peptides also accounted for the 111 residues of protein L37. The carboxyl-terminal amino acids were identified after carboxypeptidase A treatment. There is a disulfide bridge between half-cystinyl residues at positions 40 and 69. Rat liver ribosomal protein L37 is homologous with yeast YP55 and with Escherichia coli L34. Moreover, there is a segment of 17 residues in rat L37 that occurs, albeit with modifications, in yeast YP55 and in E. coli S4, L20, and L34.

Structure of chymotrypsin variant B from Atlantic cod, Gadus morhua

DEFF Research Database (Denmark)

Leth-Larsen, Rikke; Asgeirsson, B; Thórólfsson, M

1996-01-01

The amino-acid sequence of chymotrypsin variant B isolated from the pyloric caeca of Atlantic cod has been elucidated. The characterization of the primary structure is based on N-terminal Edman degradation and mass spectrometry of the native protein and enzymatically derived peptides. Chymotrypsi...... autolysis sites, cod variant B only contains a single autolysis site. The three-dimensional structures of the A- and B-variants of cod has been modelled on the known crystal structure of bovine alpha-chymotrypsin showing almost superimposable structures....

Secretory signal peptide modification for optimized antibody-fragment expression-secretion in Leishmania tarentolae

Directory of Open Access Journals (Sweden)

Klatt Stephan

2012-07-01

Full Text Available Abstract Background Secretory signal peptides (SPs are well-known sequence motifs targeting proteins for translocation across the endoplasmic reticulum membrane. After passing through the secretory pathway, most proteins are secreted to the environment. Here, we describe the modification of an expression vector containing the SP from secreted acid phosphatase 1 (SAP1 of Leishmania mexicana for optimized protein expression-secretion in the eukaryotic parasite Leishmania tarentolae with regard to recombinant antibody fragments. For experimental design the online tool SignalP was used, which predicts the presence and location of SPs and their cleavage sites in polypeptides. To evaluate the signal peptide cleavage site as well as changes of expression, SPs were N-terminally linked to single-chain Fragment variables (scFv’s. The ability of L. tarentolae to express complex eukaryotic proteins with highly diverse post-translational modifications and its easy bacteria-like handling, makes the parasite a promising expression system for secretory proteins. Results We generated four vectors with different SP-sequence modifications based on in-silico analyses with SignalP in respect to cleavage probability and location, named pLTEX-2 to pLTEX-5. To evaluate their functionality, we cloned four individual scFv-fragments into the vectors and transfected all 16 constructs into L. tarentolae. Independently from the expressed scFv, pLTEX-5 derived constructs showed the highest expression rate, followed by pLTEX-4 and pLTEX-2, whereas only low amounts of protein could be obtained from pLTEX-3 clones, indicating dysfunction of the SP. Next, we analysed the SP cleavage sites by Edman degradation. For pLTEX-2, -4, and -5 derived scFv’s, the results corresponded to in-silico predictions, whereas pLTEX-3 derived scFv’s contained one additional amino-acid (AA. Conclusions The obtained results demonstrate the importance of SP-sequence optimization for efficient

Prediction of Antimicrobial Peptides Based on Sequence Alignment and Support Vector Machine-Pairwise Algorithm Utilizing LZ-Complexity

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Xin Yi Ng

2015-01-01

Full Text Available This study concerns an attempt to establish a new method for predicting antimicrobial peptides (AMPs which are important to the immune system. Recently, researchers are interested in designing alternative drugs based on AMPs because they have found that a large number of bacterial strains have become resistant to available antibiotics. However, researchers have encountered obstacles in the AMPs designing process as experiments to extract AMPs from protein sequences are costly and require a long set-up time. Therefore, a computational tool for AMPs prediction is needed to resolve this problem. In this study, an integrated algorithm is newly introduced to predict AMPs by integrating sequence alignment and support vector machine- (SVM- LZ complexity pairwise algorithm. It was observed that, when all sequences in the training set are used, the sensitivity of the proposed algorithm is 95.28% in jackknife test and 87.59% in independent test, while the sensitivity obtained for jackknife test and independent test is 88.74% and 78.70%, respectively, when only the sequences that has less than 70% similarity are used. Applying the proposed algorithm may allow researchers to effectively predict AMPs from unknown protein peptide sequences with higher sensitivity.

Driving engineering of novel antimicrobial peptides from simulations of peptide-micelle interactions

DEFF Research Database (Denmark)

Khandelia, Himanshu; Langham, Allison A; Kaznessis, Yiannis N

2006-01-01

Simulations of antimicrobial peptides in membrane mimics can provide the high resolution, atomistic picture that is necessary to decipher which sequence and structure components are responsible for activity and toxicity. With such detailed insight, engineering new sequences that are active but non...... peptides and their interaction with membrane mimics. In this article, we discuss the promise and the challenges of widely used models and detail our recent work on peptide-micelle simulations as an attractive alternative to peptide-bilayer simulations. We detail our results with two large structural...... classes of peptides, helical and beta-sheet and demonstrate how simulations can assist in engineering of novel antimicrobials with therapeutic potential....

Aggregation and toxicity of amyloid-beta peptide in relation to peptide sequence variation

OpenAIRE

Vandersteen, A.

2012-01-01

Generally, aggregation of the amyloid-ß peptide is considered the cause of neuronal death in Alzheimer disease. The heterogenous Aß peptide occurs in various lengths in vivo: Aß40 and Aß42 are the predominant forms while both shorter and longer peptides exist. Aß40 and shorter isoforms are less aggregation-prone and hence considered less dangerous than Aß42 and longer isoforms, which are more aggregation-prone. Up to now research mainly focussed on the predominant Aß peptides and their indivi...

Peptide-membrane interactions of arginine-tryptophan peptides probed using quartz crystal microbalance with dissipation monitoring.

KAUST Repository

Rydberg, Hanna A

2014-04-18

Membrane-active peptides include peptides that can cross cellular membranes and deliver macromolecular cargo as well as peptides that inhibit bacterial growth. Some of these peptides can act as both transporters and antibacterial agents. It is desirable to combine the knowledge from these two different fields of membrane-active peptides into design of new peptides with tailored actions, as transporters of cargo or as antibacterial substances, targeting specific membranes. We have previously shown that the position of the amino acid tryptophan in the peptide sequence of three arginine-tryptophan peptides affects their uptake and intracellular localization in live mammalian cells, as well as their ability to inhibit bacterial growth. Here, we use quartz crystal microbalance with dissipation monitoring to assess the induced changes caused by binding of the three peptides to supported model membranes composed of POPC, POPC/POPG, POPC/POPG/cholesterol or POPC/lactosyl PE. Our results indicate that the tryptophan position in the peptide sequence affects the way these peptides interact with the different model membranes and that the presence of cholesterol in particular seems to affect the membrane interaction of the peptide with an even distribution of tryptophans in the peptide sequence. These results give mechanistic insight into the function of these peptides and may aid in the design of membrane-active peptides with specified cellular targets and actions.

Peptide-membrane interactions of arginine-tryptophan peptides probed using quartz crystal microbalance with dissipation monitoring.

KAUST Repository

Rydberg, Hanna A; Kunze, Angelika; Carlsson, Nils; Altgä rde, Noomi; Svedhem, Sofia; Nordé n, Bengt

2014-01-01

Membrane-active peptides include peptides that can cross cellular membranes and deliver macromolecular cargo as well as peptides that inhibit bacterial growth. Some of these peptides can act as both transporters and antibacterial agents. It is desirable to combine the knowledge from these two different fields of membrane-active peptides into design of new peptides with tailored actions, as transporters of cargo or as antibacterial substances, targeting specific membranes. We have previously shown that the position of the amino acid tryptophan in the peptide sequence of three arginine-tryptophan peptides affects their uptake and intracellular localization in live mammalian cells, as well as their ability to inhibit bacterial growth. Here, we use quartz crystal microbalance with dissipation monitoring to assess the induced changes caused by binding of the three peptides to supported model membranes composed of POPC, POPC/POPG, POPC/POPG/cholesterol or POPC/lactosyl PE. Our results indicate that the tryptophan position in the peptide sequence affects the way these peptides interact with the different model membranes and that the presence of cholesterol in particular seems to affect the membrane interaction of the peptide with an even distribution of tryptophans in the peptide sequence. These results give mechanistic insight into the function of these peptides and may aid in the design of membrane-active peptides with specified cellular targets and actions.

CycloBranch: De Novo Sequencing of Nonribosomal Peptides from Accurate Product Ion Mass Spectra

Czech Academy of Sciences Publication Activity Database

Novák, Jiří; Lemr, Karel; Schug, K. A.; Havlíček, Vladimír

2015-01-01

Roč. 26, č. 10 (2015), s. 1780-1786 ISSN 1044-0305 R&D Projects: GA ČR(CZ) GAP206/12/1150 Grant - others:OPPC(XE) CZ.2.16/3.1.00/24023 Institutional support: RVO:61388971 Keywords : De novo sequencing * Nonribosomal peptides * Linear Subject RIV: CE - Biochemistry Impact factor: 3.031, year: 2015

NNAlign: A Web-Based Prediction Method Allowing Non-Expert End-User Discovery of Sequence Motifs in Quantitative Peptide Data

DEFF Research Database (Denmark)

Andreatta, Massimo; Schafer-Nielsen, Claus; Lund, Ole

2011-01-01

Recent advances in high-throughput technologies have made it possible to generate both gene and protein sequence data at an unprecedented rate and scale thereby enabling entirely new "omics"-based approaches towards the analysis of complex biological processes. However, the amount and complexity...... to interpret large data sets. We have recently developed a method, NNAlign, which is generally applicable to any biological problem where quantitative peptide data is available. This method efficiently identifies underlying sequence patterns by simultaneously aligning peptide sequences and identifying motifs...... associated with quantitative readouts. Here, we provide a web-based implementation of NNAlign allowing non-expert end-users to submit their data (optionally adjusting method parameters), and in return receive a trained method (including a visual representation of the identified motif) that subsequently can...

Detection of discriminative sequence patterns in the neighborhood of proline cis peptide bonds and their functional annotation

Directory of Open Access Journals (Sweden)

Papaloukas Costas

2009-04-01

Full Text Available Abstract Background Polypeptides are composed of amino acids covalently bonded via a peptide bond. The majority of peptide bonds in proteins is found to occur in the trans conformation. In spite of their infrequent occurrence, cis peptide bonds play a key role in the protein structure and function, as well as in many significant biological processes. Results We perform a systematic analysis of regions in protein sequences that contain a proline cis peptide bond in order to discover non-random associations between the primary sequence and the nature of proline cis/trans isomerization. For this purpose an efficient pattern discovery algorithm is employed which discovers regular expression-type patterns that are overrepresented (i.e. appear frequently repeated in a set of sequences. Four types of pattern discovery are performed: i exact pattern discovery, ii pattern discovery using a chemical equivalency set, iii pattern discovery using a structural equivalency set and iv pattern discovery using certain amino acids' physicochemical properties. The extracted patterns are carefully validated using a specially implemented scoring function and a significance measure (i.e. log-probability estimate indicative of their specificity. The score threshold for the first three types of pattern discovery is 0.90 while for the last type of pattern discovery 0.80. Regarding the significance measure, all patterns yielded values in the range [-9, -31] which ensure that the derived patterns are highly unlikely to have emerged by chance. Among the highest scoring patterns, most of them are consistent with previous investigations concerning the neighborhood of cis proline peptide bonds, and many new ones are identified. Finally, the extracted patterns are systematically compared against the PROSITE database, in order to gain insight into the functional implications of cis prolyl bonds. Conclusion Cis patterns with matches in the PROSITE database fell mostly into two

Statistically significant dependence of the Xaa-Pro peptide bond conformation on secondary structure and amino acid sequence

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Leitner Dietmar

2005-04-01

Full Text Available Abstract Background A reliable prediction of the Xaa-Pro peptide bond conformation would be a useful tool for many protein structure calculation methods. We have analyzed the Protein Data Bank and show that the combined use of sequential and structural information has a predictive value for the assessment of the cis versus trans peptide bond conformation of Xaa-Pro within proteins. For the analysis of the data sets different statistical methods such as the calculation of the Chou-Fasman parameters and occurrence matrices were used. Furthermore we analyzed the relationship between the relative solvent accessibility and the relative occurrence of prolines in the cis and in the trans conformation. Results One of the main results of the statistical investigations is the ranking of the secondary structure and sequence information with respect to the prediction of the Xaa-Pro peptide bond conformation. We observed a significant impact of secondary structure information on the occurrence of the Xaa-Pro peptide bond conformation, while the sequence information of amino acids neighboring proline is of little predictive value for the conformation of this bond. Conclusion In this work, we present an extensive analysis of the occurrence of the cis and trans proline conformation in proteins. Based on the data set, we derived patterns and rules for a possible prediction of the proline conformation. Upon adoption of the Chou-Fasman parameters, we are able to derive statistically relevant correlations between the secondary structure of amino acid fragments and the Xaa-Pro peptide bond conformation.

Recurrent Neural Network Model for Constructive Peptide Design.

Science.gov (United States)

Müller, Alex T; Hiss, Jan A; Schneider, Gisbert

2018-02-26

We present a generative long short-term memory (LSTM) recurrent neural network (RNN) for combinatorial de novo peptide design. RNN models capture patterns in sequential data and generate new data instances from the learned context. Amino acid sequences represent a suitable input for these machine-learning models. Generative models trained on peptide sequences could therefore facilitate the design of bespoke peptide libraries. We trained RNNs with LSTM units on pattern recognition of helical antimicrobial peptides and used the resulting model for de novo sequence generation. Of these sequences, 82% were predicted to be active antimicrobial peptides compared to 65% of randomly sampled sequences with the same amino acid distribution as the training set. The generated sequences also lie closer to the training data than manually designed amphipathic helices. The results of this study showcase the ability of LSTM RNNs to construct new amino acid sequences within the applicability domain of the model and motivate their prospective application to peptide and protein design without the need for the exhaustive enumeration of sequence libraries.

Prediction of Scylla olivacea (Crustacea; Brachyura) peptide hormones using publicly accessible transcriptome shotgun assembly (TSA) sequences.

Science.gov (United States)

Christie, Andrew E

2016-05-01

The aquaculture of crabs from the genus Scylla is of increasing economic importance for many Southeast Asian countries. Expansion of Scylla farming has led to increased efforts to understand the physiology and behavior of these crabs, and as such, there are growing molecular resources for them. Here, publicly accessible Scylla olivacea transcriptomic data were mined for putative peptide-encoding transcripts; the proteins deduced from the identified sequences were then used to predict the structures of mature peptide hormones. Forty-nine pre/preprohormone-encoding transcripts were identified, allowing for the prediction of 187 distinct mature peptides. The identified peptides included isoforms of adipokinetic hormone-corazonin-like peptide, allatostatin A, allatostatin B, allatostatin C, bursicon β, CCHamide, corazonin, crustacean cardioactive peptide, crustacean hyperglycemic hormone/molt-inhibiting hormone, diuretic hormone 31, eclosion hormone, FMRFamide-like peptide, HIGSLYRamide, insulin-like peptide, intocin, leucokinin, myosuppressin, neuroparsin, neuropeptide F, orcokinin, pigment dispersing hormone, pyrokinin, red pigment concentrating hormone, RYamide, short neuropeptide F, SIFamide and tachykinin-related peptide, all well-known neuropeptide families. Surprisingly, the tissue used to generate the transcriptome mined here is reported to be testis. Whether or not the testis samples had neural contamination is unknown. However, if the peptides are truly produced by this reproductive organ, it could have far reaching consequences for the study of crustacean endocrinology, particularly in the area of reproductive control. Regardless, this peptidome is the largest thus far predicted for any brachyuran (true crab) species, and will serve as a foundation for future studies of peptidergic control in members of the commercially important genus Scylla. Copyright © 2016 Elsevier Inc. All rights reserved.

PuLSE: Quality control and quantification of peptide sequences explored by phage display libraries.

Science.gov (United States)

Shave, Steven; Mann, Stefan; Koszela, Joanna; Kerr, Alastair; Auer, Manfred

2018-01-01

The design of highly diverse phage display libraries is based on assumption that DNA bases are incorporated at similar rates within the randomized sequence. As library complexity increases and expected copy numbers of unique sequences decrease, the exploration of library space becomes sparser and the presence of truly random sequences becomes critical. We present the program PuLSE (Phage Library Sequence Evaluation) as a tool for assessing randomness and therefore diversity of phage display libraries. PuLSE runs on a collection of sequence reads in the fastq file format and generates tables profiling the library in terms of unique DNA sequence counts and positions, translated peptide sequences, and normalized 'expected' occurrences from base to residue codon frequencies. The output allows at-a-glance quantitative quality control of a phage library in terms of sequence coverage both at the DNA base and translated protein residue level, which has been missing from toolsets and literature. The open source program PuLSE is available in two formats, a C++ source code package for compilation and integration into existing bioinformatics pipelines and precompiled binaries for ease of use.

Antioxidant activity of yoghurt peptides: Part 2 – Characterisationof peptide fractions

DEFF Research Database (Denmark)

Farvin, Sabeena; Baron, Caroline; Nielsen, Nina Skall

2010-01-01

the peptides identified contained at least one proline residue. Some of the identified peptides included the hydrophobic amino acid residues Val or Leu at the N-terminus and Pro, His or Tyr in the amino acid sequence, which is characteristic of antioxidant peptides. In addition, the yoghurt contained...

Flanking signal and mature peptide residues influence signal peptide cleavage

Directory of Open Access Journals (Sweden)

Ranganathan Shoba

2008-12-01

Full Text Available Abstract Background Signal peptides (SPs mediate the targeting of secretory precursor proteins to the correct subcellular compartments in prokaryotes and eukaryotes. Identifying these transient peptides is crucial to the medical, food and beverage and biotechnology industries yet our understanding of these peptides remains limited. This paper examines the most common type of signal peptides cleavable by the endoprotease signal peptidase I (SPase I, and the residues flanking the cleavage sites of three groups of signal peptide sequences, namely (i eukaryotes (Euk (ii Gram-positive (Gram+ bacteria, and (iii Gram-negative (Gram- bacteria. Results In this study, 2352 secretory peptide sequences from a variety of organisms with amino-terminal SPs are extracted from the manually curated SPdb database for analysis based on physicochemical properties such as pI, aliphatic index, GRAVY score, hydrophobicity, net charge and position-specific residue preferences. Our findings show that the three groups share several similarities in general, but they display distinctive features upon examination in terms of their amino acid compositions and frequencies, and various physico-chemical properties. Thus, analysis or prediction of their sequences should be separated and treated as distinct groups. Conclusion We conclude that the peptide segment recognized by SPase I extends to the start of the mature protein to a limited extent, upon our survey of the amino acid residues surrounding the cleavage processing site. These flanking residues possibly influence the cleavage processing and contribute to non-canonical cleavage sites. Our findings are applicable in defining more accurate prediction tools for recognition and identification of cleavage site of SPs.

Identification of novel bacteriophage peptides using a combination of gene sequence LC-MS-MS analysis and BLASTP

Science.gov (United States)

Introduction: In an effort to characterize novel bacteriophage with lytic activity against pathogenic E.coli associated with foodborne illness, gene sequencing and mass spectrometry have been used to identify expressed peptides which differentiate isolated bacteriophage from other known phage. Here,...

Production, purification, sequencing and activity spectra of mutacins D-123.1 and F-59.1.

Science.gov (United States)

Nicolas, Guillaume G; LaPointe, Gisèle; Lavoie, Marc C

2011-04-10

The increase in bacterial resistance to antibiotics impels the development of new anti-bacterial substances. Mutacins (bacteriocins) are small antibacterial peptides produced by Streptococcus mutans showing activity against bacterial pathogens. The objective of the study was to produce and characterise additional mutacins in order to find new useful antibacterial substances. Mutacin F-59.1 was produced in liquid media by S. mutans 59.1 while production of mutacin D-123.1 by S. mutans 123.1 was obtained in semi-solid media. Mutacins were purified by hydrophobic chromatography. The amino acid sequences of the mutacins were obtained by Edman degradation and their molecular mass was determined by mass spectrometry. Mutacin F-59.1 consists of 25 amino acids, containing the YGNGV consensus sequence of pediocin-like bacteriocins with a molecular mass calculated at 2719 Da. Mutacin D-123.1 has an identical molecular mass (2364 Da) with the same first 9 amino acids as mutacin I. Mutacins D-123.1 and F-59.1 have wide activity spectra inhibiting human and food-borne pathogens. The lantibiotic mutacin D-123.1 possesses a broader activity spectrum than mutacin F-59.1 against the bacterial strains tested. Mutacin F-59.1 is the first pediocin-like bacteriocin identified and characterised that is produced by Streptococcus mutans. Mutacin D-123.1 appears to be identical to mutacin I previously identified in different strains of S. mutans.

Production, purification, sequencing and activity spectra of mutacins D-123.1 and F-59.1

Directory of Open Access Journals (Sweden)

LaPointe Gisèle

2011-04-01

Full Text Available Abstract Background The increase in bacterial resistance to antibiotics impels the development of new anti-bacterial substances. Mutacins (bacteriocins are small antibacterial peptides produced by Streptococcus mutans showing activity against bacterial pathogens. The objective of the study was to produce and characterise additional mutacins in order to find new useful antibacterial substances. Results Mutacin F-59.1 was produced in liquid media by S. mutans 59.1 while production of mutacin D-123.1 by S. mutans 123.1 was obtained in semi-solid media. Mutacins were purified by hydrophobic chromatography. The amino acid sequences of the mutacins were obtained by Edman degradation and their molecular mass was determined by mass spectrometry. Mutacin F-59.1 consists of 25 amino acids, containing the YGNGV consensus sequence of pediocin-like bacteriocins with a molecular mass calculated at 2719 Da. Mutacin D-123.1 has an identical molecular mass (2364 Da with the same first 9 amino acids as mutacin I. Mutacins D-123.1 and F-59.1 have wide activity spectra inhibiting human and food-borne pathogens. The lantibiotic mutacin D-123.1 possesses a broader activity spectrum than mutacin F-59.1 against the bacterial strains tested. Conclusion Mutacin F-59.1 is the first pediocin-like bacteriocin identified and characterised that is produced by Streptococcus mutans. Mutacin D-123.1 appears to be identical to mutacin I previously identified in different strains of S. mutans.

Investigating the microstructure of keratin extracted from wool: peptide sequence (MALDI-TOF/TOF) and protein conformation (FTIR)

Science.gov (United States)

Keratin was extracted from wool by reduction with 2-mercaptoethanol. It was isolated as intact keratin and characterized by its similar molecular weight, protein composition, and secondary structure to native keratin. Gel electrophoresis patterns and MALDI-TOF/TOF peptide sequences provided the ide...

Interpreting peptide mass spectra by VEMS

DEFF Research Database (Denmark)

Mathiesen, Rune; Lundsgaard, M.; Welinder, Karen G.

2003-01-01

the calculated and the experimental mass spectrum of the called peptide. The program package includes four accessory programs. VEMStrans creates protein databases in FASTA format from EST or cDNA sequence files. VEMSdata creates a virtual peptide database from FASTA files. VEMSdist displays the distribution......Most existing Mass Spectra (MS) analysis programs are automatic and provide limited opportunity for editing during the interpretation. Furthermore, they rely entirely on publicly available databases for interpretation. VEMS (Virtual Expert Mass Spectrometrist) is a program for interactive analysis...... of peptide MS/MS spectra imported in text file format. Peaks are annotated, the monoisotopic peaks retained, and the b-and y-ion series identified in an interactive manner. The called peptide sequence is searched against a local protein database for sequence identity and peptide mass. The report compares...

Dissociation Behavior of a TEMPO-Active Ester Cross-Linker for Peptide Structure Analysis by Free Radical Initiated Peptide Sequencing (FRIPS) in Negative ESI-MS.

Science.gov (United States)

Hage, Christoph; Ihling, Christian H; Götze, Michael; Schäfer, Mathias; Sinz, Andrea

2017-01-01

We have synthesized a homobifunctional amine-reactive cross-linking reagent, containing a TEMPO (2,2,6,6-tetramethylpiperidine-1-oxy) and a benzyl group (Bz), termed TEMPO-Bz-linker, to derive three-dimensional structural information of proteins. The aim for designing this novel cross-linker was to facilitate the mass spectrometric analysis of cross-linked products by free radical initiated peptide sequencing (FRIPS). In an initial study, we had investigated the fragmentation behavior of TEMPO-Bz-derivatized peptides upon collision activation in (+)-electrospray ionization collision-induced dissociation tandem mass spectrometry (ESI-CID-MS/MS) experiments. In addition to the homolytic NO-C bond cleavage FRIPS pathway delivering the desired odd-electron product ions, an alternative heterolytic NO-C bond cleavage, resulting in even-electron product ions mechanism was found to be relevant. The latter fragmentation route clearly depends on the protonation of the TEMPO-Bz-moiety itself, which motivated us to conduct (-)-ESI-MS, CID-MS/MS, and MS 3 experiments of TEMPO-Bz-cross-linked peptides to further clarify the fragmentation behavior of TEMPO-Bz-peptide molecular ions. We show that the TEMPO-Bz-linker is highly beneficial for conducting FRIPS in negative ionization mode as the desired homolytic cleavage of the NO-C bond is the major fragmentation pathway. Based on characteristic fragments, the isomeric amino acids leucine and isoleucine could be discriminated. Interestingly, we observed pronounced amino acid side chain losses in cross-linked peptides if the cross-linked peptides contain a high number of acidic amino acids. Graphical Abstract ᅟ.

Purification and sequencing of radish seed calmodulin antagonists phosphorylated by calcium-dependent protein kinase.

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Polya, G M; Chandra, S; Condron, R

1993-02-01

A family of radish (Raphanus sativus) calmodulin antagonists (RCAs) was purified from seeds by extraction, centrifugation, batch-wise elution from carboxymethyl-cellulose, and high performance liquid chromatography (HPLC) on an SP5PW cation-exchange column. This RCA fraction was further resolved into three calmodulin antagonist polypeptides (RCA1, RCA2, and RCA3) by denaturation in the presence of guanidinium HCl and mercaptoethanol and subsequent reverse-phase HPLC on a C8 column eluted with an acetonitrile gradient in the presence of 0.1% trifluoroacetic acid. The RCA preparation, RCA1, RCA2, RCA3, and other radish seed proteins are phosphorylated by wheat embryo Ca(2+)-dependent protein kinase (CDPK). The RCA preparation contains other CDPK substrates in addition to RCA1, RCA2, and RCA3. The RCA preparation, RCA1, RCA2, and RCA3 inhibit chicken gizzard calmodulin-dependent myosin light chain kinase assayed with a myosin-light chain-based synthetic peptide substrate (fifty percent inhibitory concentrations of RCA2 and RCA3 are about 7 and 2 microM, respectively). N-terminal sequencing by sequential Edman degradation of RCA1, RCA2, and RCA3 revealed sequences having a high homology with the small subunit of the storage protein napin from Brassica napus and with related proteins. The deduced amino acid sequences of RCA1, RCA2, RCA3, and RCA3' (a subform of RCA3) have agreement with average molecular masses from electrospray mass spectrometry of 4537, 4543, 4532, and 4560 kD, respectively. The only sites for serine phosphorylation are near or at the C termini and hence adjacent to the sites of proteolytic precursor cleavage.

[Hemoglobins, XXXII. Analysis of the primary structure of the monomeric hemoglobin CTT VIIA (erythrocruorin) or Chironomus thummi thummi, Diptera (author's transl)].

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Kleinschmidt, T; Braunitzer, G

1980-01-01

The dimeric hemoglobin CTT VIIA (erythrocruorin) was isolated from the hemolymph of the larva from Chironomus thummi thummi and purified by preparative polyacrylamide gel electrophoresis. Peptides obtained by limited tryptical digestion were sequenced by automatic Edman degradation. For the elucidation of the sequence in the C-terminal region of the chain, additional cleavages with proteinase of Staphylococcus aureus and chymotrypsin were necessary. CTT VIIA is compared with human beta-chains and other hemoglobins of Chironomus. The amino acid residues in the pocket are especially discussed. Most of them are invariant in all Chironomus hemoglobins, independent of the size of the heme pocket, which is normal in some components and enlarged in others.

Antimicrobial Peptides from Plants

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James P. Tam

2015-11-01

Full Text Available Plant antimicrobial peptides (AMPs have evolved differently from AMPs from other life forms. They are generally rich in cysteine residues which form multiple disulfides. In turn, the disulfides cross-braced plant AMPs as cystine-rich peptides to confer them with extraordinary high chemical, thermal and proteolytic stability. The cystine-rich or commonly known as cysteine-rich peptides (CRPs of plant AMPs are classified into families based on their sequence similarity, cysteine motifs that determine their distinctive disulfide bond patterns and tertiary structure fold. Cystine-rich plant AMP families include thionins, defensins, hevein-like peptides, knottin-type peptides (linear and cyclic, lipid transfer proteins, α-hairpinin and snakins family. In addition, there are AMPs which are rich in other amino acids. The ability of plant AMPs to organize into specific families with conserved structural folds that enable sequence variation of non-Cys residues encased in the same scaffold within a particular family to play multiple functions. Furthermore, the ability of plant AMPs to tolerate hypervariable sequences using a conserved scaffold provides diversity to recognize different targets by varying the sequence of the non-cysteine residues. These properties bode well for developing plant AMPs as potential therapeutics and for protection of crops through transgenic methods. This review provides an overview of the major families of plant AMPs, including their structures, functions, and putative mechanisms.

Differential infectivity of two Pseudomonas species and the immune response in the milkweed bug, Oncopeltus fasciatus (Insecta: Hemiptera).

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Schneider, M; Dorn, A

2001-10-01

Pseudomonas aeruginosa and Pseudomonas putida show a profound differential infectivity after inoculation in Oncopeltus fasciatus. Whereas P. putida has no significant impact on nymphs, P. aeruginosa kills all experimental animals within 48 h. Both Pseudomonas species, however, induce the same four hemolymph peptides in O. fasciatus. Also injection of saline solution and injury induced these peptides. In general peptide induction was stronger in nymphs than in adult males. A significantly higher number of nymphs survived a challenge with P. aeruginosa when an immunization with P. putida preceded. The antibacterial properties of the hemolymph were demonstrated in inhibition experiments with P. putida. Two of the four inducible peptides (peptides 1 and 4) could be partially sequenced after Edman degradation and were compared with known antibacterial peptides. Peptide 1, of 15 kDa, showed 47.1% identity with the glycine-rich hemiptericin of Pyrrhocoris apterus. Peptide 4, of 2 kDa, had a 77.8% identity with the proline-rich pyrrhocoricin of P. apterus and a 76.9% identity with metalnikowin 1 of Palomena prasina. Peptides 2 and 3 are also small, with molecular weights of 8 and 5 kDa.

Novel Zn2+-chelating peptides selected from a fimbria-displayed random peptide library

DEFF Research Database (Denmark)

Kjærgaard, Kristian; Schembri, Mark; Klemm, Per

2001-01-01

The display of peptide sequences on the surface of bacteria is a technology that offers exciting applications in biotechnology and medical research. Type 1 fimbriae are surface organelles of Escherichia coli which mediate D-mannose-sensitive binding to different host surfaces by virtue of the Fim......H adhesin. FimH is a component of the fimbrial organelle that can accommodate and display a diverse range of peptide sequences on the E. coli cell surface. In this study we have constructed a random peptide library in FimH. The library, consisting of similar to 40 million individual clones, was screened...

Neurospora tryptophan synthase: N-terminal analysis and the sequence of the pyridoxal phosphate active site peptide

International Nuclear Information System (INIS)

Pratt, M.L.; Hsu, P.Y.; DeMoss, J.A.

1986-01-01

Tryptophan synthase (TS), which catalyzes the final step of tryptophan biosynthesis, is a multifunctional protein requiring pyridoxal phosphate (B6P) for two of its three distinct enzyme activities. TS from Neurospora has a blocked N-terminal, is a homodimer of 150 KDa and binds one mole of B6P per mole of subunit. The authors shown the N-terminal residue to be acyl-serine. The B6P-active site of holoenzyme was labelled by reduction of the B6P-Schiff base with [ 3 H]-NaBH 4 , and resulted in a proportionate loss of activity in the two B6P-requiring reactions. SDS-polyacrylamide gel electrophoresis of CNBr-generated peptides showed the labelled, active site peptide to be 6 KDa. The sequence of this peptide, purified to apparent homogeneity by a combination of C-18 reversed phase and TSK gel filtration HPLC is: gly-arg-pro-gly-gln-leu-his-lys-ala-glu-arg-leu-thr-glu-tyr-ala-gly-gly-ala-gln-ile-xxx-leu-lys-arg-glu-asp-leu-asn-his-xxx-gly-xxx-his-/sub ***/-ile-asn-asn-ala-leu. Although four residues (xxx, /sub ***/) are unidentified, this peptide is minimally 78% homologous with the corresponding peptide from yeast TS, in which residue (/sub ***/) is the lysine that binds B6P

Novel nonphosphorylated peptides with conserved sequences selectively bind to Grb7 SH2 domain with affinity comparable to its phosphorylated ligand.

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Dan Zhang

Full Text Available The Grb7 (growth factor receptor-bound 7 protein, a member of the Grb7 protein family, is found to be highly expressed in such metastatic tumors as breast cancer, esophageal cancer, liver cancer, etc. The src-homology 2 (SH2 domain in the C-terminus is reported to be mainly involved in Grb7 signaling pathways. Using the random peptide library, we identified a series of Grb7 SH2 domain-binding nonphosphorylated peptides in the yeast two-hybrid system. These peptides have a conserved GIPT/K/N sequence at the N-terminus and G/WD/IP at the C-terminus, and the region between the N-and C-terminus contains fifteen amino acids enriched with serines, threonines and prolines. The association between the nonphosphorylated peptides and the Grb7 SH2 domain occurred in vitro and ex vivo. When competing for binding to the Grb7 SH2 domain in a complex, one synthesized nonphosphorylated ligand, containing the twenty-two amino acid-motif sequence, showed at least comparable affinity to the phosphorylated ligand of ErbB3 in vitro, and its overexpression inhibited the proliferation of SK-BR-3 cells. Such nonphosphorylated peptides may be useful for rational design of drugs targeted against cancers that express high levels of Grb7 protein.

Peptide Nucleic Acids

DEFF Research Database (Denmark)

2004-01-01

A novel class of compounds known as peptide nucleic acids, bind complementary DNA and RNA strands, and generally do so more strongly than the corresponding DNA or RNA strands while exhibiting increased sequence specificity and solubility. The peptide nucleic acids comprise ligands selected from...

EThcD Discrimination of Isomeric Leucine/Isoleucine Residues in Sequencing of the Intact Skin Frog Peptides with Intramolecular Disulfide Bond

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Samgina, Tatiana Yu; Kovalev, Sergey V.; Tolpina, Miriam D.; Trebse, Polonca; Torkar, Gregor; Lebedev, Albert T.

2018-05-01

Our scientific interests involve de novo sequencing of non-tryptic natural amphibian skin peptides including those with intramolecular S-S bond by means of exclusively mass spectrometry. Reliable discrimination of the isomeric leucine/isoleucine residues during peptide sequencing by means of mass spectrometry represents a bottleneck in the workflow for complete automation of the primary structure elucidation of these compounds. MS3 is capable of solving the problem. Earlier we demonstrated the advanced efficiency of ETD-HCD method to discriminate Leu/Ile in individual peptides by consecutive application of ETD to the polyprotonated peptides followed by HCD applied to the manually selected primary z-ions with the targeted isomeric residues at their N-termini and registration of the characteristic w-ions. Later this approach was extended to deal with several (4-7) broad band mass ranges, without special isolation of the primary z-ions. The present paper demonstrates an advanced version of this method when EThcD is applied in the whole mass range to a complex mixture of natural non-tryptic peptides without their separation and intermediate isolation of the targeted z-ions. The proposed EThcD method showed over 81% efficiency for the large natural peptides with intact disulfide ring, while the interfering process of radical site migration is suppressed. Due to higher speed and sensitivity, the proposed EThcD approach facilitates the analytical procedure and allows for the automation of the entire experiment and data processing. Moreover, in some cases it gives a chance to establish the nature of the residues in the intact intramolecular disulfide loops. [Figure not available: see fulltext.

Effect of amino acid sequence and pH on nanofiber formation of self-assembling peptides EAK16-II and EAK16-IV.

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Hong, Yooseong; Legge, Raymond L; Zhang, S; Chen, P

2003-01-01

Atomic force microscopy (AFM) and axisymmetric drop shape analysis-profile (ASDA-P) were used to investigate the mechanism of self-assembly of peptides. The peptides chosen consisted of 16 alternating hydrophobic and hydrophilic amino acids, where the hydrophilic residues possess alternating negative and positive charges. Two types of peptides, AEAEAKAKAEAEAKAK (EAK16-II) and AEAEAEAEAKAKAKAK (EAK16-IV), were investigated in terms of nanostructure formation through self-assembly. The experimental results, which focused on the effects of the amino acid sequence and pH, show that the nanostructures formed by the peptides are dependent on the amino acid sequence and the pH of the solution. For pH conditions around neutrality, one of the peptides used in this study, EAK16-IV, forms globular assemblies and has lower surface tension at air-water interfaces than another peptide, EAK16-II, which forms fibrillar assemblies at the same pH. When the pH is lowered below 6.5 or raised above 7.5, there is a transition from globular to fibrillar structures for EAK16-IV, but EAK16-II does not show any structural transition. Surface tension measurements using ADSA-P showed different surface activities of peptides at air-water interfaces. EAK16-II does not show a significant difference in surface tension for the pH range between 4 and 9. However, EAK16-IV shows a noticeable decrease in surface tension at pH around neutrality, indicating that the formation of globular assemblies is related to the molecular hydrophobicity.

Sorting of a HaloTag protein that has only a signal peptide sequence into exocrine secretory granules without protein aggregation.

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Fujita-Yoshigaki, Junko; Matsuki-Fukushima, Miwako; Yokoyama, Megumi; Katsumata-Kato, Osamu

2013-11-15

The mechanism involved in the sorting and accumulation of secretory cargo proteins, such as amylase, into secretory granules of exocrine cells remains to be solved. To clarify that sorting mechanism, we expressed a reporter protein HaloTag fused with partial sequences of salivary amylase protein in primary cultured parotid acinar cells. We found that a HaloTag protein fused with only the signal peptide sequence (Met(1)-Ala(25)) of amylase, termed SS25H, colocalized well with endogenous amylase, which was confirmed by immunofluorescence microscopy. Percoll-density gradient centrifugation of secretory granule fractions shows that the distributions of amylase and SS25H were similar. These results suggest that SS25H is transported to secretory granules and is not discriminated from endogenous amylase by the machinery that functions to remove proteins other than granule cargo from immature granules. Another reporter protein, DsRed2, that has the same signal peptide sequence also colocalized with amylase, suggesting that the sorting to secretory granules is not dependent on a characteristic of the HaloTag protein. Whereas Blue Native PAGE demonstrates that endogenous amylase forms a high-molecular-weight complex, SS25H does not participate in the complex and does not form self-aggregates. Nevertheless, SS25H was released from cells by the addition of a β-adrenergic agonist, isoproterenol, which also induces amylase secretion. These results indicate that addition of the signal peptide sequence, which is necessary for the translocation in the endoplasmic reticulum, is sufficient for the transportation and storage of cargo proteins in secretory granules of exocrine cells.

Identification of a haemolysin-like peptide with antibacterial activity using the draft genome sequence of Staphylococcus epidermidis strain A487.

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Al-Mahrous, Mohammed M; Jack, Ralph W; Sandiford, Stephanie K; Tagg, John R; Beatson, Scott A; Upton, Mathew

2011-08-01

Our interest in Staphylococcus epidermidis strain A487 was prompted by the unusual nature of its inhibitory activity in screening tests against methicillin-resistant Staphylococcus aureus isolates. The inhibitory activity was detected in deferred antagonism tests only if the agar plate was preheated for at least 35 min at ≥ 55 °C before inoculation of the indicator bacteria, this phenomenon indicating possible involvement of a heat-labile immunity agent or protease. The inhibitor was purified to homogeneity by ammonium sulphate precipitation, followed by cation-exchange and reversed-phase chromatography. Tandem MS revealed a novel peptide of molecular weight 2588.4 Da. The draft genome sequence of strain A487 was determined using 454 GS FLX technology, allowing the identification of the structural gene (hlp) encoding the mature peptide MQFITDLIKKAVDFFKGLFGNK. The deduced amino acid sequence of peptide 487 exhibited 70.8% similarity to that of a putative haemolysin from Staphylococcus cohnii. Analysis of the genome of strain A487 showed several additional inhibitor-encoding genes, including hld, the determinant for staphylococcal δ-lysin. This work indicates that potentially useful inhibitors could be overlooked in agar-based inhibitor screening programmes lacking a heat pretreatment step and also highlights the utility of draft genome sequence examination in antibacterial agent discovery. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Photodissociative Cross-Linking of Non-covalent Peptide-Peptide Ion Complexes in the Gas Phase

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Nguyen, Huong T. H.; Andrikopoulos, Prokopis C.; Rulíšek, Lubomír; Shaffer, Christopher J.; Tureček, František

2018-05-01

We report a gas-phase UV photodissociation study investigating non-covalent interactions between neutral hydrophobic pentapeptides and peptide ions incorporating a diazirine-tagged photoleucine residue. Phenylalanine (Phe) and proline (Pro) were chosen as the conformation-affecting residues that were incorporated into a small library of neutral pentapeptides. Gas-phase ion-molecule complexes of these peptides with photo-labeled pentapeptides were subjected to photodissociation. Selective photocleavage of the diazirine ring at 355 nm formed short-lived carbene intermediates that underwent cross-linking by insertion into H-X bonds of the target peptide. The cross-link positions were established from collision-induced dissociation tandem mass spectra (CID-MS3) providing sequence information on the covalent adducts. Effects of the amino acid residue (Pro or Phe) and its position in the target peptide sequence were evaluated. For proline-containing peptides, interactions resulting in covalent cross-links in these complexes became more prominent as proline was moved towards the C-terminus of the target peptide sequence. The photocross-linking yields of phenylalanine-containing peptides depended on the position of both phenylalanine and photoleucine. Density functional theory calculations were used to assign structures of low-energy conformers of the (GLPMG + GLL*LK + H)+ complex. Born-Oppenheimer molecular dynamics trajectory calculations were used to capture the thermal motion in the complexes within 100 ps and determine close contacts between the incipient carbene and the H-X bonds in the target peptide. This provided atomic-level resolution of potential cross-links that aided spectra interpretation and was in agreement with experimental data. [Figure not available: see fulltext.

Artificial neural network study on organ-targeting peptides

Science.gov (United States)

Jung, Eunkyoung; Kim, Junhyoung; Choi, Seung-Hoon; Kim, Minkyoung; Rhee, Hokyoung; Shin, Jae-Min; Choi, Kihang; Kang, Sang-Kee; Lee, Nam Kyung; Choi, Yun-Jaie; Jung, Dong Hyun

2010-01-01

We report a new approach to studying organ targeting of peptides on the basis of peptide sequence information. The positive control data sets consist of organ-targeting peptide sequences identified by the peroral phage-display technique for four organs, and the negative control data are prepared from random sequences. The capacity of our models to make appropriate predictions is validated by statistical indicators including sensitivity, specificity, enrichment curve, and the area under the receiver operating characteristic (ROC) curve (the ROC score). VHSE descriptor produces statistically significant training models and the models with simple neural network architectures show slightly greater predictive power than those with complex ones. The training and test set statistics indicate that our models could discriminate between organ-targeting and random sequences. We anticipate that our models will be applicable to the selection of organ-targeting peptides for generating peptide drugs or peptidomimetics.

Effects on in vitro and in vivo angiogenesis induced by small peptides carrying adhesion sequences.

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Conconi, Maria Teresa; Ghezzo, Francesca; Dettin, Monica; Urbani, Luca; Grandi, Claudio; Guidolin, Diego; Nico, Beatrice; Di Bello, Carlo; Ribatti, Domenico; Parnigotto, Pier Paolo

2010-07-01

It is well known that tumor growth is strictly dependent on neo-vessel formation inside the tumor mass and that cell adhesion is required to allow EC proliferation and migration inside the tumor. In this work, we have evaluated the in vitro and in vivo effects on angiogenesis of some peptides, originally designed to promote cell adhesion on biomaterials, containing RGD motif mediating cell adhesion via integrin receptors [RGD, GRGDSPK, and (GRGDSP)(4)K] or the heparin-binding sequence of human vitronectin that interacts with HSPGs [HVP(351-359)]. Cell adhesion, proliferation, migration, and capillary-like tube formation in Matrigel were determined on HUVECs, whereas the effects on in vivo angiogenesis were evaluated using the CAM assay. (GRGDSP)(4)K linear sequence inhibited cell adhesion, decreased cell proliferation, migration and morphogenesis in Matrigel, and induced anti-angiogenic responses on CAM at higher degree than that determined after incubation with RGD or GRGDSPK. Moreover, it counteracted both in vitro and in vivo the pro-angiogenic effects induced by the Fibroblast growth factor (FGF-2). On the other hand, HVP was not able to affect cell adhesion and appeared less effective than (GRGDSP)(4)K. Our data indicate that the activity of RGD-containing peptides is related to their adhesive properties, and their effects are modulated by the number of cell adhesion motifs and the aminoacidic residues next to these sequences. The anti-angiogenic properties of (GRGDSP)(4)K seem to depend on its interaction with integrins, whereas the effects of HVP may be partially due to an impairment of HSPGs/FGF-2.

