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Sample records for acid peptide designated

  1. Designing amino acids to determine the local conformations of peptides.

    PubMed Central

    Burgess, A W

    1994-01-01

    The local conformations of proteins and peptides are determined by the amino acid sequence. However, the 20 amino acids encoded by the genome allow the peptide backbone to fold into many conformations, so that even for a small peptide it becomes very difficult to predict the three-dimensional structure. By using empirical conformational energy calculations, a set of amino acids has been designed that would be expected to constrain the conformation of a peptide or a protein to one or two local minima. Most of these amino acids are based on asymmetric substitutions at the C alpha atom of each residue. The H alpha atom of alanine was replaced by various groups: -OCH3, -NCH3, -SCH3, -CONH2, -CONHCH3, -CON(CH3)2, -NH.CO.CH3, -phenyl, or -o-(OCH3)phenyl. Several of these new amino acids are predicted to fold into unique peptide conformations such as right-handed alpha-helical, left-handed alpha-helical, or extended. In an attempt to produce an amino acid that favored the C(eq)7 conformation (torsion angles: phi = -70 degrees and psi = +70 degrees), an extra amide group was added to the C beta atom of the asparagine side chain. Conformationally restricted amino acids of this type could prove useful for developing new peptide pharmaceuticals, catalysts, or polymers. Images PMID:8146170

  2. Design and characterization of an acid-activated antimicrobial peptide.

    PubMed

    Li, Lina; He, Jian; Eckert, Randal; Yarbrough, Daniel; Lux, Renate; Anderson, Maxwell; Shi, Wenyuan

    2010-01-01

    Dental caries is a microbial biofilm infection in which the metabolic activities of plaque bacteria result in a dramatic pH decrease and shift the demineralization/remineralization equilibrium on the tooth surface towards demineralization. In addition to causing a net loss in tooth minerals, creation of an acidic environment favors growth of acid-enduring and acid-generating species, which causes further reduction in the plaque pH. In this study, we developed a prototype antimicrobial peptide capable of achieving high activity exclusively at low environmental pH to target bacterial species like Streptococcus mutans that produce acid and thrive under the low pH conditions detrimental for tooth integrity. The features of clavanin A, a naturally occurring peptide rich in histidine and phenylalanine residues with pH-dependent antimicrobial activity, served as a design basis for these prototype 'acid-activated peptides' (AAPs). Employing the major cariogenic species S. mutans as a model system, the two AAPs characterized in this study exhibited a striking pH-dependent antimicrobial activity, which correlated well with the calculated charge distribution. This type of peptide represents a potential new way to combat dental caries. PMID:19878192

  3. Incorporation of Noncanonical Amino Acids into Rosetta and Use in Computational Protein-Peptide Interface Design

    PubMed Central

    Renfrew, P. Douglas; Choi, Eun Jung; Bonneau, Richard; Kuhlman, Brian

    2012-01-01

    Noncanonical amino acids (NCAAs) can be used in a variety of protein design contexts. For example, they can be used in place of the canonical amino acids (CAAs) to improve the biophysical properties of peptides that target protein interfaces. We describe the incorporation of 114 NCAAs into the protein-modeling suite Rosetta. We describe our methods for building backbone dependent rotamer libraries and the parameterization and construction of a scoring function that can be used to score NCAA containing peptides and proteins. We validate these additions to Rosetta and our NCAA-rotamer libraries by showing that we can improve the binding of a calpastatin derived peptides to calpain-1 by substituting NCAAs for native amino acids using Rosetta. Rosetta (executables and source), auxiliary scripts and code, and documentation can be found at (http://www.rosettacommons.org/). PMID:22431978

  4. Synthetic antimicrobial peptide design.

    PubMed

    Powell, W A; Catranis, C M; Maynard, C A

    1995-01-01

    To guide the design of potential plant pathogen-resistance genes, synthetic variants of naturally occurring antimicrobial gene products were evaluated. Five 20-amino acid (ESF1, ESF4, ESF5, ESF6, ESF13), one 18-amino acid (ESF12), and one 17-amino acid (ESF17) amphipathic peptide sequences were designed, synthesized, and tested with in vitro bioassays. Positive charges on the hydrophilic side of the peptide were shown to be essential for antifungal activity, yet the number of positive charges could be varied with little or no change in activity. The size could be reduced to 18 amino acids, but at 17 amino acids a significant reduction in activity was observed. ESF1, 5, 6, and 12 peptides were inhibitory to the germination of conidia from Cryphonectria parasitica, Fusarium oxysporum f. sp. lycopersici, and Septoria musiva but did not inhibit the germination of pollen from Castanea mollissima and Salix lucida. ESF12 also had no effect on the germination of Malus sylvestris and Lycopersicon esculentum pollen, but inhibited the growth of the bacteria Agrobacterium tumefaciens, Erwinia amylovora, and Pseudomonas syringae. The minimal inhibitory concentrations of the active ESF peptides were similar to those of the naturally occurring control peptides, magainin II and cecropin B. The significant differential in sensitivity between the microbes and plant cells indicated that the active ESF peptides are potentially useful models for designing plant pathogen-resistance genes. PMID:7579625

  5. Calcium Binding to Amino Acids and Small Glycine Peptides in Aqueous Solution: Toward Peptide Design for Better Calcium Bioavailability.

    PubMed

    Tang, Ning; Skibsted, Leif H

    2016-06-01

    Deprotonation of amino acids as occurs during transfer from stomach to intestines during food digestion was found by comparison of complex formation constants as determined electrochemically for increasing pH to increase calcium binding (i) by a factor of around 6 for the neutral amino acids, (ii) by a factor of around 4 for anions of the acidic amino acids aspartic and glutamic acid, and (iii) by a factor of around 5.5 for basic amino acids. Optimized structures of the 1:1 complexes and ΔHbinding for calcium binding as calculated by density functional theory (DFT) confirmed in all complexes a stronger calcium binding and shorter calcium-oxygen bond length in the deprotonated form. In addition, the stronger calcium binding was also accompanied by a binding site shift from carboxylate binding to chelation by α-amino group and carboxylate oxygen for leucine, aspartate, glutamate, alanine, and asparagine. For binary amino acid mixtures, the calcium-binding constant was close to the predicted geometric mean of the individual amino acid binding constants indicating separate binding of calcium to two amino acids when present together in solution. At high pH, corresponding to conditions for calcium absorption, the binding affinity increased in the order Lys < Arg < Cys < Gln < Gly ∼ Ala < Asn < His < Leu < Glu< Asp. In a series of glycine peptides, calcium-binding affinity was found to increase in the order Gly-Leu ∼ Gly-Gly < Ala-Gly < Gly-His ∼ Gly-Lys-Gly < Glu-Cys-Gly < Gly-Glu, an ordering confirmed by DFT calculations for the dipeptides and which also accounted for large synergistic effects in calcium binding for up to 6 kJ/mol when compared to the corresponding amino acid mixtures. PMID:27159329

  6. Toxin acidic residue evolutionary function-guided design of de novo peptide drugs for the immunotherapeutic target, the Kv1.3 channel

    PubMed Central

    Chen, Zongyun; Hu, Youtian; Hong, Jing; Hu, Jun; Yang, Weishan; Xiang, Fang; Yang, Fan; Xie, Zili; Cao, Zhijian; Li, Wenxin; Lin, Donghai; Wu, Yingliang

    2015-01-01

    During the long-term evolution of animal toxins acting on potassium channels, the acidic residues can orientate the toxin binding interfaces by adjusting the molecular polarity. Based on the evolutionary function of toxin acidic residues, de novo peptide drugs with distinct binding interfaces were designed for the immunotherapeutic target, the Kv1.3 channel. Using a natural basic toxin, BmKTX, as a template, which contains 2 acidic residues (Asp19 and Asp33), we engineered two new peptides BmKTX-19 with 1 acidic residue (Asp33), and BmKTX-196 with 2 acidic residues (Asp6 and Asp33) through only adjusting acidic residue distribution for reorientation of BmKTX binding interface. Pharmacological experiments indicated that BmKTX-19 and BmKTX-196 peptides were specific inhibitors of the Kv1.3 channel and effectively suppressed cytokine secretion. In addition to the structural similarity between the designed and native peptides, both experimental alanine-scanning mutagenesis and computational simulation further indicated that the binding interface of wild-type BmKTX was successfully reoriented in BmKTX-19 and BmKTX-196, which adopted distinct toxin surfaces as binding interfaces. Together, these findings indicate not only the promising prospect of BmKTX-19 and BmKTX-196 as drug candidates but also the desirable feasibility of the evolution-guided peptide drug design for discovering numerous peptide drugs for the Kv1.3 channel. PMID:25955787

  7. Peptides and Peptidomimetics for Antimicrobial Drug Design

    PubMed Central

    Mojsoska, Biljana; Jenssen, Håvard

    2015-01-01

    The purpose of this paper is to introduce and highlight a few classes of traditional antimicrobial peptides with a focus on structure-activity relationship studies. After first dissecting the important physiochemical properties that influence the antimicrobial and toxic properties of antimicrobial peptides, the contributions of individual amino acids with respect to the peptides antibacterial properties are presented. A brief discussion of the mechanisms of action of different antimicrobials as well as the development of bacterial resistance towards antimicrobial peptides follows. Finally, current efforts on novel design strategies and peptidomimetics are introduced to illustrate the importance of antimicrobial peptide research in the development of future antibiotics. PMID:26184232

  8. Novel Thiosemicarbazide Hybrids with Amino Acids and Peptides Against Hepatocellular Carcinoma: A Molecular Designing Approach Towards Multikinase Inhibitor.