Structure-Related Roles for the Conservation of the HIV-1 Fusion Peptide Sequence Revealed by Nuclear Magnetic Resonance.

Science.gov (United States)

Serrano, Soraya; Huarte, Nerea; Rujas, Edurne; Andreu, David; Nieva, José L; Jiménez, María Angeles

2017-10-17

Despite extensive characterization of the human immunodeficiency virus type 1 (HIV-1) hydrophobic fusion peptide (FP), the structure-function relationships underlying its extraordinary degree of conservation remain poorly understood. Specifically, the fact that the tandem repeat of the FLGFLG tripeptide is absolutely conserved suggests that high hydrophobicity may not suffice to unleash FP function. Here, we have compared the nuclear magnetic resonance (NMR) structures adopted in nonpolar media by two FP surrogates, wtFP-tag and scrFP-tag, which had equal hydrophobicity but contained wild-type and scrambled core sequences LFLGFLG and FGLLGFL, respectively. In addition, these peptides were tagged at their C-termini with an epitope sequence that folded independently, thereby allowing Western blot detection without interfering with FP structure. We observed similar α-helical FP conformations for both specimens dissolved in the low-polarity medium 25% (v/v) 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), but important differences in contact with micelles of the membrane mimetic dodecylphosphocholine (DPC). Thus, whereas wtFP-tag preserved a helix displaying a Gly-rich ridge, the scrambled sequence lost in great part the helical structure upon being solubilized in DPC. Western blot analyses further revealed the capacity of wtFP-tag to assemble trimers in membranes, whereas membrane oligomers were not observed in the case of the scrFP-tag sequence. We conclude that, beyond hydrophobicity, preserving sequence order is an important feature for defining the secondary structures and oligomeric states adopted by the HIV FP in membranes.

Computationally assisted screening and design of cell-interactive peptides by a cell-based assay using peptide arrays and a fuzzy neural network algorithm.

Science.gov (United States)

Kaga, Chiaki; Okochi, Mina; Tomita, Yasuyuki; Kato, Ryuji; Honda, Hiroyuki

2008-03-01

We developed a method of effective peptide screening that combines experiments and computational analysis. The method is based on the concept that screening efficiency can be enhanced from even limited data by use of a model derived from computational analysis that serves as a guide to screening and combining the model with subsequent repeated experiments. Here we focus on cell-adhesion peptides as a model application of this peptide-screening strategy. Cell-adhesion peptides were screened by use of a cell-based assay of a peptide array. Starting with the screening data obtained from a limited, random 5-mer library (643 sequences), a rule regarding structural characteristics of cell-adhesion peptides was extracted by fuzzy neural network (FNN) analysis. According to this rule, peptides with unfavored residues in certain positions that led to inefficient binding were eliminated from the random sequences. In the restricted, second random library (273 sequences), the yield of cell-adhesion peptides having an adhesion rate more than 1.5-fold to that of the basal array support was significantly high (31%) compared with the unrestricted random library (20%). In the restricted third library (50 sequences), the yield of cell-adhesion peptides increased to 84%. We conclude that a repeated cycle of experiments screening limited numbers of peptides can be assisted by the rule-extracting feature of FNN.

Cationic antimicrobial peptides inactivate Shiga toxin-encoding bacteriophages

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Del Cogliano, Manuel E.; Hollmann, Axel; Martinez, Melina; Semorile, Liliana; Ghiringhelli, Pablo D.; Maffía, Paulo C.; Bentancor, Leticia V.

2017-12-01

Shiga toxin (Stx) is the principal virulence factor during Shiga toxin-producing Escherichia coli (STEC) infections. We have previously reported the inactivation of bacteriophage encoding Stx after treatment with chitosan, a linear polysaccharide polymer with cationic properties. Cationic antimicrobial peptides (cAMPs) are short linear aminoacidic sequences, with a positive net charge, which display bactericidal or bacteriostatic activity against a wide range of bacterial species. They are promising novel antibiotics since they have shown bactericidal effects against multiresistant bacteria. To evaluate whether cationic properties are responsible for bacteriophage inactivation, we tested seven cationic peptides with proven antimicrobial activity as anti-bacteriophage agents, and one random sequence cationic peptide with no antimicrobial activity as a control. We observed bacteriophage inactivation after incubation with five cAMPs, but no inactivating activity was observed with the random sequence cationic peptide or with the non alpha helical cAMP Omiganan. Finally, to confirm peptide-bacteriophage interaction, zeta potential was analyzed by following changes on bacteriophage surface charges after peptide incubation. According to our results we could propose that: 1) direct interaction of peptides with phage is a necessary step for bacteriophage inactivation, 2) cationic properties are necessary but not sufficient for bacteriophage inactivation, and 3) inactivation by cationic peptides could be sequence (or structure) specific. Overall our data suggest that these peptides could be considered a new family of molecules potentially useful to decrease bacteriophage replication and Stx expression.

Cationic Antimicrobial Peptides Inactivate Shiga Toxin-Encoding Bacteriophages

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Manuel E. Del Cogliano

2017-12-01

Full Text Available Shiga toxin (Stx is the principal virulence factor during Shiga toxin-producing Escherichia coli (STEC infections. We have previously reported the inactivation of bacteriophage encoding Stx after treatment with chitosan, a linear polysaccharide polymer with cationic properties. Cationic antimicrobial peptides (cAMPs are short linear aminoacidic sequences, with a positive net charge, which display bactericidal or bacteriostatic activity against a wide range of bacterial species. They are promising novel antibiotics since they have shown bactericidal effects against multiresistant bacteria. To evaluate whether cationic properties are responsible for bacteriophage inactivation, we tested seven cationic peptides with proven antimicrobial activity as anti-bacteriophage agents, and one random sequence cationic peptide with no antimicrobial activity as a control. We observed bacteriophage inactivation after incubation with five cAMPs, but no inactivating activity was observed with the random sequence cationic peptide or with the non-alpha helical cAMP Omiganan. Finally, to confirm peptide-bacteriophage interaction, zeta potential was analyzed by following changes on bacteriophage surface charges after peptide incubation. According to our results we could propose that: (1 direct interaction of peptides with phage is a necessary step for bacteriophage inactivation, (2 cationic properties are necessary but not sufficient for bacteriophage inactivation, and (3 inactivation by cationic peptides could be sequence (or structure specific. Overall our data suggest that these peptides could be considered a new family of molecules potentially useful to decrease bacteriophage replication and Stx expression.

Novel Inhibitor Cystine Knot Peptides from Momordica charantia

Science.gov (United States)

Clark, Richard J.; Tang, Jun; Zeng, Guang-Zhi; Franco, Octavio L.; Cantacessi, Cinzia; Craik, David J.; Daly, Norelle L.; Tan, Ning-Hua

2013-01-01

Two new peptides, MCh-1 and MCh-2, along with three known trypsin inhibitors (MCTI-I, MCTI-II and MCTI-III), were isolated from the seeds of the tropical vine Momordica charantia. The sequences of the peptides were determined using mass spectrometry and NMR spectroscopy. Using a strategy involving partial reduction and stepwise alkylation of the peptides, followed by enzymatic digestion and tandem mass spectrometry sequencing, the disulfide connectivity of MCh-1 was elucidated to be CysI-CysIV, CysII-CysV and CysIII-CysVI. The three-dimensional structures of MCh-1 and MCh-2 were determined using NMR spectroscopy and found to contain the inhibitor cystine knot (ICK) motif. The sequences of the novel peptides differ significantly from peptides previously isolated from this plant. Therefore, this study expands the known peptide diversity in M. charantia and the range of sequences that can be accommodated by the ICK motif. Furthermore, we show that a stable two-disulfide intermediate is involved in the oxidative folding of MCh-1. This disulfide intermediate is structurally homologous to the proposed ancestral fold of ICK peptides, and provides a possible pathway for the evolution of this structural motif, which is highly prevalent in nature. PMID:24116036

Novel inhibitor cystine knot peptides from Momordica charantia.

Directory of Open Access Journals (Sweden)

Wen-Jun He

Full Text Available Two new peptides, MCh-1 and MCh-2, along with three known trypsin inhibitors (MCTI-I, MCTI-II and MCTI-III, were isolated from the seeds of the tropical vine Momordica charantia. The sequences of the peptides were determined using mass spectrometry and NMR spectroscopy. Using a strategy involving partial reduction and stepwise alkylation of the peptides, followed by enzymatic digestion and tandem mass spectrometry sequencing, the disulfide connectivity of MCh-1 was elucidated to be CysI-CysIV, CysII-CysV and CysIII-CysVI. The three-dimensional structures of MCh-1 and MCh-2 were determined using NMR spectroscopy and found to contain the inhibitor cystine knot (ICK motif. The sequences of the novel peptides differ significantly from peptides previously isolated from this plant. Therefore, this study expands the known peptide diversity in M. charantia and the range of sequences that can be accommodated by the ICK motif. Furthermore, we show that a stable two-disulfide intermediate is involved in the oxidative folding of MCh-1. This disulfide intermediate is structurally homologous to the proposed ancestral fold of ICK peptides, and provides a possible pathway for the evolution of this structural motif, which is highly prevalent in nature.

Trapping and partial characterization of an adduct postulated to be the covalent catalytic ternary complex of thymidylate synthetase

International Nuclear Information System (INIS)

Ahmed, F.; Moore, M.A.; Dunlap, R.B.

1986-01-01

The proposed mechanism of action of thymidylate synthetase envisages the formation of a covalent ternary complex of the enzyme via the active site cysteine with dUMP and 5,10-methylenetetrahydrofolate (CH 2 H 4 folate). The authors recent success in using trichloroacetic acid to trap the covalent enzyme-FdUMP binary and ternary (enzyme-FdUMP-CH 2 H 4 folate) complexes led to the use of this technique in attempts to trap the transient covalent catalytic ternary complex. Experiments performed with [2-C 14 ]dUMP and 3 H-CH 2 H 4 folate show that both these ligands remained bound to the enzyme after trichloroacetic acid precipitation. The trapped covalent catalytic ternary complex was subjected to CNBr fragmentation, and the peptides were fractionated by HPLC. The isolated active-site peptide was shown to retain the two ligands and was subjected to a limited sequence analysis by the dansyl-Edman procedure. The inhibitory ternary complex formed with 14 C-FdUMP and 3 H-CH 2 4 folate served as a control. The active-site peptides isolated from the CNBr treated inhibitory ternary complex and the catalytic complex exhibited identical sequences for the first four N-terminal residues, Ala-Leu-Pro-Pro, and the fifth residue was found to be associated with the labeled ligands. Sequence analysis of the active site peptide derived from the carboxymethylated enzyme confirmed this sequence and the 5th residue was shown to be Cm-Cys

Short peptides allowing preferential detection of Candida albicans hyphae.

Science.gov (United States)

Kaba, Hani E J; Pölderl, Antonia; Bilitewski, Ursula

2015-09-01

Whereas the detection of pathogens via recognition of surface structures by specific antibodies and various types of antibody mimics is frequently described, the applicability of short linear peptides as sensor molecules or diagnostic tools is less well-known. We selected peptides which were previously reported to bind to recombinant S. cerevisiae cells, expressing members of the C. albicans Agglutinin-Like-Sequence (ALS) cell wall protein family. We slightly modified amino acid sequences to evaluate peptide sequence properties influencing binding to C. albicans cells. Among the selected peptides, decamer peptides with an "AP"-N-terminus were superior to shorter peptides. The new decamer peptide FBP4 stained viable C. albicans cells more efficiently in their mature hyphal form than in their yeast form. Moreover, it allowed distinction of C. albicans from other related Candida spp. and could thus be the basis for the development of a useful tool for the diagnosis of invasive candidiasis.

Novel ZnO-binding peptides obtained by the screening of a phage display peptide library

Energy Technology Data Exchange (ETDEWEB)

Golec, Piotr [Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Laboratory of Molecular Biology (affiliated with the University of Gdansk) (Poland); Karczewska-Golec, Joanna [University of Gdansk and Medical University of Gdansk, Laboratory of Molecular Bacteriology, Intercollegiate Faculty of Biotechnology (Poland); Los, Marcin; Wegrzyn, Grzegorz, E-mail: wegrzyn@biotech.univ.gda.pl [University of Gdansk, Department of Molecular Biology (Poland)

2012-11-15

Zinc oxide (ZnO) is a semiconductor compound with a potential for wide use in various applications, including biomaterials and biosensors, particularly as nanoparticles (the size range of ZnO nanoparticles is from 2 to 100 nm, with an average of about 35 nm). Here, we report isolation of novel ZnO-binding peptides, by screening of a phage display library. Interestingly, amino acid sequences of the ZnO-binding peptides reported in this paper and those described previously are significantly different. This suggests that there is a high variability in sequences of peptides which can bind particular inorganic molecules, indicating that different approaches may lead to discovery of different peptides of generally the same activity (e.g., binding of ZnO) but having various detailed properties, perhaps crucial under specific conditions of different applications.

Identification of a highly reactive threonine residue at the active site of γ-glutamyl transpeptidase

International Nuclear Information System (INIS)

Stole, E.; Seddon, A.P.; Wellner, D.; Meister, A.

1990-01-01

γ-Glutamyl transpeptidase an enzyme of major importance in glutathione metabolism, was inactivated by treating it with L-(αS,5S)-α-amino-3-chloro-4,5-dihydro-5-[3- 14 C]isoxazoleacetic acid. This selective reagent binds stoichiometrically to the enzyme; more than 90% of the label was bound to its light subunit. Enzymatic digestion of the light subunit gave a 14 C-labeled peptide that corresponds to amino acid residues 517-527 of the enzyme and two incomplete digestion products that contain this labeled peptide moiety. The radioactivity associated with this peptide was released with threonine-523 during sequencing by the automated gas-phase Edman method. The light subunit contains 14 other threonine residues and a total of 19 serine residues; these were not labeled. Threonine-523 is situated in the enzyme in an environment that greatly increases its reactivity, indicating that other amino acid residues of the enzyme must also participate in the active-site chemistry of the enzyme

Peptides, polypeptides and peptide-polymer hybrids as nucleic acid carriers.

Science.gov (United States)

Ahmed, Marya

2017-10-24

Cell penetrating peptides (CPPs), and protein transduction domains (PTDs) of viruses and other natural proteins serve as a template for the development of efficient peptide based gene delivery vectors. PTDs are sequences of acidic or basic amphipathic amino acids, with superior membrane trespassing efficacies. Gene delivery vectors derived from these natural, cationic and cationic amphipathic peptides, however, offer little flexibility in tailoring the physicochemical properties of single chain peptide based systems. Owing to significant advances in the field of peptide chemistry, synthetic mimics of natural peptides are often prepared and have been evaluated for their gene expression, as a function of amino acid functionalities, architecture and net cationic content of peptide chains. Moreover, chimeric single polypeptide chains are prepared by a combination of multiple small natural or synthetic peptides, which imparts distinct physiological properties to peptide based gene delivery therapeutics. In order to obtain multivalency and improve the gene delivery efficacies of low molecular weight cationic peptides, bioactive peptides are often incorporated into a polymeric architecture to obtain novel 'polymer-peptide hybrids' with improved gene delivery efficacies. Peptide modified polymers prepared by physical or chemical modifications exhibit enhanced endosomal escape, stimuli responsive degradation and targeting efficacies, as a function of physicochemical and biological activities of peptides attached onto a polymeric scaffold. The focus of this review is to provide comprehensive and step-wise progress in major natural and synthetic peptides, chimeric polypeptides, and peptide-polymer hybrids for nucleic acid delivery applications.

Identification of snake bradykinin-potentiating peptides (BPPs)-simile sequences in rat brain--Potential BPP-like precursor protein?

Science.gov (United States)

Campeiro, Joana D'Arc; Neshich, Izabella P; Sant'Anna, Osvaldo A; Lopes, Robson; Ianzer, Danielle; Assakura, Marina T; Neshich, Goran; Hayashi, Mirian A F

2015-08-01

Bradykinin-potentiating peptides (BPPs) from the South American pit viper snake venom were the first natural inhibitors of the human angiotensin I-converting enzyme (ACE) described. The pioneer characterization of the BPPs precursor from the snake venom glands by our group showed for the first time the presence of the C-type natriuretic peptide (CNP) in this same viper precursor protein. The confirmation of the BPP/CNP expression in snake brain regions correlated with neuroendocrine functions stimulated us to pursue the physiological correlates of these vasoactive peptides in mammals. Notably, several snake toxins were shown to have endogenous physiological correlates in mammals. In the present work, we expressed in bacteria the BPPs domain of the snake venom gland precursor protein, and this purified recombinant protein was used to raise specific polyclonal anti-BPPs antibodies. The correspondent single protein band immune-recognized in adult rat brain cytosol was isolated by 2D-SDS/PAGE and/or HPLC, before characterization by MS fingerprint analysis, which identified this protein as superoxide dismutase (SOD, EC 1.15.1.1), a classically known enzyme with antioxidant activity and important roles in the blood pressure modulation. In silico analysis showed the exposition of the BPP-like peptide sequences on the surface of the 3D structure of rat SOD. These peptides were chemically synthesized to show the BPP-like biological activities in ex vivo and in vivo pharmacological bioassays. Taken together, our data suggest that SOD protein have the potential to be a source for putative BPP-like bioactive peptides, which once released may contribute to the blood pressure control in mammals. Copyright © 2015 Elsevier Inc. All rights reserved.

Syntheses and structures of technetium(V) and rhenium(V) oxo complexes of peptide having KYC-sequence

International Nuclear Information System (INIS)

Takayama, T.; Suzuki, K.; Sekine, T.; Kudo, H.

2000-01-01

Technetium(V) and rhenium(V) oxo complexes of a peptide having a KYC-sequence such as KYCAR (H 3 L 5 ) and KYCAREPPTRTNAYQGQG-NH 2 (H 3 L 18 ) were synthesized, and structures of the complexes were characterized by spectroscopic techniques. All of the complexes were synthesized by the ligand exchange reaction of [(n-C 4 H 9 ) 4 N][MOCl 4 ] (M = 99 Tc, Re) with peptide in methanol or dimethylformamide solution. These complexes have a square pyramidal structure with an oxo ligand at the apical position. The peptide is coordinated to a metal atom through N amine of lysine. S thiol of cysteine, and N amide of tyrosine and cysteine in the equatorial plane. A lysine (CH 2 ) 4 NH 2 group of the L 5 ligand has the syn conformation with respect to metal-oxo bonding in the complex. The syn isomer was selectively formed in the ligand exchange reaction. The conversion of the syn isomer to the anti isomer was observed only for syn-[ReO(L 5 )], in which the coordination of water to the trans position of the oxo ligand was involved. (orig.)

An insecticidal toxin from Nephila clavata spider venom.

Science.gov (United States)

Jin, Lin; Fang, Mingqian; Chen, Mengrou; Zhou, Chunling; Ombati, Rose; Hakim, Md Abdul; Mo, Guoxiang; Lai, Ren; Yan, Xiuwen; Wang, Yumin; Yang, Shilong

2017-07-01

Spiders are the most successful insect predators given that they use their venom containing insecticidal peptides as biochemical weapons for preying. Due to the high specificity and potency of peptidic toxins, discoveries of insecticidal toxins from spider venom have provided an opportunity to obtain natural compounds for agricultural applications without affecting human health. In this study, a novel insecticidal toxin (μ-NPTX-Nc1a) was identified and characterized from the venom of Nephila clavata. Its primary sequence is GCNPDCTGIQCGWPRCPGGQNPVMDKCVSCCPFCPPKSAQG which was determined by automated Edman degradation, cDNA cloning, and MS/MS analysis. BLAST search indicated that Nc1a shows no similarity with known peptides or proteins, indicating that Nc1a belongs to a novel family of insecticidal peptide. Nc1a displayed inhibitory effects on Na V and K V channels in cockroach dorsal unpaired median neurons. The median lethal dose (LD50) of Nc1a on cockroach was 573 ng/g. Herein, a study that identifies a novel insecticidal toxin, which can be a potential candidate and/or template for the development of bioinsecticides, is presented.

Hydroxyapatite-binding peptides for bone growth and inhibition

Science.gov (United States)

Bertozzi, Carolyn R [Berkeley, CA; Song, Jie [Shrewsbury, MA; Lee, Seung-Wuk [Walnut Creek, CA

2011-09-20

Hydroxyapatite (HA)-binding peptides are selected using combinatorial phage library display. Pseudo-repetitive consensus amino acid sequences possessing periodic hydroxyl side chains in every two or three amino acid sequences are obtained. These sequences resemble the (Gly-Pro-Hyp).sub.x repeat of human type I collagen, a major component of extracellular matrices of natural bone. A consistent presence of basic amino acid residues is also observed. The peptides are synthesized by the solid-phase synthetic method and then used for template-driven HA-mineralization. Microscopy reveal that the peptides template the growth of polycrystalline HA crystals .about.40 nm in size.

Enterocin TW21, a novel bacteriocin from dochi-isolated Enterococcus faecium D081821.

Science.gov (United States)

Chang, S-Y; Chen, Y-S; Pan, S-F; Lee, Y-S; Chang, C-H; Chang, C-H; Yu, B; Wu, H-C

2013-09-01

Purification and characterization of a novel bacteriocin produced by strain Enterococcus faecium D081821. Enterococcus faecium D081821, isolated from the traditional Taiwanese fermented food dochi (fermented black beans), was previously found to produce a bacteriocin against Listeria monocytogenes and some Gram-positive bacteria. This bacteriocin, termed enterocin TW21, was purified from culture supernatant by ammonium sulfate precipitation, Sep-Pak C18 cartridge, ion-exchange and gel filtration chromatography. Mass spectrometry analysis showed the mass of the peptide to be approximately 5300·6 Da. The N-terminal amino acid sequencing yielded a partial sequence NH2 -ATYYGNGVYxNTQK by Edman degradation, and it contains the consensus class IIa bacteriocin motif YGNGV in the N-terminal region. The open reading frame (ORF) encoding the bacteriocin was identified from the draft genome sequence of Enterococcus faecium D081821, and sequence analysis of this peptide indicated that enterocin TW21 is a novel bacteriocin. Enterococcus faecium D081821 produced a bacteriocin named enterocin TW21, the molecular weight and amino acid sequence both revealed it to be a novel bacteriocin. A new member of class IIa bacteriocin was identified. This bacteriocin shows great inhibitory ability against L. monocytogenes and could be applied as a natural food preservative. © 2013 The Society for Applied Microbiology.

Screening and Identification of Peptides Specifically Targeted to Gastric Cancer Cells from a Phage Display Peptide Library

Science.gov (United States)

Sahin, Deniz; Taflan, Sevket Onur; Yartas, Gizem; Ashktorab, Hassan; Smoot, Duane T

2018-04-25

Background: Gastric cancer is the second most common cancer among the malign cancer types. Inefficiency of traditional techniques both in diagnosis and therapy of the disease makes the development of alternative and novel techniques indispensable. As an alternative to traditional methods, tumor specific targeting small peptides can be used to increase the efficiency of the treatment and reduce the side effects related to traditional techniques. The aim of this study is screening and identification of individual peptides specifically targeted to human gastric cancer cells using a phage-displayed peptide library and designing specific peptide sequences by using experimentally-eluted peptide sequences. Methods: Here, MKN-45 human gastric cancer cells and HFE-145 human normal gastric epithelial cells were used as the target and control cells, respectively. 5 rounds of biopannning with a phage display 12-peptide library were applied following subtraction biopanning with HFE-145 control cells. The selected phage clones were established by enzyme-linked immunosorbent assay and immunofluorescence detection. We first obtain random phage clones after five biopanning rounds, determine the binding levels of each individual clone. Then, we analyze the frequencies of each amino acid in best binding clones to determine positively overexpressed amino acids for designing novel peptide sequences. Results: DE532 (VETSQYFRGTLS) phage clone was screened positive, showing specific binding on MKN-45 gastric cancer cells. DE-Obs (HNDLFPSWYHNY) peptide, which was designed by using amino acid frequencies of experimentally selected peptides in the 5th round of biopanning, showed specific binding in MKN-45 cells. Conclusion: Selection and characterization of individual clones may give us specifically binding peptides, but more importantly, data extracted from eluted phage clones may be used to design theoretical peptides with better binding properties than even experimentally selected ones

Dynamic peptide libraries for the discovery of supramolecular nanomaterials

Science.gov (United States)

Pappas, Charalampos G.; Shafi, Ramim; Sasselli, Ivan R.; Siccardi, Henry; Wang, Tong; Narang, Vishal; Abzalimov, Rinat; Wijerathne, Nadeesha; Ulijn, Rein V.

2016-11-01

Sequence-specific polymers, such as oligonucleotides and peptides, can be used as building blocks for functional supramolecular nanomaterials. The design and selection of suitable self-assembling sequences is, however, challenging because of the vast combinatorial space available. Here we report a methodology that allows the peptide sequence space to be searched for self-assembling structures. In this approach, unprotected homo- and heterodipeptides (including aromatic, aliphatic, polar and charged amino acids) are subjected to continuous enzymatic condensation, hydrolysis and sequence exchange to create a dynamic combinatorial peptide library. The free-energy change associated with the assembly process itself gives rise to selective amplification of self-assembling candidates. By changing the environmental conditions during the selection process, different sequences and consequent nanoscale morphologies are selected.

Characterization of synthetic peptides by mass spectrometry

DEFF Research Database (Denmark)

Prabhala, Bala Krishna; Mirza, Osman Asghar; Højrup, Peter

2015-01-01

Mass spectrometry (MS) is well suited for analysis of the identity and purity of synthetic peptides. The sequence of a synthetic peptide is most often known, so the analysis is mainly used to confirm the identity and purity of the peptide. Here, simple procedures are described for MALDI......-TOF-MS and LC-MS of synthetic peptides....

PeptideNavigator: An interactive tool for exploring large and complex data sets generated during peptide-based drug design projects.

Science.gov (United States)

Diller, Kyle I; Bayden, Alexander S; Audie, Joseph; Diller, David J

2018-01-01

There is growing interest in peptide-based drug design and discovery. Due to their relatively large size, polymeric nature, and chemical complexity, the design of peptide-based drugs presents an interesting "big data" challenge. Here, we describe an interactive computational environment, PeptideNavigator, for naturally exploring the tremendous amount of information generated during a peptide drug design project. The purpose of PeptideNavigator is the presentation of large and complex experimental and computational data sets, particularly 3D data, so as to enable multidisciplinary scientists to make optimal decisions during a peptide drug discovery project. PeptideNavigator provides users with numerous viewing options, such as scatter plots, sequence views, and sequence frequency diagrams. These views allow for the collective visualization and exploration of many peptides and their properties, ultimately enabling the user to focus on a small number of peptides of interest. To drill down into the details of individual peptides, PeptideNavigator provides users with a Ramachandran plot viewer and a fully featured 3D visualization tool. Each view is linked, allowing the user to seamlessly navigate from collective views of large peptide data sets to the details of individual peptides with promising property profiles. Two case studies, based on MHC-1A activating peptides and MDM2 scaffold design, are presented to demonstrate the utility of PeptideNavigator in the context of disparate peptide-design projects. Copyright © 2017 Elsevier Ltd. All rights reserved.

Escherichia coli Peptide Binding Protein OppA Has a Preference for Positively Charged Peptides

NARCIS (Netherlands)

Klepsch, M. M.; Kovermann, M.; Löw, C.; Balbach, J.; Permentier, H. P.; Fusetti, F.; de Gier, J. W.; Gier, Jan-Willem de; Slotboom, D. J.; Berntsson, R. P. -A.

2011-01-01

The Escherichia coli peptide binding protein OppA is an essential component of the oligopeptide transporter Opp. Based on studies on its orthologue from Salmonella typhimurium, it has been proposed that OppA binds peptides between two and five amino acids long, with no apparent sequence selectivity.

Enterocin 96, a Novel Class II Bacteriocin Produced by Enterococcus faecalis WHE 96, Isolated from Munster Cheese▿

Science.gov (United States)

Izquierdo, Esther; Wagner, Camille; Marchioni, Eric; Aoude-Werner, Dalal; Ennahar, Saïd

2009-01-01

Enterococcus faecalis WHE 96, a strain isolated from soft cheese based on its anti-Listeria activity, produced a 5,494-Da bacteriocin that was purified to homogeneity by ultrafiltration and cation-exchange and reversed-phase chromatographies. The amino acid sequence of this bacteriocin, named enterocin 96, was determined by Edman degradation, and its structural gene was sequenced, revealing a double-glycine leader peptide. After a comparison with other bacteriocins, it was shown that enterocin 96 was a new class II bacteriocin that showed very little similarity with known structures. Enterocin 96 was indeed a new bacteriocin belonging to class II bacteriocins. The activity spectrum of enterocin 96 covered a wide range of bacteria, with strong activity against most gram-positive strains but very little or no activity against gram-negative strains. PMID:19411428

Enterocin 96, a novel class II bacteriocin produced by Enterococcus faecalis WHE 96, isolated from Munster cheese.

Science.gov (United States)

Izquierdo, Esther; Wagner, Camille; Marchioni, Eric; Aoude-Werner, Dalal; Ennahar, Saïd

2009-07-01

Enterococcus faecalis WHE 96, a strain isolated from soft cheese based on its anti-Listeria activity, produced a 5,494-Da bacteriocin that was purified to homogeneity by ultrafiltration and cation-exchange and reversed-phase chromatographies. The amino acid sequence of this bacteriocin, named enterocin 96, was determined by Edman degradation, and its structural gene was sequenced, revealing a double-glycine leader peptide. After a comparison with other bacteriocins, it was shown that enterocin 96 was a new class II bacteriocin that showed very little similarity with known structures. Enterocin 96 was indeed a new bacteriocin belonging to class II bacteriocins. The activity spectrum of enterocin 96 covered a wide range of bacteria, with strong activity against most gram-positive strains but very little or no activity against gram-negative strains.

Human T-cell recognition of synthetic peptides representing conserved and variant sequences from the merozoite surface protein 2 of Plasmodium falciparum

DEFF Research Database (Denmark)

Theander, T G; Hviid, L; Dodoo, D

1997-01-01

Merozoite surface protein 2 (MSP2) is a malaria vaccine candidate currently undergoing clinical trials. We analyzed the peripheral blood mononuclear cell (PBMC) response to synthetic peptides corresponding to conserved and variant regions of the FCQ-27 allelic form of MSP2 in Ghanaian individuals....... The findings are encouraging for the development of a vaccine based on these T-epitope containing regions of MSP2, as the peptides were broadly recognized suggesting that they can bind to diverse HLA alleles and also because they include conserved MSP2 sequences. Immunisation with a vaccine construct...

Elucidation of Peptide-Directed Palladium Surface Structure for Biologically Tunable Nanocatalysts

Energy Technology Data Exchange (ETDEWEB)

Bedford, Nicholas M.; Ramezani-Dakhel, Hadi; Slocik, Joseph M.; Briggs, Beverly D.; Ren, Yang; Frenkel, Anatoly I.; Petkov, Valeri; Heinz, Hendrik; Naik, Rajesh R.; Knecht, Mark R.

2015-05-01

Peptide-enabled synthesis of inorganic nanostructures represents an avenue to access catalytic materials with tunable and optimized properties. This is achieved via peptide complexity and programmability that is missing in traditional ligands for catalytic nanomaterials. Unfortunately, there is limited information available to correlate peptide sequence to particle structure and catalytic activity to date. As such, the application of peptide-enabled nanocatalysts remains limited to trial and error approaches. In this paper, a hybrid experimental and computational approach is introduced to systematically elucidate biomolecule-dependent structure/function relationships for peptide-capped Pd nanocatalysts. Synchrotron X-ray techniques were used to uncover substantial particle surface structural disorder, which was dependent upon the amino acid sequence of the peptide capping ligand. Nanocatalyst configurations were then determined directly from experimental data using reverse Monte Carlo methods and further refined using molecular dynamics simulation, obtaining thermodynamically stable peptide-Pd nanoparticle configurations. Sequence-dependent catalytic property differences for C-C coupling and olefin hydrogenation were then eluddated by identification of the catalytic active sites at the atomic level and quantitative prediction of relative reaction rates. This hybrid methodology provides a clear route to determine peptide-dependent structure/function relationships, enabling the generation of guidelines for catalyst design through rational tailoring of peptide sequences

Peptide imprinted receptors for the determination of the small cell lung cancer associated biomarker progastrin releasing peptide

DEFF Research Database (Denmark)

Qader, A. A.; Urraca, J.; Torsetnes, S. B.

2014-01-01

Peptide imprinted polymers were developed for detection of progastrin releasing peptide (ProGRP); a low abundant blood based biomarker for small cell lung cancer. The polymers targeted the proteotypic nona-peptide sequence NLLGLIEAK and were used for selective enrichment of the proteotypic peptide...... prior to LCMS based quantification. Peptide imprinted polymers with the best affinity characteristics were first identified from a 96-polymer combinatorial library. The effects of functional monomers, crosslinker, porogen, and template on adsorption capacity and selectivity for NLLGLIEAK were...

Identification of cysteine-644 as the covalent site of attachment of dexamethasone 21-mesylate to murine glucocorticoid receptors in WEHI-7 cells

International Nuclear Information System (INIS)

Smith, L.I.; Bodwell, J.E.; Mendel, D.B.; Ciardelli, T.; North, W.G.; Munck, A.

1988-01-01

Dexamethasone 21-mesylate is a highly specific synthetic glucocorticoid derivative that binds covalently to glucocorticoid receptors via sulfhydryl groups. The authors have identified the amino acid that reacts with the dexamethasone 21-mesylate by using enzymatic digestion and microsequencing for radiolabel. Nonactivated glucocorticoid receptors obtained from labeling intact WEHI-7 mouse thymoma cells with [ 3 H]dexamethasone 21-mesylate were immunopurified and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Trypsin digestion followed by reversed-phase high-performance liquid chromatography (reversed-phase HPLC) produced a single [ 3 H]dexamethasone 21-mesylate labeled peptide. Automated Edman degradation of this peptide revealed that the [ 3 H]dexamethasone 21-mesylate was located at position 5 from the amino terminus. Dual-isotope labeling studies with [ 3 H]dexamethasone 21-mesylate and [ 35 S]methionine demonstrated that this peptide contained methionine. Staphylococcus aureus V8 protease digestion of [ 3 H]dexamethasone 21-mesylate labeled steroid-binding subunits generated a different radiolabeled peptide containing label at position 7 from the amino terminus. On the basis of the published amino acid sequence of the murine glucocorticoid receptor, their data clearly identify cysteine-644 as the single residue in the steroid-binding domain that covalently binds dexamethasone 21-mesylate. They have confirmed this finding by demonstrating that a synthetic peptide representing the amino acid sequence 640-650 of the murine glucocorticoid receptor behaves in an identical manner on reversed-phase HPLC as the trypsin-generated peptide from intact cells

Effective modification of cell death-inducing intracellular peptides by means of a photo-cleavable peptide array-based screening system.

Science.gov (United States)

Kozaki, Ikko; Shimizu, Kazunori; Honda, Hiroyuki

2017-08-01

Intracellular functional peptides that play a significant role inside cells have been receiving a lot of attention as regulators of cellular activity. Previously, we proposed a novel screening system for intracellular functional peptides; it combined a photo-cleavable peptide array system with cell-penetrating peptides (CPPs). Various peptides can be delivered into cells and intracellular functions of the peptides can be assayed by means of our system. The aim of the present study was to demonstrate that the proposed screening system can be used for assessing the intracellular activity of peptides. The cell death-inducing peptide (LNLISKLF) identified in a mitochondria-targeting domain (MTD) of the Noxa protein served as an original peptide sequence for screening of peptides with higher activity via modification of the peptide sequence. We obtained 4 peptides with higher activity, in which we substituted serine (S) at the fifth position with phenylalanine (F), valine (V), tryptophan (W), or tyrosine (Y). During analysis of the mechanism of action, the modified peptides induced an increase in intracellular calcium concentration, which was caused by the treatment with the original peptide. Higher capacity for cell death induction by the modified peptides may be caused by increased hydrophobicity or an increased number of aromatic residues. Thus, the present work suggests that the intracellular activity of peptides can be assessed using the proposed screening system. It could be used for identifying intracellular functional peptides with higher activity through comprehensive screening. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Designing anticancer peptides by constructive machine learning.

Science.gov (United States)

Grisoni, Francesca; Neuhaus, Claudia; Gabernet, Gisela; Müller, Alex; Hiss, Jan; Schneider, Gisbert

2018-04-21

Constructive machine learning enables the automated generation of novel chemical structures without the need for explicit molecular design rules. This study presents the experimental application of such a generative model to design membranolytic anticancer peptides (ACPs) de novo. A recurrent neural network with long short-term memory cells was trained on alpha-helical cationic amphipathic peptide sequences and then fine-tuned with 26 known ACPs. This optimized model was used to generate unique and novel amino acid sequences. Twelve of the peptides were synthesized and tested for their activity on MCF7 human breast adenocarcinoma cells and selectivity against human erythrocytes. Ten of these peptides were active against cancer cells. Six of the active peptides killed MCF7 cancer cells without affecting human erythrocytes with at least threefold selectivity. These results advocate constructive machine learning for the automated design of peptides with desired biological activities. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Production of peptide antisera specific for mouse and rat proinsulin C-peptide 2

DEFF Research Database (Denmark)

Blume, N; Madsen, O D; Kofod, Hans

1990-01-01

for antibody binding to the immunizing antigen. Antisera to C-peptide 2, stained islet beta-cells on mouse and rat, but not monkey pancreas sections in immunocytochemical analysis. Preabsorption to the synthetic C-peptide 2, but not the synthetic mouse and rat C-peptide 1 abolished staining. In conclusion we......Mice and rats have two functional non-allelic insulin genes. By using a synthetic peptide representing a common sequence in mouse and rat C-peptide 2 as antigen, we have produced rabbit antisera specific for an epitope which is not present in mouse or rat C-peptide 1. Long-term immunization did...... not seem to increase the end point titre as tested in direct ELISA. The specificity of the antiserum was determined by competitive ELISA and histochemistry on pancreas sections. Only the synthetic C-peptide 2, but not the homologous synthetic C-peptide 1 from mouse and rat competed efficiently in ELISA...

Therapeutic peptides for cancer therapy. Part I - peptide inhibitors of signal transduction cascades.

Science.gov (United States)

Bidwell, Gene L; Raucher, Drazen

2009-10-01

Therapeutic peptides have great potential as anticancer agents owing to their ease of rational design and target specificity. However, their utility in vivo is limited by low stability and poor tumor penetration. The authors review the development of peptide inhibitors with potential for cancer therapy. Peptides that inhibit signal transduction cascades are discussed. The authors searched Medline for articles concerning the development of therapeutic peptides and their delivery. Given our current knowledge of protein sequences, structures and interaction interfaces, therapeutic peptides that inhibit interactions of interest are easily designed. These peptides are advantageous because they are highly specific for the interaction of interest, and they are much more easily developed than small molecule inhibitors of the same interactions. The main hurdle to application of peptides for cancer therapy is their poor pharmacokinetic and biodistribution parameters. Therefore, successful development of peptide delivery vectors could potentially make possible the use of this new and very promising class of anticancer agents.

Trapping and partial characterization of an adduct postulated to be the covalent catalytic ternary complex of thymidylate synthetase

Energy Technology Data Exchange (ETDEWEB)

Ahmed, F.; Moore, M.A.; Dunlap, R.B.

1986-05-01

The proposed mechanism of action of thymidylate synthetase envisages the formation of a covalent ternary complex of the enzyme via the active site cysteine with dUMP and 5,10-methylenetetrahydrofolate (CH/sub 2/H/sub 4/folate). The authors recent success in using trichloroacetic acid to trap the covalent enzyme-FdUMP binary and ternary (enzyme-FdUMP-CH/sub 2/H/sub 4/folate) complexes led to the use of this technique in attempts to trap the transient covalent catalytic ternary complex. Experiments performed with (2-C/sup 14/)dUMP and /sup 3/H-CH/sub 2/H/sub 4/folate show that both these ligands remained bound to the enzyme after trichloroacetic acid precipitation. The trapped covalent catalytic ternary complex was subjected to CNBr fragmentation, and the peptides were fractionated by HPLC. The isolated active-site peptide was shown to retain the two ligands and was subjected to a limited sequence analysis by the dansyl-Edman procedure. The inhibitory ternary complex formed with /sup 14/C-FdUMP and /sup 3/H-CH/sub 2/ /sub 4/folate served as a control. The active-site peptides isolated from the CNBr treated inhibitory ternary complex and the catalytic complex exhibited identical sequences for the first four N-terminal residues, Ala-Leu-Pro-Pro, and the fifth residue was found to be associated with the labeled ligands. Sequence analysis of the active site peptide derived from the carboxymethylated enzyme confirmed this sequence and the 5th residue was shown to be Cm-Cys.

Syntheses and structures of technetium(V) and rhenium(V) oxo complexes of peptide having KYC-sequence

Energy Technology Data Exchange (ETDEWEB)

Takayama, T.; Suzuki, K.; Sekine, T.; Kudo, H. [Dept. of Chemistry, Tohoku Univ., Sendai (Japan)

2000-07-01

Technetium(V) and rhenium(V) oxo complexes of a peptide having a KYC-sequence such as KYCAR (H{sub 3}L{sup 5}) and KYCAREPPTRTNAYQGQG-NH{sub 2} (H{sub 3}L{sup 18}) were synthesized, and structures of the complexes were characterized by spectroscopic techniques. All of the complexes were synthesized by the ligand exchange reaction of [(n-C{sub 4}H{sub 9}){sub 4}N][MOCl{sub 4}] (M = {sup 99}Tc, Re) with peptide in methanol or dimethylformamide solution. These complexes have a square pyramidal structure with an oxo ligand at the apical position. The peptide is coordinated to a metal atom through N{sub amine} of lysine. S{sub thiol} of cysteine, and N{sub amide} of tyrosine and cysteine in the equatorial plane. A lysine (CH{sub 2}){sub 4}NH{sub 2} group of the L{sup 5} ligand has the syn conformation with respect to metal-oxo bonding in the complex. The syn isomer was selectively formed in the ligand exchange reaction. The conversion of the syn isomer to the anti isomer was observed only for syn-[ReO(L{sup 5})], in which the coordination of water to the trans position of the oxo ligand was involved. (orig.)

A Novel MS-Cleavable Azo Cross-Linker for Peptide Structure Analysis by Free Radical Initiated Peptide Sequencing (FRIPS)

Science.gov (United States)

Iacobucci, Claudio; Hage, Christoph; Schäfer, Mathias; Sinz, Andrea

2017-10-01

The chemical cross-linking/mass spectrometry (MS) approach is a growing research field in structural proteomics that allows gaining insights into protein conformations. It relies on creating distance constraints between cross-linked amino acid side chains that can further be used to derive protein structures. Currently, the most urgent task for designing novel cross-linking principles is an unambiguous and automated assignment of the created cross-linked products. Here, we introduce the homobifunctional, amine-reactive, and water soluble cross-linker azobisimidoester (ABI) as a prototype of a novel class of cross-linkers. The ABI-linker possesses an innovative modular scaffold combining the benefits of collisional activation lability with open shell chemistry. This MS-cleavable cross-linker can be efficiently operated via free radical initiated peptide sequencing (FRIPS) in positive ionization mode. Our proof-of-principle study challenges the gas phase behavior of the ABI-linker for the three amino acids, lysine, leucine, and isoleucine, as well as the model peptide thymopentin. The isomeric amino acids leucine and isoleucine could be discriminated by their characteristic side chain fragments. Collisional activation experiments were conducted via positive electrospray ionization (ESI) on two Orbitrap mass spectrometers. The ABI-mediated formation of odd electron product ions in MS/MS and MS3 experiments was evaluated and compared with a previously described azo-based cross-linker. All cross-linked products were amenable to automated analysis by the MeroX software, underlining the future potential of the ABI-linker for structural proteomics studies. [Figure not available: see fulltext.

Biomedical Applications of Self-Assembling Peptides

NARCIS (Netherlands)

Radmalekshahi, Mazda; Lempsink, Ludwijn; Amidi, Maryam; Hennink, Wim E.; Mastrobattista, Enrico

2016-01-01

Self-assembling peptides have gained increasing attention as versatile molecules to generate diverse supramolecular structures with tunable functionality. Because of the possibility to integrate a wide range of functional domains into self-assembling peptides including cell attachment sequences,

THE USE OF DEDICATED PEPTIDE LIBRARIES PERMITS THE DISCOVERY OF HIGH-AFFINITY BINDING PEPTIDES

NARCIS (Netherlands)

DEKOSTER, HS; AMONS, R; BENCKHUIJSEN, WE; FEIJLBRIEF, M; SCHELLEKENS, GA; DRIJFHOUT, JW

1995-01-01

The motif for peptide binding to monoclonal antibody mAb A16, which is known to be directed against glycoprotein D of Herpes simplex virus type 1, was determined using two dedicated peptide libraries. As a starting point for this study we used an A-16 binding lead sequence, which had previously been

Discovery of Novel Antimicrobial Peptides from Varanus komodoensis (Komodo Dragon) by Large-Scale Analyses and De-Novo-Assisted Sequencing Using Electron-Transfer Dissociation Mass Spectrometry.

Science.gov (United States)

Bishop, Barney M; Juba, Melanie L; Russo, Paul S; Devine, Megan; Barksdale, Stephanie M; Scott, Shaylyn; Settlage, Robert; Michalak, Pawel; Gupta, Kajal; Vliet, Kent; Schnur, Joel M; van Hoek, Monique L

2017-04-07

Komodo dragons are the largest living lizards and are the apex predators in their environs. They endure numerous strains of pathogenic bacteria in their saliva and recover from wounds inflicted by other dragons, reflecting the inherent robustness of their innate immune defense. We have employed a custom bioprospecting approach combining partial de novo peptide sequencing with transcriptome assembly to identify cationic antimicrobial peptides from Komodo dragon plasma. Through these analyses, we identified 48 novel potential cationic antimicrobial peptides. All but one of the identified peptides were derived from histone proteins. The antimicrobial effectiveness of eight of these peptides was evaluated against Pseudomonas aeruginosa (ATCC 9027) and Staphylococcus aureus (ATCC 25923), with seven peptides exhibiting antimicrobial activity against both microbes and one only showing significant potency against P. aeruginosa. This study demonstrates the power and promise of our bioprospecting approach to cationic antimicrobial peptide discovery, and it reveals the presence of a plethora of novel histone-derived antimicrobial peptides in the plasma of the Komodo dragon. These findings may have broader implications regarding the role that intact histones and histone-derived peptides play in defending the host from infection. Data are available via ProteomeXChange with identifier PXD005043.

Preliminary screening and identification of the hepatocarcinoma cell-binding peptide

International Nuclear Information System (INIS)

Zhu Xiaohua; Wu Hua

2004-01-01

Objective: To explore the feasibility of screening and isolating homing peptides that bind specifically, or preferentially, to hepatocarcinoma cells using phage display random peptide library and to develop a new peptide which may be potentially used as targeting delivery carrier in the biological targeted diagnosis or therapy for liver cancer. Methods: A 12-mer peptide phage display library was used to screen and isolate peptides that bind to human hepatocarcinoma cells, and four rounds of subtractive panning were carried out with the human hepatocarcinoma cell line HepG2 as the target. The affinities of selected phage clones for human hepatocarcinoma cells were determined with enzyme-linked immunosorbent assay (ELISA) and compared with that to human liver cell and other tumor cells of different tissue origins, respectively. In addition, the binding site in the tumor cells was observed with immunofluorescence analysis under confocal light microscopy. The amino acid sequences of phages that bind HepG2 specifically were deduced through DNA sequencing. Based on the results of DNA sequence, a 16-mer peptide (WH16) was designed and synthesized. Binding ability of the new peptide, WH16, was determined with competitive inhibition test. Results: After four rounds of panning, the phages that were bound to and internalized in human hepatocarcinoma cells were isolated. ELISA and immunofluorescence analysis confirmed the affinity of these phages for hepatocarcinoma cells. 56.67%(17/30) of the isolated phages displayed repeated sequence FLLEPHLMDTSM, and FLEP was defined as conservative motif . Binding of the selected phage to HepG2 cells was inhibited by synthesized peptide WH16, that strongly support that cellular binding of the phage is mediated through its displayed peptide, and WH16 can also bind to HepG2. Conclusions: It is feasible to screen and isolate homing peptides that bind specifically, or preferentially, to hepatocarcinoma cells using phage display random peptide

Preliminary screening and identification of the hepatocarcinoma cell-binding peptide

Energy Technology Data Exchange (ETDEWEB)

Xiaohua, Zhu; Hua, Wu [Department of Nuclear Medicine, Tongji Hospital, Tongji Medical College, Huazhong Univ. of Science and Technology, Wuhan (China)

2004-12-15

Objective: To explore the feasibility of screening and isolating homing peptides that bind specifically, or preferentially, to hepatocarcinoma cells using phage display random peptide library and to develop a new peptide which may be potentially used as targeting delivery carrier in the biological targeted diagnosis or therapy for liver cancer. Methods: A 12-mer peptide phage display library was used to screen and isolate peptides that bind to human hepatocarcinoma cells, and four rounds of subtractive panning were carried out with the human hepatocarcinoma cell line HepG2 as the target. The affinities of selected phage clones for human hepatocarcinoma cells were determined with enzyme-linked immunosorbent assay (ELISA) and compared with that to human liver cell and other tumor cells of different tissue origins, respectively. In addition, the binding site in the tumor cells was observed with immunofluorescence analysis under confocal light microscopy. The amino acid sequences of phages that bind HepG2 specifically were deduced through DNA sequencing. Based on the results of DNA sequence, a 16-mer peptide (WH16) was designed and synthesized. Binding ability of the new peptide, WH16, was determined with competitive inhibition test. Results: After four rounds of panning, the phages that were bound to and internalized in human hepatocarcinoma cells were isolated. ELISA and immunofluorescence analysis confirmed the affinity of these phages for hepatocarcinoma cells. 56.67%(17/30) of the isolated phages displayed repeated sequence FLLEPHLMDTSM, and FLEP was defined as conservative motif . Binding of the selected phage to HepG2 cells was inhibited by synthesized peptide WH16, that strongly support that cellular binding of the phage is mediated through its displayed peptide, and WH16 can also bind to HepG2. Conclusions: It is feasible to screen and isolate homing peptides that bind specifically, or preferentially, to hepatocarcinoma cells using phage display random peptide

Post-staining electroblotting for efficient and reliable peptide blotting.

Science.gov (United States)

Lee, Der-Yen; Chang, Geen-Dong

2015-01-01

Post-staining electroblotting has been previously described to transfer Coomassie blue-stained proteins from polyacrylamide gel onto polyvinylidene difluoride (PVDF) membranes. Actually, stained peptides can also be efficiently and reliably transferred. Because of selective staining procedures for peptides and increased retention of stained peptides on the membrane, even peptides with molecular masses less than 2 kDa such as bacitracin and granuliberin R are transferred with satisfactory results. For comparison, post-staining electroblotting is about 16-fold more sensitive than the conventional electroblotting for visualization of insulin on the membrane. Therefore, the peptide blots become practicable and more accessible to further applications, e.g., blot overlay detection or immunoblotting analysis. In addition, the efficiency of peptide transfer is favorable for N-terminal sequence analysis. With this method, peptide blotting can be normalized for further analysis such as blot overlay assay, immunoblotting, and N-terminal sequencing for identification of peptide in crude or partially purified samples.

Peptide inhibitors of appressorium development in Glomerella cingulata.

Science.gov (United States)

Al-Samarrai, Taha H; Sullivan, Patrick A; Templeton, Matthew D; Farley, Peter C

2002-04-09

The phytopathogen Glomerella cingulata (anamorph: Colletotrichum gloeosporioides) infects host tissue by means of a specialised infection structure, the appressorium. The Saccharomyces cerevisiae alpha-mating factor pheromone, the Saccharomyces kluyveri alpha-mating factor pheromone and a hendecapeptide produced by G. cingulata inhibit appressorium development. The amino acid sequence of the G. cingulata peptide, GYFSYPHGNLF, is different from that of the mature pheromone peptides of other filamentous fungi. The peptide has sequence similarity with the Saccharomyces alpha-mating factor pheromones, but is unable to elicit growth arrest in S. cerevisiae.

Enzyme-Assisted Discovery of Antioxidant Peptides from Edible Marine Invertebrates: A Review.

Science.gov (United States)

Chai, Tsun-Thai; Law, Yew-Chye; Wong, Fai-Chu; Kim, Se-Kwon

2017-02-16

Marine invertebrates, such as oysters, mussels, clams, scallop, jellyfishes, squids, prawns, sea cucumbers and sea squirts, are consumed as foods. These edible marine invertebrates are sources of potent bioactive peptides. The last two decades have seen a surge of interest in the discovery of antioxidant peptides from edible marine invertebrates. Enzymatic hydrolysis is an efficient strategy commonly used for releasing antioxidant peptides from food proteins. A growing number of antioxidant peptide sequences have been identified from the enzymatic hydrolysates of edible marine invertebrates. Antioxidant peptides have potential applications in food, pharmaceuticals and cosmetics. In this review, we first give a brief overview of the current state of progress of antioxidant peptide research, with special attention to marine antioxidant peptides. We then focus on 22 investigations which identified 32 antioxidant peptides from enzymatic hydrolysates of edible marine invertebrates. Strategies adopted by various research groups in the purification and identification of the antioxidant peptides will be summarized. Structural characteristic of the peptide sequences in relation to their antioxidant activities will be reviewed. Potential applications of the peptide sequences and future research prospects will also be discussed.

Systematic discovery of new recognition peptides mediating protein interaction networks

DEFF Research Database (Denmark)

Neduva, Victor; Linding, Rune; Su-Angrand, Isabelle

2005-01-01

Many aspects of cell signalling, trafficking, and targeting are governed by interactions between globular protein domains and short peptide segments. These domains often bind multiple peptides that share a common sequence pattern, or "linear motif" (e.g., SH3 binding to PxxP). Many domains...... by interactions between globular protein domains and short peptide segments. These domains often bind multiple peptides that share a common sequence pattern, or "linear motif" (e.g., SH3 binding to PxxP). Many domains are known, though comparatively few linear motifs have been discovered. Their short length...

Shotgun protein sequencing.

Energy Technology Data Exchange (ETDEWEB)

Faulon, Jean-Loup Michel; Heffelfinger, Grant S.

2009-06-01

A novel experimental and computational technique based on multiple enzymatic digestion of a protein or protein mixture that reconstructs protein sequences from sequences of overlapping peptides is described in this SAND report. This approach, analogous to shotgun sequencing of DNA, is to be used to sequence alternative spliced proteins, to identify post-translational modifications, and to sequence genetically engineered proteins.

B and T Cell Epitope-Based Peptides Predicted from Evolutionarily Conserved and Whole Protein Sequences of Ebola Virus as Vaccine Targets.

Science.gov (United States)

Yasmin, T; Nabi, A H M Nurun

2016-05-01

Ebola virus (EBV) has become a serious threat to public health. Different approaches were applied to predict continuous and discontinuous B cell epitopes as well as T cell epitopes from the sequence-based and available three-dimensional structural analyses of each protein of EBV. Peptides '(79) VPSATKRWGFRSGVPP(94) ' from GP1 and '(515) LHYWTTQDEGAAIGLA(530) ' from GP2 of Ebola were found to be the consensus peptidic sequences predicted as linear B cell epitope of which the latter contains a region (519) TTQDEG(524) that fulfilled all the criteria of accessibility, hydrophilicity, flexibility and beta turn region for becoming an ideal B cell epitope. Different nonamers as T cell epitopes were obtained that interacted with different numbers of MHC class I and class II alleles with a binding affinity of <100 nm. Interestingly, these alleles also bound to the MHC class I alleles mostly prevalent in African and South Asian regions. Of these, 'LANETTQAL' and 'FLYDRLAST' nonamers were predicted to be the most potent T cell epitopes and they, respectively, interacted with eight and twelve class I alleles that covered 63.79% and 54.16% of world population, respectively. These nonamers were found to be the core sequences of 15mer peptides that interacted with the most common class II allele, HLA-DRB1*01:01. They were further validated for their binding to specific class I alleles using docking technique. Thus, these predicted epitopes may be used as vaccine targets against EBV and can be validated in model hosts to verify their efficacy as vaccine. © 2016 The Foundation for the Scandinavian Journal of Immunology.

Antimicrobial peptides design by evolutionary multiobjective optimization.

Directory of Open Access Journals (Sweden)

Giuseppe Maccari

Full Text Available Antimicrobial peptides (AMPs are an abundant and wide class of molecules produced by many tissues and cell types in a variety of mammals, plant and animal species. Linear alpha-helical antimicrobial peptides are among the most widespread membrane-disruptive AMPs in nature, representing a particularly successful structural arrangement in innate defense. Recently, AMPs have received increasing attention as potential therapeutic agents, owing to their broad activity spectrum and their reduced tendency to induce resistance. The introduction of non-natural amino acids will be a key requisite in order to contrast host resistance and increase compound's life. In this work, the possibility to design novel AMP sequences with non-natural amino acids was achieved through a flexible computational approach, based on chemophysical profiles of peptide sequences. Quantitative structure-activity relationship (QSAR descriptors were employed to code each peptide and train two statistical models in order to account for structural and functional properties of alpha-helical amphipathic AMPs. These models were then used as fitness functions for a multi-objective evolutional algorithm, together with a set of constraints for the design of a series of candidate AMPs. Two ab-initio natural peptides were synthesized and experimentally validated for antimicrobial activity, together with a series of control peptides. Furthermore, a well-known Cecropin-Mellitin alpha helical antimicrobial hybrid (CM18 was optimized by shortening its amino acid sequence while maintaining its activity and a peptide with non-natural amino acids was designed and tested, demonstrating the higher activity achievable with artificial residues.

Gastrointestinal Endogenous Proteins as a Source of Bioactive Peptides - An In Silico Study

Science.gov (United States)

Dave, Lakshmi A.; Montoya, Carlos A.; Rutherfurd, Shane M.; Moughan, Paul J.

2014-01-01

Dietary proteins are known to contain bioactive peptides that are released during digestion. Endogenous proteins secreted into the gastrointestinal tract represent a quantitatively greater supply of protein to the gut lumen than those of dietary origin. Many of these endogenous proteins are digested in the gastrointestinal tract but the possibility that these are also a source of bioactive peptides has not been considered. An in silico prediction method was used to test if bioactive peptides could be derived from the gastrointestinal digestion of gut endogenous proteins. Twenty six gut endogenous proteins and seven dietary proteins were evaluated. The peptides present after gastric and intestinal digestion were predicted based on the amino acid sequence of the proteins and the known specificities of the major gastrointestinal proteases. The predicted resultant peptides possessing amino acid sequences identical to those of known bioactive peptides were identified. After gastrointestinal digestion (based on the in silico simulation), the total number of bioactive peptides predicted to be released ranged from 1 (gliadin) to 55 (myosin) for the selected dietary proteins and from 1 (secretin) to 39 (mucin-5AC) for the selected gut endogenous proteins. Within the intact proteins and after simulated gastrointestinal digestion, angiotensin converting enzyme (ACE)-inhibitory peptide sequences were the most frequently observed in both the dietary and endogenous proteins. Among the dietary proteins, after in silico simulated gastrointestinal digestion, myosin was found to have the highest number of ACE-inhibitory peptide sequences (49 peptides), while for the gut endogenous proteins, mucin-5AC had the greatest number of ACE-inhibitory peptide sequences (38 peptides). Gut endogenous proteins may be an important source of bioactive peptides in the gut particularly since gut endogenous proteins represent a quantitatively large and consistent source of protein. PMID:24901416

Gastrointestinal endogenous proteins as a source of bioactive peptides--an in silico study.

Science.gov (United States)

Dave, Lakshmi A; Montoya, Carlos A; Rutherfurd, Shane M; Moughan, Paul J

2014-01-01

Dietary proteins are known to contain bioactive peptides that are released during digestion. Endogenous proteins secreted into the gastrointestinal tract represent a quantitatively greater supply of protein to the gut lumen than those of dietary origin. Many of these endogenous proteins are digested in the gastrointestinal tract but the possibility that these are also a source of bioactive peptides has not been considered. An in silico prediction method was used to test if bioactive peptides could be derived from the gastrointestinal digestion of gut endogenous proteins. Twenty six gut endogenous proteins and seven dietary proteins were evaluated. The peptides present after gastric and intestinal digestion were predicted based on the amino acid sequence of the proteins and the known specificities of the major gastrointestinal proteases. The predicted resultant peptides possessing amino acid sequences identical to those of known bioactive peptides were identified. After gastrointestinal digestion (based on the in silico simulation), the total number of bioactive peptides predicted to be released ranged from 1 (gliadin) to 55 (myosin) for the selected dietary proteins and from 1 (secretin) to 39 (mucin-5AC) for the selected gut endogenous proteins. Within the intact proteins and after simulated gastrointestinal digestion, angiotensin converting enzyme (ACE)-inhibitory peptide sequences were the most frequently observed in both the dietary and endogenous proteins. Among the dietary proteins, after in silico simulated gastrointestinal digestion, myosin was found to have the highest number of ACE-inhibitory peptide sequences (49 peptides), while for the gut endogenous proteins, mucin-5AC had the greatest number of ACE-inhibitory peptide sequences (38 peptides). Gut endogenous proteins may be an important source of bioactive peptides in the gut particularly since gut endogenous proteins represent a quantitatively large and consistent source of protein.

StraPep: a structure database of bioactive peptides

Science.gov (United States)

Wang, Jian; Yin, Tailang; Xiao, Xuwen; He, Dan; Xue, Zhidong; Jiang, Xinnong; Wang, Yan

2018-01-01

Abstract Bioactive peptides, with a variety of biological activities and wide distribution in nature, have attracted great research interest in biological and medical fields, especially in pharmaceutical industry. The structural information of bioactive peptide is important for the development of peptide-based drugs. Many databases have been developed cataloguing bioactive peptides. However, to our knowledge, database dedicated to collect all the bioactive peptides with known structure is not available yet. Thus, we developed StraPep, a structure database of bioactive peptides. StraPep holds 3791 bioactive peptide structures, which belong to 1312 unique bioactive peptide sequences. About 905 out of 1312 (68%) bioactive peptides in StraPep contain disulfide bonds, which is significantly higher than that (21%) of PDB. Interestingly, 150 out of 616 (24%) bioactive peptides with three or more disulfide bonds form a structural motif known as cystine knot, which confers considerable structural stability on proteins and is an attractive scaffold for drug design. Detailed information of each peptide, including the experimental structure, the location of disulfide bonds, secondary structure, classification, post-translational modification and so on, has been provided. A wide range of user-friendly tools, such as browsing, sequence and structure-based searching and so on, has been incorporated into StraPep. We hope that this database will be helpful for the research community. Database URL: http://isyslab.info/StraPep PMID:29688386

High-throughput sequencing enhanced phage display identifies peptides that bind mycobacteria

CSIR Research Space (South Africa)

Ngubane, NAC

2013-11-01

Full Text Available . The displayed peptides are flanked by two cysteine residues, which are oxidized during phage assembly to a disulfide bond, resulting in a loop constrained peptide. We initially used the traditional clone picking method to identify the enriched clones... of the library, 1.236109 heptapeptides, it represented sufficient depth to measure the quantitative enrich- ment of relevant peptides. To confirm successful enrichment during selection, we characterized the reduction in diversity of the pool in the consecutive...

Optimization of antibacterial peptides by genetic algorithms and cheminformatics

DEFF Research Database (Denmark)

Fjell, Christopher D.; Jenssen, Håvard; Cheung, Warren A.

2011-01-01

Pathogens resistant to available drug therapies are a pressing global health problem. Short, cationic peptides represent a novel class of agents that have lower rates of drug resistance than derivatives of current antibiotics. Previously, we created a software system utilizing artificial neural...... 47 of the top rated 50 peptides chosen from an in silico library of nearly 100 000 sequences. Here, we report a method of generating candidate peptide sequences using the heuristic evolutionary programming method of genetic algorithms (GA), which provided a large (19-fold) improvement...

Preliminary screening and identification of the peptide binding to hepatocarcinoma cell

International Nuclear Information System (INIS)

Zhu Xiaohua; Wu Ha

2004-01-01

Objective: The present study was performed to screen and isolate homing peptides that bind specifically, or preferentially, to hepatocarcinoma cells using phage display of random peptide library with the purpose of developing a new peptide which may be potentially used as target delivery carrier in the biological target diagnosis or therapy for liver cancer. Methods: A peptide 12-mer phage display library was used to screen and isolate peptide that bind to human hepatocarcinoma cell, and four rounds subtractive panning were carried out with the human hepatocarcinoma cell line HepG2 as the target. The affinities of selected phage clones to human hepatocarcinoma cell were determined with ELISA and compared with human liver cell and other tumor cells of different tissue origins respectively. In addition, the binding site in the tumor cells was observed with immunofluorescence analysis under confocal light microscopy. The amino acid sequences of phages that bind HepG2 specifically were deduced though DNA sequencing. Based on the results of DNA sequence, a 16-mer peptide (WH16) was designed and synthesized. Binding ability of the new peptide WH16 was determined with competitive inhibition test. Results: After four rounds panning, the phages that bound to and internalized in human hepatocarcinoma cell were isolated. ELISA and immunofluorescence analysis confirmed the affinity of these phages to hepatpcarcinoma cells 56.57%(17/30) of the isolated phages displayed repeated sequence FLLEPHLMDTSM, and FLEP was defined as conservative motif. Binding of the selected phage to HepG2 cells was inhibited by synthesized peptide WH16, which strongly support that cellular binding of phage is mediated though its displayed peptide and WH16 can also bind to HepG2. Conclusion: It is feasible to screen and isolate homing peptides that bind specifically, or preferentially, to hepatocarcinoma cells using phage display of random peptide libraries. The sequence of peptide that can bind to

Preliminary screening and identification of the peptide binding to hepatocarcinoma cell

Energy Technology Data Exchange (ETDEWEB)

Xiaohua, Zhu; Ha, Wu [Department of Nuclear Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan (China)

2004-07-01

Objective: The present study was performed to screen and isolate homing peptides that bind specifically, or preferentially, to hepatocarcinoma cells using phage display of random peptide library with the purpose of developing a new peptide which may be potentially used as target delivery carrier in the biological target diagnosis or therapy for liver cancer. Methods: A peptide 12-mer phage display library was used to screen and isolate peptide that bind to human hepatocarcinoma cell, and four rounds subtractive panning were carried out with the human hepatocarcinoma cell line HepG2 as the target. The affinities of selected phage clones to human hepatocarcinoma cell were determined with ELISA and compared with human liver cell and other tumor cells of different tissue origins respectively. In addition, the binding site in the tumor cells was observed with immunofluorescence analysis under confocal light microscopy. The amino acid sequences of phages that bind HepG2 specifically were deduced though DNA sequencing. Based on the results of DNA sequence, a 16-mer peptide (WH16) was designed and synthesized. Binding ability of the new peptide WH16 was determined with competitive inhibition test. Results: After four rounds panning, the phages that bound to and internalized in human hepatocarcinoma cell were isolated. ELISA and immunofluorescence analysis confirmed the affinity of these phages to hepatpcarcinoma cells 56.57%(17/30) of the isolated phages displayed repeated sequence FLLEPHLMDTSM, and FLEP was defined as conservative motif. Binding of the selected phage to HepG2 cells was inhibited by synthesized peptide WH16, which strongly support that cellular binding of phage is mediated though its displayed peptide and WH16 can also bind to HepG2. Conclusion: It is feasible to screen and isolate homing peptides that bind specifically, or preferentially, to hepatocarcinoma cells using phage display of random peptide libraries. The sequence of peptide that can bind to

Novel anti-HIV peptides containing multiple copies of artificially designed heptad repeat motifs

International Nuclear Information System (INIS)

Shi Weiguo; Qi Zhi; Pan Chungen; Xue Na; Debnath, Asim K.; Qie Jiankun; Jiang Shibo; Liu Keliang

2008-01-01

The peptidic anti-HIV drug T20 (Fuzeon) and its analog C34 share a common heptad repeat (HR) sequence, but they have different functional domains, i.e., pocket- and lipid-binding domains (PBD and LBD, respectively). We hypothesize that novel anti-HIV peptides may be designed by using artificial sequences containing multiple copies of HR motifs plus zero, one or two functional domains. Surprisingly, we found that the peptides containing only the non-natural HR sequences could significantly inhibit HIV-1 infection, while addition of PBD and/or LBD to the peptides resulted in significant improvement of anti-HIV-1 activity. These results suggest that these artificial HR sequences, which may serve as structural domains, could be used as templates for the design of novel antiviral peptides against HIV and other viruses with class I fusion proteins

Characterization of the Organic Component of Low-Molecular-Weight Chromium-Binding Substance and Its Binding of Chromium123

Science.gov (United States)

Chen, Yuan; Watson, Heather M.; Gao, Junjie; Sinha, Sarmistha Halder; Cassady, Carolyn J.; Vincent, John B.

2011-01-01

Chromium was proposed to be an essential element over 50 y ago and was shown to have therapeutic potential in treating the symptoms of type 2 diabetes; however, its mechanism of action at a molecular level is unknown. One chromium-binding biomolecule, low-molecular weight chromium-binding substance (LMWCr or chromodulin), has been found to be biologically active in in vitro assays and proposed as a potential candidate for the in vivo biologically active form of chromium. Characterization of the organic component of LMWCr has proven difficult. Treating bovine LMWCr with trifluoroacetic acid followed by purification on a graphite powder micro-column generates a heptapeptide fragment of LMWCr. The peptide sequence of the fragment was analyzed by MS and tandem MS (MS/MS and MS/MS/MS) using collision-induced dissociation and post-source decay. Two candidate sequences, pEEEEGDD and pEEEGEDD (where pE is pyroglutamate), were identified from the MS/MS experiments; additional tandem MS suggests the sequence is pEEEEGDD. The N-terminal glutamate residues explain the inability to sequence LMWCr by the Edman method. Langmuir isotherms and Hill plots were used to analyze the binding constants of chromic ions to synthetic peptides similar in composition to apoLMWCr. The sequence pEEEEGDD was found to bind 4 chromic ions per peptide with nearly identical cooperativity and binding constants to those of apoLMWCr. This work should lead to further studies elucidating or eliminating a potential role for LMWCr in treating the symptoms of type 2 diabetes and other conditions resulting from improper carbohydrate and lipid metabolism. PMID:21593351

De novo peptide design and experimental validation of histone methyltransferase inhibitors.

Directory of Open Access Journals (Sweden)

James Smadbeck

Full Text Available Histones are small proteins critical to the efficient packaging of DNA in the nucleus. DNA–protein complexes, known as nucleosomes, are formed when the DNA winds itself around the surface of the histones. The methylation of histone residues by enhancer of zeste homolog 2 (EZH2 maintains gene repression over successive cell generations. Overexpression of EZH2 can silence important tumor suppressor genes leading to increased invasiveness of many types of cancers. This makes the inhibition of EZH2 an important target in the development of cancer therapeutics. We employed a three-stage computational de novo peptide design method to design inhibitory peptides of EZH2. The method consists of a sequence selection stage and two validation stages for fold specificity and approximate binding affinity. The sequence selection stage consists of an integer linear optimization model that was solved to produce a rank-ordered list of amino acid sequences with increased stability in the bound peptide-EZH2 structure. These sequences were validated through the calculation of the fold specificity and approximate binding affinity of the designed peptides. Here we report the discovery of novel EZH2 inhibitory peptides using the de novo peptide design method. The computationally discovered peptides were experimentally validated in vitro using dose titrations and mechanism of action enzymatic assays. The peptide with the highest in vitro response, SQ037, was validated in nucleo using quantitative mass spectrometry-based proteomics. This peptide had an IC50 of 13.5 mM, demonstrated greater potency as an inhibitor when compared to the native and K27A mutant control peptides, and demonstrated competitive inhibition versus the peptide substrate. Additionally, this peptide demonstrated high specificity to the EZH2 target in comparison to other histone methyltransferases. The validated peptides are the first computationally designed peptides that directly inhibit EZH2

De novo peptide design and experimental validation of histone methyltransferase inhibitors.

Directory of Open Access Journals (Sweden)

James Smadbeck

Full Text Available Histones are small proteins critical to the efficient packaging of DNA in the nucleus. DNA-protein complexes, known as nucleosomes, are formed when the DNA winds itself around the surface of the histones. The methylation of histone residues by enhancer of zeste homolog 2 (EZH2 maintains gene repression over successive cell generations. Overexpression of EZH2 can silence important tumor suppressor genes leading to increased invasiveness of many types of cancers. This makes the inhibition of EZH2 an important target in the development of cancer therapeutics. We employed a three-stage computational de novo peptide design method to design inhibitory peptides of EZH2. The method consists of a sequence selection stage and two validation stages for fold specificity and approximate binding affinity. The sequence selection stage consists of an integer linear optimization model that was solved to produce a rank-ordered list of amino acid sequences with increased stability in the bound peptide-EZH2 structure. These sequences were validated through the calculation of the fold specificity and approximate binding affinity of the designed peptides. Here we report the discovery of novel EZH2 inhibitory peptides using the de novo peptide design method. The computationally discovered peptides were experimentally validated in vitro using dose titrations and mechanism of action enzymatic assays. The peptide with the highest in vitro response, SQ037, was validated in nucleo using quantitative mass spectrometry-based proteomics. This peptide had an IC50 of 13.5 [Formula: see text]M, demonstrated greater potency as an inhibitor when compared to the native and K27A mutant control peptides, and demonstrated competitive inhibition versus the peptide substrate. Additionally, this peptide demonstrated high specificity to the EZH2 target in comparison to other histone methyltransferases. The validated peptides are the first computationally designed peptides that directly

Effect of stereochemistry, chain length and sequence pattern on antimicrobial properties of short synthetic β-sheet forming peptide amphiphiles.

Science.gov (United States)

Ong, Zhan Yuin; Cheng, Junchi; Huang, Yuan; Xu, Kaijin; Ji, Zhongkang; Fan, Weimin; Yang, Yi Yan

2014-01-01

In the face of mounting global antibiotics resistance, the identification and development of membrane-active antimicrobial peptides (AMPs) as an alternative class of antimicrobial agent have gained significant attention. The physical perturbation and disruption of microbial membranes by the AMPs have been proposed to be an effective means to overcome conventional mechanisms of drug resistance. Recently, we have reported the design of a series of short synthetic β-sheet folding peptide amphiphiles comprised of recurring (X1Y1X2Y2)n-NH2 sequences where X: hydrophobic amino acids, Y: cationic amino acids and n: number of repeat units. In efforts to investigate the effects of key parameters including stereochemistry, chain length and sequence pattern on antimicrobial effects, systematic d-amino acid substitutions of the lead peptides (IRIK)2-NH2 (IK8-all L) and (IRVK)3-NH2 (IK12-all L) were performed. It was found that the corresponding D-enantiomers exhibited stronger antimicrobial activities with minimal or no change in hemolytic activities, hence translating very high selectivity indices of 407.0 and >9.8 for IK8-all D and IK12-all D respectively. IK8-all D was also demonstrated to be stable to degradation by broad spectrum proteases trypsin and proteinase K. The membrane disrupting bactericidal properties of IK8-all D effectively prevented drug resistance development and inhibited the growth of various clinically isolated MRSA, VRE, Acinetobacter baumanni, Pseudomonas aeruginosa, Cryptococcus. neoformans and Mycobacterium tuberculosis. Significant reduction in intracellular bacteria counts was also observed following treatment with IK8-all D in the Staphylococcus. aureus infected mouse macrophage cell line RAW264.7 (P < 0.01). These results suggest that the d-amino acids substituted β-sheet forming peptide IK8-all D with its enhanced antimicrobial activities and improved protease stability, is a promising therapeutic candidate with potential to combat

Towards Identify Selective Antibacterial Peptides Based on Abstracts Meaning

Directory of Open Access Journals (Sweden)

Liliana I. Barbosa-Santillán

2016-01-01

Full Text Available We present an Identify Selective Antibacterial Peptides (ISAP approach based on abstracts meaning. Laboratories and researchers have significantly increased the report of their discoveries related to antibacterial peptides in primary publications. It is important to find antibacterial peptides that have been reported in primary publications because they can produce antibiotics of different generations that attack and destroy the bacteria. Unfortunately, researchers used heterogeneous forms of natural language to describe their discoveries (sometimes without the sequence of the peptides. Thus, we propose that learning the words meaning instead of the antibacterial peptides sequence is possible to identify and predict antibacterial peptides reported in the PubMed engine. The ISAP approach consists of two stages: training and discovering. ISAP founds that the 35% of the abstracts sample had antibacterial peptides and we tested in the updated Antimicrobial Peptide Database 2 (APD2. ISAP predicted that 45% of the abstracts had antibacterial peptides. That is, ISAP found that 810 antibacterial peptides were not classified like that, so they are not reported in APD2. As a result, this new search tool would complement the APD2 with a set of peptides that are candidates to be antibacterial. Finally, 20% of the abstracts were not semantic related to APD2.

Selective peptide bond hydrolysis of cysteine peptides in the presence of Ni(II) ions.

Science.gov (United States)

Protas, Anna Maria; Bonna, Arkadiusz; Kopera, Edyta; Bal, Wojciech

2011-01-01

Recently, we described a sequence-specific R1-(Ser/Thr) peptide bond hydrolysis reaction in peptides of a general sequence R1-(Ser/Thr)-Xaa-His-Zaa-R, which occurs in the presence of Ni(II) ions [A. Krężel, E. Kopera, A. M. Protas, A. Wysłouch-Cieszyńska, J. Poznański, W. Bal, J. Am. Chem. Soc. 132 (2010) 3355-3366]. In this study we explored the possibility of substituting the Ser/Thr and the His residues, necessary for the reaction to occur according to the Ni(II)-assisted acyl shift reaction mechanism, with Cys residues. We tested this concept by synthesizing three homologous peptides: R1-Ser-Arg-Cys-Trp-R2, R1-Cys-Arg-His-Trp-R2, and R1-Cys-Arg-Cys-Trp-R2, and the R1-Ser-Arg-His-Trp-R2 peptide as comparator (R1 and R2 were CH3CO-Gly-Ala and Lys-Phe-Leu-NH2, respectively). We studied their hydrolysis in the presence of Ni(II) ions, under anaerobic conditions and in the presence of TCEP as a thiol group antioxidant. We measured hydrolysis rates using HPLC and identified products of reaction using electrospray mass spectrometry. Potentiometry and UV-vis spectroscopy were used to assess Ni(II) complexation. We demonstrated that Ni(II) is not compatible with the Cys substitution of the Ser/Thr acyl acceptor residue, but the substitution of the Ni(II) binding His residue with a Cys yields a peptide susceptible to Ni(II)-related hydrolysis. The relatively high activity of the R1-Ser-Arg-Cys-Trp-R2 peptide at pH 7.0 suggests that this peptide and its Cys-containing analogs might be useful in practical applications of Ni(II)-dependent peptide bond hydrolysis. Copyright © 2010 Elsevier Inc. All rights reserved.

A sensitive mass spectrometric method for hypothesis-driven detection of peptide post-translational modifications: multiple reaction monitoring-initiated detection and sequencing (MIDAS).

Science.gov (United States)

Unwin, Richard D; Griffiths, John R; Whetton, Anthony D

2009-01-01

The application of a targeted mass spectrometric workflow to the sensitive identification of post-translational modifications is described. This protocol employs multiple reaction monitoring (MRM) to search for all putative peptides specifically modified in a target protein. Positive MRMs trigger an MS/MS experiment to confirm the nature and site of the modification. This approach, termed MIDAS (MRM-initiated detection and sequencing), is more sensitive than approaches using neutral loss scanning or precursor ion scanning methodologies, due to a more efficient use of duty cycle along with a decreased background signal associated with MRM. We describe the use of MIDAS for the identification of phosphorylation, with a typical experiment taking just a couple of hours from obtaining a peptide sample. With minor modifications, the MIDAS method can be applied to other protein modifications or unmodified peptides can be used as a MIDAS target.

Cyanine-based probe\\tag-peptide pair fluorescence protein imaging and fluorescence protein imaging methods

Science.gov (United States)

Mayer-Cumblidge, M. Uljana; Cao, Haishi

2013-01-15

A molecular probe comprises two arsenic atoms and at least one cyanine based moiety. A method of producing a molecular probe includes providing a molecule having a first formula, treating the molecule with HgOAc, and subsequently transmetallizing with AsCl.sub.3. The As is liganded to ethanedithiol to produce a probe having a second formula. A method of labeling a peptide includes providing a peptide comprising a tag sequence and contacting the peptide with a biarsenical molecular probe. A complex is formed comprising the tag sequence and the molecular probe. A method of studying a peptide includes providing a mixture containing a peptide comprising a peptide tag sequence, adding a biarsenical probe to the mixture, and monitoring the fluorescence of the mixture.

Superior Antifouling Performance of a Zwitterionic Peptide Compared to an Amphiphilic, Non-Ionic Peptide.

Science.gov (United States)

Ye, Huijun; Wang, Libing; Huang, Renliang; Su, Rongxin; Liu, Boshi; Qi, Wei; He, Zhimin

2015-10-14

The aim of this study was to explore the influence of amphiphilic and zwitterionic structures on the resistance of protein adsorption to peptide self-assembled monolayers (SAMs) and gain insight into the associated antifouling mechanism. Two kinds of cysteine-terminated heptapeptides were studied. One peptide had alternating hydrophobic and hydrophilic residues with an amphiphilic sequence of CYSYSYS. The other peptide (CRERERE) was zwitterionic. Both peptides were covalently attached onto gold substrates via gold-thiol bond formation. Surface plasmon resonance analysis results showed that both peptide SAMs had ultralow or low protein adsorption amounts of 1.97-11.78 ng/cm2 in the presence of single proteins. The zwitterionic peptide showed relatively higher antifouling ability with single proteins and natural complex protein media. We performed molecular dynamics simulations to understand their respective antifouling behaviors. The results indicated that strong surface hydration of peptide SAMs contributes to fouling resistance by impeding interactions with proteins. Compared to the CYSYSYS peptide, more water molecules were predicted to form hydrogen-bonding interactions with the zwitterionic CRERERE peptide, which is in agreement with the antifouling test results. These findings reveal a clear relation between peptide structures and resistance to protein adsorption, facilitating the development of novel peptide-containing antifouling materials.

Antimicrobial Peptides in Reptiles

Science.gov (United States)

van Hoek, Monique L.

2014-01-01

Reptiles are among the oldest known amniotes and are highly diverse in their morphology and ecological niches. These animals have an evolutionarily ancient innate-immune system that is of great interest to scientists trying to identify new and useful antimicrobial peptides. Significant work in the last decade in the fields of biochemistry, proteomics and genomics has begun to reveal the complexity of reptilian antimicrobial peptides. Here, the current knowledge about antimicrobial peptides in reptiles is reviewed, with specific examples in each of the four orders: Testudines (turtles and tortosises), Sphenodontia (tuataras), Squamata (snakes and lizards), and Crocodilia (crocodilans). Examples are presented of the major classes of antimicrobial peptides expressed by reptiles including defensins, cathelicidins, liver-expressed peptides (hepcidin and LEAP-2), lysozyme, crotamine, and others. Some of these peptides have been identified and tested for their antibacterial or antiviral activity; others are only predicted as possible genes from genomic sequencing. Bioinformatic analysis of the reptile genomes is presented, revealing many predicted candidate antimicrobial peptides genes across this diverse class. The study of how these ancient creatures use antimicrobial peptides within their innate immune systems may reveal new understandings of our mammalian innate immune system and may also provide new and powerful antimicrobial peptides as scaffolds for potential therapeutic development. PMID:24918867

Peptide-Mediated Liposome Fusion: The Effect of Anchor Positioning

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Niek S. A. Crone

2018-01-01

Full Text Available A minimal model system for membrane fusion, comprising two complementary peptides dubbed “E” and “K” joined to a cholesterol anchor via a polyethyleneglycol spacer, has previously been developed in our group. This system promotes the fusion of large unilamellar vesicles and facilitates liposome-cell fusion both in vitro and in vivo. Whilst several aspects of the system have previously been investigated to provide an insight as to how fusion is facilitated, anchor positioning has not yet been considered. In this study, the effects of placing the anchor at either the N-terminus or in the center of the peptide are investigated using a combination of circular dichroism spectroscopy, dynamic light scattering, and fluorescence assays. It was discovered that anchoring the “K” peptide in the center of the sequence had no effect on its structure, its ability to interact with membranes, or its ability to promote fusion, whereas anchoring the ‘E’ peptide in the middle of the sequence dramatically decreases fusion efficiency. We postulate that anchoring the ‘E’ peptide in the middle of the sequence disrupts its ability to form homodimers with peptides on the same membrane, leading to aggregation and content leakage.

Dynamical System Modeling to Simulate Donor T Cell Response to Whole Exome Sequencing-Derived Recipient Peptides Demonstrates Different Alloreactivity Potential in HLA-Matched and -Mismatched Donor-Recipient Pairs.

Science.gov (United States)

Abdul Razzaq, Badar; Scalora, Allison; Koparde, Vishal N; Meier, Jeremy; Mahmood, Musa; Salman, Salman; Jameson-Lee, Max; Serrano, Myrna G; Sheth, Nihar; Voelkner, Mark; Kobulnicky, David J; Roberts, Catherine H; Ferreira-Gonzalez, Andrea; Manjili, Masoud H; Buck, Gregory A; Neale, Michael C; Toor, Amir A

2016-05-01

Immune reconstitution kinetics and subsequent clinical outcomes in HLA-matched recipients of allogeneic stem cell transplantation (SCT) are variable and difficult to predict. Considering SCT as a dynamical system may allow sequence differences across the exomes of the transplant donors and recipients to be used to simulate an alloreactive T cell response, which may allow better clinical outcome prediction. To accomplish this, whole exome sequencing was performed on 34 HLA-matched SCT donor-recipient pairs (DRPs) and the nucleotide sequence differences translated to peptides. The binding affinity of the peptides to the relevant HLA in each DRP was determined. The resulting array of peptide-HLA binding affinity values in each patient was considered as an operator modifying a hypothetical T cell repertoire vector, in which each T cell clone proliferates in accordance with the logistic equation of growth. Using an iterating system of matrices, each simulated T cell clone's growth was calculated with the steady-state population being proportional to the magnitude of the binding affinity of the driving HLA-peptide complex. Incorporating competition between T cell clones responding to different HLA-peptide complexes reproduces a number of features of clinically observed T cell clonal repertoire in the simulated repertoire, including sigmoidal growth kinetics of individual T cell clones and overall repertoire, Power Law clonal frequency distribution, increase in repertoire complexity over time with increasing clonal diversity, and alteration of clonal dominance when a different antigen array is encountered, such as in SCT. The simulated, alloreactive T cell repertoire was markedly different in HLA-matched DRPs. The patterns were differentiated by rate of growth and steady-state magnitude of the simulated T cell repertoire and demonstrate a possible correlation with survival. In conclusion, exome wide sequence differences in DRPs may allow simulation of donor alloreactive T

NetMHCpan, a method for quantitative predictions of peptide binding to any HLA-A and -B locus protein of known sequence

DEFF Research Database (Denmark)

Nielsen, Morten; Lundegaard, Claus; Blicher, Thomas

2007-01-01

BACKGROUND: Binding of peptides to Major Histocompatibility Complex (MHC) molecules is the single most selective step in the recognition of pathogens by the cellular immune system. The human MHC class I system (HLA-I) is extremely polymorphic. The number of registered HLA-I molecules has now surp...... to provide new basic insights into HLA structure-function relationships. The method is available at http://www.cbs.dtu.dk/services/NetMHCpan....... surpassed 1500. Characterizing the specificity of each separately would be a major undertaking. PRINCIPAL FINDINGS: Here, we have drawn on a large database of known peptide-HLA-I interactions to develop a bioinformatics method, which takes both peptide and HLA sequence information into account...... successfully validate this method. We further demonstrate that the method can be applied to perform a clustering analysis of MHC specificities and suggest using this clustering to select particularly informative novel MHC molecules for future biochemical and functional analysis. CONCLUSIONS: Encompassing all...

Lanthanide-binding peptides with two pendant aminodiacetate arms: impact of the sequence on chelation.

Science.gov (United States)

Niedźwiecka, Agnieszka; Cisnetti, Federico; Lebrun, Colette; Gateau, Christelle; Delangle, Pascale

2012-03-21

Lanthanide complexes with a series of hexapeptides-incorporating two unnatural chelating amino acids with aminodiacetate groups, Ada(1) and Ada(2)-have been examined in terms of their speciation, structure, stability and luminescence properties. Whereas Ada(2) acts as a tridentate donor in all cases, Ada(1) may act as a tetradentate donor thanks to the coordination of the amide carbonyl function assisted by the formation of a six-membered chelate ring. The position of the Ada(1) residue in the sequence is demonstrated to be critical for the lanthanide complex speciation and structure. Ada(1) promotes the coordination of the backbone amide function to afford a highly dehydrated Ln complex and an S-shape structure of the peptide backbone, only when found in position 2.

Improvement of an enzyme-linked immunosorbent assay for equine herpesvirus type 4 by using a synthetic-peptide 24-mer repeat sequence of glycoprotein G as an antigen.

Science.gov (United States)

Bannai, Hiroshi; Nemoto, Manabu; Tsujimura, Koji; Yamanaka, Takashi; Maeda, Ken; Kondo, Takashi

2016-02-01

To increase the sensitivity of an enzyme-linked immunosorbent assay (ELISA) for equine herpesvirus type 4 (EHV-4) that uses a 12-mer peptide of glycoprotein G (gG4-12-mer: MKNNPIYSEGSL) [4], we used a longer peptide consisting of a 24-mer repeat sequence (gG4-24-mer: MKNNPIYSEGSLMLNVQHDDSIHT) as an antigen. Sera of horses experimentally infected with EHV-4 reacted much more strongly to the gG4-24-mer peptide than to the gG4-12-mer peptide. We used peptide ELISAs to test paired sera from horses naturally infected with EHV-4 (n=40). gG4-24-mer ELISA detected 37 positive samples (92.5%), whereas gG4-12-mer ELISA detected only 28 (70.0%). gG4-24-mer ELISA was much more sensitive than gG4-12-mer ELISA.

Peptide array-based screening of human mesenchymal stem cell-adhesive peptides derived from fibronectin type III domain

International Nuclear Information System (INIS)

Okochi, Mina; Nomura, Shigeyuki; Kaga, Chiaki; Honda, Hiroyuki

2008-01-01

Human mesenchymal stem cell-adhesive peptides were screened based on the amino acid sequence of fibronectin type III domain 8-11 (FN-III 8-11 ) using a peptide array synthesized by the Fmoc-chemistry. Using hexameric peptide library of FN-III 8-11 scan, we identified the ALNGR (Ala-Leu-Asn-Gly-Arg) peptide that induced cell adhesion as well as RGDS (Arg-Gly-Asp-Ser) peptide. After incubation for 2 h, approximately 68% of inoculated cells adhere to the ALNGR peptide disk. Adhesion inhibition assay with integrin antibodies showed that the ALNGR peptide interacts with integrin β1 but not with αvβ3, indicating that the receptors for ALNGR are different from RGDS. Additionally, the ALNGR peptide expressed cell specificities for adhesion: cell adhesion was promoted for fibroblasts but not for keratinocytes or endotherial cells. The ALNGR peptide induced cell adhesion and promoted cell proliferation without changing its property. It is therefore useful for the construction of functional biomaterials

Cloning of precursors for two MIH/VIH-related peptides in the prawn, Macrobrachium rosenbergii.