    PubMed

    Chacko, Shinu; Samanta, Subir

    2015-01-01

    Hepatocellular Carcinoma is the most common primary malignant tumor of the liver. Development of multidrug resistance is the main obstacle to the success of anticancer drugs. In this study, designing and docking study of thiosemicarbazide hybrids with amino acids or peptides against hepatocellular carcinoma was performed since hybrids of biologically active compounds with amino acids or peptides may show target specificity and lower toxicity. All the structures were drawn in 2D platform and converted to the 3D platform using ChemDraw 10.0. Evaluations of ADME properties were done by using QikProp 3.0 to check for the possibility of oral delivery. In silico prediction of LD50 values were performed using Pro-Tox webserver. Interestingly, it was found that conjugation with amino acids decreases toxicity and increases the therapeutic index of thiosemicarbazide. Finally, all the compounds were docked to the crystal structure of the Vascular Endothelial Growth Factor Receptor-2 and Checkpoint kinase-1 utilizing Glide 5.0, Schrödinger 8.5, to understand the interaction of ligands with the receptor. A significant number of derivatives have been found active in both the receptors and also displayed multikinase inhibitory activity similar to Sorafenib, against hepatocellular carcinoma. Further, wet lab synthesis, in vitro ADMET and biological screening studies need to be performed to prove that designed compounds are effective against hepatocellular carcinoma as predicted by molecular modeling. However, as predicted by molecular modeling, the efficacy of designed compounds against hepatocellular carcinoma, needs to be confirmed by wet lab synthesis, in vitro ADMET and biological screening studies. PMID:26526710

  9. Structure-based rational design of peptide hydroxamic acid inhibitors to target tumor necrosis factor-α converting enzyme as potential therapeutics for hepatitis.

    PubMed

    Wu, Dan; Gu, Qiuhong; Zhao, Ning; Xia, Fei; Li, Zhiwei

    2015-12-01

    The human tumor necrosis factor-α converting enzyme (TACE) has recently been raised as a new and promising therapeutic target of hepatitis and other inflammatory diseases. Here, we reported a successful application of the solved crystal structure of TACE complex with a peptide-like ligand INN for rational design of novel peptide hydroxamic acid inhibitors with high potency and selectivity to target and inhibit TACE. First, the intermolecular interactions between TACE catalytic domain and INN were characterized through an integrated bioinformatics approach, with which the key substructures of INN that dominate ligand binding were identified. Subsequently, the INN molecular structure was simplified to a chemical sketch of peptide hydroxamic acid compound, which can be regarded as a linear tripeptide capped by a N-terminal carboxybenzyl group (chemically protective group) and a C-terminal hydroxamate moiety (coordinated to the Zn(2+) at TACE active site). Based on the sketch, a virtual combinatorial library containing 180 peptide hydroxamic acids was generated, from which seven samples were identified as promising candidates by using a knowledge-based protein-peptide affinity predictor and were then tested in vitro with a standard TACE activity assay protocol. Consequently, three designed peptide hydroxamic acids, i.e. Cbz-Pro-Ile-Gln-hydroxamic acid, Cbz-Leu-Ile-Val-hydroxamic acid and Cbz-Phe-Val-Met-hydroxamic acid, exhibited moderate or high inhibitory activity against TACE, with inhibition constants Ki of 36 ± 5, 510 ± 46 and 320 ± 26 nM, respectively. We also examined the structural basis and non-bonded profile of TACE interaction with a designed peptide hydroxamic acid inhibitor, and found that the inhibitor ligand is tightly buried in the active pocket of TACE, forming a number of hydrogen bonds, hydrophobic forces and van der Waals contacts at the interaction interface, conferring both stability and specificity for TACE-inhibitor complex

  10. Design and evaluation of peptide nucleic acid probes for specific identification of Candida albicans.

    PubMed

    Kim, Hyun-Joong; Brehm-Stecher, Byron F

    2015-02-01

    Candida albicans is an important cause of systemic fungal infections, and rapid diagnostics for identifying and differentiating C. albicans from other Candida species are critical for the timely application of appropriate antimicrobial therapy, improved patient outcomes, and pharmaceutical cost savings. In this work, two 28S rRNA-directed peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) probes, P-Ca726 (targeting a novel region of the ribosome) and P-CalB2208 (targeting a previously reported region), were evaluated. Hybridization conditions were optimized by using both fluorescence microscopy (FM) and flow cytometry (FCM), and probes were screened for specificity and discriminative ability against a panel of C. albicans and various nontarget Candida spp. The performance of these PNA probes was compared quantitatively against that of DNA probes or DNA probe/helper combinations directed against the same target regions. Ratiometric analyses of FCM results indicated that both the hybridization quality and yield of the PNA probes were higher than those of the DNA probes. In FCM-based comparisons of the PNA probes, P-Ca726 was found to be highly specific, showing 2.5- to 5.5-fold-higher discriminatory power for C. albicans than P-CalB2208. The use of formamide further improved the performance of the new probe. Our results reinforce the significant practical and diagnostic advantages of PNA probes over their DNA counterparts for FISH and indicate that P-Ca726 may be used advantageously for the rapid and specific identification of C. albicans in clinical and related applications, especially when combined with FCM. PMID:25428160

  11. Design and Evaluation of Peptide Nucleic Acid Probes for Specific Identification of Candida albicans

    PubMed Central

    Kim, Hyun-Joong

    2014-01-01

    Candida albicans is an important cause of systemic fungal infections, and rapid diagnostics for identifying and differentiating C. albicans from other Candida species are critical for the timely application of appropriate antimicrobial therapy, improved patient outcomes, and pharmaceutical cost savings. In this work, two 28S rRNA-directed peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) probes, P-Ca726 (targeting a novel region of the ribosome) and P-CalB2208 (targeting a previously reported region), were evaluated. Hybridization conditions were optimized by using both fluorescence microscopy (FM) and flow cytometry (FCM), and probes were screened for specificity and discriminative ability against a panel of C. albicans and various nontarget Candida spp. The performance of these PNA probes was compared quantitatively against that of DNA probes or DNA probe/helper combinations directed against the same target regions. Ratiometric analyses of FCM results indicated that both the hybridization quality and yield of the PNA probes were higher than those of the DNA probes. In FCM-based comparisons of the PNA probes, P-Ca726 was found to be highly specific, showing 2.5- to 5.5-fold-higher discriminatory power for C. albicans than P-CalB2208. The use of formamide further improved the performance of the new probe. Our results reinforce the significant practical and diagnostic advantages of PNA probes over their DNA counterparts for FISH and indicate that P-Ca726 may be used advantageously for the rapid and specific identification of C. albicans in clinical and related applications, especially when combined with FCM. PMID:25428160

  12. Design of antimicrobial peptides conjugated biodegradable citric acid derived hydrogels for wound healing.

    PubMed

    Xie, Zhiwei; Aphale, Nikhil V; Kadapure, Tejaswi D; Wadajkar, Aniket S; Orr, Sara; Gyawali, Dipendra; Qian, Guoying; Nguyen, Kytai T; Yang, Jian

    2015-12-01

    Wound healing is usually facilitated by the use of a wound dressing that can be easily applied to cover the wound bed, maintain moisture, and avoid bacterial infection. In order to meet all of these requirements, we developed an in situ forming biodegradable hydrogel (iFBH) system composed of a newly developed combination of biodegradable poly(ethylene glycol) maleate citrate (PEGMC) and poly(ethylene glycol) diacrylate (PEGDA). The in situ forming hydrogel systems are able to conform to the wound shape in order to cover the wound completely and prevent bacterial invasion. A 2(k) factorial analysis was performed to examine the effects of polymer composition on specific properties, including the curing time, Young's modulus, swelling ratio, and degradation rate. An optimized iFBH formulation was achieved from the systematic factorial analysis. Further, in vitro biocompatibility studies using adult human dermal fibroblasts (HDFs) confirmed that the hydrogels and degradation products are not cytotoxic. The iFBH wound dressing was conjugated and functionalized with antimicrobial peptides as well. Evaluation against bacteria both in vitro and in vivo in rats demonstrated that the peptide-incorporated iFBH wound dressing offered excellent bacteria inhibition and promoted wound healing. These studies indicated that our in situ forming antimicrobial biodegradable hydrogel system is a promising candidate for wound treatment. PMID:26014899

  13. Antimicrobial Peptides Design by Evolutionary Multiobjective Optimization

    PubMed Central

    Maccari, Giuseppe; Di Luca, Mariagrazia; Nifosí, Riccardo; Cardarelli, Francesco; Signore, Giovanni; Boccardi, Claudia; Bifone, Angelo

    2013-01-01

    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. PMID:24039565

  14. Dual Peptide Nucleic Acid- and Peptide-functionalized Shell Crosslinked Nanoparticles Designed to Target mRNA toward the Diagnosis and Treatment of Acute Lung Injury

    PubMed Central

    Shrestha, Ritu; Shen, Yuefei; Pollack, Kevin A.; Taylor, John-Stephen A.; Wooley, Karen L.

    2012-01-01

    In this work, multi-functional bio-synthetic hybrid nanostructures were prepared and studied for their potential utility in the recognition and inhibition of mRNA sequences for inducible nitric oxide synthase (iNOS), which are overexpressed at sites of inflammation, such as in cases of acute lung injury. Shell crosslinked knedel-like polymer nanoparticles (SCKs) that present peptide nucleic acids, for binding to complementary mRNAs, and cell penetrating peptides (CPPs), to gain cell entry, along with fluorescent labels and sites for radiolabeling, were prepared by a series of robust, efficient and versatile synthetic steps that proceeded from monomers to polymers to functional nanoparticles. Amphiphilic block graft copolymers having combinations of methoxy- and thioacetyl-terminated poly(ethylene glycol) (PEG) and DOTA-lysine units grafted from the backbone of poly(acrylic acid) (PAA) and extending with a backbone segment of poly(octadecyl acrylate-co-decyl acrylate) (P(ODA-co-DA)) were prepared by a combination of reversible addition-fragmentation chain transfer (RAFT) polymerization and chemical modification reactions, which were then used as the building blocks for the formation of well-defined SCKs decorated with reactive thiols accessible to the surface. Fluorescent labeling with Alexa Fluor 633 hydrazide was then accomplished by amidation with residual acrylic acid residues within the SCK shells. Finally, the PNAs and CPP units were covalently conjugated to the SCKs via Michael addition of thiols on the SCKs to maleimide units on the termini of PNAs and CPPs. Confirmation of the ability of the PNAs to bind selectively to the target iNOS mRNAs when tethered to the SCK nanoparticles was determined by in vitro competition experiments. When attached to the SCKs having a hydrodynamic diameter of 60 ± 16 nm, the Kd values of the PNAs were ca. an order of magnitude greater than the free PNAs, while the mismatched PNA showed no significant binding. PMID:22372643

  15. APD2: the updated antimicrobial peptide database and its application in peptide design

    PubMed Central

    Wang, Guangshun; Li, Xia; Wang, Zhe

    2009-01-01

    The antimicrobial peptide database (APD, http://aps.unmc.edu/AP/main.php) has been updated and expanded. It now hosts 1228 entries with 65 anticancer, 76 antiviral (53 anti-HIV), 327 antifungal and 944 antibacterial peptides. The second version of our database (APD2) allows users to search peptide families (e.g. bacteriocins, cyclotides, or defensins), peptide sources (e.g. fish, frogs or chicken), post-translationally modified peptides (e.g. amidation, oxidation, lipidation, glycosylation or d-amino acids), and peptide binding targets (e.g. membranes, proteins, DNA/RNA, LPS or sugars). Statistical analyses reveal that the frequently used amino acid residues (>10%) are Ala and Gly in bacterial peptides, Cys and Gly in plant peptides, Ala, Gly and Lys in insect peptides, and Leu, Ala, Gly and Lys in amphibian peptides. Using frequently occurring residues, we demonstrate database-aided peptide design in different ways. Among the three peptides designed, GLK-19 showed a higher activity against Escherichia coli than human LL-37. PMID:18957441

  16. Ribosomal Synthesis of Peptides with Multiple β-Amino Acids.

    PubMed

    Fujino, Tomoshige; Goto, Yuki; Suga, Hiroaki; Murakami, Hiroshi

    2016-02-17

    The compatibility of β-amino acids with ribosomal translation was studied for decades, but it has been still unclear whether the ribosome can accept various β-amino acids, and whether the ribosome can introduce multiple β-amino acids in a peptide. In the present study, by using the Escherichia coli reconstituted cell-free translation system with a reprogramed genetic code, we screened β-amino acids that give high single incorporation efficiency and used them to synthesize peptides containing multiple β-amino acids. The experiments of single β-amino acid incorporation into a peptide revealed that 13 β-amino acids are compatible with ribosomal translation. Six of the tested β-amino acids (βhGly, l-βhAla, l-βhGln, l-βhPhg, l-βhMet, and d-βhPhg) showed high incorporation efficiencies, and seven (l-βhLeu, l-βhIle, l-βhAsn, l-βhPhe, l-βhLys, d-βhAla, and d-βhLeu) showed moderate incorporation efficiencies; whereas no full-length peptide was produced using other β-amino acids (l-βhPro, l-βhTrp, and l-βhGlu). Subsequent double-incorporation experiments using β-amino acids with high single incorporation efficiency revealed that elongation of peptides with successive β-amino acids is prohibited. Efficiency of the double-incorporation of the β-amino acids was restored by the insertion of Tyr or Ile between the two β-amino acids. On the basis of these experiments, we also designed mRNA sequences of peptides, and demonstrated the ribosomal synthesis of peptides containing different types of β-amino acids at multiple positions. PMID:26807980

  17. β-Peptide bundles: Design. Build. Analyze. Biosynthesize.

    PubMed

    Wang, Pam S P; Schepartz, Alanna

    2016-06-14

    Peptides containing β-amino acids are unique non-natural polymers known to assemble into protein-like tertiary and quaternary structures. When composed solely of β-amino acids, the structures formed, defined assemblies of 14-helices called β-peptide bundles, fold cooperatively in water solvent into unique and discrete quaternary assemblies that are highly thermostable, bind complex substrates and metal ion cofactors, and, in certain cases, catalyze chemical reactions. In this Perspective, we recount the design and elaboration of β-peptide bundles and provide an outlook on recent, unexpected discoveries that could influence research on β-peptides and β-peptide bundles (and β-amino acid-containing proteins) for decades to come. PMID:27146019

  18. Uncovering the design rules for peptide synthesis of metal nanoparticles.

    PubMed

    Tan, Yen Nee; Lee, Jim Yang; Wang, Daniel I C

    2010-04-28

    Peptides are multifunctional reagents (reducing and capping agents) that can be used for the synthesis of biocompatible metal nanoparticles under relatively mild conditions. However, the progress in peptide synthesis of metal nanoparticles has been slow due to the lack of peptide design rules. It is difficult to establish sequence-reactivity relationships from peptides isolated from biological sources (e.g., biomineralizing organisms) or selected by combinatorial display libraries because of their widely varying compositions and structures. The abundance of random and inactive amino acid sequences in the peptides also increases the difficulty in knowledge extraction. In this study, a "bottom-up" approach was used to formulate a set of rudimentary rules for the size- and shape-controlled peptide synthesis of gold nanoparticles from the properties of the 20 natural alpha-amino acids for AuCl(4)(-) reduction and binding to Au(0). It was discovered that the reduction capability of a peptide depends on the presence of certain reducing amino acid residues, whose activity may be regulated by neighboring residues with different Au(0) binding strengths. Another finding is the effect of peptide net charge on the nucleation and growth of the Au nanoparticles. On the basis of these understandings, several multifunctional peptides were designed to synthesize gold nanoparticles in different morphologies (nanospheres and nanoplates) and with sizes tunable by the strategic placement of selected amino acid residues in the peptide sequence. The methodology presented here and the findings are useful for establishing the scientific basis for the rational design of peptides for the synthesis of metal nanostructures. PMID:20355728

  19. Improved methods for classification, prediction, and design of antimicrobial peptides.

    PubMed

    Wang, Guangshun

    2015-01-01

    Peptides with diverse amino acid sequences, structures, and functions are essential players in biological systems. The construction of well-annotated databases not only facilitates effective information management, search, and mining but also lays the foundation for developing and testing new peptide algorithms and machines. The antimicrobial peptide database (APD) is an original construction in terms of both database design and peptide entries. The host defense antimicrobial peptides (AMPs) registered in the APD cover the five kingdoms (bacteria, protists, fungi, plants, and animals) or three domains of life (bacteria, archaea, and eukaryota). This comprehensive database ( http://aps.unmc.edu/AP ) provides useful information on peptide discovery timeline, nomenclature, classification, glossary, calculation tools, and statistics. The APD enables effective search, prediction, and design of peptides with antibacterial, antiviral, antifungal, antiparasitic, insecticidal, spermicidal, anticancer activities, chemotactic, immune modulation, or antioxidative properties. A universal classification scheme is proposed herein to unify innate immunity peptides from a variety of biological sources. As an improvement, the upgraded APD makes predictions based on the database-defined parameter space and provides a list of the sequences most similar to natural AMPs. In addition, the powerful pipeline design of the database search engine laid a solid basis for designing novel antimicrobials to combat resistant superbugs, viruses, fungi, or parasites. This comprehensive AMP database is a useful tool for both research and education. PMID:25555720

  20. Improved Methods for Classification, Prediction and Design of Antimicrobial Peptides

    PubMed Central

    Wang, Guangshun

    2015-01-01

    Peptides with diverse amino acid sequences, structures and functions are essential players in biological systems. The construction of well-annotated databases not only facilitates effective information management, search and mining, but also lays the foundation for developing and testing new peptide algorithms and machines. The antimicrobial peptide database (APD) is an original construction in terms of both database design and peptide entries. The host defense antimicrobial peptides (AMPs) registered in the APD cover the five kingdoms (bacteria, protists, fungi, plants, and animals) or three domains of life (bacteria, archaea, and eukaryota). This comprehensive database (http://aps.unmc.edu/AP) provides useful information on peptide discovery timeline, nomenclature, classification, glossary, calculation tools, and statistics. The APD enables effective search, prediction, and design of peptides with antibacterial, antiviral, antifungal, antiparasitic, insecticidal, spermicidal, anticancer activities, chemotactic, immune modulation, or anti-oxidative properties. A universal classification scheme is proposed herein to unify innate immunity peptides from a variety of biological sources. As an improvement, the upgraded APD makes predictions based on the database-defined parameter space and provides a list of the sequences most similar to natural AMPs. In addition, the powerful pipeline design of the database search engine laid a solid basis for designing novel antimicrobials to combat resistant superbugs, viruses, fungi or parasites. This comprehensive AMP database is a useful tool for both research and education. PMID:25555720

  1. Computational Design of Peptide Ligands for Ochratoxin A

    PubMed Central

    Heurich, Meike; Altintas, Zeynep; Tothill, Ibtisam E.

    2013-01-01

    In this paper, we describe a peptide library designed by computational modelling and the selection of two peptide sequences showing affinity towards the mycotoxin, ochratoxin A (OTA). A virtual library of 20 natural amino acids was used as building blocks to design a short peptide library against ochratoxin A template using the de novo design program, LeapFrog, and the dynamic modelling software, FlexiDock. Peptide sequences were ranked according to calculated binding scores in their capacity to bind to ochratoxin A. Two high scoring peptides with the sequences N'-Cys-Ser-Ile-Val-Glu-Asp-Gly-Lys-C' (octapeptide) and N'-Gly-Pro-Ala-Gly-Ile-Asp-Gly-Pro-Ala-Gly-Ile-Arg-Cys-C' (13-mer) were selected for synthesis from the resulting database. These synthesized peptides were characterized using a microtitre plate-based binding assay and a surface plasmon resonance biosensor (Biacore 3000). The binding assay confirmed that both de novo designed peptides did bind to ochratoxin A in vitro. SPR analysis confirmed that the peptides bind to ochratoxin A, with calculated KD values of ~15.7 μM (13-mer) and ~11.8 μM (octamer). The affinity of the peptides corresponds well with the molecular modelling results, as the 13-mer peptide affinity is about 1.3-times weaker than the octapeptide; this is in accordance with the binding energy values modelled by FlexiDock. This work illustrates the potential of using computational modelling to design a peptide sequence that exhibits in vitro binding affinity for a small molecular weight toxin. PMID:23793075

  2. Lasso peptide, a highly stable structure and designable multifunctional backbone.

    PubMed

    Zhao, Ning; Pan, Yongxu; Cheng, Zhen; Liu, Hongguang

    2016-06-01

    Lasso peptide belongs to a new class of natural product with highly compact and stable structure. It has varieties of biological activities, among which the most important one is its antibacterial efficacy. Novel lasso peptides have been constantly discovered and analyzed by advanced techniques, and the biosynthesis or even chemical synthesis of lasso peptide has been studied after learning its constituent amino acids and maturation process. Structural identification of lasso peptide provides information for elucidating the mechanisms of its antibacterial activity and basis for further modifications. Ring of lasso peptide is the key to both its highly compact and stable structure and its intrinsic antibacterial property. The loop has been considered as suitable modification region of lasso peptide, such as V11-S18 of MccJ25 being modifiable without disrupting the lasso structure in biosynthesis. The tail is the immunity protein that can export lasso peptide out of its produced strain and serve as a self-protection mechanism at the same time. Most of currently known lasso peptides are non-pathogenic, which implies that the modified lasso peptides are promising candidates for medical applications. Arginine, glycine, and aspartic acid as a ligands of cancer-specific receptor have been grafted to the loop of lasso peptide without losing its bioactivity, and many other targets are expected to be used for lasso peptide modification. Multi-molecular modification and large-scale production need to be studied and solved in future for designing and using multifunctional lasso peptide based on its extraordinary stable structure. PMID:27074719