Science.gov (United States)

Yang, W J; Rao, K R

2001-11-30

Two cDNA clones (634 and 1366 bp) encoding MIH/VIH (molt-inhibiting hormone/vitellogenesis-inhibiting hormone)-related peptides were isolated and sequenced from a Macrobrachium rosenbergii eyestalk ganglia cDNA library. The clones contain a 360 and 339 bp open-reading frame, and their conceptually translated peptides consist of a 41 and 34 amino acid signal peptide, respectively, and a 78 amino acid residue mature peptide hormone. The amino acid sequences of the peptides exhibit higher identities with other known MIHs and VIH (44-69%) than with CHHs (28-33%). This is the first report describing the cloning and sequencing of two MIH/VIH-related peptides in a single crustacean species. Transcription of these mRNAs was detected in the eyestalk ganglia, but not in the thoracic ganglia, hepatopancreas, gut, gill, heart, or muscle.

Smart biomaterials: Surfaces functionalized with proteolytically stable osteoblast-adhesive peptides

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Annj Zamuner

2017-09-01

Full Text Available Engineered scaffolds for bone tissue regeneration are designed to promote cell adhesion, growth, proliferation and differentiation. Recently, covalent and selective functionalization of glass and titanium surfaces with an adhesive peptide (HVP mapped on [351–359] sequence of human Vitronectin allowed to selectively increase osteoblast attachment and adhesion strength in in vitro assays, and to promote osseointegration in in vivo studies. For the first time to our knowledge, in this study we investigated the resistance of adhesion sequences to proteolytic digestion: HVP was completely cleaved after 5 h. In order to overcome the enzymatic degradation of the native peptide under physiological conditions we synthetized three analogues of HVP sequence. A retro-inverted peptide D-2HVP, composed of D amino acids, was completely stable in serum-containing medium. In addition, glass surfaces functionalized with D-2HVP increased human osteoblast adhesion as compared to the native peptide and maintained deposition of calcium. Interestingly, D-2HVP increased expression of IBSP, VTN and SPP1 genes as compared to HVP functionalized surfaces. Total internal reflection fluorescence microscope analysis showed cells with numerous filopodia spread on D-2HVP-functionalized surfaces. Therefore, the D-2HVP sequence is proposed as new osteoblast adhesive peptide with increased bioactivity and high proteolytic resistance.

Smart biomaterials: Surfaces functionalized with proteolytically stable osteoblast-adhesive peptides.

Science.gov (United States)

Zamuner, Annj; Brun, Paola; Scorzeto, Michele; Sica, Giuseppe; Castagliuolo, Ignazio; Dettin, Monica

2017-09-01

Engineered scaffolds for bone tissue regeneration are designed to promote cell adhesion, growth, proliferation and differentiation. Recently, covalent and selective functionalization of glass and titanium surfaces with an adhesive peptide (HVP) mapped on [351-359] sequence of human Vitronectin allowed to selectively increase osteoblast attachment and adhesion strength in in vitro assays, and to promote osseointegration in in vivo studies. For the first time to our knowledge, in this study we investigated the resistance of adhesion sequences to proteolytic digestion: HVP was completely cleaved after 5 h. In order to overcome the enzymatic degradation of the native peptide under physiological conditions we synthetized three analogues of HVP sequence. A retro-inverted peptide D-2HVP, composed of D amino acids, was completely stable in serum-containing medium. In addition, glass surfaces functionalized with D-2HVP increased human osteoblast adhesion as compared to the native peptide and maintained deposition of calcium. Interestingly, D-2HVP increased expression of IBSP, VTN and SPP1 genes as compared to HVP functionalized surfaces. Total internal reflection fluorescence microscope analysis showed cells with numerous filopodia spread on D-2HVP-functionalized surfaces. Therefore, the D-2HVP sequence is proposed as new osteoblast adhesive peptide with increased bioactivity and high proteolytic resistance.

[The primary structure of the alpha-amylase inhibitor Hoe 467A from Streptomyces tendae 4158. A new class of inhibitors].

Science.gov (United States)

Aschauer, H; Vértesy, L; Nesemann, G; Braunitzer, G

1983-10-01

The native or modified alpha-amylase inhibitor Hoe 467A - isolated from the culture medium of Streptomyces tendae 4158 - and overlapping peptides were degraded by the automatic Edman technique. The oxidized or aminoethylated or oxidized and maleoylated inhibitor was digested with trypsin and the native inhibitor with pepsin. Further digestion with Staphylococcus aureus proteinase was also carried out. After peptic digestion two cystin peptides were isolated, which allowed the establishment of the disulfide bonds. The alpha-amylase inhibitor is a polypeptid consisting of 74 amino-acid residues with a molecular mass of 7958 Da. The inhibitor is composed of all naturally occurring amino acids except methionine and phenylalanine and shows no sequence homology to known inhibitors. The clinical and pharmacological importance in respect to the inhibitors ability for inactivation of human salivary and pancreatic alpha-amylase is discussed. Especially the proteinase resistance of the inhibitor enables a clinical application in human (e.g. Diabetes mellitus) per os.

Homologies between the amino acid sequences of some vertebrate peptide hormones and peptides isolated from invertebrate sources.

Science.gov (United States)

De Loof, A; Schoofs, L

1990-01-01

1. The 4K-prothoracicotropic hormone (PTTH) or bombyxin and the melanization-reddish coloration hormone of the silkworm Bombyx mori resemble insulin and insulin-like growth factors. 2. The family of adipokinetic/red pigment concentrating hormones has some similarity with glucagon. 3. Members of the FMRFamide family are found in vertebrates as well as in invertebrates. 4. In Locusta, a molecule immunologically and biologically related to amphibian melanophore stimulating hormone has been partially characterized. 5. Enkephalins and enkephalin-related peptides occur in insects and other invertebrates. 6. Peptides belonging to the tachykinin family have been isolated from molluscan (Octopus) salivary glands and from insect nervous tissue (Locusta migratoria). 7. Invertebrate arginine-vasotocin homologs have been isolated from an insect (Locusta migratoria) and from a mollusc (Conus). 8. In Leucophaea, Locusta and Drosophila, peptides resembling those of the vertebrate gastrin/cholecystokinin family have been identified. 9. As the number of different neuro-/gut peptides with possible function(s) as hormone, neurotransmitter or neuromodulator is now estimated to be of the order of a few hundred, more similarities will probably show up in the near future.

Massively parallel amplicon sequencing reveals isotype-specific variability of antimicrobial peptide transcripts in Mytilus galloprovincialis.

Directory of Open Access Journals (Sweden)

Umberto Rosani

Full Text Available BACKGROUND: Effective innate responses against potential pathogens are essential in the living world and possibly contributed to the evolutionary success of invertebrates. Taken together, antimicrobial peptide (AMP precursors of defensin, mytilin, myticin and mytimycin can represent about 40% of the hemocyte transcriptome in mussels injected with viral-like and bacterial preparations, and unique profiles of myticin C variants are expressed in single mussels. Based on amplicon pyrosequencing, we have ascertained and compared the natural and Vibrio-induced diversity of AMP transcripts in mussel hemocytes from three European regions. METHODOLOGY/PRINCIPAL FINDINGS: Hemolymph was collected from mussels farmed in the coastal regions of Palavas (France, Vigo (Spain and Venice (Italy. To represent the AMP families known in M. galloprovincialis, nine transcript sequences have been selected, amplified from hemocyte RNA and subjected to pyrosequencing. Hemolymph from farmed (offshore and wild (lagoon Venice mussels, both injected with 10(7 Vibrio cells, were similarly processed. Amplicon pyrosequencing emphasized the AMP transcript diversity, with Single Nucleotide Changes (SNC minimal for mytilin B/C and maximal for arthropod-like defensin and myticin C. Ratio of non-synonymous vs. synonymous changes also greatly differed between AMP isotypes. Overall, each amplicon revealed similar levels of nucleotidic variation across geographical regions, with two main sequence patterns confirmed for mytimycin and no substantial changes after immunostimulation. CONCLUSIONS/SIGNIFICANCE: Barcoding and bidirectional pyrosequencing allowed us to map and compare the transcript diversity of known mussel AMPs. Though most of the genuine cds variation was common to the analyzed samples we could estimate from 9 to 106 peptide variants in hemolymph pools representing 100 mussels, depending on the AMP isoform and sampling site. In this study, no prevailing SNC patterns related

Radio-immunoassays for glucagon-like peptides 1 and 2 (GLP-1 and GLP-2)

DEFF Research Database (Denmark)

Orskov, C; Holst, J J

1987-01-01

Gene-sequencing studies have shown that the glucagon precursor contains two additional glucagon-like sequences, the so-called glucagon-like peptides 1 and 2 (GLP-1 and GLP-2). We developed radio-immunoassays against synthetic peptides corresponding to these sequences. Antisera were raised in rabb...

Cyanine-based probe\\tag-peptide pair for fluorescence protein imaging and fluorescence protein imaging methods

Science.gov (United States)

Mayer-Cumblidge, M Uljana [Richland, WA; Cao, Haishi [Richland, WA

2010-08-17

A molecular probe comprises two arsenic atoms and at least one cyanine based moiety. A method of producing a molecular probe includes providing a molecule having a first formula, treating the molecule with HgOAc, and subsequently transmetallizing with AsCl.sub.3. The As is liganded to ethanedithiol to produce a probe having a second formula. A method of labeling a peptide includes providing a peptide comprising a tag sequence and contacting the peptide with a biarsenical molecular probe. A complex is formed comprising the tag sequence and the molecular probe. A method of studying a peptide includes providing a mixture containing a peptide comprising a peptide tag sequence, adding a biarsenical probe to the mixture, and monitoring the fluorescence of the mixture.

Design, synthesis, and validation of an in vitro platform peptide-whole cell screening assay using MTT reagent

Directory of Open Access Journals (Sweden)

Sahar Ahmed

2017-05-01

Full Text Available An in vitro platform to perform peptide screening against different cancer cell lines was designed. The strategy for this screening relied on the design and detection of high-affinity cancer-targeting peptides based on the sequences of NGR and P160. Evaluation of the best binding peptides was performed via incubation of the peptide array-bounded cells with MTT reagent, which is reduced to purple formazan in living cells and further quantified using an Elispot and Kodak imager. For proof of concept, a peptide library (132 spots, and 66 different peptides was designed, synthesized, and screened against different cancer cell lines. The current strategy assists in the identification of positive and negative peptides as well as the relative binding between positive ones. Better binding peptide sequences of the NGR motif were demonstrated to show up to a 2.6-fold increase in CD13+ cell lines with insignificant binding to CD13− ones. Comparable results were observed for P160 peptide sequences, to which different peptides had increased binding, with an up to 3-fold increase relative to the native P160 peptide. Based on our results, new peptide sequences for cancer targeting were identified, and the developed strategy was applied to two different peptide libraries.

Chimeric opioid peptides: tools for identifying opioid receptor types.

OpenAIRE

Xie, G X; Miyajima, A; Yokota, T; Arai, K; Goldstein, A

1990-01-01

We synthesized several chimeric peptides in which the N-terminal nine residues of dynorphin-32, a peptide selective for the kappa opioid receptor, were replaced by opioid peptides selective for other opioid receptor types. Each chimeric peptide retained the high affinity and type selectivity characteristic of its N-terminal sequence. The common C-terminal two-thirds of the chimeric peptides served as an epitope recognized by the same monoclonal antibody. When bound to receptors on a cell surf...

Serodiagnosis of Leishmania donovani infections: assessment of enzyme-linked immunosorbent assays using recombinant L. donovani gene B protein (GBP) and a peptide sequence of L. donovani GBP

DEFF Research Database (Denmark)

Jensen, A T; Gasim, S; Moller, T

1999-01-01

The repetitive sequence of Leishmania major gene B protein (GBP) has previously been shown to be a useful tool in the diagnosis of cutaneous leishmaniasis (CL). Here, we have assessed enzyme-linked immunosorbent assays (ELISAs) using recombinant L. donovani GBP (rGBP) and a peptide sequence of L...... for malaria but free of leishmaniasis was negative in both assays....

Facilitating protein solubility by use of peptide extensions

Science.gov (United States)

Freimuth, Paul I; Zhang, Yian-Biao; Howitt, Jason

2013-09-17

Expression vectors for expression of a protein or polypeptide of interest as a fusion product composed of the protein or polypeptide of interest fused at one terminus to a solubility enhancing peptide extension are provided. Sequences encoding the peptide extensions are provided. The invention further comprises antibodies which bind specifically to one or more of the solubility enhancing peptide extensions.

Revisiting and re-engineering the classical zinc finger peptide: consensus peptide-1 (CP-1).

Science.gov (United States)

Besold, Angelique N; Widger, Leland R; Namuswe, Frances; Michalek, Jamie L; Michel, Sarah L J; Goldberg, David P

2016-04-01

Zinc plays key structural and catalytic roles in biology. Structural zinc sites are often referred to as zinc finger (ZF) sites, and the classical ZF contains a Cys2His2 motif that is involved in coordinating Zn(II). An optimized Cys2His2 ZF, named consensus peptide 1 (CP-1), was identified more than 20 years ago using a limited set of sequenced proteins. We have reexamined the CP-1 sequence, using our current, much larger database of sequenced proteins that have been identified from high-throughput sequencing methods, and found the sequence to be largely unchanged. The CCHH ligand set of CP-1 was then altered to a CAHH motif to impart hydrolytic activity. This ligand set mimics the His2Cys ligand set of peptide deformylase (PDF), a hydrolytically active M(II)-centered (M = Zn or Fe) protein. The resultant peptide [CP-1(CAHH)] was evaluated for its ability to coordinate Zn(II) and Co(II) ions, adopt secondary structure, and promote hydrolysis. CP-1(CAHH) was found to coordinate Co(II) and Zn(II) and a pentacoordinate geometry for Co(II)-CP-1(CAHH) was implicated from UV-vis data. This suggests a His2Cys(H2O)2 environment at the metal center. The Zn(II)-bound CP-1(CAHH) was shown to adopt partial secondary structure by 1-D (1)H NMR spectroscopy. Both Zn(II)-CP-1(CAHH) and Co(II)-CP-1(CAHH) show good hydrolytic activity toward the test substrate 4-nitrophenyl acetate, exhibiting faster rates than most active synthetic Zn(II) complexes.

New Potent Membrane-Targeting Antibacterial Peptides from Viral Capsid Proteins

Science.gov (United States)

Dias, Susana A.; Freire, João M.; Pérez-Peinado, Clara; Domingues, Marco M.; Gaspar, Diana; Vale, Nuno; Gomes, Paula; Andreu, David; Henriques, Sónia T.; Castanho, Miguel A. R. B.; Veiga, Ana S.

2017-01-01

The increasing prevalence of multidrug-resistant bacteria urges the development of new antibacterial agents. With a broad spectrum activity, antimicrobial peptides have been considered potential antibacterial drug leads. Using bioinformatic tools we have previously shown that viral structural proteins are a rich source for new bioactive peptide sequences, namely antimicrobial and cell-penetrating peptides. Here, we test the efficacy and mechanism of action of the most promising peptides among those previously identified against both Gram-positive and Gram-negative bacteria. Two cell-penetrating peptides, vCPP 0769 and vCPP 2319, have high antibacterial activity against Staphylococcus aureus, MRSA, Escherichia coli, and Pseudomonas aeruginosa, being thus multifunctional. The antibacterial mechanism of action of the two most active viral protein-derived peptides, vAMP 059 and vCPP 2319, was studied in detail. Both peptides act on both Gram-positive S. aureus and Gram-negative P. aeruginosa, with bacterial cell death occurring within minutes. Also, these peptides cause bacterial membrane permeabilization and damage of the bacterial envelope of P. aeruginosa cells. Overall, the results show that structural viral proteins are an abundant source for membrane-active peptides sequences with strong antibacterial properties. PMID:28522994

Improvement of an enzyme-linked immunosorbent assay for equine herpesvirus type 4 by using a synthetic-peptide 24-mer repeat sequence of glycoprotein G as an antigen

Science.gov (United States)

BANNAI, Hiroshi; NEMOTO, Manabu; TSUJIMURA, Koji; YAMANAKA, Takashi; MAEDA, Ken; KONDO, Takashi

2015-01-01

To increase the sensitivity of an enzyme-linked immunosorbent assay (ELISA) for equine herpesvirus type 4 (EHV-4) that uses a 12-mer peptide of glycoprotein G (gG4-12-mer: MKNNPIYSEGSL) [4], we used a longer peptide consisting of a 24-mer repeat sequence (gG4-24-mer: MKNNPIYSEGSLMLNVQHDDSIHT) as an antigen. Sera of horses experimentally infected with EHV-4 reacted much more strongly to the gG4-24-mer peptide than to the gG4-12-mer peptide. We used peptide ELISAs to test paired sera from horses naturally infected with EHV-4 (n=40). gG4-24-mer ELISA detected 37 positive samples (92.5%), whereas gG4-12-mer ELISA detected only 28 (70.0%). gG4-24-mer ELISA was much more sensitive than gG4-12-mer ELISA. PMID:26424485

Design of Tail-Clamp Peptide Nucleic Acid Tethered with Azobenzene Linker for Sequence-Specific Detection of Homopurine DNA

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Shinjiro Sawada

2017-10-01

Full Text Available DNA carries genetic information in its sequence of bases. Synthetic oligonucleotides that can sequence-specifically recognize a target gene sequence are a useful tool for regulating gene expression or detecting target genes. Among the many synthetic oligonucleotides, tail-clamp peptide nucleic acid (TC-PNA offers advantages since it has two homopyrimidine PNA strands connected via a flexible ethylene glycol-type linker that can recognize complementary homopurine sequences via Watson-Crick and Hoogsteen base pairings and form thermally-stable PNA/PNA/DNA triplex structures. Here, we synthesized a series of TC-PNAs that can possess different lengths of azobenzene-containing linkers and studied their binding behaviours to homopurine single-stranded DNA. Introduction of azobenzene at the N-terminus amine of PNA increased the thermal stability of PNA-DNA duplexes. Further extension of the homopyrimidine PNA strand at the N-terminus of PNA-AZO further increased the binding stability of the PNA/DNA/PNA triplex to the target homopurine sequence; however, it induced TC-PNA/DNA/TC-PNA complex formation. Among these TC-PNAs, 9W5H-C4-AZO consisting of nine Watson-Crick bases and five Hoogsteen bases tethered with a beta-alanine conjugated azobenzene linker gave a stable 1:1 TC-PNA/ssDNA complex and exhibited good mismatch recognition. Our design for TC-PNA-AZO can be utilized for detecting homopurine sequences in various genes.

Isolation and characterization of a new bacteriocin, termed enterocin M, produced by environmental isolate Enterococcus faecium AL41.

Science.gov (United States)

Mareková, Mária; Lauková, Andrea; Skaugen, Morten; Nes, Ingolf

2007-08-01

The new bacteriocin, termed enterocin M, produced by Enterococcus faecium AL 41 showed a wide spectrum of inhibitory activity against the indicator organisms from different sources. It was purified by (NH4)2SO4 precipitation, cation-exchange chromatography and reverse phase chromatography (FPLC). The purified peptide was sequenced by N-terminal amino acid Edman degradation and a mass spectrometry analysis was performed. By combining the data obtained from amino acid sequence (39 N-terminal amino acid residues was determined) and the molecular weight (determined to be 4628 Da) it was concluded that the purified enterocin M is a new bacteriocin, which is very similar to enterocin P. However, its molecular weight is different from enterocin P (4701.25). Of the first 39 N-terminal residues of enterocin M, valine was found in position 20 and a lysine in position 35, while enterocin P has tryptophane residues in these positions.

Selection of staphylococcal enterotoxin B (SEB)-binding peptide using phage display technology

International Nuclear Information System (INIS)

Soykut, Esra Acar; Dudak, Fahriye Ceyda; Boyaci, Ismail Hakki

2008-01-01

In this study, peptides were selected to recognize staphylococcal enterotoxin B (SEB) which cause food intoxication and can be used as a biological war agent. By using commercial M13 phage library, single plaque isolation of 38 phages was done and binding affinities were investigated with phage-ELISA. The specificities of the selected phage clones showing high affinity to SEB were checked by using different protein molecules which can be found in food samples. Furthermore, the affinities of three selected phage clones were determined by using surface plasmon resonance (SPR) sensors. Sequence analysis was realized for three peptides showing high binding affinity to SEB and WWRPLTPESPPA, MNLHDYHRLFWY, and QHPQINQTLYRM amino acid sequences were obtained. The peptide sequence with highest affinity to SEB was synthesized with solid phase peptide synthesis technique and thermodynamic constants of the peptide-SEB interaction were determined by using isothermal titration calorimetry (ITC) and compared with those of antibody-SEB interaction. The binding constant of the peptide was determined as 4.2 ± 0.7 x 10 5 M -1 which indicates a strong binding close to that of antibody

Bioinformatics Tools for the Discovery of New Nonribosomal Peptides

DEFF Research Database (Denmark)

Leclère, Valérie; Weber, Tilmann; Jacques, Philippe

2016-01-01

-dimensional structure of the peptides can be compared with the structural patterns of all known NRPs. The presented workflow leads to an efficient and rapid screening of genomic data generated by high throughput technologies. The exploration of such sequenced genomes may lead to the discovery of new drugs (i......This chapter helps in the use of bioinformatics tools relevant to the discovery of new nonribosomal peptides (NRPs) produced by microorganisms. The strategy described can be applied to draft or fully assembled genome sequences. It relies on the identification of the synthetase genes...... and the deciphering of the domain architecture of the nonribosomal peptide synthetases (NRPSs). In the next step, candidate peptides synthesized by these NRPSs are predicted in silico, considering the specificity of incorporated monomers together with their isomery. To assess their novelty, the two...

Turn stability in beta-hairpin peptides: Investigation of peptides containing 3:5 type I G1 bulge turns.

Science.gov (United States)

Blandl, Tamas; Cochran, Andrea G; Skelton, Nicholas J

2003-02-01

The turn-forming ability of a series of three-residue sequences was investigated by substituting them into a well-characterized beta-hairpin peptide. The starting scaffold, bhpW, is a disulfide-cyclized 10-residue peptide that folds into a stable beta-hairpin with two antiparallel strands connected by a two-residue reverse turn. Substitution of the central two residues with the three-residue test sequences leads to less stable hairpins, as judged by thiol-disulfide equilibrium measurements. However, analysis of NMR parameters indicated that each molecule retains a significant folded population, and that the type of turn adopted by the three-residue sequence is the same in all cases. The solution structure of a selected peptide with a PDG turn contained an antiparallel beta-hairpin with a 3:5 type I + G1 bulge turn. Analysis of the energetic contributions of individual turn residues in the series of peptides indicates that substitution effects have significant context dependence, limiting the predictive power of individual amino acid propensities for turn formation. The most stable and least stable sequences were also substituted into a more stable disulfide-cyclized scaffold and a linear beta-hairpin scaffold. The relative stabilities remained the same, suggesting that experimental measurements in the bhpW context are a useful way to evaluate turn stability for use in protein design projects. Moreover, these scaffolds are capable of displaying a diverse set of turns, which can be exploited for the mimicry of protein loops or for generating libraries of reverse turns.

Peptide Nucleic Acids Having Amino Acid Side Chains

DEFF Research Database (Denmark)

1998-01-01

A novel class of compounds, known as peptide nucleic acids, bind complementary DNA and RNA strands more strongly than the corresponding DNA or RNA strands, and exhibit increased sequence specificity and solubility. The peptide nucleic acids comprise ligands selected from a group consisting...

A highly scalable peptide-based assay system for proteomics.

Directory of Open Access Journals (Sweden)

Igor A Kozlov

Full Text Available We report a scalable and cost-effective technology for generating and screening high-complexity customizable peptide sets. The peptides are made as peptide-cDNA fusions by in vitro transcription/translation from pools of DNA templates generated by microarray-based synthesis. This approach enables large custom sets of peptides to be designed in silico, manufactured cost-effectively in parallel, and assayed efficiently in a multiplexed fashion. The utility of our peptide-cDNA fusion pools was demonstrated in two activity-based assays designed to discover protease and kinase substrates. In the protease assay, cleaved peptide substrates were separated from uncleaved and identified by digital sequencing of their cognate cDNAs. We screened the 3,011 amino acid HCV proteome for susceptibility to cleavage by the HCV NS3/4A protease and identified all 3 known trans cleavage sites with high specificity. In the kinase assay, peptide substrates phosphorylated by tyrosine kinases were captured and identified by sequencing of their cDNAs. We screened a pool of 3,243 peptides against Abl kinase and showed that phosphorylation events detected were specific and consistent with the known substrate preferences of Abl kinase. Our approach is scalable and adaptable to other protein-based assays.

HIPdb: a database of experimentally validated HIV inhibiting peptides.

Science.gov (United States)

Qureshi, Abid; Thakur, Nishant; Kumar, Manoj

2013-01-01

Besides antiretroviral drugs, peptides have also demonstrated potential to inhibit the Human immunodeficiency virus (HIV). For example, T20 has been discovered to effectively block the HIV entry and was approved by the FDA as a novel anti-HIV peptide (AHP). We have collated all experimental information on AHPs at a single platform. HIPdb is a manually curated database of experimentally verified HIV inhibiting peptides targeting various steps or proteins involved in the life cycle of HIV e.g. fusion, integration, reverse transcription etc. This database provides experimental information of 981 peptides. These are of varying length obtained from natural as well as synthetic sources and tested on different cell lines. Important fields included are peptide sequence, length, source, target, cell line, inhibition/IC(50), assay and reference. The database provides user friendly browse, search, sort and filter options. It also contains useful services like BLAST and 'Map' for alignment with user provided sequences. In addition, predicted structure and physicochemical properties of the peptides are also included. HIPdb database is freely available at http://crdd.osdd.net/servers/hipdb. Comprehensive information of this database will be helpful in selecting/designing effective anti-HIV peptides. Thus it may prove a useful resource to researchers for peptide based therapeutics development.

An efficient method for isolating antibody fragments against small peptides by antibody phage display

DEFF Research Database (Denmark)

Duan, Zhi; Siegumfeldt, Henrik

2010-01-01

We generated monoclonal scFv (single chain variable fragment) antibodies from an antibody phage display library towards three small synthetic peptides derived from the sequence of s1-casein. Key difficulties for selection of scFv-phages against small peptides were addressed. Small peptides do....... The scFvs were sequenced and characterized, and specificity was characterized by ELISA. The methods developed in this study are universally applicable for antibody phage display to efficiently produce antibody fragments against small peptides....

Histidine-lysine peptides as carriers of nucleic acids.

Science.gov (United States)

Leng, Qixin; Goldgeier, Lisa; Zhu, Jingsong; Cambell, Patricia; Ambulos, Nicholas; Mixson, A James

2007-03-01

With their biodegradability and diversity of permutations, peptides have significant potential as carriers of nucleic acids. This review will focus on the sequence and branching patterns of peptide carriers composed primarily of histidines and lysines. While lysines within peptides are important for binding to the negatively charged phosphates, histidines are critical for endosomal lysis enabling nucleic acids to reach the cytosol. Histidine-lysine (HK) polymers by either covalent or ionic bonds with liposomes augment transfection compared to liposome carriers alone. More recently, we have examined peptides as sole carriers of nucleic acids because of their intrinsic advantages compared to the bipartite HK/liposome carriers. With a protocol change and addition of a histidine-rich tail, HK peptides as sole carriers were more effective than liposomes alone in several cell lines. While four-branched polymers with a primary repeating sequence pattern of -HHK- were more effective as carriers of plasmids, eight-branched polymers with a sequence pattern of -HHHK- were more effective as carriers of siRNA. Compared to polyethylenimine, HK carriers of siRNA and plasmids had reduced toxicity. When injected intravenously, HK polymers in complex with plasmids encoding antiangiogenic proteins significantly decreased tumor growth. Furthermore, modification of HK polymers with polyethylene glycol and vascular-specific ligands increased specificity of the polyplex to the tumor by more than 40-fold. Together with further development and insight on the structure of HK polyplexes, HK peptides may prove to be useful as carriers of different forms of nucleic acids both in vitro and in vivo.

PEPTIDE SOLUBILITY, STRUCTURE AND CHARGE POSITION EFFECT ON ADSORPTION BY ALUMINIUM HYDROXIDE

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Mary Trujillo

2008-04-01

Full Text Available Solubility, structure and position of charges in a peptide antigen sequence can be mentioned as being amongst the basic features of adsorption. In order to study their effect on adsorption, seven analogue series were synthesized from a MSP-1 peptide sequence by systematically replacing each one of the positions in the peptide sequence by aspartic acid, glutamic acid, serine, alanine, asparagine, glutamine or lysine. Such modifications in analogue peptide sequences showed a non-regular tendency regarding solubility and adsorption data. Aspartic acid and Glutamic acid analogue series showed great improvements in adsorption, especially in peptides where Lysine in position 6 and Arginine in position 13 were replaced. Solubility of position 5 analogue was greater than the position 6 analogue in Aspartic acid series; however, the position 6 analogue showed best adsorption results whilst the Aspartic acid in position 5 analogue showed no adsorption in the same conditions. Nuclear Magnetic Resonance structural analysis revealed differences in the -helical structureextension between these analogues. The Aspartic acid in position 6, located in the polar side of the helix, may allow this analogueto fit better onto the adsorption regions suggesting that the local electrostatic charge is responsible for this behavior.

Peptide ligands for targeting the extracellular domain of EGFR: Comparison between linear and cyclic peptides.

Science.gov (United States)

Williams, Tyrslai M; Sable, Rushikesh; Singh, Sitanshu; Vicente, Maria Graca H; Jois, Seetharama D

2018-02-01

Colorectal cancer (CRC) is the third most common solid internal malignancy among cancers. Early detection of cancer is key to increasing the survival rate of colorectal cancer patients. Overexpression of the EGFR protein is associated with CRC. We have designed a series of peptides that are highly specific for the extracellular domain of EGFR, based on our earlier studies on linear peptides. The previously reported linear peptide LARLLT, known to bind to EGFR, was modified with the goals of increasing its stability and its specificity toward EGFR. Peptide modifications, including D-amino acid substitution, cyclization, and chain reversal, were investigated. In addition, to facilitate labeling of the peptide with a fluorescent dye, an additional lysine residue was introduced onto the linear (KLARLLT) and cyclic peptides cyclo(KLARLLT) (Cyclo.L1). The lysine residue was also converted into an azide group in both a linear and reversed cyclic peptide sequences cyclo(K(N3)larllt) (Cyclo.L1.1) to allow for subsequent "click" conjugation. The cyclic peptides showed enhanced binding to EGFR by SPR. NMR and molecular modeling studies suggest that the peptides acquire a β-turn structure in solution. In vitro stability studies in human serum show that the cyclic peptide is more stable than the linear peptide. © 2017 John Wiley & Sons A/S.

Peptide Nucleic Acids Having 2,6-Diaminopurine Nucleobases

DEFF Research Database (Denmark)

2003-01-01

A novel class of compounds, known as peptide nucleic acids, bind complementary DNA and RNA strands more strongly than a corresponding DNA strand, and exhibit increased sequence specificity and binding affinity. The peptide nucleic acids of the invention comprise ligands selected from a group...

Biopanning and characterization of peptides with Fe3O4 nanoparticles-binding capability via phage display random peptide library technique.

Science.gov (United States)

You, Fei; Yin, Guangfu; Pu, Ximing; Li, Yucan; Hu, Yang; Huang, Zhongbin; Liao, Xiaoming; Yao, Yadong; Chen, Xianchun

2016-05-01

Functionalization of inorganic nanoparticles (NPs) play an important role in biomedical applications. A proper functionalization of NPs can improve biocompatibility, avoid a loss of bioactivity, and further endow NPs with unique performances. Modification with vairous specific binding biomolecules from random biological libraries has been explored. In this work, two 7-mer peptides with sequences of HYIDFRW and TVNFKLY were selected from a phage display random peptide library by using ferromagnetic NPs as targets, and were verified to display strong binding affinity to Fe3O4 NPs. Fourier transform infrared spectrometry, fluorescence microscopy, thermal analysis and X-ray photoelectron spectroscopy confirmed the presence of peptides on the surface of Fe3O4 NPs. Sequence analyses revealed that the probable binding mechanism between the peptide and Fe3O4 NPs might be driven by Pearson hard acid-hard base specific interaction and hydrogen bonds, accompanied with hydrophilic interactions and non-specific electrostatic attractions. The cell viability assay indicated a good cytocompatibility of peptide-bound Fe3O4 NPs. Furthermore, TVNFKLY peptide and an ovarian tumor cell A2780 specific binding peptide (QQTNWSL) were conjugated to afford a liner 14-mer peptide (QQTNWSLTVNFKLY). The binding and targeting studies showed that 14-mer peptide was able to retain both the strong binding ability to Fe3O4 NPs and the specific binding ability to A2780 cells. The results suggested that the Fe3O4-binding peptides would be of great potential in the functionalization of Fe3O4 NPs for the tumor-targeted drug delivery and magnetic hyperthermia. Copyright © 2016 Elsevier B.V. All rights reserved.

Purification and Characterization of Antioxidant Peptide from Sunflower Protein Hydrolysate

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Xi-Qun Zheng

2010-01-01

Full Text Available Sunflower proteins were hydrolyzed with Flavourzyme for the production of antioxidant peptide. DEAE-Sepharose Fast Flow, Sephadex G-25 gel filtration chromatography and reversed-phase HPLC were consecutively employed to purify a novel sunflower antioxidant peptide, and the ability to inhibit the autoxidation of pyrogallol was expressed as the antioxidative activity of the peptide. The amino acid sequence was identified as Ala-Cys-Ala-His-Asp-Lys-Val by a Q-Tof2 mass spectrometer. This novel peptide exhibited a high antioxidative activity of 79.42 U/mL, which is expected to protect against oxidative damage in living systems in relation to aging and carcinogenesis. Higher antioxidative activities were presumed mainly due to the presence of hydrophobic amino acids in its sequence.

Expression of the cationic antimicrobial peptide lactoferricin fused with the anionic peptide in Escherichia coli.

Science.gov (United States)

Kim, Ha-Kun; Chun, Dae-Sik; Kim, Joon-Sik; Yun, Cheol-Ho; Lee, Ju-Hoon; Hong, Soon-Kwang; Kang, Dae-Kyung

2006-09-01

Direct expression of lactoferricin, an antimicrobial peptide, is lethal to Escherichia coli. For the efficient production of lactoferricin in E. coli, we developed an expression system in which the gene for the lysine- and arginine-rich cationic lactoferricin was fused to an anionic peptide gene to neutralize the basic property of lactoferricin, and successfully overexpressed the concatemeric fusion gene in E. coli. The lactoferricin gene was linked to a modified magainin intervening sequence gene by a recombinational polymerase chain reaction, thus producing an acidic peptide-lactoferricin fusion gene. The monomeric acidic peptide-lactoferricin fusion gene was multimerized and expressed in E. coli BL21(DE3) upon induction with isopropyl-beta-D-thiogalactopyranoside. The expression levels of the fusion peptide reached the maximum at the tetramer, while further increases in the copy number of the fusion gene substantially reduced the peptide expression level. The fusion peptides were isolated and cleaved to generate the separate lactoferricin and acidic peptide. About 60 mg of pure recombinant lactoferricin was obtained from 1 L of E. coli culture. The purified recombinant lactoferricin was found to have a molecular weight similar to that of chemically synthesized lactoferricin. The recombinant lactoferricin showed antimicrobial activity and disrupted bacterial membrane permeability, as the native lactoferricin peptide does.

AcT-2: a novel myotropic and antimicrobial type 2 tryptophyllin from the skin secretion of the Central American red-eyed leaf frog, Agalychnis callidryas.

Science.gov (United States)

Ge, Lilin; Lyu, Peng; Zhou, Mei; Zhang, Huiling; Wan, Yuantai; Li, Bin; Li, Renjie; Wang, Lei; Chen, Tianbao; Shaw, Chris

2014-01-01

Tryptophyllins are a diverse family of amphibian peptides originally found in extracts of phyllomedusine frog skin by chemical means. Their biological activities remain obscure. Here we describe the isolation and preliminary pharmacological characterization of a novel type 2 tryptophyllin, named AcT-2, from the skin secretion of the red-eyed leaf frog, Agalychnis callidryas. The peptide was initially identified during smooth muscle pharmacological screening of skin secretion HPLC fractions and the unique primary structure--GMRPPWF-NH2--was established by both Edman degradation and electrospray MS/MS fragmentation sequencing. A. cDNA encoding the biosynthetic precursor of AcT-2 was successfully cloned from a skin secretion-derived cDNA library by means of RACE PCR and this contained an open-reading frame consisting of 62 amino acid residues with a single AcT-2 encoding sequence located towards the C-terminus. A synthetic replicate of AcT-2 was found to relax arterial smooth muscle (EC50 = 5.1 nM) and to contract rat urinary bladder smooth muscle (EC50 = 9.3 μ M). The peptide could also inhibit the growth of the microorganisms, Staphylococcus aureus, (MIC = 256 mg/L) Escherichia coli (MIC = 512 mg/L), and Candida albicans (128 mg/L). AcT-2 is thus the first amphibian skin tryptophyllin found to possess both myotropic and antimicrobial activities.

Chagas disease-specific antigens: characterization of epitopes in CRA/FRA by synthetic peptide mapping and evaluation by ELISA-peptide assay.

Science.gov (United States)

Bottino, Carolina G; Gomes, Luciano P; Pereira, José B; Coura, José R; Provance, David William; De-Simone, Salvatore G

2013-12-03

The identification of epitopes in proteins recognized by medically relevant antibodies is useful for the development of peptide-based diagnostics and vaccines. In this study, epitopes in the cytoplasmic repetitive antigen (CRA) and flagellar repetitive antigen (FRA) proteins from Trypanosoma cruzi were identified using synthetic peptide techniques and pooled sera from Chagasic patients. The epitopes were further assayed with an ELISA assay based on synthetic peptides. Twenty-two overlapping synthetic peptides representing the coding sequence of the T. cruzi CRA and FRA proteins were assessed by a Spot-synthesis array analysis using sera donated by patients with Chagas disease. Shorter peptides were selected that represented the determined epitopes and synthesized by solid phase synthesis to evaluate the patterns of cross-reactivities and discrimination through an ELISA-diagnostic assay. The peptide Spot-synthesis array successfully identified two IgG antigenic determinants in the CRA protein and four in FRA. Bioinformatics suggested that the CRA antigens were unique to T. cruzi while the FRA antigen showed similarity with sequences present within various proteins from Leishmania sp. Subsequently, shorter peptides representing the CRA-1, CRA-2 and FRA-1 epitopes were synthesized by solid phase synthesis and assayed by an ELISA-diagnostic assay. The CRA antigens gave a high discrimination between Chagasic, Leishmaniasis and T. cruzi-uninfected serum. A sensitivity and specificity of 100% was calculated for CRA. While the FRA antigen showed a slightly lower sensitivity (91.6%), its specificity was only 60%. The epitopes recognized by human anti-T. cruzi antibodies have been precisely located in two biomarkers of T. cruzi, CRA and FRA. The results from screening a panel of patient sera through an ELISA assay based on peptides representing these epitopes strongly suggest that the sequences from CRA would be useful for the development of diagnostic reagents that could

The differences in heparin binding for the C-terminal basic-sequence-rich peptides of HPV-16 and HPV-18 capsid protein L1

International Nuclear Information System (INIS)

Sun Jian; Yu Jisheng; Yu Zhiwu; Zha Xiao; Wu Yuqing

2012-01-01

Graphial abstract: The differences in heparin binding for the C-terminal basic-sequence-rich peptides of HPV-16 and HPV-18 capsid protein L1. Highlights: ► Several driving forces contribute to the interaction between heparin and peptides. ► C-terminal of HPV L1 is a potential candidate for the attachment to host cells. ► The C-terminal peptides of HPV-16 and -18 L1 have different heparin-binding. ► The different heparin-binding provides an explanation for the distinct prevalences. - Abstract: The high-risk types of human papillomaviruses (HPV) HPV-16 and -18 are the predominant types associated with cervical cancer. HPV-16 and -18 account for about 50% and 20%, respectively, of cervical cancers worldwide. While the reason and molecular mechanism of the distinct prevalence and distributions between them remain poorly understood, the binding affinity of cell surface receptor with capsid proteins, especially L1, may be involved. We examined heparin binding with two synthetic peptides corresponding to the 14 amino acid C-terminal peptides of HPV-16 and -18 L1 with the goal of comparing the equivalent residues in different HPV types. Using isothermal titration calorimetry (ITC) and static right-angle light scattering (SLS), we determined the binding constant K, reaction enthalpy ΔH, and other thermodynamic parameters in the interaction. Especially, we assessed the role of specific residues in binding with heparin by comparing the NMR spectra of free and heparin-bound peptides.

Contribution of peptide backbone to Anti-citrulline-dependent antibody reactivity

DEFF Research Database (Denmark)

Trier, Nicole Hartwig; Dam, Catharina; Olsen, Dorthe

2015-01-01

for ACPA reactivity and to be cross-reactive between the selected citrullinated peptides. The remaining amino acids within the citrullinated peptides were found to be of less importance for antibody reactivity. Moreover, these findings indicated that the Cit-Gly motif in combination with peptide backbone...... found in up to 70% of RA patients’ sera, have received much attention. Several citrullinated proteins are associated with RA, suggesting that ACPAs may react with different sequence patterns, separating them from traditional antibodies, whose reactivity usually is specific towards a single target...... homology rather than sequence homology are favored between citrullinated epitopes. These findings are important in relation to clarifying the etiology of RA and to determine the nature of ACPAs, e.g. why some Cit-Gly-containing sequences are not targeted by ACPAs....

Amphiphilic cationic peptides mediate cell adhesion to plastic surfaces.

Science.gov (United States)

Rideout, D C; Lambert, M; Kendall, D A; Moe, G R; Osterman, D G; Tao, H P; Weinstein, I B; Kaiser, E T

1985-09-01

Four amphiphilic peptides, each with net charges of +2 or more at neutrality and molecular weights under 4 kilodaltons, were found to mediate the adhesion of normal rat kidney fibroblasts to polystyrene surfaces. Two of these peptides, a model for calcitonin (peptide 1, MCT) and melittin (peptide 2, MEL), form amphiphilic alpha-helical structures at aqueous/nonpolar interfaces. The other two, a luteinizing hormone-releasing hormone model (peptide 3, LHM) and a platelet factor model (peptide 4, MPF) form beta-strand structures in amphiphilic environments. Although it contains only 10 residues, LHM mediated adhesion to surfaces coated with solutions containing as little as 10 pmoles/ml of peptide. All four of these peptides were capable of forming monolayers at air-buffer interfaces with collapse pressures greater than 20 dynes/cm. None of these four peptides contains the tetrapeptide sequence Arg-Gly-Asp-Ser, which has been associated with fibronectin-mediated cell adhesion. Ten polypeptides that also lacked the sequence Arg-Gly-Asp-Ser but were nonamphiphilic and/or had net charges less than +2 at neutrality were all incapable of mediating cell adhesion (Pierschbacher and Ruoslahti, 1984). The morphologies of NRK cells spread on polystyrene coated with peptide LHM resemble the morphologies on fibronectin-coated surfaces, whereas cells spread on surfaces coated with MCT or MEL exhibit strikingly different morphologies. The adhesiveness of MCT, MEL, LHM, and MPF implies that many amphiphilic cationic peptides could prove useful as well defined adhesive substrata for cell culture and for studies of the mechanism of cell adhesion.

Peptides in headlock ? a novel high-affinity and versatile peptide-binding nanobody for proteomics and microscopy

OpenAIRE

Braun, Michael B.; Traenkle, Bjoern; Koch, Philipp A.; Emele, Felix; Weiss, Frederik; Poetz, Oliver; Stehle, Thilo; Rothbauer, Ulrich

2016-01-01

Nanobodies are highly valuable tools for numerous bioanalytical and biotechnical applications. Here, we report the characterization of a nanobody that binds a short peptide epitope with extraordinary affinity. Structural analysis reveals an unusual binding mode where the extended peptide becomes part of a ?-sheet structure in the nanobody. This interaction relies on sequence-independent backbone interactions augmented by a small number of specificity-determining side chain contacts. Once boun...

Molecular evolution of a peptide GPCR ligand driven by artificial neural networks.

Directory of Open Access Journals (Sweden)

Sebastian Bandholtz

Full Text Available Peptide ligands of G protein-coupled receptors constitute valuable natural lead structures for the development of highly selective drugs and high-affinity tools to probe ligand-receptor interaction. Currently, pharmacological and metabolic modification of natural peptides involves either an iterative trial-and-error process based on structure-activity relationships or screening of peptide libraries that contain many structural variants of the native molecule. Here, we present a novel neural network architecture for the improvement of metabolic stability without loss of bioactivity. In this approach the peptide sequence determines the topology of the neural network and each cell corresponds one-to-one to a single amino acid of the peptide chain. Using a training set, the learning algorithm calculated weights for each cell. The resulting network calculated the fitness function in a genetic algorithm to explore the virtual space of all possible peptides. The network training was based on gradient descent techniques which rely on the efficient calculation of the gradient by back-propagation. After three consecutive cycles of sequence design by the neural network, peptide synthesis and bioassay this new approach yielded a ligand with 70fold higher metabolic stability compared to the wild type peptide without loss of the subnanomolar activity in the biological assay. Combining specialized neural networks with an exploration of the combinatorial amino acid sequence space by genetic algorithms represents a novel rational strategy for peptide design and optimization.

Exhaustive search of linear information encoding protein-peptide recognition.

Science.gov (United States)

Kelil, Abdellali; Dubreuil, Benjamin; Levy, Emmanuel D; Michnick, Stephen W

2017-04-01

High-throughput in vitro methods have been extensively applied to identify linear information that encodes peptide recognition. However, these methods are limited in number of peptides, sequence variation, and length of peptides that can be explored, and often produce solutions that are not found in the cell. Despite the large number of methods developed to attempt addressing these issues, the exhaustive search of linear information encoding protein-peptide recognition has been so far physically unfeasible. Here, we describe a strategy, called DALEL, for the exhaustive search of linear sequence information encoded in proteins that bind to a common partner. We applied DALEL to explore binding specificity of SH3 domains in the budding yeast Saccharomyces cerevisiae. Using only the polypeptide sequences of SH3 domain binding proteins, we succeeded in identifying the majority of known SH3 binding sites previously discovered either in vitro or in vivo. Moreover, we discovered a number of sites with both non-canonical sequences and distinct properties that may serve ancillary roles in peptide recognition. We compared DALEL to a variety of state-of-the-art algorithms in the blind identification of known binding sites of the human Grb2 SH3 domain. We also benchmarked DALEL on curated biological motifs derived from the ELM database to evaluate the effect of increasing/decreasing the enrichment of the motifs. Our strategy can be applied in conjunction with experimental data of proteins interacting with a common partner to identify binding sites among them. Yet, our strategy can also be applied to any group of proteins of interest to identify enriched linear motifs or to exhaustively explore the space of linear information encoded in a polypeptide sequence. Finally, we have developed a webserver located at http://michnick.bcm.umontreal.ca/dalel, offering user-friendly interface and providing different scenarios utilizing DALEL.

Peptide-membrane Interactions by Spin-labeling EPR

Science.gov (United States)

Smirnova, Tatyana I.; Smirnov, Alex I.

2016-01-01

Site-directed spin labeling (SDSL) in combination with Electron Paramagnetic Resonance (EPR) spectroscopy is a well-established method that has recently grown in popularity as an experimental technique, with multiple applications in protein and peptide science. The growth is driven by development of labeling strategies, as well as by considerable technical advances in the field, that are paralleled by an increased availability of EPR instrumentation. While the method requires an introduction of a paramagnetic probe at a well-defined position in a peptide sequence, it has been shown to be minimally destructive to the peptide structure and energetics of the peptide-membrane interactions. In this chapter, we describe basic approaches for using SDSL EPR spectroscopy to study interactions between small peptides and biological membranes or membrane mimetic systems. We focus on experimental approaches to quantify peptide-membrane binding, topology of bound peptides, and characterize peptide aggregation. Sample preparation protocols including spin-labeling methods and preparation of membrane mimetic systems are also described. PMID:26477253

Physicochemical properties determining the detection probability of tryptic peptides in Fourier transform mass spectrometry. A correlation study

DEFF Research Database (Denmark)

Nielsen, Michael L; Savitski, Mikhail M; Kjeldsen, Frank

2004-01-01

Sequence verification and mapping of posttranslational modifications require nearly 100% sequence coverage in the "bottom-up" protein analysis. Even in favorable cases, routine liquid chromatography-mass spectrometry detects from protein digests peptides covering 50-90% of the sequence. Here we...... investigated the reasons for limited peptide detection, considering various physicochemical aspects of peptide behavior in liquid chromatography-Fourier transform mass spectrometry (LC-FTMS). No overall correlation was found between the detection probability and peptide mass. In agreement with literature data...... between pI and signal response. An explanation of this paradoxal behavior was found through the observation that more acidic tryptic peptide lengths tend to be longer. Longer peptides tend to acquire higher average charge state in positive mode electrospray ionization than more basic but shorter...

Water-Floating Giant Nanosheets from Helical Peptide Pentamers

Science.gov (United States)

Lee, Jaehun; Nam, Ki Tae

One of the important challenges in the development of protein-mimetic materials is to understand the sequence specific assembly behavior and the dynamic folding change. Conventional strategies to construct two dimensional nanostructures from the peptides have been limited to beta-sheet forming sequences in use of basic building blocks because of their natural tendency to form sheet like aggregations. Here we identified a new peptide sequence, YFCFY that can form dimers by the disulfide bridge, fold into helix and assemble into macroscopic flat sheet at the air/water interface. Because of large driving force for two dimensional assembly and high elastic modulus of the resulting sheet, the peptide assembly induces the flattening of initially round water droplet. Additionally, we found that stabilization of helix by the dimerization is a key determinant for maintaining macroscopic flatness over a few tens centimeter even with a uniform thickness below 10 nm. Furthermore, the capability to transfer 2D film from water droplet to other substrates allows for the multiple stacking of 2D peptide nanostructure, suggesting possible applications in the biomimetic catalysts, biosensor and 2D related electronic devices. This work was supported by Samsung Research Funding Center of Samsung Electronics under Project Number SRFC-MA1401-01.

Predicting binding within disordered protein regions to structurally characterised peptide-binding domains.

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Waqasuddin Khan

Full Text Available Disordered regions of proteins often bind to structured domains, mediating interactions within and between proteins. However, it is difficult to identify a priori the short disordered regions involved in binding. We set out to determine if docking such peptide regions to peptide binding domains would assist in these predictions.We assembled a redundancy reduced dataset of SLiM (Short Linear Motif containing proteins from the ELM database. We selected 84 sequences which had an associated PDB structures showing the SLiM bound to a protein receptor, where the SLiM was found within a 50 residue region of the protein sequence which was predicted to be disordered. First, we investigated the Vina docking scores of overlapping tripeptides from the 50 residue SLiM containing disordered regions of the protein sequence to the corresponding PDB domain. We found only weak discrimination of docking scores between peptides involved in binding and adjacent non-binding peptides in this context (AUC 0.58.Next, we trained a bidirectional recurrent neural network (BRNN using as input the protein sequence, predicted secondary structure, Vina docking score and predicted disorder score. The results were very promising (AUC 0.72 showing that multiple sources of information can be combined to produce results which are clearly superior to any single source.We conclude that the Vina docking score alone has only modest power to define the location of a peptide within a larger protein region known to contain it. However, combining this information with other knowledge (using machine learning methods clearly improves the identification of peptide binding regions within a protein sequence. This approach combining docking with machine learning is primarily a predictor of binding to peptide-binding sites, and is not intended as a predictor of specificity of binding to particular receptors.

Tumor penetrating peptides

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Tambet eTeesalu

2013-08-01

Full Text Available Tumor-homing peptides can be used to deliver drugs into tumors. Phage library screening in live mice has recently identified homing peptides that specifically recognize the endothelium of tumor vessels, extravasate, and penetrate deep into the extravascular tumor tissue. The prototypic peptide of this class, iRGD (CRGDKGPDC, contains the integrin-binding RGD motif. RGD mediates tumor homing through binding to αv integrins, which are selectively expressed on various cells in tumors, including tumor endothelial cells. The tumor-penetrating properties of iRGD are mediated by a second sequence motif, R/KXXR/K. This C-end Rule (or CendR motif is active only when the second basic residue is exposed at the C-terminus of the peptide. Proteolytic processing of iRGD in tumors activates the cryptic CendR motif, which then binds to neuropilin-1 activating an endocytic bulk transport pathway through tumor tissue. Phage screening has also yielded tumor-penetrating peptides that function like iRGD in activating the CendR pathway, but bind to a different primary receptor. Moreover, novel tumor-homing peptides can be constructed from tumor-homing motifs, CendR elements and protease cleavage sites. Pathologies other than tumors can be targeted with tissue-penetrating peptides, and the primary receptor can also be a vascular zip code of a normal tissue. The CendR technology provides a solution to a major problem in tumor therapy, poor penetration of drugs into tumors. The tumor-penetrating peptides are capable of taking a payload deep into tumor tissue in mice, and they also penetrate into human tumors ex vivo. Targeting with these peptides specifically increases the accumulation in tumors of a variety of drugs and contrast agents, such as doxorubicin, antibodies and nanoparticle-based compounds. Remarkably the drug to be targeted does not have to be coupled to the peptide; the bulk transport system activated by the peptide sweeps along any compound that is

Gene cluster analysis for the biosynthesis of elgicins, novel lantibiotics produced by paenibacillus elgii B69

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Teng Yi

2012-03-01

Full Text Available Abstract Background The recent increase in bacterial resistance to antibiotics has promoted the exploration of novel antibacterial materials. As a result, many researchers are undertaking work to identify new lantibiotics because of their potent antimicrobial activities. The objective of this study was to provide details of a lantibiotic-like gene cluster in Paenibacillus elgii B69 and to produce the antibacterial substances coded by this gene cluster based on culture screening. Results Analysis of the P. elgii B69 genome sequence revealed the presence of a lantibiotic-like gene cluster composed of five open reading frames (elgT1, elgC, elgT2, elgB, and elgA. Screening of culture extracts for active substances possessing the predicted properties of the encoded product led to the isolation of four novel peptides (elgicins AI, AII, B, and C with a broad inhibitory spectrum. The molecular weights of these peptides were 4536, 4593, 4706, and 4820 Da, respectively. The N-terminal sequence of elgicin B was Leu-Gly-Asp-Tyr, which corresponded to the partial sequence of the peptide ElgA encoded by elgA. Edman degradation suggested that the product elgicin B is derived from ElgA. By correlating the results of electrospray ionization-mass spectrometry analyses of elgicins AI, AII, and C, these peptides are deduced to have originated from the same precursor, ElgA. Conclusions A novel lantibiotic-like gene cluster was shown to be present in P. elgii B69. Four new lantibiotics with a broad inhibitory spectrum were isolated, and these appear to be promising antibacterial agents.

Chimeric opioid peptides: Tools for identifying opioid receptor types

International Nuclear Information System (INIS)

Xie, G.; Miyajima, A.; Yokota, T.; Arai, K.; Goldstein, A.

1990-01-01

The authors synthesized several chimeric [125J-labelled] peptides in which the N-terminal nine residues of dynorphin-32, a peptide selective for the κ opioid receptor, were replaced by opioid peptides selective for other opioid receptor types. Each chimeric peptide retained the high affinity and type selectivity characteristic of its N-terminal sequence. The common C-terminal two-thirds of the chimeric peptides served as an epitope recognized by the same monoclonal antibody. When bound to receptors on a cell surface or membrane preparation, these peptides could still bind specifically to the monoclonal antibody. These chimeric peptides should be useful for isolating μ, δ, and κ opioid receptors and for identifying opioid receptors on transfected cells in expression cloning procedures. The general approach using chimeric peptides should be applicable to other peptide receptors

Applying the Concept of Peptide Uniqueness to Anti-Polio Vaccination

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Darja Kanduc

2015-01-01

Full Text Available Background. Although rare, adverse events may associate with anti-poliovirus vaccination thus possibly hampering global polio eradication worldwide. Objective. To design peptide-based anti-polio vaccines exempt from potential cross-reactivity risks and possibly able to reduce rare potential adverse events such as the postvaccine paralytic poliomyelitis due to the tendency of the poliovirus genome to mutate. Methods. Proteins from poliovirus type 1, strain Mahoney, were analyzed for amino acid sequence identity to the human proteome at the pentapeptide level, searching for sequences that (1 have zero percent of identity to human proteins, (2 are potentially endowed with an immunologic potential, and (3 are highly conserved among poliovirus strains. Results. Sequence analyses produced a set of consensus epitopic peptides potentially able to generate specific anti-polio immune responses exempt from cross-reactivity with the human host. Conclusion. Peptide sequences unique to poliovirus proteins and conserved among polio strains might help formulate a specific and universal anti-polio vaccine able to react with multiple viral strains and exempt from the burden of possible cross-reactions with human proteins. As an additional advantage, using a peptide-based vaccine instead of current anti-polio DNA vaccines would eliminate the rare post-polio poliomyelitis cases and other disabling symptoms that may appear following vaccination.

Applying the Concept of Peptide Uniqueness to Anti-Polio Vaccination.

Science.gov (United States)

Kanduc, Darja; Fasano, Candida; Capone, Giovanni; Pesce Delfino, Antonella; Calabrò, Michele; Polimeno, Lorenzo

2015-01-01

Although rare, adverse events may associate with anti-poliovirus vaccination thus possibly hampering global polio eradication worldwide. To design peptide-based anti-polio vaccines exempt from potential cross-reactivity risks and possibly able to reduce rare potential adverse events such as the postvaccine paralytic poliomyelitis due to the tendency of the poliovirus genome to mutate. Proteins from poliovirus type 1, strain Mahoney, were analyzed for amino acid sequence identity to the human proteome at the pentapeptide level, searching for sequences that (1) have zero percent of identity to human proteins, (2) are potentially endowed with an immunologic potential, and (3) are highly conserved among poliovirus strains. Sequence analyses produced a set of consensus epitopic peptides potentially able to generate specific anti-polio immune responses exempt from cross-reactivity with the human host. Peptide sequences unique to poliovirus proteins and conserved among polio strains might help formulate a specific and universal anti-polio vaccine able to react with multiple viral strains and exempt from the burden of possible cross-reactions with human proteins. As an additional advantage, using a peptide-based vaccine instead of current anti-polio DNA vaccines would eliminate the rare post-polio poliomyelitis cases and other disabling symptoms that may appear following vaccination.

Novel angiotensin-converting enzyme (ACE) inhibitory peptides derived from boneless chicken leg meat.

Science.gov (United States)

Terashima, Masaaki; Baba, Takako; Ikemoto, Narumi; Katayama, Midori; Morimoto, Tomoko; Matsumura, Saki

2010-06-23

Four peptides that inhibit angiotensin-converting enzyme (ACE) were separated from the hydorlysate of boneless chicken leg meat digested with artificial gastric juice (pepsin). Two peptides were identified as the peptides encrypted in myosin heavy chain. The peptide P1 (MNVKHWPWMK) corresponds to the amino acid sequence from amino acids 825 to 834 of myosin heavy chain, and the peptide P4 (VTVNPYKWLP) corresponds to the amino acid sequence from amino acids 125 to 135 of myosin heavy chain. They are novel ACE inhibitory peptides derived from chicken, and IC(50) values of P1 and P4 were determined as 228 and 5.5 microM, respectively. Although these values were much larger than 0.022 microM for captopril, a typical synthetic ACE inhibitor, they are comparable to IC(50) values reported for various ACE inhibitory peptides derived from foods. Because the peptide P4 has a relatively low IC(50) value, it is a good starting substance for designing food supplements for hypertensive patients.

PinaColada: peptide-inhibitor ant colony ad-hoc design algorithm.

Science.gov (United States)

Zaidman, Daniel; Wolfson, Haim J

2016-08-01

Design of protein-protein interaction (PPI) inhibitors is a major challenge in Structural Bioinformatics. Peptides, especially short ones (5-15 amino acid long), are natural candidates for inhibition of protein-protein complexes due to several attractive features such as high structural compatibility with the protein binding site (mimicking the surface of one of the proteins), small size and the ability to form strong hotspot binding connections with the protein surface. Efficient rational peptide design is still a major challenge in computer aided drug design, due to the huge space of possible sequences, which is exponential in the length of the peptide, and the high flexibility of peptide conformations. In this article we present PinaColada, a novel computational method for the design of peptide inhibitors for protein-protein interactions. We employ a version of the ant colony optimization heuristic, which is used to explore the exponential space ([Formula: see text]) of length n peptide sequences, in combination with our fast robotics motivated PepCrawler algorithm, which explores the conformational space for each candidate sequence. PinaColada is being run in parallel, on a DELL PowerEdge 2.8 GHZ computer with 20 cores and 256 GB memory, and takes up to 24 h to design a peptide of 5-15 amino acids length. An online server available at: http://bioinfo3d.cs.tau.ac.il/PinaColada/. danielza@post.tau.ac.il; wolfson@tau.ac.il. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Structures and related properties of helical, disulfide-stabilized peptides

Energy Technology Data Exchange (ETDEWEB)

Pagel, Mark D. [Univ. of California, Berkeley, CA (United States). Dept. of Chemistry

1993-11-01

The three dimensional structure of several peptides were determined by NMR spectroscopy and distance geometry calculations. Each peptide formed a predictable, rigid structure, consisting of an α-helix, a "scaffold" region which packed along one face of the helix, and two disulfide bridges which covalently connect the helix and scaffold regions. The peptide Apa-M5 was designed to constrain the M5 peptide from MLCK in a helical geometry using the apamin disulfide scaffold. This scaffold constrains the N- terminal end of the helix with two disulfide bridges and a reverse turn. Like the M5 peptide, Apa-M5 was found to bind calmodulin in a Ca²⁺-dependent 1:1 stoichiometry. However, the dissociation constant of the (Apa-M5)-calmodulin complex, 107 nM, was 100-fold higher than the dissociation constant of the M5-calmodulin complex. This difference was due to a putative steric overlap between the Apa-M5 scaffold and calmodulin. The peptide Apa-Cro was designed to replace the large structural protein matrix of λ Cro with the apamin disulfide scaffold. However, Apa-Cro did not bind the consensus DNA operator half-site of λ Cro, probably due to a steric overlap between the Apa-Cro disulfide framework and the DNA. The amino acid sequence of the scaffold-disulfide bridge arrangement of the peptide Max was derived from the core sequence of scyllatoxin, which contains an α-helix constrained at the C-terminal end by two disulfide bridges and a two-stranded βsheet scaffold. Max was shown to fold with >84% yield to form a predictable, stable structure that is similar to scyllatoxin. The folding and stability properties of Max make this scaffold and disulfide bridge arrangement an ideal candidate for the development of hybrid sequence peptides. The dynamics of a fraying C-terminal end of the helix of the peptide Apa-AlaN was determined by analysis of ¹⁵N NMR relaxation properties.

What can machine learning do for antimicrobial peptides, and what can antimicrobial peptides do for machine learning?

Science.gov (United States)

Lee, Ernest Y; Lee, Michelle W; Fulan, Benjamin M; Ferguson, Andrew L; Wong, Gerard C L

2017-12-06

Antimicrobial peptides (AMPs) are a diverse class of well-studied membrane-permeating peptides with important functions in innate host defense. In this short review, we provide a historical overview of AMPs, summarize previous applications of machine learning to AMPs, and discuss the results of our studies in the context of the latest AMP literature. Much work has been recently done in leveraging computational tools to design new AMP candidates with high therapeutic efficacies for drug-resistant infections. We show that machine learning on AMPs can be used to identify essential physico-chemical determinants of AMP functionality, and identify and design peptide sequences to generate membrane curvature. In a broader scope, we discuss the implications of our findings for the discovery of membrane-active peptides in general, and uncovering membrane activity in new and existing peptide taxonomies.

Structure of HLA-A*1101 in complex with a hepatitis B peptide homologue

DEFF Research Database (Denmark)

Blicher, Thomas; Kastrup, Jette Sandholm; Pedersen, Lars Østergaard

2006-01-01

A high-resolution structure of the human MHC-I molecule HLA-A*1101 is presented in which it forms a complex with a sequence homologue of a peptide that occurs naturally in hepatitis B virus DNA polymerase. The sequence of the bound peptide is AIMPARFYPK, while that of the corresponding natural...

Towards generation of bioactive peptides from meat industry waste proteins: Generation of peptides using commercial microbial proteases.

Science.gov (United States)

Ryder, Kate; Bekhit, Alaa El-Din; McConnell, Michelle; Carne, Alan

2016-10-01

Five commercially available food-grade microbial protease preparations were evaluated for their ability to hydrolyse meat myofibrillar and connective tissue protein extracts to produce bioactive peptides. A bacterial-derived protease (HT) extensively hydrolysed both meat protein extracts, producing peptide hydrolysates with significant in vitro antioxidant and ACE inhibitor activities. The hydrolysates retained bioactivity after simulated gastrointestinal hydrolysis challenge. Gel permeation chromatography sub-fractionation of the crude protein hydrolysates showed that the smaller peptide fractions exhibited the highest antioxidant and ACE inhibitor activities. OFFGEL electrophoresis of the small peptides of both hydrolysates showed that low isoelectric point peptides had antioxidant activity; however, no consistent relationship was observed between isoelectric point and ACE inhibition. Cell-based assays indicated that the hydrolysates present no significant cytotoxicity towards Vero cells. The results indicate that HT protease hydrolysis of meat myofibrillar and connective tissue protein extracts produces bioactive peptides that are non-cytotoxic, should be stable in the gastrointestinal tract and may contain novel bioactive peptide sequences. Copyright © 2016 Elsevier Ltd. All rights reserved.

Halogenation dictates the architecture of amyloid peptide nanostructures.

Science.gov (United States)

Pizzi, Andrea; Pigliacelli, Claudia; Gori, Alessandro; Nonappa; Ikkala, Olli; Demitri, Nicola; Terraneo, Giancarlo; Castelletto, Valeria; Hamley, Ian W; Baldelli Bombelli, Francesca; Metrangolo, Pierangelo

2017-07-20

Amyloid peptides yield a plethora of interesting nanostructures though difficult to control. Here we report that depending on the number, position, and nature of the halogen atoms introduced into either one or both phenylalanine benzene rings of the amyloid β peptide-derived core-sequence KLVFF, four different architectures were obtained in a controlled manner. Our findings demonstrate that halogenation may develop as a general strategy to engineer amyloidal peptide self-assembly and obtain new amyloidal nanostructures.

Affinity selection of Nipah and Hendra virus-related vaccine candidates from a complex random peptide library displayed on bacteriophage virus-like particles

Energy Technology Data Exchange (ETDEWEB)

Peabody, David S.; Chackerian, Bryce; Ashley, Carlee; Carnes, Eric; Negrete, Oscar

2017-01-24

The invention relates to virus-like particles of bacteriophage MS2 (MS2 VLPs) displaying peptide epitopes or peptide mimics of epitopes of Nipah Virus envelope glycoprotein that elicit an immune response against Nipah Virus upon vaccination of humans or animals. Affinity selection on Nipah Virus-neutralizing monoclonal antibodies using random sequence peptide libraries on MS2 VLPs selected peptides with sequence similarity to peptide sequences found within the envelope glycoprotein of Nipah itself, thus identifying the epitopes the antibodies recognize. The selected peptide sequences themselves are not necessarily identical in all respects to a sequence within Nipah Virus glycoprotein, and therefore may be referred to as epitope mimics VLPs displaying these epitope mimics can serve as vaccine. On the other hand, display of the corresponding wild-type sequence derived from Nipah Virus and corresponding to the epitope mapped by affinity selection, may also be used as a vaccine.

Peptide profiling of bovine kefir reveals 236 unique peptides released from caseins during its production by starter culture or kefir grains.

Science.gov (United States)

Ebner, Jennifer; Aşçı Arslan, Ayşe; Fedorova, Maria; Hoffmann, Ralf; Küçükçetin, Ahmet; Pischetsrieder, Monika

2015-03-18

Kefir has a long tradition in human nutrition due to its presupposed health promoting effects. To investigate the potential contribution of bioactive peptides to the physiological effects of kefir, comprehensive analysis of the peptide profile was performed by nano-ESI-LTQ-Orbitrap MS coupled to nano-ultrahigh-performance liquid chromatography. Thus, 257 peptides were identified, mainly released from β-casein, followed by αS1-, κ-, and αS2-casein. Most (236) peptides were uniquely detected in kefir, but not in raw milk indicating that the fermentation step does not only increase the proteolytic activity 1.7- to 2.4-fold compared to unfermented milk, but also alters the composition of the peptide fraction. The influence of the microflora was determined by analyzing kefir produced from traditional kefir grains or commercial starter culture. Kefir from starter culture featured 230 peptide sequences and showed a significantly, 1.4-fold higher proteolytic activity than kefir from kefir grains with 127 peptides. A match of 97 peptides in both varieties indicates the presence of a typical kefir peptide profile that is not influenced by the individual composition of the microflora. Sixteen of the newly identified peptides were previously described as bioactive, including angiotensin-converting enzyme (ACE)-inhibitory, antimicrobial, immunomodulating, opioid, mineral binding, antioxidant, and antithrombotic effects. The present study describes a comprehensive peptide profile of kefir comprising 257 sequences. The peptide list was used to identify 16 bioactive peptides with ACE-inhibitory, antioxidant, antithrombotic, mineral binding, antimicrobial, immunomodulating and opioid activity in kefir. Furthermore, it was shown that a majority of the kefir peptides were not endogenously present in the raw material milk, but were released from milk caseins by proteases of the microbiota and are therefore specific for the product. Consequently, the proteolytic activity and the

Synthesis of Mikto-Arm Star Peptide Conjugates.

Science.gov (United States)

Koo, Jin Mo; Su, Hao; Lin, Yi-An; Cui, Honggang

2018-01-01

Mikto-arm star peptide conjugates are an emerging class of self-assembling peptide-based structural units that contain three or more auxiliary segments of different chemical compositions and/or functionalities. This group of molecules exhibit interesting self-assembly behavior in solution due to their chemically asymmetric topology. Here we describe the detailed procedure for synthesis of an ABC Mikto-arm star peptide conjugate in which two immiscible entities (a saturated hydrocarbon and a hydrophobic and lipophobic fluorocarbon) are conjugated onto a short β-sheet forming peptide sequence, GNNQQNY, derived from the Sup35 prion, through a lysine junction. Automated and manual Fmoc-solid phase synthesis techniques are used to synthesize the Mikto-arm star peptide conjugates, followed by HPLC purification. We envision that this set of protocols can afford a versatile platform to synthesize a new class of peptidic building units for diverse applications.

Use of synthetic peptide libraries for the H-2Kd binding motif identification.

Science.gov (United States)

Quesnel, A; Casrouge, A; Kourilsky, P; Abastado, J P; Trudelle, Y

1995-01-01

To identify Kd-binding peptides, an approach based on small peptide libraries has been developed. These peptide libraries correspond to all possible single-amino acid variants of a particular Kd-binding peptide, SYIPSAEYI, an analog of the Plasmodium berghei 252-260 antigenic peptide SYIPSAEKI. In the parent sequence, each position is replaced by all the genetically encoded amino acids (except cysteine). The multiple analog syntheses are performed either by the Divide Couple and Recombine method or by the Single Resin method and generate mixtures containing 19 peptides. The present report deals with the synthesis, the purification, the chemical characterization by amino acid analysis and electrospray mass spectrometry (ES-MS), and the application of such mixtures in binding tests with a soluble, functionally empty, single-chain H-2Kd molecule denoted SC-Kd. For each mixture, bound peptides were eluted and analyzed by sequencing. Since the binding tests were realized in noncompetitive conditions, our results show that a much broader set of peptides bind to Kd than expected from previous studies. This may be of practical importance when looking for low affinity peptides such as tumor peptides capable of eliciting protective immune response.

Procedures of amino acid sequencing of peptides in natural proteins collection of knowledge and intelligence for construction of reliable chemical inference system

OpenAIRE

Kudo, Yoshihiro; Kanaya, Shigehiko

1994-01-01

In order to establish a reliable chemical inference system on amino acid sequencing of natural peptides, as various kinds of relevant knowledge and intelligence as possible are collected. Topics are on didemnins, dolastatin 3, TL-119 and/or A-3302-B, mycosubtilin, patellamide A, duramycin (and cinnamycin), bottoromycin A 2, A19009, galantin I, vancomycin, stenothricin, calf speleen profilin, neocarzinostatin, pancreatic spasmolytic polypeptide, cerebratulus toxin B-IV, RNAase U 2, ferredoxin ...

Amino acid sequence and posttranslational modifications of human factor VIIa from plasma and transfected baby hamster kidney cells

International Nuclear Information System (INIS)

Thim, L.; Bjoern, S.; Christensen, M.; Nicolaisen, E.M.; Lund-Hansen, T.; Pedersen, A.H.; Hedner, U.

1988-01-01

Blood coagulation factor VII is a vitamin K dependent glycoprotein which in its activated form, factor VII a , participates in the coagulation process by activating factor X and/or factor IX in the presence of Ca 2+ and tissue factor. Three types of potential posttranslational modifications exist in the human factor VII a molecule, namely, 10 γ-carboxylated, N-terminally located glutamic acid residues, 1 β-hydroxylated aspartic acid residue, and 2 N-glycosylated asparagine residues. In the present study, the amino acid sequence and posttranslational modifications of recombinant factor VII a as purified from the culture medium of a transfected baby hamster kidney cell line have been compared to human plasma factor VII a . By use of HPLC, amino acid analysis, peptide mapping, and automated Edman degradation, the protein backbone of recombinant factor VII a was found to be identical with human factor VII a . Asparagine residues 145 and 322 were found to be fully N-glycosylated in human plasma factor VII a . In the recombinant factor VII a , asparagine residue 322 was fully glycosylated whereas asparagine residue 145 was only partially (approximately 66%) glycosylated. Besides minor differences in the sialic acid and fucose contents, the overall carbohydrate compositions were nearly identical in recombinant factor VII a and human plasma factor VII a . These results show that factor VII a as produced in the transfected baby hamster kidney cells is very similar to human plasma factor VII a and that this cell line thus might represent an alternative source for human factor VII a

Dual-functioning peptides discovered by phage display increase the magnitude and specificity of BMSC attachment to mineralized biomaterials.

Science.gov (United States)

Ramaraju, Harsha; Miller, Sharon J; Kohn, David H

2017-07-01

Design of biomaterials for cell-based therapies requires presentation of specific physical and chemical cues to cells, analogous to cues provided by native extracellular matrices (ECM). We previously identified a peptide sequence with high affinity towards apatite (VTKHLNQISQSY, VTK) using phage display. The aims of this study were to identify a human MSC-specific peptide sequence through phage display, combine it with the apatite-specific sequence, and verify the specificity of the combined dual-functioning peptide to both apatite and human bone marrow stromal cells. In this study, a combinatorial phage display identified the cell binding sequence (DPIYALSWSGMA, DPI) which was combined with the mineral binding sequence to generate the dual peptide DPI-VTK. DPI-VTK demonstrated significantly greater binding affinity (1/K D ) to apatite surfaces compared to VTK, phosphorylated VTK (VTK phos ), DPI-VTK phos , RGD-VTK, and peptide-free apatite surfaces (p biomaterial surfaces and subsequently increase cell proliferation and differentiation. These new peptides expand biomaterial design methodology for cell-based regeneration of bone defects. This strategy of combining cell and material binding phage display derived peptides is broadly applicable to a variety of systems requiring targeted adhesion of specific cell populations, and may be generalized to the engineering of any adhesion surface. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cardioprotective peptides from marine sources.

Science.gov (United States)

Harnedy, Padraigín A; FitzGerald, Richard J

2013-05-01

Elevated blood pressure or hypertension is one of the fastest growing health problems worldwide. Although the etiology of essential hypertension has a genetic component, dietary factors play an important role. With the high costs and adverse side-effects associated with synthetic antihypertensive drugs and the awareness of the link between diet and health there has been increased focus on identification of food components that may contribute to cardiovascular health. In recent years special interest has been paid to the cardioprotective activity of peptides derived from food proteins including marine proteins. These peptides are latent within the sequence of the parent protein and only become active when released by proteolytic digestion during gastrointestinal digestion or through food processing. Current data on antihypertensive activity of marine-derived protein hydrolysates/peptides in animal and human studies is reviewed herein. Furthermore, products containing protein hydrolysates/peptides from marine origin with antihypertensive effects are discussed.

Competition between bound and free peptides in an ELISA-based procedure that assays peptides derived from protein digests

Directory of Open Access Journals (Sweden)

Pace Umberto

2006-05-01

Full Text Available Abstract Background We describe an ELISA-based method that can be used to identify and quantitate proteins in biological samples. In this method, peptides in solution, derived from proteolytic digests of the sample, compete with substrate-attached synthetic peptides for antibodies, also in solution, generated against the chosen peptides. The peptides used for the ELISA are chosen on the basis of their being (i products of the proteolytic (e.g. tryptic digestion of the protein to be identified and (ii unique to the target protein, as far as one can know from the published sequences. Results In this paper we describe the competition assay and we define the optimal conditions for the most effective assay. We have performed an analysis of the kinetics of interaction between the four components of the assay: the plastic substratum to which the peptide is bound, the bound peptide itself, the competing added peptide, and the antibody that is specific for the peptide and we compare the results of theoretical simulations to the actual data in some model systems. Conclusion The data suggest that the peptides bind to the plastic substratum in more than one conformation and that, once bound, the peptide displays different affinities for the antibody, depending on how it has bound to the plate

Morintides: cargo-free chitin-binding peptides from Moringa oleifera.

Science.gov (United States)

Kini, Shruthi G; Wong, Ka H; Tan, Wei Liang; Xiao, Tianshu; Tam, James P

2017-03-31

Hevein-like peptides are a family of cysteine-rich and chitin-binding peptides consisting of 29-45 amino acids. Their chitin-binding property is essential for plant defense against fungi. Based on the number of cysteine residues in their sequences, they are divided into three sub-families: 6C-, 8C- and 10C-hevein-like peptides. All three subfamilies contain a three-domain precursor comprising a signal peptide, a mature hevein-like peptide and a C-terminal domain comprising a hinge region with protein cargo in 8C- and 10C-hevein-like peptides. Here we report the isolation and characterization of two novel 8C-hevein-like peptides, designated morintides (mO1 and mO2), from the drumstick tree Moringa oleifera, a drought-resistant tree belonging to the Moringaceae family. Proteomic analysis revealed that morintides comprise 44 amino acid residues and are rich in cysteine, glycine and hydrophilic amino acid residues such as asparagine and glutamine. Morintides are resistant to thermal and enzymatic degradation, able to bind to chitin and inhibit the growth of phyto-pathogenic fungi. Transcriptomic analysis showed that they contain a three-domain precursor comprising an endoplasmic reticulum (ER) signal sequence, a mature peptide domain and a C-terminal domain. A striking feature distinguishing morintides from other 8C-hevein-like peptides is a short and protein-cargo-free C-terminal domain. Previously, a similar protein-cargo-free C-terminal domain has been observed only in ginkgotides, the 8C-hevein-like peptides from a gymnosperm Ginkgo biloba. Thus, morintides, with a cargo-free C-terminal domain, are a stand-alone class of 8C-hevein-like peptides from angiosperms. Our results expand the existing library of hevein-like peptides and shed light on molecular diversity within the hevein-like peptide family. Our work also sheds light on the anti-fungal activity and stability of 8C-hevein-like peptides.

Evaluation of peptides release using a natural rubber latex biomembrane as a carrier.

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Miranda, M C R; Borges, F A; Barros, N R; Santos Filho, N A; Mendonça, R J; Herculano, R D; Cilli, E M

2018-05-01

The biomembrane natural (NRL-Natural Rubber Latex), manipulated from the latex obtained from the rubber tree Hevea brasiliensis, has shown great potential for application in biomedicine and biomaterials. Reflecting the biocompatibility and low bounce rate of this material, NRL has been used as a physical barrier to infectious agents and for the controlled release of drugs and extracts. The aim of the present study was to evaluate the incorporation and release of peptides using a latex biomembrane carrier. After incorporation, the release of material from the membrane was observed using spectrophotometry. Analyses using HPLC and mass spectroscopy did not confirm the release of the antimicrobial peptide [W 6 ]Hylin a1 after 24 h. In addition, analysis of the release solution showed new compounds, indicating the degradation of the peptide by enzymes contained in the latex. Additionally, the release of a peptide with a shorter sequence (Ac-WAAAA) was evaluated, and degradation was not observed. These results showed that the use of NRL as solid matrices as delivery systems of peptide are sequence dependent and could to be evaluated for each sequence.

Peptide Vaccine: Progress and Challenges

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Weidang Li

2014-07-01

Full Text Available Conventional vaccine strategies have been highly efficacious for several decades in reducing mortality and morbidity due to infectious diseases. The bane of conventional vaccines, such as those that include whole organisms or large proteins, appear to be the inclusion of unnecessary antigenic load that, not only contributes little to the protective immune response, but complicates the situation by inducing allergenic and/or reactogenic responses. Peptide vaccines are an attractive alternative strategy that relies on usage of short peptide fragments to engineer the induction of highly targeted immune responses, consequently avoiding allergenic and/or reactogenic sequences. Conversely, peptide vaccines used in isolation are often weakly immunogenic and require particulate carriers for delivery and adjuvanting. In this article, we discuss the specific advantages and considerations in targeted induction of immune responses by peptide vaccines and progresses in the development of such vaccines against various diseases. Additionally, we also discuss the development of particulate carrier strategies and the inherent challenges with regard to safety when combining such technologies with peptide vaccines.

A novel peptide from the ACEI/BPP-CNP precursor in the venom of Crotalus durissus collilineatus.

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Higuchi, Shigesada; Murayama, Nobuhiro; Saguchi, Ken-ichi; Ohi, Hiroaki; Fujita, Yoshiaki; da Silva, Nelson Jorge; de Siqueira, Rodrigo José Bezerra; Lahlou, Saad; Aird, Steven D

2006-10-01

In crotaline venoms, angiotensin-converting enzyme inhibitors [ACEIs, also known as bradykinin potentiating peptides (BPPs)], are products of a gene coding for an ACEI/BPP-C-type natriuretic peptide (CNP) precursor. In the genes from Bothrops jararaca and Gloydius blomhoffii, ACEI/BPP sequences are repeated. Sequencing of a cDNA clone from venom glands of Crotalus durissus collilineatus showed that two ACEIs/BPPs are located together at the N-terminus, but without repeats. An additional sequence for CNP was unexpectedly found at the C-terminus. Homologous genes for the ACEI/BPP-CNP precursor suggest that most crotaline venoms contain both ACEIs/BPPs and CNP. The sequence of ACEIs/BPPs is separated from the CNP sequence by a long spacer sequence. Previously, there was no evidence that this spacer actually coded any expressed peptides. Aird and Kaiser (1986, unpublished) previously isolated and sequenced a peptide of 11 residues (TPPAGPDVGPR) from Crotalus viridis viridis venom. In the present study, analysis of the cDNA clone from C. d. collilineatus revealed a nearly identical sequence in the ACEI/BPP-CNP spacer. Fractionation of the crude venom by reverse phase HPLC (C(18)), and analysis of the fractions by mass spectrometry (MS) indicated a component of 1020.5 Da. Amino acid sequencing by MS/MS confirmed that C. d. collilineatus venom contains the peptide TPPAGPDGGPR. Its high proline content and paired proline residues are typical of venom hypotensive peptides, although it lacks the usual N-terminal pyroglutamate. It has no demonstrable hypotensive activity when injected intravenously in rats; however, its occurrence in the venoms of dissimilar species suggests that its presence is not accidental. Evidence suggests that these novel toxins probably activate anaphylatoxin C3a receptors.

Structure of genes for dermaseptins B, antimicrobial peptides from frog skin. Exon 1-encoded prepropeptide is conserved in genes for peptides of highly different structures and activities.

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Vouille, V; Amiche, M; Nicolas, P

1997-09-01

We cloned the genes of two members of the dermaseptin family, broad-spectrum antimicrobial peptides isolated from the skin of the arboreal frog Phyllomedusa bicolor. The dermaseptin gene Drg2 has a 2-exon coding structure interrupted by a small 137-bp intron, wherein exon 1 encoded a 22-residue hydrophobic signal peptide and the first three amino acids of the acidic propiece; exon 2 contained the 18 additional acidic residues of the propiece plus a typical prohormone processing signal Lys-Arg and a 32-residue dermaseptin progenitor sequence. The dermaseptin genes Drg2 and Drg1g2 have conserved sequences at both untranslated ends and in the first and second coding exons. In contrast, Drg1g2 comprises a third coding exon for a short version of the acidic propiece and a second dermaseptin progenitor sequence. Structural conservation between the two genes suggests that Drg1g2 arose recently from an ancestral Drg2-like gene through amplification of part of the second coding exon and 3'-untranslated region. Analysis of the cDNAs coding precursors for several frog skin peptides of highly different structures and activities demonstrates that the signal peptides and part of the acidic propieces are encoded by conserved nucleotides encompassed by the first coding exon of the dermaseptin genes. The organization of the genes that belong to this family, with the signal peptide and the progenitor sequence on separate exons, permits strikingly different peptides to be directed into the secretory pathway. The recruitment of such a homologous 'secretory' exon by otherwise non-homologous genes may have been an early event in the evolution of amphibian.

The preparation and characterization of peptide's lung cancer imaging agent

International Nuclear Information System (INIS)

Liu Jianfeng; Chu Liping; Wang Yan; Wang Yueying; Liu Jinjian; Wu Hongying

2010-01-01

Objective: To screen in vivo lung cancer specific binding seven peptides by T7 phage display peptide library, so as to prepare peptide's lung cancer early diagnostic agent. Methods: Use phage display in vivo technology, the 7-peptide phage that binding the lung cancer specifically was obtained, then the DNA sequence was measured and the seven peptide was synthesized. After labeled by 125 I, the seven peptide was injected into mice via vein and the distribution was observed. Results: One peptide was obtained by four rounds screening, and the peptide can bind lung cancer tissue specifically. Two hours after injection get the best imaging of lung cancer, metabolism of peptide in mice is fast, the distribution in vivo is decrease six hours and almost disappear 20 hours after injection. Conclusion: The peptide can image and diagnose lung cancer better. (authors)

Peptide drugs to target G protein-coupled receptors.

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Bellmann-Sickert, Kathrin; Beck-Sickinger, Annette G

2010-09-01

Major indications for use of peptide-based therapeutics include endocrine functions (especially diabetes mellitus and obesity), infectious diseases, and cancer. Whereas some peptide pharmaceuticals are drugs, acting as agonists or antagonists to directly treat cancer, others (including peptide diagnostics and tumour-targeting pharmaceuticals) use peptides to 'shuttle' a chemotherapeutic agent or a tracer to the tumour and allow sensitive imaging or targeted therapy. Significant progress has been made in the last few years to overcome disadvantages in peptide design such as short half-life, fast proteolytic cleavage, and low oral bioavailability. These advances include peptide PEGylation, lipidisation or multimerisation; the introduction of peptidomimetic elements into the sequences; and innovative uptake strategies such as liposomal, capsule or subcutaneous formulations. This review focuses on peptides targeting G protein-coupled receptors that are promising drug candidates or that have recently entered the pharmaceutical market. Copyright 2010 Elsevier Ltd. All rights reserved.

Structural Characterization of Peptide Antibodies

DEFF Research Database (Denmark)

Chailyan, Anna; Marcatili, Paolo

2015-01-01

The role of proteins as very effective immunogens for the generation of antibodies is indisputable. Nevertheless, cases in which protein usage for antibody production is not feasible or convenient compelled the creation of a powerful alternative consisting of synthetic peptides. Synthetic peptides...... can be modified to obtain desired properties or conformation, tagged for purification, isotopically labeled for protein quantitation or conjugated to immunogens for antibody production. The antibodies that bind to these peptides represent an invaluable tool for biological research and discovery....... To better understand the underlying mechanisms of antibody-antigen interaction here we present a pipeline developed by us to structurally classify immunoglobulin antigen binding sites and to infer key sequence residues and other variables that have a prominent role in each structural class....

Sequential immunization with V3 peptides from primary human immunodeficiency virus type 1 produces cross-neutralizing antibodies against primary isolates with a matching narrow-neutralization sequence motif.

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Eda, Yasuyuki; Takizawa, Mari; Murakami, Toshio; Maeda, Hiroaki; Kimachi, Kazuhiko; Yonemura, Hiroshi; Koyanagi, Satoshi; Shiosaki, Kouichi; Higuchi, Hirofumi; Makizumi, Keiichi; Nakashima, Toshihiro; Osatomi, Kiyoshi; Tokiyoshi, Sachio; Matsushita, Shuzo; Yamamoto, Naoki; Honda, Mitsuo

2006-06-01

An antibody response capable of neutralizing not only homologous but also heterologous forms of the CXCR4-tropic human immunodeficiency virus type 1 (HIV-1) MNp and CCR5-tropic primary isolate HIV-1 JR-CSF was achieved through sequential immunization with a combination of synthetic peptides representing HIV-1 Env V3 sequences from field and laboratory HIV-1 clade B isolates. In contrast, repeated immunization with a single V3 peptide generated antibodies that neutralized only type-specific laboratory-adapted homologous viruses. To determine whether the cross-neutralization response could be attributed to a cross-reactive antibody in the immunized animals, we isolated a monoclonal antibody, C25, which neutralized the heterologous primary viruses of HIV-1 clade B. Furthermore, we generated a humanized monoclonal antibody, KD-247, by transferring the genes of the complementary determining region of C25 into genes of the human V region of the antibody. KD-247 bound with high affinity to the "PGR" motif within the HIV-1 Env V3 tip region, and, among the established reference antibodies, it most effectively neutralized primary HIV-1 field isolates possessing the matching neutralization sequence motif, suggesting its promise for clinical applications involving passive immunizations. These results demonstrate that sequential immunization with B-cell epitope peptides may contribute to a humoral immune-based HIV vaccine strategy. Indeed, they help lay the groundwork for the development of HIV-1 vaccine strategies that use sequential immunization with biologically relevant peptides to overcome difficulties associated with otherwise poorly immunogenic epitopes.