  3. Computationally designed peptides for self-assembly of nanostructured lattices.

    PubMed

    Zhang, Huixi Violet; Polzer, Frank; Haider, Michael J; Tian, Yu; Villegas, Jose A; Kiick, Kristi L; Pochan, Darrin J; Saven, Jeffery G

    2016-09-01

    Folded peptides present complex exterior surfaces specified by their amino acid sequences, and the control of these surfaces offers high-precision routes to self-assembling materials. The complexity of peptide structure and the subtlety of noncovalent interactions make the design of predetermined nanostructures difficult. Computational methods can facilitate this design and are used here to determine 29-residue peptides that form tetrahelical bundles that, in turn, serve as building blocks for lattice-forming materials. Four distinct assemblies were engineered. Peptide bundle exterior amino acids were designed in the context of three different interbundle lattices in addition to one design to produce bundles isolated in solution. Solution assembly produced three different types of lattice-forming materials that exhibited varying degrees of agreement with the chosen lattices used in the design of each sequence. Transmission electron microscopy revealed the nanostructure of the sheetlike nanomaterials. In contrast, the peptide sequence designed to form isolated, soluble, tetrameric bundles remained dispersed and did not form any higher-order assembled nanostructure. Small-angle neutron scattering confirmed the formation of soluble bundles with the designed size. In the lattice-forming nanostructures, the solution assembly process is robust with respect to variation of solution conditions (pH and temperature) and covalent modification of the computationally designed peptides. Solution conditions can be used to control micrometer-scale morphology of the assemblies. The findings illustrate that, with careful control of molecular structure and solution conditions, a single peptide motif can be versatile enough to yield a wide range of self-assembled lattice morphologies across many length scales (1 to 1000 nm). PMID:27626071

  4. Computationally designed peptides for self-assembly of nanostructured lattices

    PubMed Central

    Zhang, Huixi Violet; Polzer, Frank; Haider, Michael J.; Tian, Yu; Villegas, Jose A.; Kiick, Kristi L.; Pochan, Darrin J.; Saven, Jeffery G.

    2016-01-01

    Folded peptides present complex exterior surfaces specified by their amino acid sequences, and the control of these surfaces offers high-precision routes to self-assembling materials. The complexity of peptide structure and the subtlety of noncovalent interactions make the design of predetermined nanostructures difficult. Computational methods can facilitate this design and are used here to determine 29-residue peptides that form tetrahelical bundles that, in turn, serve as building blocks for lattice-forming materials. Four distinct assemblies were engineered. Peptide bundle exterior amino acids were designed in the context of three different interbundle lattices in addition to one design to produce bundles isolated in solution. Solution assembly produced three different types of lattice-forming materials that exhibited varying degrees of agreement with the chosen lattices used in the design of each sequence. Transmission electron microscopy revealed the nanostructure of the sheetlike nanomaterials. In contrast, the peptide sequence designed to form isolated, soluble, tetrameric bundles remained dispersed and did not form any higher-order assembled nanostructure. Small-angle neutron scattering confirmed the formation of soluble bundles with the designed size. In the lattice-forming nanostructures, the solution assembly process is robust with respect to variation of solution conditions (pH and temperature) and covalent modification of the computationally designed peptides. Solution conditions can be used to control micrometer-scale morphology of the assemblies. The findings illustrate that, with careful control of molecular structure and solution conditions, a single peptide motif can be versatile enough to yield a wide range of self-assembled lattice morphologies across many length scales (1 to 1000 nm). PMID:27626071

  5. Design, Synthesis, and Actions of a Novel Chimeric Natriuretic Peptide: CD-NP

    PubMed Central

    Lisy, Ondrej; Huntley, Brenda K.; McCormick, Daniel J.; Kurlansky, Paul A.; Burnett, John C.

    2008-01-01

    Objectives Our aim was to design, synthesize and test in vivo and in vitro a new chimeric peptide that would combine the beneficial properties of 2 distinct natriuretic peptides with a biological profile that goes beyond native peptides. Background Studies have established the beneficial vascular and antiproliferative properties of C-type natriuretic peptide (CNP). While lacking renal actions, CNP is less hypotensive than the cardiac peptides atrial natriuretic peptide and B-type natriuretic peptide but unloads the heart due to venodilation. Dendroaspis natriuretic peptide is a potent natriuretic and diuretic peptide that is markedly hypotensive and functions via a separate guanylyl cyclase receptor compared with CNP. Methods Here we engineered a novel chimeric peptide CD-NP that represents the fusion of the 22-amino acid peptide CNP together with the 15-amino acid linear C-terminus of Dendroaspis natriuretic peptide. We also determined in vitro in cardiac fibroblasts cyclic guanosine monophosphate-activating and antiproliferative properties of CD-NP. Results Our studies demonstrate in vivo that CD-NP is natriuretic and diuretic, glomerular filtration rate enhancing, cardiac unloading, and renin inhibiting. CD-NP also demonstrates less hypotensive properties when compared with B-type natriuretic peptide. In addition, CD-NP in vitro activates cyclic guanosine monophosphate and inhibits cardiac fibroblast proliferation. Conclusions The current findings advance an innovative design strategy in natriuretic peptide drug discovery and development to create therapeutic peptides with favorable properties that may be preferable to those associated with native natriuretic peptides. PMID:18582636

  6. Design of Asymmetric Peptide Bilayer Membranes.

    PubMed

    Li, Sha; Mehta, Anil K; Sidorov, Anton N; Orlando, Thomas M; Jiang, Zhigang; Anthony, Neil R; Lynn, David G

    2016-03-16

    Energetic insights emerging from the structural characterization of peptide cross-β assemblies have enabled the design and construction of robust asymmetric bilayer peptide membranes. Two peptides differing only in their N-terminal residue, phosphotyrosine vs lysine, coassemble as stacks of antiparallel β-sheets with precisely patterned charged lattices stabilizing the bilayer leaflet interface. Either homogeneous or mixed leaflet composition is possible, and both create nanotubes with dense negative external and positive internal solvent exposed surfaces. Cross-seeding peptide solutions with a preassembled peptide nanotube seed leads to domains of different leaflet architecture within single nanotubes. Architectural control over these cross-β assemblies, both across the bilayer membrane and along the nanotube length, provides access to highly ordered asymmetric membranes for the further construction of functional mesoscale assemblies. PMID:26942690

  7. Rational design of mirror-like peptides with alanine regulation.

    PubMed

    Li, Weizhong; Tan, Tingting; Xu, Wei; Xu, Lin; Dong, Na; Ma, Deying; Shan, Anshan

    2016-02-01

    To generate effective antimicrobial peptides (AMPs) with good antimicrobial activities and cell selectivity, many synthetic strategies have been implemented to facilitate the development of AMPs. However, these synthetic strategies represent only a small proportion of the methods used for the development of AMPs and are not optimal with the requirements needed for the design of AMPs. In this investigation, we designed a mirror-like structure with a lower charge and a higher number of hydrophobic amino acids. The amino acid sequence of the designed mirror-like peptides was XXYXXXYXXXYXX [X represents L (Leu) and/or A (Ala); Y represents K (Lys)]. These mirror-like peptides displayed antimicrobial activity against both Gram-positive and Gram-negative bacteria. Hemolysis activity and cytotoxicity, detected by using human red blood cells (hRBCs) and human embryonic kidney cells (HEK293), respectively, demonstrated that the frequency of Ala residues in this structure had a regulatory effect on the high hydrophobic region. In particular, KL4A6 showed a greater antimicrobial potency than the other three mirror-like peptides, folded into an α-helical structure, and displayed the highest therapeutic index, suggesting its good cell selectivity. Observations from fluorescence spectroscopy, flow cytometry, and electron microscopy experiments indicated that KL4A6 exhibited good membrane penetration potential by inducing membrane blebbing, disruption and lysis. Therefore, generating mirror-like peptides is a promising strategy for designing effective AMPs with regions of high hydrophobicity. PMID:26385363

  8. Design, synthesis, and characterization of new cyclic d,l-α-alternate amino acid peptides by capillary electrophoresis coupled to electrospray ionization mass spectrometry.

    PubMed

    Cortez-Díaz, María Dámaris; d'Orlyé, Fanny; Gutierrez-Granados, Silvia; de Leon-Rodriguez, Luis Manuel; Varenne, Anne

    2016-06-01

    The self-assembly of peptide nanotubes (PNTs) depends on the structure and chemistry of cyclic peptide (CP) monomers, having an impact on their properties, making the choice of their monomers and their characterization a great challenge. We synthesized for the first time a new set of eight original CP sequences of 8, 10, and 12 d,l-α-alternate amino acids with a controlled internal diameter from 7 to 13 Å. They present various properties (e.g., diameter, global surface charge, hydrophobicity) that can open the way to new applications. Their structure and purity were determined thanks to a capillary electrophoresis coupled to electrospray ionization mass spectrometry (CE-ESI-MS) methodology developed for the first time for this purpose. The CPs were successfully separated in a basic hydro-organic background electrolyte (BGE, pH 8.0, H2O/EtOH 50:50, v/v) and analyzed in MS positive mode. The effect of CP structure on electrophoretic mobility was studied, and the mass spectra were deeply analyzed. This methodology allowed verifying their purity and the absence of linear peptide precursors as well as their stability when stored over several months. Therefore, we have developed a new CE-ESI-MS methodology for the structure and purity control of interesting potential precursors for PNTs that could be employed as nanoplatforms in diagnostics or as pseudo sieving tools for separative purposes. PMID:26969790

  9. Design and Engineering Strategies for Synthetic Antimicrobial Peptides

    NASA Astrophysics Data System (ADS)

    Tossi, Alessandro

    Thousands of antimicrobial peptides (AMPs) of prokaryotic, fungal, plant, or animal origin have been identified, and their potential as lead compounds for the design of novel therapeutic agents in the treatment of infection, for stimulating the immune system, or in countering septic shock has been widely recognized. Added to this is their possible use in prophylaxis of infectious diseases for animal or plant protection, for disinfection of surgical instruments or industrial surfaces, and for food preservation among other commercially important applications. Since the early eighties, AMPs have been subject to a vast number of studies aimed at understanding what determines their potency and spectrum of activities against bacterial or fungal pathogens, and at maximizing these while limiting cytotoxic activities toward host cells. Much research has also been directed toward understanding specific mechanisms of action underlying the antimicrobial activity and selectivity, to be able to redesign the peptides for optimal performance. A central theme in the mode of action of many AMPs is their dynamic interaction with biological membranes, which involves various properties of these peptides such as, among others, surface hydrophobicity and polarity, charge, structure, and induced conformational variations. These features are often intimately interconnected so that engineering peptides to independently adjust any one property in particular is not an easy task. However, solid-phase peptide synthesis allows the use of a large repertoire of nonproteinogenic amino acids that can be used in the rational design of peptides to finely tune structural and physicochemical properties and precisely probe structure-function relationships.