Microwave heating in solid-phase peptide synthesis

DEFF Research Database (Denmark)

Pedersen, Søren Ljungberg; Shelton, Anne Pernille Tofteng; Malik, Leila

2012-01-01

synthesis, precise microwave irradiation to heat the reaction mixture during coupling and N(a)-deprotection has become increasingly popular. It has often provided dramatic reductions in synthesis times, accompanied by an increase in the crude peptide purity. Microwave heating has been proven especially...... relevant for sequences which might form ß-sheet type structures and for sterically difficult couplings. The beneficial effect of microwave heating appears so far to be due to the precise nature of this type of heating, rather than a peptide-specific microwave effect. However, microwave heating...... in microwave heating for peptide synthesis, with a focus on systematic studies and general protocols, as well as important applications. The assembly of ß-peptides, peptoids and pseudopeptides are also evaluated in this critical review (254 references)....

Meat and fermented meat products as a source of bioactive peptides.

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Stadnik, Joanna; Kęska, Paulina

2015-01-01

Bioactive peptides are short amino acid sequences, that upon release from the parent protein may play different physiological roles, including antioxidant, antihypertensive, antimicrobial, and other bioactivities. They have been identified from a range of foods, including those of animal origin, e.g., milk and muscle sources (with pork, beef, or chicken and various species of fish and marine organism). Bioactive peptides are encrypted within the sequence of the parent protein molecule and latent until released and activated by enzymatic proteolysis, e.g. during gastrointestinal digestion or food processing. Bioactive peptides derived from food sources have the potential for incorporation into functional foods and nutraceuticals. The aim of this paper is to present an overview of the muscle-derived bioactive peptides, especially those of fermented meats and the potential benefits of these bioactive compounds to human health.

Antimicrobial peptide evolution in the Asiatic honey bee Apis cerana.

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Peng Xu

Full Text Available The Asiatic honeybee, Apis cerana Fabricius, is an important honeybee species in Asian countries. It is still found in the wild, but is also one of the few bee species that can be domesticated. It has acquired some genetic advantages and significantly different biological characteristics compared with other Apis species. However, it has been less studied, and over the past two decades, has become a threatened species in China. We designed primers for the sequences of the four antimicrobial peptide cDNA gene families (abaecin, defensin, apidaecin, and hymenoptaecin of the Western honeybee, Apis mellifera L. and identified all the antimicrobial peptide cDNA genes in the Asiatic honeybee for the first time. All the sequences were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR. In all, 29 different defensin cDNA genes coding 7 different defensin peptides, 11 different abaecin cDNA genes coding 2 different abaecin peptides, 13 different apidaecin cDNA genes coding 4 apidaecin peptides and 34 different hymenoptaecin cDNA genes coding 13 different hymenoptaecin peptides were cloned and identified from the Asiatic honeybee adult workers. Detailed comparison of these four antimicrobial peptide gene families with those of the Western honeybee revealed that there are many similarities in the quantity and amino acid components of peptides in the abaecin, defensin and apidaecin families, while many more hymenoptaecin peptides are found in the Asiatic honeybee than those in the Western honeybee (13 versus 1. The results indicated that the Asiatic honeybee adult generated more variable antimicrobial peptides, especially hymenoptaecin peptides than the Western honeybee when stimulated by pathogens or injury. This suggests that, compared to the Western honeybee that has a longer history of domestication, selection on the Asiatic honeybee has favored the generation of more variable antimicrobial peptides as protection against pathogens.

Antimicrobial peptide evolution in the Asiatic honey bee Apis cerana.

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Xu, Peng; Shi, Min; Chen, Xue-Xin

2009-01-01

The Asiatic honeybee, Apis cerana Fabricius, is an important honeybee species in Asian countries. It is still found in the wild, but is also one of the few bee species that can be domesticated. It has acquired some genetic advantages and significantly different biological characteristics compared with other Apis species. However, it has been less studied, and over the past two decades, has become a threatened species in China. We designed primers for the sequences of the four antimicrobial peptide cDNA gene families (abaecin, defensin, apidaecin, and hymenoptaecin) of the Western honeybee, Apis mellifera L. and identified all the antimicrobial peptide cDNA genes in the Asiatic honeybee for the first time. All the sequences were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR). In all, 29 different defensin cDNA genes coding 7 different defensin peptides, 11 different abaecin cDNA genes coding 2 different abaecin peptides, 13 different apidaecin cDNA genes coding 4 apidaecin peptides and 34 different hymenoptaecin cDNA genes coding 13 different hymenoptaecin peptides were cloned and identified from the Asiatic honeybee adult workers. Detailed comparison of these four antimicrobial peptide gene families with those of the Western honeybee revealed that there are many similarities in the quantity and amino acid components of peptides in the abaecin, defensin and apidaecin families, while many more hymenoptaecin peptides are found in the Asiatic honeybee than those in the Western honeybee (13 versus 1). The results indicated that the Asiatic honeybee adult generated more variable antimicrobial peptides, especially hymenoptaecin peptides than the Western honeybee when stimulated by pathogens or injury. This suggests that, compared to the Western honeybee that has a longer history of domestication, selection on the Asiatic honeybee has favored the generation of more variable antimicrobial peptides as protection against pathogens.

Peptide and nucleotide sequences of rat CD4 (W3/25) antigen: evidence for derivation from a structure with four immunoglobulin-related domains

International Nuclear Information System (INIS)

Clark, S.J.; Jefferies, W.A.; Barclay, A.N.; Gagnon, J.; Williams, A.F.

1987-01-01

The rat W3/25 antigen was the first marker antigen of helper T lymphocytes to be identified. Subsequently, the human OKT4 antigen (now called CD4) was described, and cell distribution and functional data suggested that W3/25 and OKT4 antigens were homologous. This is now confirmed by the matching of peptide sequences from W3/25 antigen with sequence predicted from rat cDNA clones detected by cross-hybridization with a cDNA probe for human CD4. Analysis of the two sequences suggests an evolutionary origin from a structure with four immunoglobulin-related domains, although only domain 1 at the NH 2 terminus meets the standard criteria for an immunoglobulin-related sequence. CD4 domains 2 and 4 contain disulfide bonds but seem like truncated immunoglobulin domains, whereas domain 3 may have a pattern of β-strands like an immunoglobulin variable domain, but without the disulfide bond

Assessing high affinity binding to HLA-DQ2.5 by a novel peptide library based approach

DEFF Research Database (Denmark)

Jüse, Ulrike; Arntzen, Magnus; Højrup, Peter

2011-01-01

Here we report on a novel peptide library based method for HLA class II binding motif identification. The approach is based on water soluble HLA class II molecules and soluble dedicated peptide libraries. A high number of different synthetic peptides are competing to interact with a limited amount...... library. The eluted sequences fit very well with the previously described HLA-DQ2.5 peptide binding motif. This novel method, limited by library complexity and sensitivity of mass spectrometry, allows the analysis of several thousand synthetic sequences concomitantly in a simple water soluble format....

A family of cell-adhering peptides homologous to fibrinogen C-termini

International Nuclear Information System (INIS)

Levy-Beladev, Liron; Levdansky, Lilia; Gaberman, Elena; Friedler, Assaf; Gorodetsky, Raphael

2010-01-01

Research highlights: → Cell-adhesive sequences homologous to fibrinogen C-termini exist in other proteins. → The extended homologous cell-adhesive C-termini peptides family is termed Haptides. → In membrane-like environment random coiled Haptides adopt a helical conformation. → Replacing positively charged residues with alanine reduces Haptides activity. -- Abstract: A family of cell-adhesive peptides homologous to sequences on different chains of fibrinogen was investigated. These homologous peptides, termed Haptides, include the peptides Cβ, preCγ, and CαE, corresponding to sequences on the C-termini of fibrinogen chains β, γ, and αE, respectively. Haptides do not affect cell survival and rate of proliferation of the normal cell types tested. The use of new sensitive assays of cell adhesion clearly demonstrated the ability of Haptides, bound to inert matrices, to mediate attachment of different matrix-dependent cell types including normal fibroblasts, endothelial, and smooth muscle cells. Here we present new active Haptides bearing homologous sequences derived from the C-termini of other proteins, such as angiopoietin 1 and 2, tenascins C and X, and microfibril-associated glycoprotein-4. The cell adhesion properties of all the Haptides were found to be associated mainly with their 11 N-terminal residues. Mutated preCγ peptides revealed that positively charged residues account for their attachment effect. These results suggest a mechanism of direct electrostatic interaction of Haptides with the cell membrane. The extended Haptides family may be applied in modulating adhesion of cells to scaffolds for tissue regeneration and for enhancement of nanoparticulate transfection into cells.

Machine learning-enabled discovery and design of membrane-active peptides.

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Lee, Ernest Y; Wong, Gerard C L; Ferguson, Andrew L

2017-07-08

Antimicrobial peptides are a class of membrane-active peptides that form a critical component of innate host immunity and possess a diversity of sequence and structure. Machine learning approaches have been profitably employed to efficiently screen sequence space and guide experiment towards promising candidates with high putative activity. In this mini-review, we provide an introduction to antimicrobial peptides and summarize recent advances in machine learning-enabled antimicrobial peptide discovery and design with a focus on a recent work Lee et al. Proc. Natl. Acad. Sci. USA 2016;113(48):13588-13593. This study reports the development of a support vector machine classifier to aid in the design of membrane active peptides. We use this model to discover membrane activity as a multiplexed function in diverse peptide families and provide interpretable understanding of the physicochemical properties and mechanisms governing membrane activity. Experimental validation of the classifier reveals it to have learned membrane activity as a unifying signature of antimicrobial peptides with diverse modes of action. Some of the discriminating rules by which it performs classification are in line with existing "human learned" understanding, but it also unveils new previously unknown determinants and multidimensional couplings governing membrane activity. Integrating machine learning with targeted experimentation can guide both antimicrobial peptide discovery and design and new understanding of the properties and mechanisms underpinning their modes of action. Copyright © 2017 Elsevier Ltd. All rights reserved.

Sense-antisense (complementary) peptide interactions and the proteomic code; potential opportunities in biology and pharmaceutical science.

Science.gov (United States)

Miller, Andrew D

2015-02-01

A sense peptide can be defined as a peptide whose sequence is coded by the nucleotide sequence (read 5' → 3') of the sense (positive) strand of DNA. Conversely, an antisense (complementary) peptide is coded by the corresponding nucleotide sequence (read 5' → 3') of the antisense (negative) strand of DNA. Research has been accumulating steadily to suggest that sense peptides are capable of specific interactions with their corresponding antisense peptides. Unfortunately, although more and more examples of specific sense-antisense peptide interactions are emerging, the very idea of such interactions does not conform to standard biology dogma and so there remains a sizeable challenge to lift this concept from being perceived as a peripheral phenomenon if not worse, into becoming part of the scientific mainstream. Specific interactions have now been exploited for the inhibition of number of widely different protein-protein and protein-receptor interactions in vitro and in vivo. Further, antisense peptides have also been used to induce the production of antibodies targeted to specific receptors or else the production of anti-idiotypic antibodies targeted against auto-antibodies. Such illustrations of utility would seem to suggest that observed sense-antisense peptide interactions are not just the consequence of a sequence of coincidental 'lucky-hits'. Indeed, at the very least, one might conclude that sense-antisense peptide interactions represent a potentially new and different source of leads for drug discovery. But could there be more to come from studies in this area? Studies on the potential mechanism of sense-antisense peptide interactions suggest that interactions may be driven by amino acid residue interactions specified from the genetic code. If so, such specified amino acid residue interactions could form the basis for an even wider amino acid residue interaction code (proteomic code) that links gene sequences to actual protein structure and function, even

Coiled-coil forming peptides for the induction of silver nanoparticles

International Nuclear Information System (INIS)

Božič Abram, Sabina; Aupič, Jana; Dražić, Goran; Gradišar, Helena; Jerala, Roman

2016-01-01

Biopolymers with defined sequence patterns offer an attractive alternative for the formation of silver nanoparticle (AgNP). A set of coiled-coil dimer forming peptides was tested for their AgNP formation ability. Seventeen of those peptides mediated the formation of AgNPs in aqueous solution at neutral pH, while the formation of a coiled-coil dimer inhibited the nanoparticle generation. A QSAR regression model on the relationship between sequence and function suggests that in this peptide type the patterns KXQQ and KXEE are favorable, whereas Ala residues appear to have an inhibitory effect. UV–VIS spectra of the obtained nanoparticles gave a peak at around 420 nm, typical for AgNPs in the size range around 40 nm, which was confirmed by dynamic light scattering and transmission electron microscopy. Peptide-induced AgNPs exhibited good antibacterial activity, even after a 15 min contact time, while they had low toxicity to human cells at the same concentrations. These results show that our designed peptides generate AgNPs with antibacterial activity at mild conditions and might be used for antibacterial coatings. - Highlights: • 17 of the 30 tested coiled-coil forming peptides induce AgNP formation. • Coiled-coil dimer formation suppresses AgNP generation of individual peptides. • Size of the peptide-induced silver nanoparticles is around 40 nm. • QSAR analysis points to the importance of KXQQ and KXEE motifs for AgNP generation. • Peptide-induced silver nanoparticles exhibit antibacterial activity.

Coiled-coil forming peptides for the induction of silver nanoparticles

Energy Technology Data Exchange (ETDEWEB)

Božič Abram, Sabina [Department of Synthetic Biology and Immunology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana (Slovenia); Graduate School of Biomedicine, University of Ljubljana, Ljubljana 1000 (Slovenia); Aupič, Jana [Department of Synthetic Biology and Immunology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana (Slovenia); Doctoral Programme in Chemical Sciences, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana 1000 (Slovenia); Dražić, Goran [Laboratory for Materials Chemistry, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana (Slovenia); Gradišar, Helena [Department of Synthetic Biology and Immunology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana (Slovenia); EN-FIST, Centre of Excellence, Trg Osvobodilne fronte 13, Ljubljana 1000 (Slovenia); Jerala, Roman, E-mail: roman.jerala@ki.si [Department of Synthetic Biology and Immunology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana (Slovenia); EN-FIST, Centre of Excellence, Trg Osvobodilne fronte 13, Ljubljana 1000 (Slovenia)

2016-04-08

Biopolymers with defined sequence patterns offer an attractive alternative for the formation of silver nanoparticle (AgNP). A set of coiled-coil dimer forming peptides was tested for their AgNP formation ability. Seventeen of those peptides mediated the formation of AgNPs in aqueous solution at neutral pH, while the formation of a coiled-coil dimer inhibited the nanoparticle generation. A QSAR regression model on the relationship between sequence and function suggests that in this peptide type the patterns KXQQ and KXEE are favorable, whereas Ala residues appear to have an inhibitory effect. UV–VIS spectra of the obtained nanoparticles gave a peak at around 420 nm, typical for AgNPs in the size range around 40 nm, which was confirmed by dynamic light scattering and transmission electron microscopy. Peptide-induced AgNPs exhibited good antibacterial activity, even after a 15 min contact time, while they had low toxicity to human cells at the same concentrations. These results show that our designed peptides generate AgNPs with antibacterial activity at mild conditions and might be used for antibacterial coatings. - Highlights: • 17 of the 30 tested coiled-coil forming peptides induce AgNP formation. • Coiled-coil dimer formation suppresses AgNP generation of individual peptides. • Size of the peptide-induced silver nanoparticles is around 40 nm. • QSAR analysis points to the importance of KXQQ and KXEE motifs for AgNP generation. • Peptide-induced silver nanoparticles exhibit antibacterial activity.

AcT-2: A Novel Myotropic and Antimicrobial Type 2 Tryptophyllin from the Skin Secretion of the Central American Red-Eyed Leaf Frog, Agalychnis callidryas

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Lilin Ge

2014-01-01

Full Text Available Tryptophyllins are a diverse family of amphibian peptides originally found in extracts of phyllomedusine frog skin by chemical means. Their biological activities remain obscure. Here we describe the isolation and preliminary pharmacological characterization of a novel type 2 tryptophyllin, named AcT-2, from the skin secretion of the red-eyed leaf frog, Agalychnis callidryas. The peptide was initially identified during smooth muscle pharmacological screening of skin secretion HPLC fractions and the unique primary structure—GMRPPWF-NH2—was established by both Edman degradation and electrospray MS/MS fragmentation sequencing. A. cDNA encoding the biosynthetic precursor of AcT-2 was successfully cloned from a skin secretion-derived cDNA library by means of RACE PCR and this contained an open-reading frame consisting of 62 amino acid residues with a single AcT-2 encoding sequence located towards the C-terminus. A synthetic replicate of AcT-2 was found to relax arterial smooth muscle (EC50 = 5.1 nM and to contract rat urinary bladder smooth muscle (EC50 = 9.3 μM. The peptide could also inhibit the growth of the microorganisms, Staphylococcus aureus, (MIC = 256 mg/L Escherichia coli (MIC = 512 mg/L, and Candida albicans (128 mg/L. AcT-2 is thus the first amphibian skin tryptophyllin found to possess both myotropic and antimicrobial activities.

Peptidomic analysis of the venom of the solitary bee Xylocopa appendiculata circumvolans.

Science.gov (United States)

Kazuma, Kohei; Ando, Kenji; Nihei, Ken-Ichi; Wang, Xiaoyu; Rangel, Marisa; Franzolin, Marcia Regina; Mori-Yasumoto, Kanami; Sekita, Setsuko; Kadowaki, Makoto; Satake, Motoyoshi; Konno, Katsuhiro

2017-01-01

Among the hymenopteran insect venoms, those from social wasps and bees - such as honeybee, hornets and paper wasps - have been well documented. Their venoms are composed of a number of peptides and proteins and used for defending their nests and themselves from predators. In contrast, the venoms of solitary wasps and bees have not been the object of further research. In case of solitary bees, only major peptide components in a few venoms have been addressed. Therefore, the aim of the present study was to explore the peptide component profile of the venom from the solitary bee Xylocopa appendiculata circumvolans by peptidomic analysis with using LC-MS. A reverse-phase HPLC connected to ESI-OrbiTrap MS was used for LC-MS. On-line mass fingerprinting was made from TIC, and data-dependent tandem mass spectrometry gave MSMS spectra. A major peptide component was isolated by reverse-phase HPLC by conventional way, and its sequence was determined by Edman degradation, which was finally corroborated by solid phase synthesis. Using the synthetic specimen, biological activities (antimicrobial activity, mast cell devaluation, hemolysis, leishmanicidal activity) and pore formation in artificial lipid bilayer were evaluated. On-line mass fingerprinting revealed that the crude venom contained 124 components. MS/MS analysis gave 75 full sequences of the peptide components. Most of these are related to the major and novel peptide, xylopin. Its sequence, GFVALLKKLPLILKHLH-NH 2 , has characteristic features of linear cationic α-helical peptides; rich in hydrophobic and basic amino acids with no disulfide bond, and accordingly, it can be predicted to adopt an amphipathic α-helix secondary structure. In biological evaluation, xylopin exhibited broad-spectrum antimicrobial activity, and moderate mast cell degranulation and leishmanicidal activities, but showed virtually no hemolytic activity. Additionally, the peptide was able to incorporate pores in artificial lipid bilayers of

Serum stabilities of short tryptophan- and arginine-rich antimicrobial peptide analogs.

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Leonard T Nguyen

2010-09-01

Full Text Available Several short antimicrobial peptides that are rich in tryptophan and arginine residues were designed with a series of simple modifications such as end capping and cyclization. The two sets of hexapeptides are based on the Trp- and Arg-rich primary sequences from the "antimicrobial centre" of bovine lactoferricin as well as an antimicrobial sequence obtained through the screening of a hexapeptide combinatorial library.HPLC, mass spectrometry and antimicrobial assays were carried out to explore the consequences of the modifications on the serum stability and microbicidal activity of the peptides. The results show that C-terminal amidation increases the antimicrobial activity but that it makes little difference to its proteolytic degradation in human serum. On the other hand, N-terminal acetylation decreases the peptide activities but significantly increases their protease resistance. Peptide cyclization of the hexameric peptides was found to be highly effective for both serum stability and antimicrobial activity. However the two cyclization strategies employed have different effects, with disulfide cyclization resulting in more active peptides while backbone cyclization results in more proteolytically stable peptides. However, the benefit of backbone cyclization did not extend to longer 11-mer peptides derived from the same region of lactoferricin. Mass spectrometry data support the serum stability assay results and allowed us to determine preferred proteolysis sites in the peptides. Furthermore, isothermal titration calorimetry experiments showed that the peptides all had weak interactions with albumin, the most abundant protein in human serum.Taken together, the results provide insight into the behavior of the peptides in human serum and will therefore aid in advancing antimicrobial peptide design towards systemic applications.

Cell-Penetrating Ability of Peptide Hormones: Key Role of Glycosaminoglycans Clustering

Directory of Open Access Journals (Sweden)

Armelle Tchoumi Neree

2015-11-01

Full Text Available Over the last two decades, the potential usage of cell-penetrating peptides (CPPs for the intracellular delivery of various molecules has prompted the identification of novel peptidic identities. However, cytotoxic effects and unpredicted immunological responses have often limited the use of various CPP sequences in the clinic. To overcome these issues, the usage of endogenous peptides appears as an appropriate alternative approach. The hormone pituitary adenylate-cyclase-activating polypeptide (PACAP38 has been recently identified as a novel and very efficient CPP. This 38-residue polycationic peptide is a member of the secretin/glucagon/growth hormone-releasing hormone (GHRH superfamily, with which PACAP38 shares high structural and conformational homologies. In this study, we evaluated the cell-penetrating ability of cationic peptide hormones in the context of the expression of cell surface glycosaminoglycans (GAGs. Our results indicated that among all peptides evaluated, PACAP38 was unique for its potent efficiency of cellular uptake. Interestingly, the abilities of the peptides to reach the intracellular space did not correlate with their binding affinities to sulfated GAGs, but rather to their capacity to clustered heparin in vitro. This study demonstrates that the uptake efficiency of a given cationic CPP does not necessarily correlate with its affinity to sulfated GAGs and that its ability to cluster GAGs should be considered for the identification of novel peptidic sequences with potent cellular penetrating properties.

Peptidomic Identification of Cysteine-Rich Peptides from Plants.

Science.gov (United States)

Hemu, Xinya; Serra, Aida; Darwis, Dina A; Cornvik, Tobias; Sze, Siu Kwan; Tam, James P

2018-01-01

Plant cysteine-rich peptides (CRPs) constitute a majority of plant-derived peptides with high molecular diversity. This protocol describes a rapid and efficient peptidomic approach to identify a whole spectrum of CRPs in a plant extract and decipher their molecular diversity and bioprocessing mechanism. Cyclotides from C. ternatea are used as the model CRPs to demonstrate our methodology. Cyclotides exist naturally in both cyclic and linear forms, although the linear forms (acyclotide) are generally present at much lower concentrations. Both cyclotides and acyclotides require linearization of their backbone prior to fragmentation and sequencing. A novel and practical three-step chemoenzymatic treatment was developed to linearize and distinguish both forms: (1) N-terminal acetylation that pre-labels the acyclotides; (2) conversion of Cys into pseudo-Lys through aziridine-mediated S-alkylation to reduce disulfide bonds and to increase the net charge of peptides; and (3) opening of cyclic backbones by the novel asparaginyl endopeptidase butelase 2 that cleaves at the native bioprocessing site. The treated peptides are subsequently analyzed by liquid chromatography coupled to mass spectrometry using electron transfer dissociation fragmentation and sequences are identified by matching the MS/MS spectra directly with the transcriptomic database.

BlockLogo: Visualization of peptide and sequence motif conservation

DEFF Research Database (Denmark)

Olsen, Lars Rønn; Kudahl, Ulrich Johan; Simon, Christian

2013-01-01

BlockLogo is a web-server application for the visualization of protein and nucleotide fragments, continuous protein sequence motifs, and discontinuous sequence motifs using calculation of block entropy from multiple sequence alignments. The user input consists of a multiple sequence alignment, se...

Mung bean proteins and peptides: nutritional, functional and bioactive properties

Directory of Open Access Journals (Sweden)

Zhu Yi-Shen

2018-02-01

Full Text Available To date, no extensive literature review exists regarding potential uses of mung bean proteins and peptides. As mung bean has long been widely used as a food source, early studies evaluated mung bean nutritional value against the Food and Agriculture Organization of the United Nations (FAO/the World Health Organization (WHO amino acids dietary recommendations. The comparison demonstrated mung bean to be a good protein source, except for deficiencies in sulphur-containing amino acids, methionine and cysteine. Methionine and cysteine residues have been introduced into the 8S globulin through protein engineering technology. Subsequently, purified mung bean proteins and peptides have facilitated the study of their structural and functional properties. Two main types of extraction methods have been reported for isolation of proteins and peptides from mung bean flours, permitting sequencing of major proteins present in mung bean, including albumins and globulins (notably 8S globulin. However, the sequence for albumin deposited in the UniProt database differs from other sequences reported in the literature. Meanwhile, a limited number of reports have revealed other useful bioactivities for proteins and hydrolysed peptides, including angiotensin-converting enzyme inhibitory activity, anti-fungal activity and trypsin inhibitory activity. Consequently, several mung bean hydrolysed peptides have served as effective food additives to prevent proteolysis during storage. Ultimately, further research will reveal other nutritional, functional and bioactive properties of mung bean for uses in diverse applications.

Optimizing the identification of citrullinated peptides by mass spectrometry

DEFF Research Database (Denmark)

Bennike, Tue; Lauridsen, Kasper B.; Olesen, Michael Kruse

2013-01-01

Citrullinated proteins have been associated with several diseases and citrullination can most likely function as a target for novel diagnostic agents and unravel disease etiologies. The correct identification of citrullinated proteins is therefore of most importance. Mass spectrometry (MS) driven...... of trypsin, digestion was performed on synthetic peptide sets containing either arginine or citrulline. The peptide sequences originated from disease-associated in vivo citrullinated proteins; some reported as being C-terminal tryptic citrullinated peptides. Furthermore, the proteolytic activity was verified...

Gephyrin-binding peptides visualize postsynaptic sites and modulate neurotransmission

DEFF Research Database (Denmark)

Maric, Hans Michael; Hausrat, Torben Johann; Neubert, Franziska

2017-01-01

is associated with perturbation of the basic physiological action. Here we pursue a fundamentally different approach, by instead targeting the intracellular receptor-gephyrin interaction. First, we defined the gephyrin peptide-binding consensus sequence, which facilitated the development of gephyrin super......-binding peptides and later effective affinity probes for the isolation of native gephyrin. Next, we demonstrated that fluorescent super-binding peptides could be used to directly visualize inhibitory postsynaptic sites for the first time in conventional and super-resolution microscopy. Finally, we demonstrate...

A method for selecting cis-acting regulatory sequences that respond to small molecule effectors

Directory of Open Access Journals (Sweden)

Allas Ülar

2010-08-01

Full Text Available Abstract Background Several cis-acting regulatory sequences functioning at the level of mRNA or nascent peptide and specifically influencing transcription or translation have been described. These regulatory elements often respond to specific chemicals. Results We have developed a method that allows us to select cis-acting regulatory sequences that respond to diverse chemicals. The method is based on the β-lactamase gene containing a random sequence inserted into the beginning of the ORF. Several rounds of selection are used to isolate sequences that suppress β-lactamase expression in response to the compound under study. We have isolated sequences that respond to erythromycin, troleandomycin, chloramphenicol, meta-toluate and homoserine lactone. By introducing synonymous and non-synonymous mutations we have shown that at least in the case of erythromycin the sequences act at the peptide level. We have also tested the cross-activities of the constructs and found that in most cases the sequences respond most strongly to the compound on which they were isolated. Conclusions Several selected peptides showed ligand-specific changes in amino acid frequencies, but no consensus motif could be identified. This is consistent with previous observations on natural cis-acting peptides, showing that it is often impossible to demonstrate a consensus. Applying the currently developed method on a larger scale, by selecting and comparing an extended set of sequences, might allow the sequence rules underlying the activity of cis-acting regulatory peptides to be identified.

The neXtProt peptide uniqueness checker: a tool for the proteomics community.

Science.gov (United States)

Schaeffer, Mathieu; Gateau, Alain; Teixeira, Daniel; Michel, Pierre-André; Zahn-Zabal, Monique; Lane, Lydie

2017-11-01

The neXtProt peptide uniqueness checker allows scientists to define which peptides can be used to validate the existence of human proteins, i.e. map uniquely versus multiply to human protein sequences taking into account isobaric substitutions, alternative splicing and single amino acid variants. The pepx program is available at https://github.com/calipho-sib/pepx and can be launched from the command line or through a cgi web interface. Indexing requires a sequence file in FASTA format. The peptide uniqueness checker tool is freely available on the web at https://www.nextprot.org/tools/peptide-uniqueness-checker and from the neXtProt API at https://api.nextprot.org/. lydie.lane@sib.swiss. © The Author(s) 2017. Published by Oxford University Press.

SANDPUMA: ensemble predictions of nonribosomal peptide chemistry reveal biosynthetic diversity across Actinobacteria

NARCIS (Netherlands)

Chevrette, Marc G.; Aicheler, Fabian; Kohlbacher, Oliver; Currie, Cameron R.; Medema, M.H.

2017-01-01

Nonribosomally synthesized peptides (NRPs) are natural products with widespread applications in medicine and biotechnology. Many algorithms have been developed to predict the substrate specificities of nonribosomal peptide synthetase adenylation (A) domains from DNA sequences, which enables

Engineering signal peptides for enhanced protein secretion from Lactococcus lactis.

Science.gov (United States)

Ng, Daphne T W; Sarkar, Casim A

2013-01-01

Lactococcus lactis is an attractive vehicle for biotechnological production of proteins and clinical delivery of therapeutics. In many such applications using this host, it is desirable to maximize secretion of recombinant proteins into the extracellular space, which is typically achieved by using the native signal peptide from a major secreted lactococcal protein, Usp45. In order to further increase protein secretion from L. lactis, inherent limitations of the Usp45 signal peptide (Usp45sp) must be elucidated. Here, we performed extensive mutagenesis on Usp45sp to probe the effects of both the mRNA sequence (silent mutations) and the peptide sequence (amino acid substitutions) on secretion. We screened signal peptides based on their resulting secretion levels of Staphylococcus aureus nuclease and further evaluated them for secretion of Bacillus subtilis α-amylase. Silent mutations alone gave an increase of up to 16% in the secretion of α-amylase through a mechanism consistent with relaxed mRNA folding around the ribosome binding site and enhanced translation. Targeted amino acid mutagenesis in Usp45sp, combined with additional silent mutations from the best clone in the initial screen, yielded an increase of up to 51% in maximum secretion of α-amylase while maintaining secretion at lower induction levels. The best sequence from our screen preserves the tripartite structure of the native signal peptide but increases the positive charge of the n-region. Our study presents the first example of an engineered L. lactis signal peptide with a higher secretion yield than Usp45sp and, more generally, provides strategies for further enhancing protein secretion in bacterial hosts.

Fungicidal activity of peptides encoded by immunoglobulin genes

OpenAIRE

Polonelli, Luciano; Ciociola, Tecla; Sperind?, Martina; Giovati, Laura; D?Adda, Tiziana; Galati, Serena; Travassos, Luiz R.; Magliani, Walter; Conti, Stefania

2017-01-01

Evidence from previous works disclosed the antimicrobial, antiviral, anti-tumour and/or immunomodulatory activity exerted, through different mechanisms of action, by peptides expressed in the complementarity-determining regions or even in the constant region of antibodies, independently from their specificity and isotype. Presently, we report the selection, from available databases, of peptide sequences encoded by immunoglobulin genes for the evaluation of their potential biological activitie...

Complete primary structure of a Lolium perenne (perennial rye grass) pollen allergen, Lol p III: comparison with known Lol p I and II sequences.

Science.gov (United States)

Ansari, A A; Shenbagamurthi, P; Marsh, D G

1989-10-17

The complete amino acid sequence of a Lolium perenne (rye grass) pollen allergen, Lol p III, determined by the automated Edman degradation of the protein and its selected fragments, is reported in this paper. Cleavage by enzymatic and chemical techniques established unambiguously the sequence for this 97-residue protein (Mr = 10,909), which lacks cysteine and shows no evidence of glycosylation. The sequence of Lol p III is very similar to that of another L. perenne allergen, Lol p II, which was sequenced recently; of the 97 positions in the two proteins, 57 are occupied by identical amino acids (59% identity). In addition, both allergens share a similar structure with an antibody-binding fragment of a third L. perenne allergen, Lol p I. Since human antibody responsiveness to all these three allergens is associated with HLA-DR3, and since the structure common to the three molecules shows high degrees of amphipathicity in Lol p II and III, we speculate that this common segment in the three molecules might contain or contribute to the respectively Ia/T-cell sites.

Improved surface bioactivity of stainless steel substrates using osteocalcin mimetic peptide

International Nuclear Information System (INIS)

Hosseini, Samaneh; Naderi-Manesh, Hossein; Vali, Hojatollah; Faghihi, Shahab

2014-01-01

Although stainless steel has a good biocompatibility for most clinical cases, the higher tissue response (bone bonding property) is required in orthopedic field. In this study, to improve bone-bonding ability of stainless steel substrates, a specific sequence of osteocalcin mimetic peptide is used as bioactive coating material to biochemically modify the surface of metallic samples. This sequence consists of thirteen amino acids present in the first helix of osteocalcin is synthesized in amidic form and physically adsorbed on the surface of 316LS (316 low carbon surgical grade) stainless steel substrates. Atomic force microscopy (AFM) and scanning electron microscopy (SEM) are used to characterize the surface of peptide coated and uncoated substrates. The bioactivity and bone bonding ability of coated and uncoated substrates are assessed by level of hydroxyapatite formation, using transmission electron microscopy (TEM), energy-dispersive x-ray (EDS), and scanning electron microscopy (SEM). The pre-osteoblast cell attachment and proliferation are also evaluated by MTT assay. The results show that the surface of coated sample is homogenously covered by the peptide and display a rougher surface relative to uncoated sample. TEM images reveal the formation of plate-like hydroxyapatite crystals in the presence of the peptide and an amorphous calcium phosphate phase without the peptide. Pre-osteoblast cells proliferation is significantly higher on the surface of peptide coated substrate, while cell attachment remains unaffected by the peptide coatings. Pre-osteoblast cells also demonstrate a higher degree of spreading on the surface of coated sample. It is believed that osteocalcin mimetic peptide improve surface bioactivity and promote hydroxyapatite crystal formation may lead to increased mineralization and bone formation on the surface of metallic biomedical devices. - Graphical abstract: A peptide sequence located in the first helix of OC is selected based on its

Improved surface bioactivity of stainless steel substrates using osteocalcin mimetic peptide

Energy Technology Data Exchange (ETDEWEB)

Hosseini, Samaneh [Department of Nanobiotechnology, Faculty of Biological Sciences, Tarbiat Modares University, Tehran (Iran, Islamic Republic of); Tissue Engineering and Biomaterials Division, National Institute of Genetic Engineering and Biotechnology, Tehran 14965/161 (Iran, Islamic Republic of); Naderi-Manesh, Hossein, E-mail: naderman@modares.ac.ir [Department of Nanobiotechnology, Faculty of Biological Sciences, Tarbiat Modares University, Tehran (Iran, Islamic Republic of); Vali, Hojatollah [Department of Anatomy and Cell Biology, McGill University, 3640 University Street, Montréal, QC H3A 0C7 (Canada); Faghihi, Shahab, E-mail: sfaghihi@nigeb.ac.ir [Tissue Engineering and Biomaterials Division, National Institute of Genetic Engineering and Biotechnology, Tehran 14965/161 (Iran, Islamic Republic of)

2014-02-14

Although stainless steel has a good biocompatibility for most clinical cases, the higher tissue response (bone bonding property) is required in orthopedic field. In this study, to improve bone-bonding ability of stainless steel substrates, a specific sequence of osteocalcin mimetic peptide is used as bioactive coating material to biochemically modify the surface of metallic samples. This sequence consists of thirteen amino acids present in the first helix of osteocalcin is synthesized in amidic form and physically adsorbed on the surface of 316LS (316 low carbon surgical grade) stainless steel substrates. Atomic force microscopy (AFM) and scanning electron microscopy (SEM) are used to characterize the surface of peptide coated and uncoated substrates. The bioactivity and bone bonding ability of coated and uncoated substrates are assessed by level of hydroxyapatite formation, using transmission electron microscopy (TEM), energy-dispersive x-ray (EDS), and scanning electron microscopy (SEM). The pre-osteoblast cell attachment and proliferation are also evaluated by MTT assay. The results show that the surface of coated sample is homogenously covered by the peptide and display a rougher surface relative to uncoated sample. TEM images reveal the formation of plate-like hydroxyapatite crystals in the presence of the peptide and an amorphous calcium phosphate phase without the peptide. Pre-osteoblast cells proliferation is significantly higher on the surface of peptide coated substrate, while cell attachment remains unaffected by the peptide coatings. Pre-osteoblast cells also demonstrate a higher degree of spreading on the surface of coated sample. It is believed that osteocalcin mimetic peptide improve surface bioactivity and promote hydroxyapatite crystal formation may lead to increased mineralization and bone formation on the surface of metallic biomedical devices. - Graphical abstract: A peptide sequence located in the first helix of OC is selected based on its

β-Casein(94-123)-derived peptides differently modulate production of mucins in intestinal goblet cells.

Science.gov (United States)

Plaisancié, Pascale; Boutrou, Rachel; Estienne, Monique; Henry, Gwénaële; Jardin, Julien; Paquet, Armelle; Léonil, Joëlle

2015-02-01

We recently reported the identification of a peptide from yoghurts with promising potential for intestinal health: the sequence (94-123) of bovine β-casein. This peptide, composed of 30 amino acid residues, maintains intestinal homoeostasis through production of the secreted mucin MUC2 and of the transmembrane-associated mucin MUC4. Our study aimed to search for the minimal sequence responsible for the biological activity of β-CN(94-123) by using several strategies based on (i) known bioactive peptides encrypted in β-CN(94-123), (ii) in silico prediction of peptides reactivity and (iii) digestion of β-CN(94-123) by enzymes of intestinal brush border membranes. The revealed sequences were tested in vitro on human intestinal mucus-producing HT29-MTX cells. We demonstrated that β-CN(108-113) (an ACE-inhibitory peptide) and β-CN(114-119) (an opioid peptide named neocasomorphin-6) up-regulated MUC4 expression whereas levels of the secreted mucins MUC2 and MUC5AC remained unchanged. The digestion of β-CN(94-123) by intestinal enzymes showed that the peptides β-CN(94-108) and β-CN(117-123) were present throughout 1·5 to 3 h of digestion, respectively. These two peptides raised MUC5AC expression while β-CN(117-123) also induced a decrease in the level of MUC2 mRNA and protein. In addition, this inhibitory effect was reproduced in airway epithelial cells. In conclusion, β-CN(94-123) is a multifunctional molecule but only the sequence of 30 amino acids has a stimulating effect on the production of MUC2, a crucial factor of intestinal protection.

A Novel Peptide from Soybean Protein Isolate Significantly Enhances Resistance of the Organism under Oxidative Stress.

Directory of Open Access Journals (Sweden)

Heran Ma

Full Text Available Recent studies have indicated that protein hydrolysates have broad biological effects. In the current study we describe a novel antioxidative peptide, FDPAL, from soybean protein isolate (SPI. The aim of this study was to purify and characterize an antioxidative peptide from SPI and determine its antioxidative mechanism. LC-MS/MS was used to isolate and identify the peptide from SPI. The sequence of the peptide was determined to be Phe-Asp-Pro-Ala-Leu (FDPAL, 561 Da. FDPAL can cause significant enhancement of resistance to oxidative stress both in cells as well as simple organisms. In Caenorhabditis elegans (C. elegans, FDPAL can up-regulate the expression of certain genes associated with resistance. The antioxidant activity of this peptide can be attributed to the presence of a specific amino acid sequence. Results from our work suggest that FDPAL can facilitate potential applications of proteins carrying this sequence in the nutraceutical, bioactive material and clinical medicine areas, as well as in cosmetics and health care products.

Analysis of skin derived peptides from the Cuyaba Dwarf Frog Physalaemus nattereri by off-line LC MALDI MS/MS

DEFF Research Database (Denmark)

Castro, Mariana S; Pires Júnior, Osmindo Rodrigues; Fontes, Wagner

2017-01-01

We have investigated the potential for analysis of the complex peptide mixtures secreted from frog skin using off-line LC-MALDI MS/MS. Since only limited information about the sequence of such peptides is available, de novo sequencing followed by Blast search was needed. An automated workflow has...... for future studies of peptides secreted from frogs....

Technetium-99m somatostatin analogues: effect of labelling methods and peptide sequence

International Nuclear Information System (INIS)

Decristoforo, C.; Mather, S.J.

1999-01-01

In this paper the preclinical evaluation of the somatostatin analogue RC160 labelled with technetium-99m using bifunctional chelators (BFCs) based on the hydrazinonicotinamide (HYNIC) and N 3 S system is described and a comparison made with [Tyr 3 ]-octreotide (TOC). Conjugates of both peptides with HYNIC, and of RC160 with benzoyl-MAG 3 and an N 3 S-adipate derivative were prepared and radiolabelling performed at high specific activities using tricine, tricine/nicotinic acid and ethylenediamine-N,N'-diacetic adic (EDDA) as co-ligands for HYNIC conjugates. All conjugates and 99m Tc-labelled peptides showed preserved binding affinity for the somatostatin receptor (IC50, Kd 99m Tc-RC160 derivatives compared with 99m Tc-EDDA/HYNIC-[Tyr 3 ]-octreotide (0.2%-3.5%ID/g vs 9.7%ID/g) and correlated well with the reduced internalisation rate for RC160 derivatives. Our results show that the selection of the labelling approach as well as the right choice of the peptide structure are crucial for labelling peptides with 99m Tc to achieve complexes with favourable biodistribution. Despite the relatively low tumour uptake compared with 99m Tc-EDDA/HYNIC-[Tyr 3 ]-octreotide, 99m Tc-RC160 could play a role in imaging tumours that do not bind octreotide derivatives. (orig.)

Peptides in headlock--a novel high-affinity and versatile peptide-binding nanobody for proteomics and microscopy.

Science.gov (United States)

Braun, Michael B; Traenkle, Bjoern; Koch, Philipp A; Emele, Felix; Weiss, Frederik; Poetz, Oliver; Stehle, Thilo; Rothbauer, Ulrich

2016-01-21

Nanobodies are highly valuable tools for numerous bioanalytical and biotechnical applications. Here, we report the characterization of a nanobody that binds a short peptide epitope with extraordinary affinity. Structural analysis reveals an unusual binding mode where the extended peptide becomes part of a β-sheet structure in the nanobody. This interaction relies on sequence-independent backbone interactions augmented by a small number of specificity-determining side chain contacts. Once bound, the peptide is fastened by two nanobody side chains that clamp it in a headlock fashion. Exploiting this unusual binding mode, we generated a novel nanobody-derived capture and detection system. Matrix-coupled nanobody enables the fast and efficient isolation of epitope-tagged proteins from prokaryotic and eukaryotic expression systems. Additionally, the fluorescently labeled nanobody visualizes subcellular structures in different cellular compartments. The high-affinity-binding and modifiable peptide tag of this system renders it a versatile and robust tool to combine biochemical analysis with microscopic studies.

Hydrophobic and electrostatic interactions between cell penetrating peptides and plasmid DNA are important for stable non-covalent complexation and intracellular delivery.

Science.gov (United States)

Upadhya, Archana; Sangave, Preeti C

2016-10-01

Cell penetrating peptides are useful tools for intracellular delivery of nucleic acids. Delivery of plasmid DNA, a large nucleic acid, poses a challenge for peptide mediated transport. The paper investigates and compares efficacy of five novel peptide designs for complexation of plasmid DNA and subsequent delivery into cells. The peptides were designed to contain reported DNA condensing agents and basic cell penetrating sequences, octa-arginine (R 8 ) and CHK 6 HC coupled to cell penetration accelerating peptides such as Bax inhibitory mutant peptide (KLPVM) and a peptide derived from the Kaposi fibroblast growth factor (kFGF) membrane translocating sequence. A tryptophan rich peptide, an analogue of Pep-3, flanked with CH 3 on either ends was also a part of the study. The peptides were analysed for plasmid DNA complexation, protection of peptide-plasmid DNA complexes against DNase I, serum components and competitive ligands by simple agarose gel electrophoresis techniques. Hemolysis of rat red blood corpuscles (RBCs) in the presence of the peptides was used as a measure of peptide cytotoxicity. Plasmid DNA delivery through the designed peptides was evaluated in two cell lines, human cervical cancer cell line (HeLa) and (NIH/3 T3) mouse embryonic fibroblasts via expression of the secreted alkaline phosphatase (SEAP) reporter gene. The importance of hydrophobic sequences in addition to cationic sequences in peptides for non-covalent plasmid DNA complexation and delivery has been illustrated. An alternative to the employment of fatty acid moieties for enhanced gene transfer has been proposed. Comparison of peptides for plasmid DNA complexation and delivery of peptide-plasmid DNA complexes to cells estimated by expression of a reporter gene, SEAP. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Topological disposition of the sequences -QRKIVE- and -KETYY in native (Na+ + K+)-ATPase

International Nuclear Information System (INIS)

Bayer, R.