  10. Anginex, a designed peptide that inhibits angiogenesis.

    PubMed Central

    Griffioen, A W; van der Schaft, D W; Barendsz-Janson, A F; Cox, A; Struijker Boudier, H A; Hillen, H F; Mayo, K H

    2001-01-01

    Novel beta-sheet-forming peptide 33-mers, betapep peptides, have been designed by using a combination approach employing basic folding principles and incorporating short sequences from the beta-sheet domains of anti-angiogenic proteins. One of these designed peptides (betapep-25), named anginex, was observed to be potently anti-angiogenic. Anginex specifically inhibits vascular endothelial cell proliferation and induces apoptosis in these cells, as shown by flow-cytometric detection of sub-diploid cells, TUNEL (terminal deoxyribonucleotidyl transferase-mediated dUTP-nick-end labelling) analysis and cell morphology. Anginex also inhibits endothelial cell adhesion to and migration on different extracellular matrix components. Inhibition of angiogenesis in vitro is demonstrated in the sprout-formation assay and in vivo in the chick embryo chorio-allantoic membrane angiogenesis assay. Comparison of active and inactive betapep sequences allows structure-function relationships to be deduced. Five hydrophobic residues and two lysines appear to be crucial to activity. This is the first report of a designed peptide having a well-defined biological function as a novel cytokine, which may be an effective anti-angiogenic agent for therapeutic use against various pathological disorders, such as neoplasia, rheumatoid arthritis, diabetic retinopathy and restenosis. PMID:11171099

  11. Solid Phase Synthesis of C-Terminal Boronic Acid Peptides.

    PubMed

    Behnam, Mira A M; Sundermann, Tom R; Klein, Christian D

    2016-05-01

    Peptides and peptidomimetics with a C-terminal boronic acid group have prolific applications in numerous fields of research, but their synthetic accessibility remains problematic. A convenient, high yield synthesis of peptide-boronic acids on a solid support is described here, using commercially available 1-glycerol polystyrene resin. The method is compatible with Fmoc chemistry and offers a versatile approach to aryl and alkyl aminoboronic acids without additional purification steps. PMID:27104613

  12. Site-specific control of silica mineralization on DNA using a designed peptide.

    PubMed

    Ozaki, Makoto; Nagai, Kazuma; Nishiyama, Hiroto; Tsuruoka, Takaaki; Fujii, Satoshi; Endoh, Tamaki; Imai, Takahito; Tomizaki, Kin-Ya; Usui, Kenji

    2016-03-01

    We developed a site-specific method for precipitating inorganic compounds using organic compounds, DNA, and designed peptides with peptide nucleic acids (PNAs). Such a system for site-specific precipitation represents a powerful tool for use in nanobiochemistry and materials chemistry. PMID:26690695

  13. Design of a novel microtubule targeted peptide vesicle for delivering different anticancer drugs.

    PubMed

    Adak, Anindyasundar; Mohapatra, Saswat; Mondal, Prasenjit; Jana, Batakrishna; Ghosh, Surajit

    2016-06-18

    A microtubule targeted peptide-based delivery vehicle has been designed using two oppositely charged peptides, which targets tubulin/microtubules, delivers both hydrophilic and hydrophobic drugs into their target site through lysosome at acidic pH. Drug loaded vesicles show a significant anticancer effect compared to control drugs in a 2D monolayer and a 3D spheroid cell. PMID:27153208

  14. Rational Evolution of Antimicrobial Peptides Containing Unnatural Amino Acids to Combat Burn Wound Infections.

    PubMed

    Xiong, Meng; Chen, Ming; Zhang, Jue

    2016-09-01

    Antimicrobial peptides have long been raised as a promising strategy to combat bacterial infection in burn wounds. Here, we attempted to rationally design small antimicrobial peptides containing unnatural amino acids by integrating in silico analysis and in vitro assay. Predictive quantitative sequence-activity models were established and validated rigorously based on a large panel of nonamer antimicrobial peptides with known antibacterial activity. The best quantitative sequence-activity model predictor was employed to guide genetic evolution of a peptide population. In the evolution procedure, a number of unnatural amino acids with desired physicochemical properties were introduced, resulting in a genetic evolution-improved population, from which seven peptide candidates with top scores, containing 1-3 unnatural amino acids, and having diverse structures were successfully identified, and their antibacterial potencies against two antibiotic-resistant bacterial strains isolated from infected burn wounds were measured using in vitro susceptibility test. Consequently, four (WL-Orn-LARKIV-NH2 , ARKRWF-Dab-FL-NH2 , KFI-Hag-IWR-Orn-R-NH2 and YW-Hag-R-Cit-RF-Orn-N-NH2 ) of the seven tested peptides were found to be more potent than reference Bac2A, the smallest naturally occurring broad spectrum antimicrobial peptide. Molecular dynamics simulations revealed that the designed peptides can fold into amphipathic helical structure that allows them to interact directly with microbial membranes. PMID:27062533

  15. Interfacial Templating of Inorganic Nanostructures Using Rationally Designed Peptide Molecules

    NASA Astrophysics Data System (ADS)

    Leon Gibbons, Lorraine

    In nature, biological molecules form interfaces that assemble patterns of chemical functionality with exceptional precision. The role of dynamics during the assembly of biological molecules appears to be important for mineralization processes. The work presented in this dissertation applies model sheet-forming peptides at interfaces to explore the dynamics of assembly in order to template mineral growth. The peptide molecules are rationally designed to have amphiphilic properties and a propensity for sheet-like secondary structure. These designed peptides are deposited at the air/water interface to explore the dynamics of their self-assembly and investigate their 2D order. To characterize the phase behavior, techniques such as Langmuir Blodgett and Brewster Angle Microscopy are used. In addition, we verify the hypothesized sheet-forming propensity using both Circular Dichroism and Attenuated Total Reflection Fourier Transform Infrared Spectroscopy, while the characterization of the inorganic phase is done using Transmission Electron Microscopy, Electron Diffraction, and Atomic Force Microscopy. Thermodynamic analysis of structure formation with increasing pressure allows us to understand the nature of self-assembly with iterative changes in the peptide sequence. Additionally, we look at the dynamics of the self-assembled state, where the organic phase switches between short- and long-range order as a function of surface pressure. We use this model system to explore the influence of electrostatic interactions on self-assembly, and additionally, the influence of short- and long-range order on the nucleation and growth of inorganic material. This is in contrast to a system that starts with a well-ordered preformed template that defines the epitaxial growth of the mineral phase. Two versions of our model peptides are constructed by substituting histidine for glutamic acid in order to nucleate Au nanocrystals in both the short and long range ordered organic matrix, to

  16. Design, Synthesis and Affinity Properties of Biologically Active Peptide and Protein Conjugates of Cotton Cellulose

    SciTech Connect

    Edwards, J. V.; Goheen, Steven C.

    2002-11-30

    The formation of peptide and protein conjugates of cellulose on cotton fabrics provides promising leads for the development of wound healing, antibacterial, and decontaminating textiles. An approach to the design, synthesis, and analysis of bioconjugates containing cellulose peptide and protein conjugates includes: 1) computer graphic modeling for a rationally designed structure; 2) attachment of the peptide or protein to cotton cellulose through a linker amino acid, and 3) characterization of the resulting bioconjugate. Computer graphic simulation of protein and peptide cellulose conjugates gives a rationally designed biopolymer to target synthetic modifications to the cotton cellulose. Techniques for preparing these types of conjugates involve both sequential assembly of the peptide on the fabric and direct crosslinking of the peptide or protein as cellulose bound esters or carboxymethylcellulose amides.

  17. α-Helix peptides designed from EBV-gH protein display higher antigenicity and induction of monocyte apoptosis than the native peptide

    PubMed Central

    Melo-Cardenas, Johanna; Guevara, Tatiana; Echeverria, Ignacia; Rodriguez, Isabel C.; Vanegas, Magnolia; Amzel, Mario; Patarroyo, Manuel E.

    2010-01-01

    We tested the hypothesis that stabilizing α-helix of Epstein–Barr virus gH-derived peptide 11438 used for binding human cells will increase its biological activity. Non-stable α-helix of peptide 11438 was unfolded in an entropy-driven process, despite the opposing effect of the enthalpy factor. Adding and/or changing amino acids in peptide 11438 allowed the designing of peptides 33207, 33208 and 33210; peptides 33208 and 33210 displayed higher helical content due to a decreased unfolding entropy change as was determined by AGADIR, molecular dynamics and circular dichroism analysis. Peptides 33207, 33208 and 33210 inhibited EBV invasion of peripheral blood mononuclear cells and displayed epitopes more similar to native protein than peptide 11438; these peptides could be useful for detecting antibodies induced by native gH protein since they displayed high reactivity with anti-EBV antibodies. Anti-peptide 33207 antibodies showed higher reactivity with EBV than anti-peptide 11438 antibodies being useful for inducing antibodies against EBV. Anti-peptide 33210 antibodies inhibit EBV invasion of epithelial cells better than anti-peptide 11438 antibodies. Peptide 33210 bound to normal T lymphocytes and Raji cells stronger than peptide 11438 and also induced apoptosis of monocytes and Raji cells but not of normal T cells in a similar way to EBV-gH. Peptide 33210 inhibited the monocytes’ development toward dendritic cells better than EBV and peptide 11438. In conclusion, stabilizing the α-helix in peptides 33208 and 33210 designed from peptide 11438 increased the antigenicity and the ability of the antibodies induced by peptides of inhibiting EBV invasion of host cells. PMID:20473772

  18. Peptides containing β-amino acid patterns: challenges and successes in medicinal chemistry.

    PubMed

    Cabrele, Chiara; Martinek, Tamás A; Reiser, Oliver; Berlicki, Łukasz

    2014-12-11

    The construction of bioactive peptides using β-amino acid-containing sequence patterns is a very promising strategy to obtain analogues that exhibit properties of high interest for medicinal chemistry applications. β-Amino acids have been shown to modulate the conformation, dynamics, and proteolytic susceptibility of native peptides. They can be either combined with α-amino acids by following specific patterns, which results in backbone architectures with well-defined orientations of the side chain functional groups, or assembled in de novo-designed bioactive β- or α,β-peptidic sequences. Such peptides display various biological functions, including antimicrobial activity, inhibition of protein-protein interactions, agonism/antagonism of GPCR ligands, and anti-angiogenic activity. PMID:25207470

  19. Synthesis and biological properties of amino acids and peptides containing a tetrazolyl moiety

    NASA Astrophysics Data System (ADS)

    Popova, E. A.; Trifonov, R. E.

    2015-09-01

    Literature data published mainly in the last 15 years on the synthesis and biological properties of amino acid analogues and derivatives containing tetrazolyl moieties are analyzed. Tetrazolyl analogues and derivatives of amino acids and peptides are shown to be promising for medicinal chemistry. Being polynitrogen heterocyclic systems comprising four endocyclic nitrogen atoms, tetrazoles can behave as acids and bases and form strong hydrogen bonds with proton donors (more rarely, with acceptors). They have high metabolic stability and are able to penetrate biological membranes. The review also considers the synthesis and properties of linear and cyclic peptides based on modified amino acids incorporating a tetrazolyl moiety. A special issue is the discussion of the biological properties of tetrazole-containing amino acids and peptides, which exhibit high biological activity and can be used to design new drugs. The bibliography includes 200 references.

  20. Antioxidant peptides from corn gluten meal: Orthogonal design evaluation.

    PubMed

    Zhou, Cunshan; Hu, Jiali; Ma, Haile; Yagoub, Abu ElGasim A; Yu, Xiaojie; Owusu, John; Ma, Haiyan; Qin, Xiaopei

    2015-11-15

    Protamex catalyzed corn gluten meal (CGM) hydrolysis peptides (CHP) were prepared. Orthogonal design L16 (4(5)) was used to optimize processing variables of CGM concentration, CGM heat pretreatment (121 °C) time, and enzymolysis pH, temperature, and time. Degree of hydrolysis (DH), undigested residue ratio, molecular weight (MW) distribution and DPPH radical inhibition were selected as analysis indicators. Optimum variables were CGM concentration of 18%, heat pretreatment time of 40 min, and enzymolysis pH, temperature and time of 7.5, 55 °C and 24h, respectively. Verification test showed that CHP IC50 for scavenging hydroxyl radical was the best and then followed by reducing power. Oligopeptides improved after hydrolysis at the expense of di- and tripeptides, suggesting formation of soluble aggregates from low MW peptides. The increase in the DH, oligopeptides, Alanyl-Tyrosine, and antioxidant free amino acids coincided with the improvement in the antioxidant activity of CHP. PMID:25977026

  1. Modifications of Natural Peptides for Nanoparticle and Drug Design

    PubMed Central

    Jallouk, Andrew P.; Palekar, Rohun U.; Pan, Hua; Schlesinger, Paul H.; Wickline, Samuel A.

    2016-01-01

    Natural products serve as an important source of novel compounds for drug development. Recently, peptides have emerged as a new class of therapeutic agents due to their versatility and specificity for biological targets. Yet, their effective application often requires use of a nanoparticle delivery system. In this chapter, we review the role of natural peptides in the design and creation of nanomedicines, with a particular focus on cell-penetrating peptides, antimicrobial peptides, and peptide toxins. The use of natural peptides in conjunction with nanoparticle delivery systems holds great promise for the development of new therapeutic formulations as well as novel platforms for the delivery of various cargoes. PMID:25819276

  2. Targeting DNA G-Quadruplex Structures with Peptide Nucleic Acids

    PubMed Central

    Panyutin, Igor G.; Onyshchenko, Mykola I.; Englund, Ethan A.; Appella, Daniel H.; Neumann, Ronald D.

    2012-01-01

    Regulation of genetic functions based on targeting DNA or RNA sequences with complementary oligonucleotides is especially attractive in the post-genome era. Oligonucleotides can be rationally designed to bind their targets based on simple nucleic acid base pairing rules. However, the use of natural DNA and RNA oligonucleotides as targeting probes can cause numerous off-target effects. In addition, natural nucleic acids are prone to degradation in vivo by various nucleases. To address these problems, nucleic acid mimics such as peptide nucleic acids (PNA) have been developed. They are more stable, show less off-target effects, and, in general, have better binding affinity to their targets. However, their high affinity to DNA can reduce their sequence-specificity. The formation of alternative DNA secondary structures, such as the G-quadruplex, provides an extra level of specificity as targets for PNA oligomers. PNA probes can target the loops of G-quadruplex, invade the core by forming PNA-DNA guanine-tetrads, or bind to the open bases on the complementary cytosine-rich strand. Not only could the development of such G-quadruplex-specific probes allow regulation of gene expression, but it will also provide a means to clarify the biological roles G-quadruplex structures may possess. PMID:22376112

  3. Occurrence and function of D-amino acid-containing peptides and proteins: antimicrobial peptides.

    PubMed

    Mignogna, G; Simmaco, M; Barra, D

    1998-01-01

    Antimicrobial peptides are widely distributed in living organisms, where they represent a constitutive defence system acting as a first line of response against infections. The number of such peptides discovered has increased rapidly in the last few years, and more than 100 have been described from different sources. So far, antimicrobial peptides containing a D-amino acid have only been found in the skin secretions of frogs belonging to the genus Bombina. In the second position of the sequence of the mature peptides either D-alloisoleucine or D-leucine were detected. The D-amino acids are derived from the corresponding L forms by an as yet unknown posttranslational reaction. PMID:9949866

  4. HomoSAR: bridging comparative protein modeling with quantitative structural activity relationship to design new peptides.

    PubMed

    Borkar, Mahesh R; Pissurlenkar, Raghuvir R S; Coutinho, Evans C

    2013-11-15

    Peptides play significant roles in the biological world. To optimize activity for a specific therapeutic target, peptide library synthesis is inevitable; which is a time consuming and expensive. Computational approaches provide a promising way to simply elucidate the structural basis in the design of new peptides. Earlier, we proposed a novel methodology termed HomoSAR to gain insight into the structure activity relationships underlying peptides. Based on an integrated approach, HomoSAR uses the principles of homology modeling in conjunction with the quantitative structural activity relationship formalism to predict and design new peptide sequences with the optimum activity. In the present study, we establish that the HomoSAR methodology can be universally applied to all classes of peptides irrespective of sequence length by studying HomoSAR on three peptide datasets viz., angiotensin-converting enzyme inhibitory peptides, CAMEL-s antibiotic peptides, and hAmphiphysin-1 SH3 domain binding peptides, using a set of descriptors related to the hydrophobic, steric, and electronic properties of the 20 natural amino acids. Models generated for all three datasets have statistically significant correlation coefficients (r(2)) and predictive r2 (r(pred)2) and cross validated coefficient ( q(LOO)2). The daintiness of this technique lies in its simplicity and ability to extract all the information contained in the peptides to elucidate the underlying structure activity relationships. The difficulties of correlating both sequence diversity and variation in length of the peptides with their biological activity can be addressed. The study has been able to identify the preferred or detrimental nature of amino acids at specific positions in the peptide sequences. PMID:24105965

  5. Modifying the electronic properties of single-walled carbon nanotubes using designed surfactant peptides

    NASA Astrophysics Data System (ADS)

    Samarajeewa, Dinushi R.; Dieckmann, Gregg R.; Nielsen, Steven O.; Musselman, Inga H.

    2012-07-01

    The electronic properties of carbon nanotubes can be altered significantly by modifying the nanotube surface. In this study, single-walled carbon nanotubes (SWCNTs) were functionalized noncovalently using designed surfactant peptides, and the resultant SWCNT electronic properties were investigated. These peptides have a common amino acid sequence of X(Valine)5(Lysine)2, where X indicates an aromatic amino acid containing either an electron-donating or electron-withdrawing functional group (i.e. p-amino-phenylalanine or p-cyano-phenylalanine). Circular dichroism spectra showed that the surfactant peptides primarily have random coil structures in an aqueous medium, both alone and in the presence of SWCNTs, simplifying analysis of the peptide/SWCNT interaction. The ability of the surfactant peptides to disperse individual SWCNTs in solution was verified using atomic force microscopy and ultraviolet-visible-near-infrared spectroscopy. The electronic properties of the surfactant peptide/SWCNT composites were examined using the observed nanotube Raman tangential band shifts and the observed additional features near the Fermi level in the scanning tunneling spectroscopy dI/dV spectra. The results revealed that SWCNTs functionalized with surfactant peptides containing electron-donor or electron-acceptor functional groups showed n-doped or p-doped altered electronic properties, respectively. This work unveils a facile and versatile approach to modify the intrinsic electronic properties of SWCNTs using a simple peptide structure, which is easily adaptable to obtain peptide/SWCNT composites for the design of tunable nanoscale electronic devices.The electronic properties of carbon nanotubes can be altered significantly by modifying the nanotube surface. In this study, single-walled carbon nanotubes (SWCNTs) were functionalized noncovalently using designed surfactant peptides, and the resultant SWCNT electronic properties were investigated. These peptides have a common amino

  6. Design and characterization of short antimicrobial peptides using leucine zipper templates with selectivity towards microorganisms.