1990-01-01

The dispositions with respect to the plane of the membrane of lysine-905 in the internal sequence -EQRKIVE- and of lysine-1012 in the carboxy-terminal sequence -RRPGGWVEKETYY of the α-polypeptide of sodium and potassium ion activated adenosinetriphosphatase have been determined. These lysines are found in peptides released from the intact α-polypeptide by the extracellular protease from Staphylococcus aureus strain V8 and by trypsin, respectively. Synthetic peptides containing terminal sequences of these were used to prepare polyclonal antibodies, which were then used to prepare immunoadsorbents directed against the respective peptides. Sealed, right-side-out membrane vesicles containing native (Na + + K + )-ATPase were labeled with pyridoxal phosphate and sodium [ 3 H]borohydride in the absence or presence of saponin. The labeled α-polypeptide was isolated from these vesicles and digested with appropriate proteases. The incorporation of radioactivity into the peptides binding to the immunoadsorbent directed against the sequence pyrERXIVE increased 3-fold int the presence of saponin as a result of the increased accessibility of this portion of the protein to the reagent when the vesicles were breached by saponin; hence, this sequence is located on the cytoplasmic face of the membrane. It was inferred that the carboxy-terminal sequence -KETYY is on the extracytoplasmic face since the incorporation of radioactivity into peptides binding to the immunoadsorbent directed against the sequence -ETYY did not change when the vesicles were breached with saponin

Antimicrobial activity predictors benchmarking analysis using shuffled and designed synthetic peptides.

Science.gov (United States)

Porto, William F; Pires, Állan S; Franco, Octavio L

2017-08-07

The antimicrobial activity prediction tools aim to help the novel antimicrobial peptides (AMP) sequences discovery, utilizing machine learning methods. Such approaches have gained increasing importance in the generation of novel synthetic peptides by means of rational design techniques. This study focused on predictive ability of such approaches to determine the antimicrobial sequence activities, which were previously characterized at the protein level by in vitro studies. Using four web servers and one standalone software, we evaluated 78 sequences generated by the so-called linguistic model, being 40 designed and 38 shuffled sequences, with ∼60 and ∼25% of identity to AMPs, respectively. The ab initio molecular modelling of such sequences indicated that the structure does not affect the predictions, as both sets present similar structures. Overall, the systems failed on predicting shuffled versions of designed peptides, as they are identical in AMPs composition, which implies in accuracies below 30%. The prediction accuracy is negatively affected by the low specificity of all systems here evaluated, as they, on the other hand, reached 100% of sensitivity. Our results suggest that complementary approaches with high specificity, not necessarily high accuracy, should be developed to be used together with the current systems, overcoming their limitations. Copyright © 2017 Elsevier Ltd. All rights reserved.

Characterization of foot-and-mouth disease virus's viral peptides with LC-ESI-MS

International Nuclear Information System (INIS)

Pzdemir, Z.O.; Bulut, E.K.; Mustafeva, Z.; Karahan, M.

2010-01-01

Peptides and proteins play a central role in numerous biological and physiological processes in living organisms. Viral capsid peptides are part of the viruses' outer shell of genetic materials. Viruses are recognized by immune system via capsid peptides. Depending on this property of capsid peptides, prototypes synthetic peptide-based vaccine can be developed. In this work, we synthesized three different viral peptide sequences of foot-and-mouth disease virus with microwave enhanced solid phase synthesis method. These peptides were characterized by using liquid chromatography electro spray interface mass spectrometry (LC-ESI-MS) with electro spray ionization. We briefly describe the essential facts for peptide characterization. (author)

Dinosaur peptides suggest mechanisms of protein survival.

Science.gov (United States)

San Antonio, James D; Schweitzer, Mary H; Jensen, Shane T; Kalluri, Raghu; Buckley, Michael; Orgel, Joseph P R O

2011-01-01

Eleven collagen peptide sequences recovered from chemical extracts of dinosaur bones were mapped onto molecular models of the vertebrate collagen fibril derived from extant taxa. The dinosaur peptides localized to fibril regions protected by the close packing of collagen molecules, and contained few acidic amino acids. Four peptides mapped to collagen regions crucial for cell-collagen interactions and tissue development. Dinosaur peptides were not represented in more exposed parts of the collagen fibril or regions mediating intermolecular cross-linking. Thus functionally significant regions of collagen fibrils that are physically shielded within the fibril may be preferentially preserved in fossils. These results show empirically that structure-function relationships at the molecular level could contribute to selective preservation in fossilized vertebrate remains across geological time, suggest a 'preservation motif', and bolster current concepts linking collagen structure to biological function. This non-random distribution supports the hypothesis that the peptides are produced by the extinct organisms and suggests a chemical mechanism for survival.

Dinosaur Peptides Suggest Mechanisms of Protein Survival

Energy Technology Data Exchange (ETDEWEB)

San Antonio, James D.; Schweitzer, Mary H.; Jensen, Shane T.; Kalluri, Raghu; Buckley, Michael; Orgel, Joseph P.R.O. (Harvard-Med); (IIT); (NCSU); (UPENN); (Manchester); (Orthovita)

2011-09-16

Eleven collagen peptide sequences recovered from chemical extracts of dinosaur bones were mapped onto molecular models of the vertebrate collagen fibril derived from extant taxa. The dinosaur peptides localized to fibril regions protected by the close packing of collagen molecules, and contained few acidic amino acids. Four peptides mapped to collagen regions crucial for cell-collagen interactions and tissue development. Dinosaur peptides were not represented in more exposed parts of the collagen fibril or regions mediating intermolecular cross-linking. Thus functionally significant regions of collagen fibrils that are physically shielded within the fibril may be preferentially preserved in fossils. These results show empirically that structure-function relationships at the molecular level could contribute to selective preservation in fossilized vertebrate remains across geological time, suggest a 'preservation motif', and bolster current concepts linking collagen structure to biological function. This non-random distribution supports the hypothesis that the peptides are produced by the extinct organisms and suggests a chemical mechanism for survival.

ArrayPitope: Automated Analysis of Amino Acid Substitutions for Peptide Microarray-Based Antibody Epitope Mapping

DEFF Research Database (Denmark)

Hansen, Christian Skjødt; Østerbye, Thomas; Marcatili, Paolo

2017-01-01

and characterization of linear B cell epitopes. Using exhaustive amino acid substitution analysis of peptides originating from target antigens, these microarrays can be used to address the specificity of polyclonal antibodies raised against such antigens containing hundreds of epitopes. However, the interpretation....... The application takes as input quantitative peptide data of fully or partially substituted overlapping peptides from a given antigen sequence and identifies epitope residues (residues that are significantly affected by substitutions) and visualize the selectivity towards each residue by sequence logo plots...

The preparation and identification of peptide imaging agent of lung cancer

International Nuclear Information System (INIS)

Chu Liping; Wang Yan; Wang Yueying; Liu Jinjian; Wu Hongying; Liu Jianfeng

2010-01-01

Objective: To screen in vivo lung cancer specific binding 7-peptide from T7 phage display random peptide library and prepare peptide imaging agent in early in early diagnosis of lung cancer. Methods: Used phage display in vivo technology to get the 7-peptide phage that can bind the lung cancer specifically, then sequenced and synthesized 7-peptide. After being labeled by 125 I, this 7-peptide was injected into mice via vein and the distribution in the mice tumor mold was observed. Results: One 7-peptide was obtained after four rounds of screening, and the peptide could bind lung cancer tissue specifically. Metabolism of this peptide in mice was fast and imaging of lung cancer was best two hours later after injection. The distribution in vivo decreased and almost disappeared after six hours. Conclusion: This 7-peptide could be used to image and diagnose of lung cancer effectively. (authors)

Double quick, double click reversible peptide "stapling".

Science.gov (United States)

Grison, Claire M; Burslem, George M; Miles, Jennifer A; Pilsl, Ludwig K A; Yeo, David J; Imani, Zeynab; Warriner, Stuart L; Webb, Michael E; Wilson, Andrew J

2017-07-01

The development of constrained peptides for inhibition of protein-protein interactions is an emerging strategy in chemical biology and drug discovery. This manuscript introduces a versatile, rapid and reversible approach to constrain peptides in a bioactive helical conformation using BID and RNase S peptides as models. Dibromomaleimide is used to constrain BID and RNase S peptide sequence variants bearing cysteine (Cys) or homocysteine ( h Cys) amino acids spaced at i and i + 4 positions by double substitution. The constraint can be readily removed by displacement of the maleimide using excess thiol. This new constraining methodology results in enhanced α-helical conformation (BID and RNase S peptide) as demonstrated by circular dichroism and molecular dynamics simulations, resistance to proteolysis (BID) as demonstrated by trypsin proteolysis experiments and retained or enhanced potency of inhibition for Bcl-2 family protein-protein interactions (BID), or greater capability to restore the hydrolytic activity of the RNAse S protein (RNase S peptide). Finally, use of a dibromomaleimide functionalized with an alkyne permits further divergent functionalization through alkyne-azide cycloaddition chemistry on the constrained peptide with fluorescein, oligoethylene glycol or biotin groups to facilitate biophysical and cellular analyses. Hence this methodology may extend the scope and accessibility of peptide stapling.

Inhibition of duck hepatitis B virus replication by mimic peptides in vitro.

Science.gov (United States)

Jia, Hongyu; Liu, Changhong; Yang, Ying; Zhu, Haihong; Chen, Feng; Liu, Jihong; Zhou, Linfu

2015-11-01

The aim of the present study was to investigate the inhibitory effect of specific mimic peptides targeting duck hepatitis B virus polymerase (DHBVP) on duck hepatitis B virus (DHBV) replication in primary duck hepatocytes. Phage display technology (PDT) was used to screen for mimic peptides specifically targeting DHBVP and the associated coding sequences were determined using DNA sequencing. The selected mimic peptides were then used to treat primary duck hepatocytes infected with DHBV in vitro. Infected hepatocytes expressing the mimic peptides intracellularly were also prepared. The cells were divided into mimic peptide groups (EXP groups), an entecavir-treated group (positive control) and a negative control group. The medium was changed every 48 h. Following a 10-day incubation, the cell supernatants were collected. DHBV-DNA in the cellular nucleus, cytoplasm and culture supernatant was analyzed by quantitative polymerase chain reaction (qPCR). Eight mimic peptides were selected following three PDT screening rounds for investigation in the DHBV-infected primary duck hepatocytes. The qPCR results showed that following direct treatment with mimic peptide 2 or 7, intracellular expression of mimic peptide 2 or 7, or treatment with entecavir, the DHBV-DNA levels in the culture supernatant and cytoplasm of duck hepatocytes were significantly lower than those in the negative control (Pmimic peptide 7 was lower than that in the other groups (Pmimic peptide 7 was significantly lower than that in the other groups (PMimic peptides specifically targeting DHBVP, administered directly or expressed intracellularly, can significantly inhibit DHBV replication in vitro .

[Molecular cloning of activin betaA subunit mature peptide from peafowl and its application in taxonomy and phylogeny].

Science.gov (United States)

Zou, Fang-Dong; Tong, Xin-Xin; Yue, Bi-Song

2005-03-01

The sequences of activin gene betaA subunit mature peptide have been amplified from white peafowl, blue peafowl (pavo cristatus) and green peafowl (pavo muticus) genomic DNA by polymerase chain reaction (PCR) with a pair of degenerate primers. The target fragments were cloned into the vector pMD18-T and sequenced. The length of activin gene betaA subunit mature peptide is 345bp, which encoded a peptide of 115 amino acid residues. Sequence analysis of activin gene betaA subunit mature peptide demonstrated that the identity of nucleotide is 98.0% between blue peaflowl and green peafowl, and the identity of that is 98.8% between blue peaflowl and white peafow. Sequences comparison in NCBI revealed that the sequences of activin gene betaA subunit mature peptides of different species are highly conserved during evolution process. In addition, the restriction enzyme map of activins is high similar between white peafowl and blue peafowl. Phylogenetic tree was constructed with Mega 2 and Clustalxldx software. The result showed that white peafowl has a closer relationship to blue peafowl than to green peafowl. Considered the nucleotide differences of peafowls' activin gene betaA subunit mature peptides, a highly conserved region, we supported that white peafowl was derived from blue peafowl, and it is more possible the hybrid but just the product of color mutation, or maybe as a subspecies of Pavo genus.

Origin of anti-tumor activity of the cysteine-containing GO peptides and further optimization of their cytotoxic properties

Science.gov (United States)

Tyuryaeva, Irina I.; Lyublinskaya, Olga G.; Podkorytov, Ivan S.; Skrynnikov, Nikolai R.

2017-01-01

Antitumor GO peptides have been designed as dimerization inhibitors of prominent oncoprotein mucin 1. In this study we demonstrate that activity of GO peptides is independent of the level of cellular expression of mucin 1. Furthermore, these peptides prove to be broadly cytotoxic, causing cell death also in normal cells such as dermal fibroblasts and endometrial mesenchymal stem cells. To explore molecular mechanism of their cytotoxicity, we have designed and tested a number of new peptide sequences containing the key CxC or CxxC motifs. Of note, these sequences bear no similarity to mucin 1 except that they also contain a pair of proximal cysteines. Several of the new peptides turned out to be significantly more potent than their GO prototypes. The results suggest that cytotoxicity of these peptides stems from their (moderate) activity as disulfide oxidoreductases. It is expected that such peptides, which we have termed DO peptides, are involved in disulfide-dithiol exchange reaction, resulting in formation of adventitious disulfide bridges in cell proteins. In turn, this leads to a partial loss of protein function and rapid onset of apoptosis. We anticipate that coupling DO sequences with tumor-homing transduction domains can create a potentially valuable new class of tumoricidal peptides.

A novel chimeric peptide with antimicrobial activity.

Science.gov (United States)

Alaybeyoglu, Begum; Akbulut, Berna Sariyar; Ozkirimli, Elif

2015-04-01

Beta-lactamase-mediated bacterial drug resistance exacerbates the prognosis of infectious diseases, which are sometimes treated with co-administration of beta-lactam type antibiotics and beta-lactamase inhibitors. Antimicrobial peptides are promising broad-spectrum alternatives to conventional antibiotics in this era of evolving bacterial resistance. Peptides based on the Ala46-Tyr51 beta-hairpin loop of beta-lactamase inhibitory protein (BLIP) have been previously shown to inhibit beta-lactamase. Here, our goal was to modify this peptide for improved beta-lactamase inhibition and cellular uptake. Motivated by the cell-penetrating pVEC sequence, which includes a hydrophobic stretch at its N-terminus, our approach involved the addition of LLIIL residues to the inhibitory peptide N-terminus to facilitate uptake. Activity measurements of the peptide based on the 45-53 loop of BLIP for enhanced inhibition verified that the peptide was a competitive beta-lactamase inhibitor with a K(i) value of 58 μM. Incubation of beta-lactam-resistant cells with peptide decreased the number of viable cells, while it had no effect on beta-lactamase-free cells, indicating that this peptide had antimicrobial activity via beta-lactamase inhibition. To elucidate the molecular mechanism by which this peptide moves across the membrane, steered molecular dynamics simulations were carried out. We propose that addition of hydrophobic residues to the N-terminus of the peptide affords a promising strategy in the design of novel antimicrobial peptides not only against beta-lactamase but also for other intracellular targets. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

Identification and the primary structure of equine alpha-lactalbumin B and C (Equus caballus, Perissodactyla).

Science.gov (United States)

Godovac-Zimmermann, J; Shaw, D; Conti, A; McKenzie, H

1987-04-01

The presence of two new alpha-lactalbumins has been demonstrated in the colostrum of a single mare (Equus caballus, Persian Arab). They have been designated equine alpha-lactalbumin B and C, and that isolated previously from the milk of Australian horses (English Thoroughbred) as alpha-lactalbumin A. The primary structures of B/C have been determined by automatic Edman degradation of enzymatic cleavage of the oxidized protein. Cyanogen bromide cleavage of S-carbamoyl-methylated protein provided necessary overlapping peptides. Comparison of the sequences of B and C with that of A indicates 3 and 4 amino-acid exchanges, respectively. The phylogenetic difference of equine alpha-lactalbumin B/C from bovine alpha-lactalbumin B is indicated by 39 and 40 amino-acid exchanges, respectively. The structure-function relationship, calcium binding sites and variants of alpha-lactalbumin are discussed.

Sequence-independent control of peptide conformation in liposomal vaccines for targeting protein misfolding diseases

NARCIS (Netherlands)

Hickman, D.T.; Nand, D.; Baldus, M.; Muhs, A.

2011-01-01

Synthetic peptide immunogens which mimic the conformation of a target epitope of pathological relevance offer the possibility to precisely control the immune response specificity. Here, we performed conformational analyses using a panel of peptides in order to investigate the key parameters

Angiotensin I-Converting Enzyme Inhibitor Derived from Cross-Linked Oyster Protein

Directory of Open Access Journals (Sweden)

Cheng-Liang Xie

2014-01-01

Full Text Available Following cross-linking by microbial transglutaminase, modified oyster proteins were hydrolyzed to improve inhibitory activity against angiotensin-converting enzyme (ACE inhibitory activity with the use of a single protease, or a combination of six proteases. The oyster hydrolysate with the lowest 50% ACE inhibitory concentration (IC50 of 0.40 mg/mL was obtained by two-step hydrolysis of the cross-linked oyster protein using Protamex and Neutrase. Five ACE inhibitory peptides were purified from the oyster hydrolysate using a multistep chromatographic procedure comprised of ion-exchange, size exclusion, and reversed-phase liquid chromatography. Their sequences were identified as TAY, VK, KY, FYN, and YA, using automated Edman degradation and mass spectrometry. These peptides were synthesized, and their IC50 values were measured to be 16.7, 29.0, 51.5, 68.2, and 93.9 μM, respectively. Toxicity of the peptides on the HepG2 cell line was not detected. The oyster hydrolysate also significantly decreased the systolic blood pressure of spontaneously hypertensive rats (SHR. The antihypertensive effect of the oyster hydrolysate on SHR was rapid and long-lasting, compared to commercially obtained sardine hydrolysate. These results suggest that the oyster hydrolysate could be a source of effective nutraceuticals against hypertension.

Interactions of photoactive DNAs with terminal deoxynucleotidyl transferase: Identification of peptides in the DNA binding domain

International Nuclear Information System (INIS)

Farrar, Y.J.K.; Evans, R.K.; Beach, C.M.; Coleman, M.S.

1991-01-01

Terminal deoxynucleotidyl transferase (terminal transferase) was specifically modified in the DNA binding site by a photoactive DNA substrate (hetero-40-mer duplex containing eight 5-azido-dUMP residues at one 3' end). Under optimal photolabeling conditions, 27-40% of the DNA was covalently cross-linked to terminal transferase. The specificity of the DNA and protein interaction was demonstrated by protection of photolabeling at the DNA binding domain with natural DNA substrates. In order to recover high yields of modified peptides from limited amounts of starting material, protein modified with 32 P-labeled photoactive DNA and digested with trypsin was extracted 4 times with phenol followed by gel filtration chromatography. All peptides not cross-linked to DNA were extracted into the phenol phase while the photolyzed DNA and the covalently cross-linked peptides remained in the aqueous phase. The 32 P-containing peptide-DNA fraction was subjected to amino acid sequence analysis. Two sequences, Asp 221 -Lys 231 (peptide B8) and Cys 234 -Lys 249 (peptide B10), present in similar yield, were identified. Structure predictions placed the two peptides in an α-helical array of 39 angstrom which would accommodate a DNA helix span of 11 nucleotides. These peptides share sequence similarity with a region in DNA polymerase β that has been implicated in the binding of DNA template

Protein sequences from mastodon and Tyrannosaurus rex revealed by mass spectrometry.

Science.gov (United States)

Asara, John M; Schweitzer, Mary H; Freimark, Lisa M; Phillips, Matthew; Cantley, Lewis C

2007-04-13

Fossilized bones from extinct taxa harbor the potential for obtaining protein or DNA sequences that could reveal evolutionary links to extant species. We used mass spectrometry to obtain protein sequences from bones of a 160,000- to 600,000-year-old extinct mastodon (Mammut americanum) and a 68-million-year-old dinosaur (Tyrannosaurus rex). The presence of T. rex sequences indicates that their peptide bonds were remarkably stable. Mass spectrometry can thus be used to determine unique sequences from ancient organisms from peptide fragmentation patterns, a valuable tool to study the evolution and adaptation of ancient taxa from which genomic sequences are unlikely to be obtained.

The property distance index PD predicts peptides that cross-react with IgE antibodies

Science.gov (United States)

Ivanciuc, Ovidiu; Midoro-Horiuti, Terumi; Schein, Catherine H.; Xie, Liping; Hillman, Gilbert R.; Goldblum, Randall M.; Braun, Werner

2009-01-01

Similarities in the sequence and structure of allergens can explain clinically observed cross-reactivities. Distinguishing sequences that bind IgE in patient sera can be used to identify potentially allergenic protein sequences and aid in the design of hypo-allergenic proteins. The property distance index PD, incorporated in our Structural Database of Allergenic Proteins (SDAP, http://fermi.utmb.edu/SDAP/), may identify potentially cross-reactive segments of proteins, based on their similarity to known IgE epitopes. We sought to obtain experimental validation of the PD index as a quantitative predictor of IgE cross-reactivity, by designing peptide variants with predetermined PD scores relative to three linear IgE epitopes of Jun a 1, the dominant allergen from mountain cedar pollen. For each of the three epitopes, 60 peptides were designed with increasing PD values (decreasing physicochemical similarity) to the starting sequence. The peptides synthesized on a derivatized cellulose membrane were probed with sera from patients who were allergic to Jun a 1, and the experimental data were interpreted with a PD classification method. Peptides with low PD values relative to a given epitope were more likely to bind IgE from the sera than were those with PD values larger than 6. Control sequences, with PD values between 18 and 20 to all the three epitopes, did not bind patient IgE, thus validating our procedure for identifying negative control peptides. The PD index is a statistically validated method to detect discrete regions of proteins that have a high probability of cross-reacting with IgE from allergic patients. PMID:18950868

Lipopolysaccharide interactions of C-terminal peptides from human thrombin.

Science.gov (United States)

Singh, Shalini; Kalle, Martina; Papareddy, Praveen; Schmidtchen, Artur; Malmsten, Martin

2013-05-13

Interactions with bacterial lipopolysaccharide (LPS), both in aqueous solution and in lipid membranes, were investigated for a series of amphiphilic peptides derived from the C-terminal region of human thrombin, using ellipsometry, dual polarization interferometry, fluorescence spectroscopy, circular dichroism (CD), dynamic light scattering, and z-potential measurements. The ability of these peptides to block endotoxic effects caused by LPS, monitored through NO production in macrophages, was compared to peptide binding to LPS and its endotoxic component lipid A, and to size, charge, and secondary structure of peptide/LPS complexes. While the antiendotoxic peptide GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE) displayed significant binding to both LPS and lipid A, so did two control peptides with either selected D-amino acid substitutions or with maintained composition but scrambled sequence, both displaying strongly attenuated antiendotoxic effects. Hence, the extent of LPS or lipid A binding is not the sole discriminant for the antiendotoxic effect of these peptides. In contrast, helix formation in peptide/LPS complexes correlates to the antiendotoxic effect of these peptides and is potentially linked to this functionality. Preferential binding to LPS over lipid membrane was furthermore demonstrated for these peptides and preferential binding to the lipid A moiety within LPS inferred.

Nucleotide sequence of the triosephosphate isomerase gene from Macaca mulatta

Energy Technology Data Exchange (ETDEWEB)

Old, S.E.; Mohrenweiser, H.W. (Univ. of Michigan, Ann Arbor (USA))

1988-09-26

The triosephosphate isomerase gene from a rhesus monkey, Macaca mulatta, charon 34 library was sequenced. The human and chimpanzee enzymes differ from the rhesus enzyme at ASN 20 and GLU 198. The nucleotide sequence identity between rhesus and human is 97% in the coding region and >94% in the flanking regions. Comparison of the rhesus and chimp genes, including the intron and flanking sequences, does not suggest a mechanism for generating the two TPI peptides of proliferating cells from hominoids and a single peptide from the rhesus gene.

Peptide bond detection via graphene nanogaps: a proof of principle study.

Science.gov (United States)

Rossini, Aldo Eugenio; Gala, Fabrizio; Chinappi, Mauro; Zollo, Giuseppe

2018-03-29

Solid-state nanopores and nanogaps are emerging as promising tools for single molecule analysis. 2D materials, such as graphene, can potentially reach the spatial resolution needed for nucleic acid and protein sequencing. In the context of the density functional theory, atomistic modeling and non-equilibrium Green's function calculation, we show that glycine based polypeptide chains translocating across a nano-gap between two semi-infinite graphene nano-ribbons leave a specific transverse current signature for each peptide bond. The projected density of states and bond current analyses reveal a complex scenario with a role played by the adjacent α-carbons and side chains and by the orbitals of the partially resonant double bond involving C, N and O atoms of the peptide bond. In this context, specific fingerprints of the atoms involved in the peptide bonds are found. The same scenario is evidenced also for peptides involving alanine residues. The signal measured can be considered as a specific fingerprint of peptide bonds between small and neutral amino acids with no polar/charge effects. On this basis, a newly conceived nano-device made of a graphene based array of nano-gap is proposed as a possible route to approach peptide sequencing with atomic resolution.

Peptide chemistry toolbox - Transforming natural peptides into peptide therapeutics.

Science.gov (United States)

Erak, Miloš; Bellmann-Sickert, Kathrin; Els-Heindl, Sylvia; Beck-Sickinger, Annette G

2018-06-01

The development of solid phase peptide synthesis has released tremendous opportunities for using synthetic peptides in medicinal applications. In the last decades, peptide therapeutics became an emerging market in pharmaceutical industry. The need for synthetic strategies in order to improve peptidic properties, such as longer half-life, higher bioavailability, increased potency and efficiency is accordingly rising. In this mini-review, we present a toolbox of modifications in peptide chemistry for overcoming the main drawbacks during the transition from natural peptides to peptide therapeutics. Modifications at the level of the peptide backbone, amino acid side chains and higher orders of structures are described. Furthermore, we are discussing the future of peptide therapeutics development and their impact on the pharmaceutical market. Copyright © 2018 Elsevier Ltd. All rights reserved.

Cloning and sequencing of the bovine gastrin gene

DEFF Research Database (Denmark)

Lund, T; Rehfeld, J F; Olsen, Jørgen

1989-01-01

In order to deduce the primary structure of bovine preprogastrin we therefore sequenced a gastrin DNA clone isolated from a bovine liver cosmid library. Bovine preprogastrin comprises 104 amino acids and consists of a signal peptide, a 37 amino acid spacer-sequence, the gastrin-34 sequence followed...

Prediction of Nucleotide Binding Peptides Using Star Graph Topological Indices.

Science.gov (United States)

Liu, Yong; Munteanu, Cristian R; Fernández Blanco, Enrique; Tan, Zhiliang; Santos Del Riego, Antonino; Pazos, Alejandro

2015-11-01

The nucleotide binding proteins are involved in many important cellular processes, such as transmission of genetic information or energy transfer and storage. Therefore, the screening of new peptides for this biological function is an important research topic. The current study proposes a mixed methodology to obtain the first classification model that is able to predict new nucleotide binding peptides, using only the amino acid sequence. Thus, the methodology uses a Star graph molecular descriptor of the peptide sequences and the Machine Learning technique for the best classifier. The best model represents a Random Forest classifier based on two features of the embedded and non-embedded graphs. The performance of the model is excellent, considering similar models in the field, with an Area Under the Receiver Operating Characteristic Curve (AUROC) value of 0.938 and true positive rate (TPR) of 0.886 (test subset). The prediction of new nucleotide binding peptides with this model could be useful for drug target studies in drug development. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Radiopharmaceutical development of radiolabelled peptides

Energy Technology Data Exchange (ETDEWEB)

Fani, Melpomeni; Maecke, Helmut R. [University Hospital Freiburg, Department of Nuclear Medicine, Freiburg (Germany)

2012-02-15

Receptor targeting with radiolabelled peptides has become very important in nuclear medicine and oncology in the past few years. The overexpression of many peptide receptors in numerous cancers, compared to their relatively low density in physiological organs, represents the molecular basis for in vivo imaging and targeted radionuclide therapy with radiolabelled peptide-based probes. The prototypes are analogs of somatostatin which are routinely used in the clinic. More recent developments include somatostatin analogs with a broader receptor subtype profile or with antagonistic properties. Many other peptide families such as bombesin, cholecystokinin/gastrin, glucagon-like peptide-1 (GLP-1)/exendin, arginine-glycine-aspartic acid (RGD) etc. have been explored during the last few years and quite a number of potential radiolabelled probes have been derived from them. On the other hand, a variety of strategies and optimized protocols for efficient labelling of peptides with clinically relevant radionuclides such as {sup 99m}Tc, M{sup 3+} radiometals ({sup 111}In, {sup 86/90}Y, {sup 177}Lu, {sup 67/68}Ga), {sup 64/67}Cu, {sup 18}F or radioisotopes of iodine have been developed. The labelling approaches include direct labelling, the use of bifunctional chelators or prosthetic groups. The choice of the labelling approach is driven by the nature and the chemical properties of the radionuclide. Additionally, chemical strategies, including modification of the amino acid sequence and introduction of linkers/spacers with different characteristics, have been explored for the improvement of the overall performance of the radiopeptides, e.g. metabolic stability and pharmacokinetics. Herein, we discuss the development of peptides as radiopharmaceuticals starting from the choice of the labelling method and the conditions to the design and optimization of the peptide probe, as well as some recent developments, focusing on a selected list of peptide families, including somatostatin

An Unusual Family of Glycosylated Peptides Isolated from Dendroaspis angusticeps Venom and Characterized by Combination of Collision Induced and Electron Transfer Dissociation

Science.gov (United States)

Quinton, Loïc; Gilles, Nicolas; Smargiasso, Nicolas; Kiehne, Andrea; de Pauw, Edwin

2011-11-01

This study describes the structural characterization of a totally new family of peptides from the venom of the snake green mamba ( Dendroaspis angusticeps). Interestingly, these peptides differ in several points from other already known mamba toxins. First of all, they exhibit very small molecular masses, ranging from 1.3 to 2.4 kDa. The molecular mass of classical mamba toxins is in the range of 7 to 25 kDa. Second, the new peptides do not contain disulfide bonds, a post-translational modification commonly encountered in animal toxins. The third difference is the very high proportion of proline residues in the sequence accounting for about one-third of the sequence. Finally, these new peptides reveal a carbohydrate moiety, indicating a glycosylation in the sequence. The last two features have made the structural characterization of the new peptides by mass spectrometry a real analytical challenge. Peptides were characterized by a combined use of MALDI- TOF/TOF and nanoESI-IT-ETD experiments to determine not only the peptide sequence but also the composition and the position of the carbohydrate moiety. Anyway, such small glycosylated and proline-rich toxins are totally different from any other known snake peptide and form, as a consequence, a new family of peptides.

Peptides in headlock – a novel high-affinity and versatile peptide-binding nanobody for proteomics and microscopy

Science.gov (United States)

Braun, Michael B.; Traenkle, Bjoern; Koch, Philipp A.; Emele, Felix; Weiss, Frederik; Poetz, Oliver; Stehle, Thilo; Rothbauer, Ulrich

2016-01-01

Nanobodies are highly valuable tools for numerous bioanalytical and biotechnical applications. Here, we report the characterization of a nanobody that binds a short peptide epitope with extraordinary affinity. Structural analysis reveals an unusual binding mode where the extended peptide becomes part of a β-sheet structure in the nanobody. This interaction relies on sequence-independent backbone interactions augmented by a small number of specificity-determining side chain contacts. Once bound, the peptide is fastened by two nanobody side chains that clamp it in a headlock fashion. Exploiting this unusual binding mode, we generated a novel nanobody-derived capture and detection system. Matrix-coupled nanobody enables the fast and efficient isolation of epitope-tagged proteins from prokaryotic and eukaryotic expression systems. Additionally, the fluorescently labeled nanobody visualizes subcellular structures in different cellular compartments. The high-affinity-binding and modifiable peptide tag of this system renders it a versatile and robust tool to combine biochemical analysis with microscopic studies. PMID:26791954

DeepRT: deep learning for peptide retention time prediction in proteomics

OpenAIRE

Ma, Chunwei; Zhu, Zhiyong; Ye, Jun; Yang, Jiarui; Pei, Jianguo; Xu, Shaohang; Zhou, Ruo; Yu, Chang; Mo, Fan; Wen, Bo; Liu, Siqi

2017-01-01

Accurate predictions of peptide retention times (RT) in liquid chromatography have many applications in mass spectrometry-based proteomics. Herein, we present DeepRT, a deep learning based software for peptide retention time prediction. DeepRT automatically learns features directly from the peptide sequences using the deep convolutional Neural Network (CNN) and Recurrent Neural Network (RNN) model, which eliminates the need to use hand-crafted features or rules. After the feature learning, pr...

Self-assembling peptide nanofiber hydrogels in tissue engineering and regenerative medicine: Progress, design guidelines, and applications.

Science.gov (United States)

Koutsopoulos, Sotirios

2016-04-01

Until the mid-1980s, mainly biologists were conducting peptide research. This changed with discoveries that opened new paths of research involving the use of peptides in bioengineering, biotechnology, biomedicine, nanotechnology, and bioelectronics. Peptide engineering and rational design of novel peptide sequences with unique and tailor-made properties further expanded the field. The discovery of short self-assembling peptides, which upon association form well-defined supramolecular architectures, created new and exciting areas of research. Depending on the amino acid sequence, the pH, and the type of the electrolyte in the medium, peptide self-assembly leads to the formation of nanofibers, which are further organized to form a hydrogel. In this review, the application of ionic complementary peptides which self-assemble to form nanofiber hydrogels for tissue engineering and regenerative medicine will be discussed through a selective presentation of the most important work performed during the last 25 years. © 2016 Wiley Periodicals, Inc.

Structural organization and spectroscopy of peptide-actinide(IV) complexes

International Nuclear Information System (INIS)

Dahou, S.

2010-01-01

The contamination of living organisms by actinide elements is at the origin of both radiological and chemical toxicity that may lead to severe dysfunction. Most of the data available on the actinide interaction with biological systems are macroscopic physiological measurements and are lacking a molecular description of the systems. Because of the intricacy of these systems, classical biochemical methods are difficult to implement. Our strategy consisted in designing simplified biomimetic peptides, and describing the corresponding intramolecular interactions with actinides. A carboxylic pentapeptide of the form DDPDD has been at the starting point of this work in order to further assess the influence of the peptide sequence on the topology of the complexes.To do so, various linear (Asp/Ala permutations, peptoids) and cyclic analogues have been synthesized. Furthermore, in order to include the hydroxamic function (with a high affinity for Fe(III)) in the peptide, both desferrioxamine and acetohydroxamic acid have been investigated. However because of difficulties in synthesis, we have not been able to test these peptides. Three actinide cations have been considered at oxidation state +IV (Th, Np, Pu) and compared to Fe(III), often considered as a biological surrogate of Pu(IV). The spatial arrangement of the peptide around the cation has been probed by spectrophotometry and X-ray Absorption Spectroscopy. The spectroscopic data and EXAFS data adjustment lead us to rationalize the topology of the complexes as a function of the peptide sequence: mix hydroxy polynuclear species for linear and cyclic peptides, mononuclear for the desferrioxamine complexes. Furthermore, significant differences have appeared between Fe(III) and actinide(IV), related to differences of reactivity in aqueous medium. (author)

A novel algorithm for validating peptide identification from a shotgun proteomics search engine.

Science.gov (United States)

Jian, Ling; Niu, Xinnan; Xia, Zhonghang; Samir, Parimal; Sumanasekera, Chiranthani; Mu, Zheng; Jennings, Jennifer L; Hoek, Kristen L; Allos, Tara; Howard, Leigh M; Edwards, Kathryn M; Weil, P Anthony; Link, Andrew J

2013-03-01

Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has revolutionized the proteomics analysis of complexes, cells, and tissues. In a typical proteomic analysis, the tandem mass spectra from a LC-MS/MS experiment are assigned to a peptide by a search engine that compares the experimental MS/MS peptide data to theoretical peptide sequences in a protein database. The peptide spectra matches are then used to infer a list of identified proteins in the original sample. However, the search engines often fail to distinguish between correct and incorrect peptides assignments. In this study, we designed and implemented a novel algorithm called De-Noise to reduce the number of incorrect peptide matches and maximize the number of correct peptides at a fixed false discovery rate using a minimal number of scoring outputs from the SEQUEST search engine. The novel algorithm uses a three-step process: data cleaning, data refining through a SVM-based decision function, and a final data refining step based on proteolytic peptide patterns. Using proteomics data generated on different types of mass spectrometers, we optimized the De-Noise algorithm on the basis of the resolution and mass accuracy of the mass spectrometer employed in the LC-MS/MS experiment. Our results demonstrate De-Noise improves peptide identification compared to other methods used to process the peptide sequence matches assigned by SEQUEST. Because De-Noise uses a limited number of scoring attributes, it can be easily implemented with other search engines.

Anticancer activities of bovine and human lactoferricin-derived peptides.

Science.gov (United States)

Arias, Mauricio; Hilchie, Ashley L; Haney, Evan F; Bolscher, Jan G M; Hyndman, M Eric; Hancock, Robert E W; Vogel, Hans J

2017-02-01

Lactoferrin (LF) is a mammalian host defense glycoprotein with diverse biological activities. Peptides derived from the cationic region of LF possess cytotoxic activity against cancer cells in vitro and in vivo. Bovine lactoferricin (LFcinB), a peptide derived from bovine LF (bLF), exhibits broad-spectrum anticancer activity, while a similar peptide derived from human LF (hLF) is not as active. In this work, several peptides derived from the N-terminal regions of bLF and hLF were studied for their anticancer activities against leukemia and breast-cancer cells, as well as normal peripheral blood mononuclear cells. The cyclized LFcinB-CLICK peptide, which possesses a stable triazole linkage, showed improved anticancer activity, while short peptides hLF11 and bLF10 were not cytotoxic to cancer cells. Interestingly, hLF11 can act as a cell-penetrating peptide; when combined with the antimicrobial core sequence of LFcinB (RRWQWR) through either a Pro or Gly-Gly linker, toxicity to Jurkat cells increased. Together, our work extends the library of LF-derived peptides tested for anticancer activity, and identified new chimeric peptides with high cytotoxicity towards cancerous cells. Additionally, these results support the notion that short cell-penetrating peptides and antimicrobial peptides can be combined to create new adducts with increased potency.

Biochemical characterization of an anti-Candida factor produced by Enterococcus faecalis

Directory of Open Access Journals (Sweden)

Shekh Raeesh M

2012-07-01

Full Text Available Abstract Background Because Candida albicans is resistant to several antifungal antibiotics, there is a need to identify other less toxic natural products, particularly antimicrobial proteins, peptides or bacteriocin like inhibitory substances. An attempt has been made to purify and characterise an anti-Candida compound produced by Enterococcus faecalis. Results An anti-Candida protein (ACP produced by E. faecalis active against 8 C. albicans strains was characterised and partially purified. The ACP showed a broad-spectrum activity against multidrug resistant C. albicans MTCC 183, MTCC 7315, MTCC 3958, NCIM 3557, NCIM 3471 and DI. It was completely inactivated by treatment with proteinase K and partially by pronase E. The ACP retained biological stability after heat-treatment at 90°C for 20 min, maintained activity over a pH range 6–10, and remained active after treatment with α-amylase, lipase, organic solvents, and detergents. The antimicrobial activity of the E. faecalis strain was found exclusively in the extracellular filtrate produced in the late logarithmic growth phase. The highest activity (1600 AU mL-1 against C. albicans MTCC 183 was recorded at 48 h of incubation, and activity decreased thereafter. The peptide showed very low haemagglutination and haemolytic activities against human red blood cells. The antimicrobial substance was purified by salt-fractionation and chromatography. Partially purified ACP had a molecular weight of approximately 43 KDa in Tricine-PAGE analysis. The 12 amino acid N terminal sequence was obtained by Edman degradation. The peptide was de novo sequenced by ESI-MS, and the deduced combined sequence when compared to other bacteriocins and antimicrobial peptide had no significant sequence similarity. Conclusions The inhibitory activity of the test strain is due to the synthesis of an antimicrobial protein. To our knowledge, this is the first report on the isolation of a promising non-haemolytic anti

Vaccination with recombinant heat shock protein 60 from Histoplasma capsulatum protects mice against pulmonary histoplasmosis.

Science.gov (United States)

Gomez, F J; Allendoerfer, R; Deepe, G S

1995-07-01

HIS-62 is a glycoprotein that has been isolated from the cell wall and cell membrane fraction of the pathogenic fungus Histoplasma capsulatum. It is a target of the cellular immune response to this fungus, and it protects mice against a lethal intravenous inoculum of H. capsulatum yeast cells. In this study, we cloned the gene encoding this antigen to reveal its biological nature and studied the immunological activity of recombinant antigen. The amino acid sequences of the NH2 terminus and internal peptides were obtained by Edman degradation. Degenerate oligonucleotides were used to isolate a gene fragment of HIS-62 by PCR. One 680-bp segment that corresponded to the known peptide sequence was amplified from H. capsulatum DNA. This DNA was used to screen a genomic library, and the full-length gene was isolated and sequenced. The deduced amino acid sequence of the gene demonstrated approximately 70 and approximately 50% identity to heat shock protein 60 (hsp 60) from Saccharomyces cerevisiae and hsp 60 from Escherichia coli, respectively. A cDNA was synthesized by reverse transcription PCR and was expressed in E. coli. Recombinant protein reacted with a monospecific polyclonal rabbit antiserum raised against native HIS-62, with monoclonal HIS-62-reactive T cells, and with splenocytes from mice immunized with viable yeast cells. Moreover, vaccination with the recombinant protein conferred protection in mice against a lethal intranasal inoculation with yeast cells. Thus, HIS-62 is a member of the hsp 60 family, and the recombinant hsp 60 is protective against pulmonary histoplasmosis in mice.

Functional characterization of a synthetic hydrophilic antifungal peptide derived from the marine snail Cenchritis muricatus.

Science.gov (United States)

López-Abarrategui, Carlos; Alba, Annia; Silva, Osmar N; Reyes-Acosta, Osvaldo; Vasconcelos, Ilka M; Oliveira, Jose T A; Migliolo, Ludovico; Costa, Maysa P; Costa, Carolina R; Silva, Maria R R; Garay, Hilda E; Dias, Simoni C; Franco, Octávio L; Otero-González, Anselmo J

2012-04-01

Antimicrobial peptides have been found in mollusks and other sea animals. In this report, a crude extract of the marine snail Cenchritis muricatus was evaluated against human pathogens responsible for multiple deleterious effects and diseases. A peptide of 1485.26 Da was purified by reversed-phase HPLC and functionally characterized. This trypsinized peptide was sequenced by MS/MS technology, and a sequence (SRSELIVHQR), named Cm-p1 was recovered, chemically synthesized and functionally characterized. This peptide demonstrated the capacity to prevent the development of yeasts and filamentous fungi. Otherwise, Cm-p1 displayed no toxic effects against mammalian cells. Molecular modeling analyses showed that this peptide possible forms a single hydrophilic α-helix and the probable cationic residue involved in antifungal activity action is proposed. The data reported here demonstrate the importance of sea animals peptide discovery for biotechnological tools development that could be useful in solving human health and agribusiness problems. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Cloning and heterologous expression of the antibiotic peptide (ABP) genes from Rhizopus oligosporus NBRC 8631.

Science.gov (United States)

Yamada, Osamu; Sakamoto, Kazutoshi; Tominaga, Mihoko; Nakayama, Tasuku; Koseki, Takuya; Fujita, Akiko; Akita, Osamu

2005-03-01

We carried out protein sequencing of purified Antibiotic Peptide (ABP), and cloned two genes encoding this peptide as abp1 and abp2, from Rhizopus oligosporus NBRC 8631. Both genes contain an almost identical 231-bp segment, with only 3 nucleotide substitutions, encoding a 77 amino acid peptide. The abp gene product comprises a 28 amino acid signal sequence and a 49 amino acid mature peptide. Northern blot analysis showed that at least one of the abp genes is transcribed in R. oligosporus NBRC 8631. A truncated form of abp1 encoding only the mature peptide was fused with the alpha-factor signal peptide and engineered for expression in Pichia pastoris SMD1168H. Culture broth of the recombinant Pichia displayed ABP activity against Bacillus subtilis NBRC 3335 after induction of heterologous gene expression. This result indicates that mature ABP formed the active structure without the aid of other factors from R. oligosporus, and was secreted.

Dynamical system modeling to simulate donor T cell response to whole exome sequencing-derived recipient peptides: Understanding randomness in alloreactivity incidence following stem cell transplantation.

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Vishal Koparde

Full Text Available Quantitative relationship between the magnitude of variation in minor histocompatibility antigens (mHA and graft versus host disease (GVHD pathophysiology in stem cell transplant (SCT donor-recipient pairs (DRP is not established. In order to elucidate this relationship, whole exome sequencing (WES was performed on 27 HLA matched related (MRD, & 50 unrelated donors (URD, to identify nonsynonymous single nucleotide polymorphisms (SNPs. An average 2,463 SNPs were identified in MRD, and 4,287 in URD DRP (p<0.01; resulting peptide antigens that may be presented on HLA class I molecules in each DRP were derived in silico (NetMHCpan ver2.0 and the tissue expression of proteins these were derived from determined (GTex. MRD DRP had an average 3,670 HLA-binding-alloreactive peptides, putative mHA (pmHA with an IC50 of <500 nM, and URD, had 5,386 (p<0.01. To simulate an alloreactive donor cytotoxic T cell response, the array of pmHA in each patient was considered as an operator matrix modifying a hypothetical cytotoxic T cell clonal vector matrix; each responding T cell clone's proliferation was determined by the logistic equation of growth, accounting for HLA binding affinity and tissue expression of each alloreactive peptide. The resulting simulated organ-specific alloreactive T cell clonal growth revealed marked variability, with the T cell count differences spanning orders of magnitude between different DRP. Despite an estimated, uniform set of constants used in the model for all DRP, and a heterogeneously treated group of patients, higher total and organ-specific T cell counts were associated with cumulative incidence of moderate to severe GVHD in recipients. In conclusion, exome wide sequence differences and the variable alloreactive peptide binding to HLA in each DRP yields a large range of possible alloreactive donor T cell responses. Our findings also help understand the apparent randomness observed in the development of alloimmune responses.