    PubMed

    Ahmad, Aqeel; Azmi, Sarfuddin; Srivastava, Saurabh; Kumar, Amit; Tripathi, Jitendra Kumar; Mishra, Nripendra N; Shukla, Praveen K; Ghosh, Jimut Kanti

    2014-11-01

    Design of antimicrobial peptides with selective activity towards microorganisms is an important step towards the development of new antimicrobial agents. Leucine zipper sequence has been implicated in cytotoxic activity of naturally occurring antimicrobial peptides; moreover, this motif has been utilized for the design of novel antimicrobial peptides with modulated cytotoxicity. To understand further the impact of substitution of amino acids at 'a' and/or 'd' position of a leucine zipper sequence of an antimicrobial peptides on its antimicrobial and cytotoxic properties four short peptides (14-residue) were designed on the basis of a leucine zipper sequence without or with replacement of leucine residues in its 'a' and 'd' positions with D-leucine or alanine or proline residue. The original short leucine zipper peptide (SLZP) and its D-leucine substituted analog, DLSA showed comparable activity against the tested Gram-positive and negative bacteria and the fungal strains. The alanine substituted analog (ASA) though showed appreciable activity against the tested bacteria, it showed to some extent lower activity against the tested fungi. However, the proline substituted analog (PSA) showed lower activity against the tested bacterial or fungal strains. Interestingly, DLSA, ASA and PSA showed significantly lower cytotoxicity than SLZP against both human red blood cells (hRBCs) and murine 3T3 cells. Cytotoxic and bactericidal properties of these peptides matched with peptide-induced damage/permeabilization of mammalian cells and bacteria or their mimetic lipid vesicles suggesting cell membrane could be the target of these peptides. As evidenced by tryptophan fluorescence and acrylamide quenching studies the peptides showed similarities either in interaction or in their localization within the bacterial membrane mimetic negatively charged lipid vesicles. Only SLZP showed localization inside the mammalian membrane mimetic zwitterionic lipid vesicles. The results show

  7. NHLBI-AbDesigner: an online tool for design of peptide-directed antibodies.

    PubMed

    Pisitkun, Trairak; Hoffert, Jason D; Saeed, Fahad; Knepper, Mark A

    2012-01-01

    Investigation of physiological mechanisms at a cellular level often requires production of high-quality antibodies, frequently using synthetic peptides as immunogens. Here we describe a new, web-based software tool called NHLBI-AbDesigner that allows the user to visualize the information needed to choose optimal peptide sequences for peptide-directed antibody production (http://helixweb.nih.gov/AbDesigner/). The choice of an immunizing peptide is generally based on a need to optimize immunogenicity, antibody specificity, multispecies conservation, and robustness in the face of posttranslational modifications (PTMs). AbDesigner displays information relevant to these criteria as follows: 1) "Immunogenicity Score," based on hydropathy and secondary structure prediction; 2) "Uniqueness Score," a predictor of specificity of an antibody against all proteins expressed in the same species; 3) "Conservation Score," a predictor of ability of the antibody to recognize orthologs in other animal species; and 4) "Protein Features" that show structural domains, variable regions, and annotated PTMs that may affect antibody performance. AbDesigner displays the information online in an interactive graphical user interface, which allows the user to recognize the trade-offs that exist for alternative synthetic peptide choices and to choose the one that is best for a proposed application. Several examples of the use of AbDesigner for the display of such trade-offs are presented, including production of a new antibody to Slc9a3. We also used the program in large-scale mode to create a database listing the 15-amino acid peptides with the highest Immunogenicity Scores for all known proteins in five animal species, one plant species (Arabidopsis thaliana), and Saccharomyces cerevisiae. PMID:21956165

  8. “De Novo” Design of Peptides with Specific Lipid-Binding Properties

    PubMed Central

    Lins, L.; Charloteaux, B.; Heinen, C.; Thomas, A.; Brasseur, R.

    2006-01-01

    In this study, we describe an in silico method to design peptides that can be made of non-natural amino acids and elicit specific membrane-interacting properties. The originality of the method holds in the capacities developed to design peptides from any non-natural amino acids as easily as from natural ones, and to test the structure stability by an angular dynamics rather than the currently-used molecular dynamics. The goal of this study was to design a non-natural tilted peptide. Tilted peptides are short protein fragments able to destabilize lipid membranes and characterized by an asymmetric distribution of hydrophobic residues along their helix structure axis. The method is based on the random generation of peptides and their selection on three main criteria: mean hydrophobicity and the presence of at least one polar residue; tilted insertion at the level of the acyl chains of lipids of a membrane; and conformational stability in that hydrophobic phase. From 10,000,000 randomly-generated peptides, four met all the criteria. One was synthesized and tested for its lipid-destabilizing properties. Biophysical assays showed that the “de novo” peptide made of non-natural amino acids is helical either in solution or into lipids as tested by Fourier transform infrared spectroscopy and is able to induce liposome fusion. These results are in agreement with the calculations and validate the theoretical approach. PMID:16275638

  9. De-Novo Design of Antimicrobial Peptides for Plant Protection

    PubMed Central

    Zeitler, Benjamin; Herrera Diaz, Areli; Dangel, Alexandra; Thellmann, Martha; Meyer, Helge; Sattler, Michael; Lindermayr, Christian

    2013-01-01

    This work describes the de-novo design of peptides that inhibit a broad range of plant pathogens. Four structurally different groups of peptides were developed that differ in size and position of their charged and hydrophobic clusters and were assayed for their ability to inhibit bacterial growth and fungal spore germination. Several peptides are highly active at concentrations between 0,1 and 1 µg/ml against plant pathogenic bacteria, such as Pseudomonas syringae, Pectobacterium carotovorum, and Xanthomonas vesicatoria. Importantly, no hemolytic activity could be detected for these peptides at concentrations up to 200 µg/ml. Moreover, the peptides are also active after spraying on the plant surface demonstrating a possible way of application. In sum, our designed peptides represent new antimicrobial agents and with the increasing demand for antimicrobial compounds for production of “healthy” food, these peptides might serve as templates for novel antibacterial and antifungal agents. PMID:23951222

  10. A peptide & peptide nucleic acid synthesis technology for transporter molecules and theranostics--the SPPS.

    PubMed

    Pipkorn, Ruediger; Braun, Klaus; Wiessler, Manfred; Waldeck, Waldemar; Schrenk, Hans-Hermann; Koch, Mario; Semmler, Wolfhard; Komljenovic, Dorde

    2014-01-01

    Advances in imaging diagnostics using magnetic resonance tomography (MRT), positron emission tomography (PET) and fluorescence imaging including near infrared (NIR) imaging methods are facilitated by constant improvement of the concepts of peptide synthesis. Feasible patient-specific theranostic platforms in the personalized medicine are particularly dependent on efficient and clinically applicable peptide constructs. The role of peptides in the interrelations between the structure and function of proteins is widely investigated, especially by using computer-assisted methods. Nowadays the solid phase synthesis (SPPS) chemistry emerges as a key technology and is considered as a promising methodology to design peptides for the investigation of molecular pharmacological processes at the transcriptional level. SPPS syntheses could be carried out in core facilities producing peptides for large-scale scientific implementations as presented here. PMID:24843319

  11. A Peptide & Peptide Nucleic Acid Synthesis Technology for Transporter Molecules and Theranostics - The SPPS

    PubMed Central

    Pipkorn, Ruediger; Braun, Klaus; Wiessler, Manfred; Waldeck, Waldemar; Schrenk, Hans-Hermann; Koch, Mario; Semmler, Wolfhard; Komljenovic, Dorde

    2014-01-01

    Advances in imaging diagnostics using magnetic resonance tomography (MRT), positron emission tomography (PET) and fluorescence imaging including near infrared (NIR) imaging methods are facilitated by constant improvement of the concepts of peptide synthesis. Feasible patient-specific theranostic platforms in the personalized medicine are particularly dependent on efficient and clinically applicable peptide constructs. The role of peptides in the interrelations between the structure and function of proteins is widely investigated, especially by using computer-assisted methods. Nowadays the solid phase synthesis (SPPS) chemistry emerges as a key technology and is considered as a promising methodology to design peptides for the investigation of molecular pharmacological processes at the transcriptional level. SPPS syntheses could be carried out in core facilities producing peptides for large-scale scientific implementations as presented here. PMID:24843319

  12. Design and expression of a short peptide as an HIV detection probe

    SciTech Connect

    Lines, Jamie A.; Yu, Zhiqiang; Dedkova, Larisa M.; Chen, Shengxi

    2014-01-03

    Highlights: •We designed a short fusion peptide (FP-50) for in vivo expression. •This peptide is a very promising component for detection of gp120 protein. •The detectable level is about 20–200 times lower than previously published methods. •It is a novel probe to detect HIV-1 gp120 during early stages of HIV infection. -- Abstract: To explore a low-cost novel probe for HIV detection, we designed and prepared a 50-amino acid-length short fusion peptide (FP-50) via Escherichia coli in vivo expression. It was employed as a novel probe to detect HIV-1 gp120 protein. The detectable level of gp120 protein using the FP-50 peptide was approximately 20–200 times lower than previously published methods that used a pair of monoclonal antibodies. Thus, this short peptide is a very promising component for detection of gp120 protein during early stages of HIV infection.

  13. Self-Assembling Peptide-Polymer Hydrogels Designed From the Coiled Coil Region of Fibrin

    PubMed Central

    Jing, Peng; Rudra, Jai S.; Herr, Andrew B.; Collier, Joel H.

    2010-01-01

    Biomaterials constructed from self-assembling peptides, peptide derivatives, and peptide-polymer conjugates are receiving increasing attention as defined matrices for tissue engineering, controlled therapeutic release, and in vitro cell expansion, but many are constructed from peptide structures not typically found in the human extracellular matrix. Here we report a self-assembling biomaterial constructed from a designed peptide inspired by the coiled coil domain of human fibrin, the major protein constituent of blood clots and the provisional scaffold of wound healing. Targeted substitutions were made in the residues forming the interface between coiled coil strands for a 37-amino acid peptide from human fibrinogen to stabilize the coiled coil peptide bundle, while the solvent-exposed residues were left unchanged to provide a surface similar to that of the native protein. This peptide, which self-assembled into coiled coil dimers and tetramers, was then used to produce triblock peptide-PEG-peptide bioconjugates that self-assembled into viscoelastic hydrogel biomaterials. PMID:18712921

  14. Design, preparation and characterization of cyclic RGDfK peptide modified poly(ethylene glycol)-block-poly(lactic acid) micelle for targeted delivery.