Chameleon sequences in neurodegenerative diseases.

Science.gov (United States)

Bahramali, Golnaz; Goliaei, Bahram; Minuchehr, Zarrin; Salari, Ali

2016-03-25

Chameleon sequences can adopt either alpha helix sheet or a coil conformation. Defining chameleon sequences in PDB (Protein Data Bank) may yield to an insight on defining peptides and proteins responsible in neurodegeneration. In this research, we benefitted from the large PDB and performed a sequence analysis on Chameleons, where we developed an algorithm to extract peptide segments with identical sequences, but different structures. In order to find new chameleon sequences, we extracted a set of 8315 non-redundant protein sequences from the PDB with an identity less than 25%. Our data was classified to "helix to strand (HE)", "helix to coil (HC)" and "strand to coil (CE)" alterations. We also analyzed the occurrence of singlet and doublet amino acids and the solvent accessibility in the chameleon sequences; we then sorted out the proteins with the most number of chameleon sequences and named them Chameleon Flexible Proteins (CFPs) in our dataset. Our data revealed that Gly, Val, Ile, Tyr and Phe, are the major amino acids in Chameleons. We also found that there are proteins such as Insulin Degrading Enzyme IDE and GTP-binding nuclear protein Ran (RAN) with the most number of chameleons (640 and 405 respectively). These proteins have known roles in neurodegenerative diseases. Therefore it can be inferred that other CFP's can serve as key proteins in neurodegeneration, and a study on them can shed light on curing and preventing neurodegenerative diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

Chameleon sequences in neurodegenerative diseases

International Nuclear Information System (INIS)

Bahramali, Golnaz; Goliaei, Bahram; Minuchehr, Zarrin; Salari, Ali

2016-01-01

Chameleon sequences can adopt either alpha helix sheet or a coil conformation. Defining chameleon sequences in PDB (Protein Data Bank) may yield to an insight on defining peptides and proteins responsible in neurodegeneration. In this research, we benefitted from the large PDB and performed a sequence analysis on Chameleons, where we developed an algorithm to extract peptide segments with identical sequences, but different structures. In order to find new chameleon sequences, we extracted a set of 8315 non-redundant protein sequences from the PDB with an identity less than 25%. Our data was classified to “helix to strand (HE)”, “helix to coil (HC)” and “strand to coil (CE)” alterations. We also analyzed the occurrence of singlet and doublet amino acids and the solvent accessibility in the chameleon sequences; we then sorted out the proteins with the most number of chameleon sequences and named them Chameleon Flexible Proteins (CFPs) in our dataset. Our data revealed that Gly, Val, Ile, Tyr and Phe, are the major amino acids in Chameleons. We also found that there are proteins such as Insulin Degrading Enzyme IDE and GTP-binding nuclear protein Ran (RAN) with the most number of chameleons (640 and 405 respectively). These proteins have known roles in neurodegenerative diseases. Therefore it can be inferred that other CFP's can serve as key proteins in neurodegeneration, and a study on them can shed light on curing and preventing neurodegenerative diseases.

Chameleon sequences in neurodegenerative diseases

Energy Technology Data Exchange (ETDEWEB)

Bahramali, Golnaz [Institute of Biochemistry and Biophysics, University of Tehran, Tehran (Iran, Islamic Republic of); Goliaei, Bahram, E-mail: goliaei@ut.ac.ir [Institute of Biochemistry and Biophysics, University of Tehran, Tehran (Iran, Islamic Republic of); Minuchehr, Zarrin, E-mail: minuchehr@nigeb.ac.ir [Department of Systems Biotechnology, National Institute of Genetic Engineering and Biotechnology, (NIGEB), Tehran (Iran, Islamic Republic of); Salari, Ali [Department of Systems Biotechnology, National Institute of Genetic Engineering and Biotechnology, (NIGEB), Tehran (Iran, Islamic Republic of)

2016-03-25

Chameleon sequences can adopt either alpha helix sheet or a coil conformation. Defining chameleon sequences in PDB (Protein Data Bank) may yield to an insight on defining peptides and proteins responsible in neurodegeneration. In this research, we benefitted from the large PDB and performed a sequence analysis on Chameleons, where we developed an algorithm to extract peptide segments with identical sequences, but different structures. In order to find new chameleon sequences, we extracted a set of 8315 non-redundant protein sequences from the PDB with an identity less than 25%. Our data was classified to “helix to strand (HE)”, “helix to coil (HC)” and “strand to coil (CE)” alterations. We also analyzed the occurrence of singlet and doublet amino acids and the solvent accessibility in the chameleon sequences; we then sorted out the proteins with the most number of chameleon sequences and named them Chameleon Flexible Proteins (CFPs) in our dataset. Our data revealed that Gly, Val, Ile, Tyr and Phe, are the major amino acids in Chameleons. We also found that there are proteins such as Insulin Degrading Enzyme IDE and GTP-binding nuclear protein Ran (RAN) with the most number of chameleons (640 and 405 respectively). These proteins have known roles in neurodegenerative diseases. Therefore it can be inferred that other CFP's can serve as key proteins in neurodegeneration, and a study on them can shed light on curing and preventing neurodegenerative diseases.

Peptides of the constant region of antibodies display fungicidal activity.

Directory of Open Access Journals (Sweden)

Luciano Polonelli

Full Text Available Synthetic peptides with sequences identical to fragments of the constant region of different classes (IgG, IgM, IgA of antibodies (Fc-peptides exerted a fungicidal activity in vitro against pathogenic yeasts, such as Candida albicans, Candida glabrata, Cryptococcus neoformans, and Malassezia furfur, including caspofungin and triazole resistant strains. Alanine-substituted derivatives of fungicidal Fc-peptides, tested to evaluate the critical role of each residue, displayed unaltered, increased or decreased candidacidal activity in vitro. An Fc-peptide, included in all human IgGs, displayed a therapeutic effect against experimental mucosal and systemic candidiasis in mouse models. It is intriguing to hypothesize that some Fc-peptides may influence the antifungal immune response and constitute the basis for devising new antifungal agents.

Peptides of the Constant Region of Antibodies Display Fungicidal Activity

Science.gov (United States)

Polonelli, Luciano; Ciociola, Tecla; Magliani, Walter; Zanello, Pier Paolo; D'Adda, Tiziana; Galati, Serena; De Bernardis, Flavia; Arancia, Silvia; Gabrielli, Elena; Pericolini, Eva; Vecchiarelli, Anna; Arruda, Denise C.; Pinto, Marcia R.; Travassos, Luiz R.; Pertinhez, Thelma A.; Spisni, Alberto; Conti, Stefania

2012-01-01

Synthetic peptides with sequences identical to fragments of the constant region of different classes (IgG, IgM, IgA) of antibodies (Fc-peptides) exerted a fungicidal activity in vitro against pathogenic yeasts, such as Candida albicans, Candida glabrata, Cryptococcus neoformans, and Malassezia furfur, including caspofungin and triazole resistant strains. Alanine-substituted derivatives of fungicidal Fc-peptides, tested to evaluate the critical role of each residue, displayed unaltered, increased or decreased candidacidal activity in vitro. An Fc-peptide, included in all human IgGs, displayed a therapeutic effect against experimental mucosal and systemic candidiasis in mouse models. It is intriguing to hypothesize that some Fc-peptides may influence the antifungal immune response and constitute the basis for devising new antifungal agents. PMID:22470523

Antimicrobial Peptides: An Introduction.

Science.gov (United States)

Haney, Evan F; Mansour, Sarah C; Hancock, Robert E W

2017-01-01

The "golden era" of antibiotic discovery has long passed, but the need for new antibiotics has never been greater due to the emerging threat of antibiotic resistance. This urgency to develop new antibiotics has motivated researchers to find new methods to combat pathogenic microorganisms resulting in a surge of research focused around antimicrobial peptides (AMPs; also termed host defense peptides) and their potential as therapeutics. During the past few decades, more than 2000 AMPs have been identified from a diverse range of organisms (animals, fungi, plants, and bacteria). While these AMPs share a number of common features and a limited number of structural motifs; their sequences, activities, and targets differ considerably. In addition to their antimicrobial effects, AMPs can also exhibit immunomodulatory, anti-biofilm, and anticancer activities. These diverse functions have spurred tremendous interest in research aimed at understanding the activity of AMPs, and various protocols have been described to assess different aspects of AMP function including screening and evaluating the activities of natural and synthetic AMPs, measuring interactions with membranes, optimizing peptide function, and scaling up peptide production. Here, we provide a general overview of AMPs and introduce some of the methodologies that have been used to advance AMP research.

Prediction of monomer isomery in Florine: a workflow dedicated to nonribosomal peptide discovery.

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Thibault Caradec

Full Text Available Nonribosomal peptides represent a large variety of natural active compounds produced by microorganisms. Due to their specific biosynthesis pathway through large assembly lines called NonRibosomal Peptide Synthetases (NRPSs, they often display complex structures with cycles and branches. Moreover they often contain non proteogenic or modified monomers, such as the D-monomers produced by epimerization. We investigate here some sequence specificities of the condensation (C and epimerization (E domains of NRPS that can be used to predict the possible isomeric state (D or L of each monomer in a putative peptide. We show that C- and E- domains can be divided into 2 sub-regions called Up-Seq and Down-Seq. The Up-Seq region corresponds to an InterPro domain (IPR001242 and is shared by C- and E-domains. The Down-Seq region is specific to the enzymatic activity of the domain. Amino-acid signatures (represented as sequence logos previously described for complete C-and E-domains have been restricted to the Down-Seq region and amplified thanks to additional sequences. Moreover a new Down-Seq signature has been found for Ct-domains found in fungi and responsible for terminal cyclization of the peptides. The identification of these signatures has been included in a workflow named Florine, aimed to predict nonribosomal peptides from NRPS sequence analyses. In some cases, the prediction of isomery is guided by genus-specific rules. Florine was used on a Pseudomonas genome to allow the determination of the type of pyoverdin produced, the update of syringafactin structure and the identification of novel putative products.

Studies on lactoferricin-derived Escherichia coli membrane-active peptides reveal differences in the mechanism of N-acylated versus nonacylated peptides.

Science.gov (United States)

Zweytick, Dagmar; Deutsch, Günter; Andrä, Jörg; Blondelle, Sylvie E; Vollmer, Ekkehard; Jerala, Roman; Lohner, Karl

2011-06-17

To improve the low antimicrobial activity of LF11, an 11-mer peptide derived from human lactoferricin, mutant sequences were designed based on the defined structure of LF11 in the lipidic environment. Thus, deletion of noncharged polar residues and strengthening of the hydrophobic N-terminal part upon adding a bulky hydrophobic amino acid or N-acylation resulted in enhanced antimicrobial activity against Escherichia coli, which correlated with the peptides' degree of perturbation of bacterial membrane mimics. Nonacylated and N-acylated peptides exhibited different effects at a molecular level. Nonacylated peptides induced segregation of peptide-enriched and peptide-poor lipid domains in negatively charged bilayers, although N-acylated peptides formed small heterogeneous domains resulting in a higher degree of packing defects. Additionally, only N-acylated peptides perturbed the lateral packing of neutral lipids and exhibited increased permeability of E. coli lipid vesicles. The latter did not correlate with the extent of improvement of the antimicrobial activity, which could be explained by the fact that elevated binding of N-acylated peptides to lipopolysaccharides of the outer membrane of gram-negative bacteria seems to counteract the elevated membrane permeabilization, reflected in the respective minimal inhibitory concentration for E. coli. The antimicrobial activity of the peptides correlated with an increase of membrane curvature stress and hence bilayer instability. Transmission electron microscopy revealed that only the N-acylated peptides induced tubular protrusions from the outer membrane, whereas all peptides caused detachment of the outer and inner membrane of E. coli bacteria. Viability tests demonstrated that these bacteria were dead before onset of visible cell lysis.

Studies on Lactoferricin-derived Escherichia coli Membrane-active Peptides Reveal Differences in the Mechanism of N-Acylated Versus Nonacylated Peptides*

Science.gov (United States)

Zweytick, Dagmar; Deutsch, Günter; Andrä, Jörg; Blondelle, Sylvie E.; Vollmer, Ekkehard; Jerala, Roman; Lohner, Karl

2011-01-01

To improve the low antimicrobial activity of LF11, an 11-mer peptide derived from human lactoferricin, mutant sequences were designed based on the defined structure of LF11 in the lipidic environment. Thus, deletion of noncharged polar residues and strengthening of the hydrophobic N-terminal part upon adding a bulky hydrophobic amino acid or N-acylation resulted in enhanced antimicrobial activity against Escherichia coli, which correlated with the peptides' degree of perturbation of bacterial membrane mimics. Nonacylated and N-acylated peptides exhibited different effects at a molecular level. Nonacylated peptides induced segregation of peptide-enriched and peptide-poor lipid domains in negatively charged bilayers, although N-acylated peptides formed small heterogeneous domains resulting in a higher degree of packing defects. Additionally, only N-acylated peptides perturbed the lateral packing of neutral lipids and exhibited increased permeability of E. coli lipid vesicles. The latter did not correlate with the extent of improvement of the antimicrobial activity, which could be explained by the fact that elevated binding of N-acylated peptides to lipopolysaccharides of the outer membrane of Gram-negative bacteria seems to counteract the elevated membrane permeabilization, reflected in the respective minimal inhibitory concentration for E. coli. The antimicrobial activity of the peptides correlated with an increase of membrane curvature stress and hence bilayer instability. Transmission electron microscopy revealed that only the N-acylated peptides induced tubular protrusions from the outer membrane, whereas all peptides caused detachment of the outer and inner membrane of E. coli bacteria. Viability tests demonstrated that these bacteria were dead before onset of visible cell lysis. PMID:21515687

Peptide-conjugated micelles as a targeting nanocarrier for gene delivery

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Lin, Wen Jen, E-mail: wjlin@ntu.edu.tw; Chien, Wei Hsuan [National Taiwan University, School of Pharmacy, Graduate Institute of Pharmaceutical Sciences (China)

2015-09-15

The aim of this study was to develop peptide-conjugated micelles possessing epidermal growth factor receptor (EGFR) targeting ability for gene delivery. A sequence-modified dodecylpeptide, GE11(2R), with enhancing EGF receptor binding affinity, was applied in this study as a targeting ligand. The active targeting micelles were composed of poly(d,l-lactide-co-glycolide)-poly(ethylene glycol) (PLGA-PEG) copolymer conjugated with GE11(2R)-peptide. The particle sizes of peptide-free and peptide-conjugated micelles were 277.0 ± 5.1 and 308.7 ± 14.5 nm, respectively. The peptide-conjugated micelles demonstrated the cellular uptake significantly higher than peptide-free micelles in EGFR high-expressed MDA-MB-231 and MDA-MB-468 cells due to GE11(2R)-peptide specificity. Furthermore, the peptide-conjugated micelles were able to encapsulate plasmid DNA and expressed cellular transfection higher than peptide-free micelles in EGFR high-expressed cells. The EGFR-targeting delivery micelles enhanced DNA internalized into cells and achieved higher cellular transfection in EGFR high-expressed cells.

The fsr Quorum-Sensing System and Cognate Gelatinase Orchestrate the Expression and Processing of Proprotein EF_1097 into the Mature Antimicrobial Peptide Enterocin O16.

Science.gov (United States)

Dundar, Halil; Brede, Dag A; La Rosa, Sabina Leanti; El-Gendy, Ahmed Osama; Diep, Dzung B; Nes, Ingolf F

2015-07-01

A novel antimicrobial peptide designated enterocin O16 was purified from Enterococcus faecalis. Mass spectrometry showed a monoisotopic mass of 7,231 Da, and N-terminal Edman degradation identified a 29-amino-acid sequence corresponding to residues 90 to 119 of the EF_1097 protein. Bioinformatic analysis showed that enterocin O16 is composed of the 68 most C-terminal residues of the EF_1097 protein. Introduction of an in-frame isogenic deletion in the ef1097 gene abolished the production of enterocin O16. Enterocin O16 has a narrow inhibitory spectrum, as it inhibits mostly lactobacilli. Apparently, E. faecalis is intrinsically resistant to the antimicrobial peptide, as no immunity connected to the production of enterocin O16 could be identified. ef1097 has previously been identified as one of three loci regulated by the fsr quorum-sensing system. The introduction of a nonsense mutation into fsrB consistently impaired enterocin O16 production, but externally added gelatinase biosynthesis-activating pheromone restored the antimicrobial activity. Functional genetic analysis showed that the EF_1097 proprotein is processed extracellularly into enterocin O16 by the metalloprotease GelE. Thus, it is evident that the fsr quorum-sensing system constitutes the regulatory unit that controls the expression of the EF_1097 precursor protein and the protease GelE and that the latter is required for the formation of enterocin O16. On the basis of these results, this study identified antibacterial antagonism as a novel aspect related to the function of fsr and provides a rationale for why ef1097 is part of the fsr regulon. The fsr quorum-sensing system modulates important physiological functions in E. faecalis via the activity of GelE. The present study presents a new facet of fsr signaling. The system controls the expression of three primary target operons (fsrABCD, gelE-sprE, and ef1097-ef1097b). We demonstrate that the concerted expression of these operons constitutes the

Design, synthesis and DNA interactions of a chimera between a platinum complex and an IHF mimicking peptide.

Science.gov (United States)

Rao, Harita; Damian, Mariana S; Alshiekh, Alak; Elmroth, Sofi K C; Diederichsen, Ulf

2015-12-28

Conjugation of metal complexes with peptide scaffolds possessing high DNA binding affinity has shown to modulate their biological activities and to enhance their interaction with DNA. In this work, a platinum complex/peptide chimera was synthesized based on a model of the Integration Host Factor (IHF), an architectural protein possessing sequence specific DNA binding and bending abilities through its interaction with a minor groove. The model peptide consists of a cyclic unit resembling the minor grove binding subdomain of IHF, a positively charged lysine dendrimer for electrostatic interactions with the DNA phosphate backbone and a flexible glycine linker tethering the two units. A norvaline derived artificial amino acid was designed to contain a dimethylethylenediamine as a bidentate platinum chelating unit, and introduced into the IHF mimicking peptides. The interaction of the chimeric peptides with various DNA sequences was studied by utilizing the following experiments: thermal melting studies, agarose gel electrophoresis for plasmid DNA unwinding experiments, and native and denaturing gel electrophoresis to visualize non-covalent and covalent peptide-DNA adducts, respectively. By incorporation of the platinum metal center within the model peptide mimicking IHF we have attempted to improve its specificity and DNA targeting ability, particularly towards those sequences containing adjacent guanine residues.

Antimicrobial Peptides for Therapeutic Applications: A Review

Directory of Open Access Journals (Sweden)

Tsogbadrakh Mishig-Ochir

2012-10-01

Full Text Available Antimicrobial peptides (AMPs have been considered as potential therapeutic sources of future antibiotics because of their broad-spectrum activities and different mechanisms of action compared to conventional antibiotics. Although AMPs possess considerable benefits as new generation antibiotics, their clinical and commercial development still have some limitations, such as potential toxicity, susceptibility to proteases, and high cost of peptide production. In order to overcome those obstacles, extensive efforts have been carried out. For instance, unusual amino acids or peptido-mimetics are introduced to avoid the proteolytic degradation and the design of short peptides retaining antimicrobial activities is proposed as a solution for the cost issue. In this review, we focus on small peptides, especially those with less than twelve amino acids, and provide an overview of the relationships between their three-dimensional structures and antimicrobial activities. The efforts to develop highly active AMPs with shorter sequences are also described.

Comprehensive computational design of ordered peptide macrocycles

Science.gov (United States)

Hosseinzadeh, Parisa; Bhardwaj, Gaurav; Mulligan, Vikram Khipple; Shortridge, Matthew D.; Craven, Timothy W.; Pardo-Avila, Fátima; Rettie, Stephen A.; Kim, David E.; Silva, Daniel-Adriano; Ibrahim, Yehia M.; Webb, Ian K.; Cort, John R.; Adkins, Joshua N.; Varani, Gabriele; Baker, David

2018-01-01

Mixed-chirality peptide macrocycles such as cyclosporine are among the most potent therapeutics identified to date, but there is currently no way to systematically search the structural space spanned by such compounds. Natural proteins do not provide a useful guide: Peptide macrocycles lack regular secondary structures and hydrophobic cores, and can contain local structures not accessible with L-amino acids. Here, we enumerate the stable structures that can be adopted by macrocyclic peptides composed of L- and D-amino acids by near-exhaustive backbone sampling followed by sequence design and energy landscape calculations. We identify more than 200 designs predicted to fold into single stable structures, many times more than the number of currently available unbound peptide macrocycle structures. Nuclear magnetic resonance structures of 9 of 12 designed 7- to 10-residue macrocycles, and three 11- to 14-residue bicyclic designs, are close to the computational models. Our results provide a nearly complete coverage of the rich space of structures possible for short peptide macrocycles and vastly increase the available starting scaffolds for both rational drug design and library selection methods. PMID:29242347

Designed graphene-peptide nanocomposites for biosensor applications: A review

International Nuclear Information System (INIS)

Wang, Li; Zhang, Yujie; Wu, Aiguo; Wei, Gang

2017-01-01

The modification of graphene with biomacromolecules like DNA, protein, peptide, and others extends the potential applications of graphene materials in various fields. The bound biomacromolecules could improve the biocompatibility and bio-recognition ability of graphene-based nanocomposites, therefore could greatly enhance their biosensing performances on both selectivity and sensitivity. In this review, we presented a comprehensive introduction and discussion on recent advance in the synthesis and biosensor applications of graphene-peptide nanocomposites. The biofunctionalization of graphene with specifically designed peptides, and the synthesis strategies of graphene-peptide (monomer, nanofibrils, and nanotubes) nanocomposites were demonstrated. On the other hand, the fabrication of graphene-peptide nanocomposite based biosensor architectures for electrochemical, fluorescent, electronic, and spectroscopic biosensing were further presented. This review includes nearly all the studies on the fabrication and applications of graphene-peptide based biosensors recently, which will promote the future developments of graphene-based biosensors in biomedical detection and environmental analysis. - Highlights: • A comprehensive review on the fabrication and application of graphene-peptide nanocomposites was presented. • The design of peptide sequences for biofunctionalization of various graphene materials was presented. • Multi-strategies on the fabrication of biosensors with graphene-peptide nanocomposites were discussed. • Designed graphene-peptide nanocomposites showed wide biosensor applications.

Binding of cholesterol and inhibitory peptide derivatives with the fusogenic hydrophobic sequence of F-glycoprotein of HVJ (Sendai virus): possible implication in the fusion reaction

International Nuclear Information System (INIS)

Asano, K.; Asano, A.

1988-01-01

Specificity of the binding of sterols and related compounds with purified F-protein (fusion protein) of the HVJ (Sendai virus) was studied by binding competition with [ 3 H] cholesterol. Requirement for cholesterol or the A/B ring trans structure and nonrequirement for the 3-hydroxyl group were found in this binding. Binding of 125 I-labeled Z-Phe-Tyr, an inhibitory peptide of viral membrane-cell membrane fusion, was studied by using purified proteins and virions. F-Protein and virions showed a specific binding with the peptide, whereas the result was negative with hemagglutinin and neuraminidase protein. Thermolysin-truncated F-protein (an F-protein derivative deprived of a 2.5-kDa fragment from the N-terminal of the F 1 subunit and without fusogenic activity) exhibited a considerably diminished binding ability both to cholesterol and to inhibitory peptides. Therefore, the N-terminal hydrophobic sequence that was previously assigned as fusogenic seems to be the binding site of these molecules. In support of this, the binding of cholesterol with F-protein was inhibited by Z-Phe-Tyr and other fusion inhibitory peptides, whereas it was not affected with non-fusion-inhibitory Z-Gly-Phe. These results are discussed in relation to the notion that the binding of the N-terminal portion of the F 1 subunit of F-protein with cholesterol in the target cell membranes facilitiates the fusion reaction

Identification of Cyclin A Binders with a Fluorescent Peptide Sensor.

Science.gov (United States)

Pazos, Elena; Mascareñas, José L; Vázquez, M Eugenio

2016-01-01

A peptide sensor that integrates the 4-dimethylaminophthalimide (4-DMAP) fluorophore in a short cyclin A binding sequence displays a large fluorescence emission increase upon interacting with the cyclin A Binding Groove (CBG). Competitive displacement assays of this probe allow the straightforward identification of peptides that interact with the CBG, which could potentially block the recognition of CDK/cyclin A kinase substrates.

Topological disposition of the sequences -QRKIVE- and -KETYY in native (Na sup + + K sup + )-ATPase

Energy Technology Data Exchange (ETDEWEB)

Bayer, R. (Univ. of California, San Diego, La Jolla (USA))

1990-03-06

The dispositions with respect to the plane of the membrane of lysine-905 in the internal sequence -EQRKIVE- and of lysine-1012 in the carboxy-terminal sequence -RRPGGWVEKETYY of the {alpha}-polypeptide of sodium and potassium ion activated adenosinetriphosphatase have been determined. These lysines are found in peptides released from the intact {alpha}-polypeptide by the extracellular protease from Staphylococcus aureus strain V8 and by trypsin, respectively. Synthetic peptides containing terminal sequences of these were used to prepare polyclonal antibodies, which were then used to prepare immunoadsorbents directed against the respective peptides. Sealed, right-side-out membrane vesicles containing native (Na{sup +} + K{sup +})-ATPase were labeled with pyridoxal phosphate and sodium ({sup 3}H)borohydride in the absence or presence of saponin. The labeled {alpha}-polypeptide was isolated from these vesicles and digested with appropriate proteases. The incorporation of radioactivity into the peptides binding to the immunoadsorbent directed against the sequence pyrERXIVE increased 3-fold int the presence of saponin as a result of the increased accessibility of this portion of the protein to the reagent when the vesicles were breached by saponin; hence, this sequence is located on the cytoplasmic face of the membrane. It was inferred that the carboxy-terminal sequence -KETYY is on the extracytoplasmic face since the incorporation of radioactivity into peptides binding to the immunoadsorbent directed against the sequence -ETYY did not change when the vesicles were breached with saponin.

Expansion for the Brachylophosaurus canadensis Collagen I Sequence and Additional Evidence of the Preservation of Cretaceous Protein.

Science.gov (United States)

Schroeter, Elena R; DeHart, Caroline J; Cleland, Timothy P; Zheng, Wenxia; Thomas, Paul M; Kelleher, Neil L; Bern, Marshall; Schweitzer, Mary H

2017-02-03

Sequence data from biomolecules such as DNA and proteins, which provide critical information for evolutionary studies, have been assumed to be forever outside the reach of dinosaur paleontology. Proteins, which are predicted to have greater longevity than DNA, have been recovered from two nonavian dinosaurs, but these results remain controversial. For proteomic data derived from extinct Mesozoic organisms to reach their greatest potential for investigating questions of phylogeny and paleobiology, it must be shown that peptide sequences can be reliably and reproducibly obtained from fossils and that fragmentary sequences for ancient proteins can be increasingly expanded. To test the hypothesis that peptides can be repeatedly detected and validated from fossil tissues many millions of years old, we applied updated extraction methodology, high-resolution mass spectrometry, and bioinformatics analyses on a Brachylophosaurus canadensis specimen (MOR 2598) from which collagen I peptides were recovered in 2009. We recovered eight peptide sequences of collagen I: two identical to peptides recovered in 2009 and six new peptides. Phylogenetic analyses place the recovered sequences within basal archosauria. When only the new sequences are considered, B. canadensis is grouped more closely to crocodylians, but when all sequences (current and those reported in 2009) are analyzed, B. canadensis is placed more closely to basal birds. The data robustly support the hypothesis of an endogenous origin for these peptides, confirm the idea that peptides can survive in specimens tens of millions of years old, and bolster the validity of the 2009 study. Furthermore, the new data expand the coverage of B. canadensis collagen I (a 33.6% increase in collagen I alpha 1 and 116.7% in alpha 2). Finally, this study demonstrates the importance of reexamining previously studied specimens with updated methods and instrumentation, as we obtained roughly the same amount of sequence data as the

Isolation and structure elucidation of a neuropeptide from three ...

African Journals Online (AJOL)

1993-03-03

Mar 3, 1993 ... determination of the primary structure by pulsed·liquid phase sequencing employing Edman chemistry .... in cockroaches and haemo1ymph lipids in locusts (Glide ..... 15 ()()() described species), and higher classification is still.

A consistent nomenclature of antimicrobial peptides isolated from frogs of the subfamily Phyllomedusinae.

Science.gov (United States)

Amiche, Mohamed; Ladram, Ali; Nicolas, Pierre

2008-11-01

A growing number of cationic antimicrobial peptides have been isolated from the skin of hylid frogs belonging to the Phyllomedusinae subfamily. The amino acid sequences of these peptides are currently located in several databases under identifiers with no consistent system of nomenclature to describe them. In order to provide a workable terminology for antimicrobial peptides from Phyllomedusid frogs, we have made a systematic effort to collect, analyze, and classify all the Phyllomedusid peptide sequences available in databases. We propose that frogs belonging to the Phyllomedusinae subfamily should be described by the species names set out in Amphibian Species of the World: http://research.amnh.org/herpetology/amphibia/index.php, American Museum of Natural History, New York, USA. Multiple alignments analysis of at least 80 antimicrobial peptides isolated from 12 Phyllomedusinae species were distributed in seven distinct peptide families including dermaseptin, phylloseptin, plasticin, dermatoxin, phylloxin, hyposin and orphan peptides, and will be considered as the name of the headgroup of each family. The parent peptide's name should be followed by the first upper letter of the species for orthologous peptides and publication date determines priority. For example, the abbreviation B for bicolor and H for hypochondrialis. When two species begin with the same letter, two letters in upper case should be used (the first letter followed by the second or the third letter and so on). For example, the abbreviation DI for distincta, DU for duellmani, VA for vaillanti and VN for vanzolinii. Paralogous peptides should bear letter(s) in upper case followed by numbers.

A novel monoclonal antibody to a defined peptide epitope in MUC16

DEFF Research Database (Denmark)

Marcos-Silva, Lara; Ricardo, Sara; Chen, Kowa

2015-01-01

with the tandem-repeat region, their epitopes appear to be conformational dependent and not definable by a short peptide. Aberrant glycoforms of MUC16 may constitute promising targets for diagnostic and immunotherapeutic intervention, and it is important to develop well-defined immunogens for induction of potent...... immunodominant linear peptide epitopes within the tandem repeat. We developed one monoclonal antibody, 5E11, reactive with a minimum epitope with the sequence FNTTER. This sequence contains potential N- and O-glycosylation sites and, interestingly, glycosylation blocked binding of 5E11. In immunochemistry...

Dual peptide conjugation strategy for improved cellular uptake and mitochondria targeting.

Science.gov (United States)

Lin, Ran; Zhang, Pengcheng; Cheetham, Andrew G; Walston, Jeremy; Abadir, Peter; Cui, Honggang

2015-01-21

Mitochondria are critical regulators of cellular function and survival. Delivery of therapeutic and diagnostic agents into mitochondria is a challenging task in modern pharmacology because the molecule to be delivered needs to first overcome the cell membrane barrier and then be able to actively target the intracellular organelle. Current strategy of conjugating either a cell penetrating peptide (CPP) or a subcellular targeting sequence to the molecule of interest only has limited success. We report here a dual peptide conjugation strategy to achieve effective delivery of a non-membrane-penetrating dye 5-carboxyfluorescein (5-FAM) into mitochondria through the incorporation of both a mitochondrial targeting sequence (MTS) and a CPP into one conjugated molecule. Notably, circular dichroism studies reveal that the combined use of α-helix and PPII-like secondary structures has an unexpected, synergistic contribution to the internalization of the conjugate. Our results suggest that although the use of positively charged MTS peptide allows for improved targeting of mitochondria, with MTS alone it showed poor cellular uptake. With further covalent linkage of the MTS-5-FAM conjugate to a CPP sequence (R8), the dually conjugated molecule was found to show both improved cellular uptake and effective mitochondria targeting. We believe these results offer important insight into the rational design of peptide conjugates for intracellular delivery.

Sequence-specific unusual (1-->2)-type helical turns in alpha/beta-hybrid peptides.

Science.gov (United States)

Prabhakaran, Panchami; Kale, Sangram S; Puranik, Vedavati G; Rajamohanan, P R; Chetina, Olga; Howard, Judith A K; Hofmann, Hans-Jörg; Sanjayan, Gangadhar J

2008-12-31

This article describes novel conformationally ordered alpha/beta-hybrid peptides consisting of repeating l-proline-anthranilic acid building blocks. These oligomers adopt a compact, right-handed helical architecture determined by the intrinsic conformational preferences of the individual amino acid residues. The striking feature of these oligomers is their ability to display an unusual periodic pseudo beta-turn network of nine-membered hydrogen-bonded rings formed in the forward direction of the sequence by 1-->2 amino acid interactions both in solid-state and in solution. Conformational investigations of several of these oligomers by single-crystal X-ray diffraction, solution-state NMR, and ab initio MO theory suggest that the characteristic steric and dihedral angle restraints exerted by proline are essential for stabilizing the unusual pseudo beta-turn network found in these oligomers. Replacing proline by the conformationally flexible analogue alanine (Ala) or by the conformationally more constrained alpha-amino isobutyric acid (Aib) had an adverse effect on the stabilization of this structural architecture. These findings increase the potential to design novel secondary structure elements profiting from the steric and dihedral angle constraints of the amino acid constituents and help to augment the conformational space available for synthetic oligomer design with diverse backbone structures.

Thermodynamics of sequence-specific binding of PNA to DNA

DEFF Research Database (Denmark)

Ratilainen, T; Holmén, A; Tuite, E

2000-01-01

For further characterization of the hybridization properties of peptide nucleic acids (PNAs), the thermodynamics of hybridization of mixed sequence PNA-DNA duplexes have been studied. We have characterized the binding of PNA to DNA in terms of binding affinity (perfectly matched duplexes) and seq......For further characterization of the hybridization properties of peptide nucleic acids (PNAs), the thermodynamics of hybridization of mixed sequence PNA-DNA duplexes have been studied. We have characterized the binding of PNA to DNA in terms of binding affinity (perfectly matched duplexes...

cDNA sequences of two inducible T-cell genes

Energy Technology Data Exchange (ETDEWEB)

Kwon, B.S. (Indiana Univ. School of Medicine, Indianapolis (USA) Guthrie Research Institute, Sayre, PA (USA)); Weissman, S.M. (Yale Univ., New Haven, CT (USA))

1989-03-01

The authors have previously described a set of human T-lymphocyte-specific cDNA clones isolated by a modified differential screening procedure. Apparent full-length cDNAs containing the sequences of 14 of the 16 initial isolates were sequenced and were found to represent five different species of mRNA; three of the five species were identical to previously reported cDNA sequences of preproenkephalin, T-cell-replacing factor, and a serine esterase, respectively. The other two species, 4-1BB and L2G25B, were inducible sequences found in mRNA from both a cytolytic T-lymphocyte and a helper T-lymphocyte clone and were not previously described in T-cell mRNA; these mRNA sequences encode peptides of 256 and 92 amino acids, respectively. Both peptides contain putative leader sequences. The protein encoded by 4-1BB also has a potential membrane anchor segment and other features also seen in known receptor proteins.

A peptide-binding assay for the disease-associated HLA-DQ8 molecule

DEFF Research Database (Denmark)

Straumfors, A; Johansen, B H; Vartdal, F

1998-01-01

The study of peptide binding to HLA class II molecules has mostly concentrated on DR molecules. Since many autoimmune diseases show a primary association to particular DQ molecules rather than DR molecules, it is also important to study the peptide-binding properties of DQ molecules. Here we repo......-affinity binders, whereas peptides derived from myelin basic protein were among the low-affinity binders. The sequence of the high-affinity peptides conformed with a previously published peptide-binding motif of DQ8.......The study of peptide binding to HLA class II molecules has mostly concentrated on DR molecules. Since many autoimmune diseases show a primary association to particular DQ molecules rather than DR molecules, it is also important to study the peptide-binding properties of DQ molecules. Here we report...

Identification and screening of potent antimicrobial peptides in arthropod genomes.

Science.gov (United States)

Duwadi, Deepesh; Shrestha, Anishma; Yilma, Binyam; Kozlovski, Itamar; Sa-Eed, Munaya; Dahal, Nikesh; Jukosky, James

2018-05-01

Using tBLASTn and BLASTp searches, we queried recently sequenced arthropod genomes and expressed sequence tags (ESTs) using a database of known arthropod cecropins, defensins, and attacins. We identified and synthesized 6 potential AMPs and screened them for antimicrobial activity. Using radial diffusion assays and microtiter antimicrobial assays, we assessed the in vitro antimicrobial effects of these peptides against several human pathogens including Gram-positive and Gram-negative bacteria and fungi. We also conducted hemolysis assays to examine the cytotoxicity of these peptides to mammalian cells. Four of the six peptides identified showed antimicrobial effects in these assays. We also created truncated versions of these four peptides to assay their antimicrobial activity. Two cecropins derived from the monarch butterfly genome (Danaus plexippus), DAN1 and DAN2, showed minimum inhibitory concentrations (MICs) in the range of 2-16 μg/ml when screened against Gram-negative bacteria. HOLO1 and LOUDEF1, two defensin-like peptides derived from red flour beetle (Tribolium castaneum) and human body louse (Pediculus humanus humanus), respectively, exhibited MICs in the range of 13-25 μg/ml against Gram-positive bacteria. Furthermore, HOLO1 showed an MIC less than 5 μg/ml against the fungal species Candida albicans. These peptides exhibited no hemolytic activity at concentrations up to 200 μg/ml. The truncated peptides derived from DAN2 and HOLO1 showed very little antimicrobial activity. Our experiments show that the peptides DAN1, DAN2, HOLO1, and LOUDEF1 showed potent antimicrobial activity in vitro against common human pathogens, did not lyse mammalian red blood cells, and indicates their potential as templates for novel therapeutic agents against microbial infection. Copyright © 2018 Elsevier Inc. All rights reserved.

Antimicrobial peptides: the role of hydrophobicity in the alpha helical structure

Directory of Open Access Journals (Sweden)

Pandurangan Perumal

2013-12-01

Full Text Available The antimicrobial peptides (AMPs are a class of molecule obtained from plants, insects, animals, and humans. These peptides have been classified into five categories: 1. Anionic peptide, 2. Linear alpha helical cationic peptide, 3. Cationic peptide, 4. Anionic and cationic peptides with disulphide bonds, and 5. Anionic and cationic peptide fragments of larger proteins. Factors affecting AMPs are sequence, size, charge, hydrophobicity, amphipathicity, structure and conformation. Synthesis of these peptides is convenient by using solid phase peptide synthesis by using FMOC chemistry protocol. The secondary structures of three synthetic peptides were determined by circular dichroism. Also, it was compared the stability of the α-helical structure and confirmed the percentage of helix of these peptides by using circular dichroism. Some of these AMPs show therapeutic properties like antimicrobial, antiviral, contraceptive, and anticancer. The formulations of some peptides have been entered into the phase I, II, or III of clinical trials. This article to review briefly the sources, classification, factors affecting AMPs activity, synthesis, characterization, mechanism of action and therapeutic concern of AMPs and mainly focussed on percentage of α-helical structure in various medium.

Antibacterial activity of synthetic peptides derived from lactoferricin against Escherichia coli ATCC 25922 and Enterococcus faecalis ATCC 29212.

Science.gov (United States)

León-Calvijo, María A; Leal-Castro, Aura L; Almanzar-Reina, Giovanni A; Rosas-Pérez, Jaiver E; García-Castañeda, Javier E; Rivera-Monroy, Zuly J

2015-01-01

Peptides derived from human and bovine lactoferricin were designed, synthesized, purified, and characterized using RP-HPLC and MALDI-TOF-MS. Specific changes in the sequences were designed as (i) the incorporation of unnatural amino acids in the sequence, the (ii) reduction or (iii) elongation of the peptide chain length, and (iv) synthesis of molecules with different number of branches containing the same sequence. For each peptide, the antibacterial activity against Escherichia coli ATCC 25922 and Enterococcus faecalis ATCC 29212 was evaluated. Our results showed that Peptides I.2 (RWQWRWQWR) and I.4 ((RRWQWR)4K2Ahx2C2) exhibit bigger or similar activity against E. coli (MIC 4-33 μM) and E. faecalis (MIC 10-33 μM) when they were compared with lactoferricin protein (LF) and some of its derivate peptides as II.1 (FKCRRWQWRMKKLGA) and IV.1 (FKCRRWQWRMKKLGAPSITCVRRAE). It should be pointed out that Peptides I.2 and I.4, containing the RWQWR motif, are short and easy to synthesize; our results demonstrate that it is possible to design and obtain synthetic peptides that exhibit enhanced antibacterial activity using a methodology that is fast and low-cost and that allows obtaining products with a high degree of purity and high yield.

Antibacterial Activity of Synthetic Peptides Derived from Lactoferricin against Escherichia coli ATCC 25922 and Enterococcus faecalis ATCC 29212

Directory of Open Access Journals (Sweden)

María A. León-Calvijo

2015-01-01

Full Text Available Peptides derived from human and bovine lactoferricin were designed, synthesized, purified, and characterized using RP-HPLC and MALDI-TOF-MS. Specific changes in the sequences were designed as (i the incorporation of unnatural amino acids in the sequence, the (ii reduction or (iii elongation of the peptide chain length, and (iv synthesis of molecules with different number of branches containing the same sequence. For each peptide, the antibacterial activity against Escherichia coli ATCC 25922 and Enterococcus faecalis ATCC 29212 was evaluated. Our results showed that Peptides I.2 (RWQWRWQWR and I.4 ((RRWQWR4K2Ahx2C2 exhibit bigger or similar activity against E. coli (MIC 4–33 μM and E. faecalis (MIC 10–33 μM when they were compared with lactoferricin protein (LF and some of its derivate peptides as II.1 (FKCRRWQWRMKKLGA and IV.1 (FKCRRWQWRMKKLGAPSITCVRRAE. It should be pointed out that Peptides I.2 and I.4, containing the RWQWR motif, are short and easy to synthesize; our results demonstrate that it is possible to design and obtain synthetic peptides that exhibit enhanced antibacterial activity using a methodology that is fast and low-cost and that allows obtaining products with a high degree of purity and high yield.

Sequence, taste and umami-enhancing effect of the peptides separated from soy sauce.

Science.gov (United States)

Zhuang, Mingzhu; Lin, Lianzhu; Zhao, Mouming; Dong, Yi; Sun-Waterhouse, Dongxiao; Chen, Huiping; Qiu, Chaoying; Su, Guowan

2016-09-01

Five tasty peptides were separated from soy sauce, by sensory-guided fractionation, using macroporous resin, medium-pressure liquid chromatography and reverse phase-high performance liquid chromatography, and identified by ultra-performance liquid chromatography tandem mass-spectrometry as ALPEEV, LPEEV, AQALQAQA, EQQQQ and EAGIQ (which originated from glycinin A1bB2-445, glycinin A1bB2-445, cobyric acid synthase, leucine-tRNA ligase and glycoprotein glucosyltransferase, respectively). LPEEV, AQALQAQA and EQQQQ tasted umami with threshold values of 0.43, 1.25 and 0.76mmol/l, respectively. ALPEEV and EAGIQ had minimal umami taste, but ALPEEV, EAGIQ and LPEEV showed umami-enhancement with a threshold estimated at 1.52, 1.94 and 3.41mmol/l, respectively. In addition, the synthetic peptides showed much better sensory taste than mixtures of their constitutive amino acids. It indicated that peptides might play an important role in the umami taste of soy sauce. Copyright © 2016 Elsevier Ltd. All rights reserved.

A large synthetic peptide and phosphopeptide reference library for mass spectrometry–based proteomics

NARCIS (Netherlands)

Marx, H.; Lemeer, S.; Schliep, J.E.; Matheron, L.I.; Mohammed, S.; Cox, J.; Mann, M.; Heck, A.J.R.; Kuster, B.

2013-01-01

We present a peptide library and data resource of >100,000 synthetic, unmodified peptides and their phosphorylated counterparts with known sequences and phosphorylation sites. Analysis of the library by mass spectrometry yielded a data set that we used to evaluate the merits of different search

Bioactive self-assembled peptide nanofibers for corneal stroma regeneration.

Science.gov (United States)

Uzunalli, G; Soran, Z; Erkal, T S; Dagdas, Y S; Dinc, E; Hondur, A M; Bilgihan, K; Aydin, B; Guler, M O; Tekinay, A B

2014-03-01

Defects in the corneal stroma caused by trauma or diseases such as macular corneal dystrophy and keratoconus can be detrimental for vision. Development of therapeutic methods to enhance corneal regeneration is essential for treatment of these defects. This paper describes a bioactive peptide nanofiber scaffold system for corneal tissue regeneration. These nanofibers are formed by self-assembling peptide amphiphile molecules containing laminin and fibronectin inspired sequences. Human corneal keratocyte cells cultured on laminin-mimetic peptide nanofibers retained their characteristic morphology, and their proliferation was enhanced compared with cells cultured on fibronectin-mimetic nanofibers. When these nanofibers were used for damaged rabbit corneas, laminin-mimetic peptide nanofibers increased keratocyte migration and supported stroma regeneration. These results suggest that laminin-mimetic peptide nanofibers provide a promising injectable, synthetic scaffold system for cornea stroma regeneration. Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Peptide inhibitors of botulinum neurotoxin by mRNA display

International Nuclear Information System (INIS)