    PubMed

    Li, Caixia; Wang, Wenlong; Xi, Yuewei; Wang, Jiexin; Chen, Jian-Feng; Yun, Jimmy; Le, Yuan

    2016-07-01

    Molecular targeted cancer therapy is a promising strategy to overcome the lack of specificity of anticancer drug. While the binding of c(RGDfK) (cyclic Arginine-Glycine-Aspartic acid-Phenylalanine-Lysine) to αvβ3 over-expressed on tumor cell has been validated, the underlying interaction remains poorly understood. In this work, docking calculation was applied to investigate the interactions between c(RGDfK)/c(RGDfK)-PEG and αvβ3. The calculated results indicated that c(RGDfK) interacted with αvβ3 mainly by electrostatic interaction, stabilization interaction, and hydrophobic interaction. Conjugation of PEG chain to the c(RGDfK) weakened the binding affinity of c(RGDfK) to αvβ3. Accordingly, docetaxel(DTX)-loaded target micelles (c(RGDfK)-PEG-PLA/PEG-PLA/DTX) were designed, characterized and evaluated using HeLa cells. In vitro release studies demonstrated both target and non-target micelles displayed almost the same profiles, which best fit in Ritger-Peppas model. Cellular uptake and MTT studies revealed that the target micelles with the presence of c(RGDfK) were more efficiently taken up by HeLa cells and significantly improved the cytotoxicity compared to that of non-target micelles. Cell inhibition rate of target micelles was improved by 20% after 24h. Our findings suggest that target micelles may be a potential anticancer drug delivery system in the treatment of integrin αvβ3 over-expressed on tumor cell. PMID:27127057

  15. Systematic screening of the cellular uptake of designed alpha-helix peptides.

    PubMed

    Usui, Kenji; Kikuchi, Takuya; Mie, Masayasu; Kobatake, Eiry; Mihara, Hisakazu

    2013-05-01

    The cellular penetration (CP) activity of functional molecules has attracted significant attention as one of the most promising new approaches for drug delivery. In particular, cell-penetrating peptides (CPPs) have been studied extensively in cellular engineering. Because there have been few large-scale systematic studies to identify peptide sequences with optimal CP activity or that are suitable for further applications in cell engineering, such as cell-specific penetration and cell-selective culture, we screened and compared the cellular uptake (CU) activity of 54 systematically designed α-helical peptides in HeLa cells. Furthermore, the CU activity of 24 designed peptides was examined in four cell lines using a cell fingerprinting technique and statistical approaches. The CU activities in various cells depended on amino acid residues of peptide sequences as well as charge, α-helical content and hydrophobicity of the peptides. Notably, the mutation of a single residue significantly altered the CU ability of a peptide, highlighting the variability of cell uptake mechanisms. Moreover, these results demonstrated the feasibility of cell-selective culture by conducting cell-selective permeation and death in cultures containing two cell types. These studies may lead to further peptide library design and screening for new classes of CPPs with useful functions. PMID:23498920

  16. Heat capacities of amino acids, peptides and proteins.

    PubMed

    Makhatadze, G I

    1998-04-20

    The heat capacity is one of the fundamental parameters describing thermodynamic properties of a system. It has wide applications in a number of areas such as polymer chemistry, protein folding and DNA stability. To aid the scientific community in the analysis of such data, I have compiled a database on the experimentally measured heat capacities of amino acids, polyamino acids, peptides, and proteins in solid state and in aqueous solutions. PMID:9648205

  17. Effective Design of Multifunctional Peptides by Combining Compatible Functions

    PubMed Central

    Diener, Christian; Garza Ramos Martínez, Georgina; Moreno Blas, Daniel; Castillo González, David A.; Corzo, Gerardo; Castro-Obregon, Susana; Del Rio, Gabriel

    2016-01-01

    Multifunctionality is a common trait of many natural proteins and peptides, yet the rules to generate such multifunctionality remain unclear. We propose that the rules defining some protein/peptide functions are compatible. To explore this hypothesis, we trained a computational method to predict cell-penetrating peptides at the sequence level and learned that antimicrobial peptides and DNA-binding proteins are compatible with the rules of our predictor. Based on this finding, we expected that designing peptides for CPP activity may render AMP and DNA-binding activities. To test this prediction, we designed peptides that embedded two independent functional domains (nuclear localization and yeast pheromone activity), linked by optimizing their composition to fit the rules characterizing cell-penetrating peptides. These peptides presented effective cell penetration, DNA-binding, pheromone and antimicrobial activities, thus confirming the effectiveness of our computational approach to design multifunctional peptides with potential therapeutic uses. Our computational implementation is available at http://bis.ifc.unam.mx/en/software/dcf. PMID:27096600

  18. Nutritional value of D-amino acids, D-peptides, and amino acid derivatives in mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This paper describes a method for determining the nutritional value of D-amino acids, D-peptides, and amino acid derivatives using a growth assay in mice fed a synthetic all-amino acid diet. A large number of experiments were carried out in which a molar equivalent of the test compound replaced a n...

  19. Pharmacokinetic properties of tandem d-peptides designed for treatment of Alzheimer's disease.

    PubMed

    Leithold, Leonie H E; Jiang, Nan; Post, Julia; Niemietz, Nicole; Schartmann, Elena; Ziehm, Tamar; Kutzsche, Janine; Shah, N Jon; Breitkreutz, Jörg; Langen, Karl-Josef; Willuweit, Antje; Willbold, Dieter

    2016-06-30

    Peptides are more and more considered for the development of drug candidates. However, they frequently exhibit severe disadvantages such as instability and unfavourable pharmacokinetic properties. Many peptides are rapidly cleared from the organism and oral bioavailabilities as well as in vivo half-lives often remain low. In contrast, some peptides consisting solely of d-enantiomeric amino acid residues were shown to combine promising therapeutic properties with high proteolytic stability and enhanced pharmacokinetic parameters. Recently, we have shown that D3 and RD2 have highly advantageous pharmacokinetic properties. Especially D3 has already proven promising properties suitable for treatment of Alzheimer's disease. Here, we analyse the pharmacokinetic profiles of D3D3 and RD2D3, which are head-to-tail tandem d-peptides built of D3 and its derivative RD2. Both D3D3 and RD2D3 show proteolytic stability in mouse plasma and organ homogenates for at least 24h and in murine and human liver microsomes for 4h. Notwithstanding their high affinity to plasma proteins, both peptides are taken up into the brain following i.v. as well as i.p. administration. Although both peptides contain identical d-amino acid residues, they are arranged in a different sequence order and the peptides show differences in pharmacokinetic properties. After i.p. administration RD2D3 exhibits lower plasma clearance and higher bioavailability than D3D3. We therefore concluded that the amino acid sequence of RD2 leads to more favourable pharmacokinetic properties within the tandem peptide, which underlines the importance of particular sequence motifs, even in short peptides, for the design of further therapeutic d-peptides. PMID:27086111

  20. Behavior of amino acids and peptides exposed in Earth orbit

    NASA Astrophysics Data System (ADS)

    Barbier, Bernard; Boillot, François; Chabin, Annie; Venet, Michel; Bure, Corinne; Jacquet, Romain; Bertrand-Urbaniak, Marylène; Brack, André

    2001-08-01

    In order to understand the chemical comportment of organic molecules of prebiotic interest when exposed to space conditions, amino acids, derivatives and peptides where exposed in Earth orbit during the CNES "Perseus-Exobiologie" mission. Dry films of samples were exposed free or associated with mineral powders to vacuum and to solar light down to 120 nm during three months outside the MIR station. After the mission, the remaining products were analyzed with respect of chemical degradation, racemization and polymerization. The analyses revealed a higher sensitivity of amino acids comparatively to peptides. The identification of by-products has allowed determining some photolysis pathways where decarboxylation and decarbonylation were found to be the major chemical reactions for amino acids and peptides, respectively. The study of associated minerals have shown that meteoritic powder was the most efficient to protect samples against UV light. The exposure of different peptides associated to meteorite powder of various thickness have allowed to determine that 5μm films were at least necessary to protect associated organics. Implications for the exogenous origin of organics are discussed.

  1. How Amino Acids and Peptides Shaped the RNA World

    PubMed Central

    van der Gulik, Peter T.S.; Speijer, Dave

    2015-01-01

    The “RNA world” hypothesis is seen as one of the main contenders for a viable theory on the origin of life. Relatively small RNAs have catalytic power, RNA is everywhere in present-day life, the ribosome is seen as a ribozyme, and rRNA and tRNA are crucial for modern protein synthesis. However, this view is incomplete at best. The modern protein-RNA ribosome most probably is not a distorted form of a “pure RNA ribosome” evolution started out with. Though the oldest center of the ribosome seems “RNA only”, we cannot conclude from this that it ever functioned in an environment without amino acids and/or peptides. Very small RNAs (versatile and stable due to basepairing) and amino acids, as well as dipeptides, coevolved. Remember, it is the amino group of aminoacylated tRNA that attacks peptidyl-tRNA, destroying the bond between peptide and tRNA. This activity of the amino acid part of aminoacyl-tRNA illustrates the centrality of amino acids in life. With the rise of the “RNA world” view of early life, the pendulum seems to have swung too much towards the ribozymatic part of early biochemistry. The necessary presence and activity of amino acids and peptides is in need of highlighting. In this article, we try to bring the role of the peptide component of early life back into focus. We argue that an RNA world completely independent of amino acids never existed. PMID:25607813

  2. Recent Developments in Peptide-Based Nucleic Acid Delivery

    PubMed Central

    Veldhoen, Sandra; Laufer, Sandra D.; Restle, Tobias

    2008-01-01

    Despite the fact that non-viral nucleic acid delivery systems are generally considered to be less efficient than viral vectors, they have gained much interest in recent years due to their superior safety profile compared to their viral counterpart. Among these synthetic vectors are cationic polymers, branched dendrimers, cationic liposomes and cell-penetrating peptides (CPPs). The latter represent an assortment of fairly unrelated sequences essentially characterised by a high content of basic amino acids and a length of 10–30 residues. CPPs are capable of mediating the cellular uptake of hydrophilic macromolecules like peptides and nucleic acids (e.g. siRNAs, aptamers and antisense-oligonucleotides), which are internalised by cells at a very low rate when applied alone. Up to now, numerous sequences have been reported to show cell-penetrating properties and many of them have been used to successfully transport a variety of different cargos into mammalian cells. In recent years, it has become apparent that endocytosis is a major route of internalisation even though the mechanisms underlying the cellular translocation of CPPs are poorly understood and still subject to controversial discussions. In this review, we will summarise the latest developments in peptide-based cellular delivery of nucleic acid cargos. We will discuss different mechanisms of entry, the intracellular fate of the cargo, correlation studies of uptake versus biological activity of the cargo as well as technical problems and pitfalls. PMID:19325804

  3. Recent developments in peptide-based nucleic acid delivery.

    PubMed

    Veldhoen, Sandra; Laufer, Sandra D; Restle, Tobias

    2008-06-01

    Despite the fact that non-viral nucleic acid delivery systems are generally considered to be less efficient than viral vectors, they have gained much interest in recent years due to their superior safety profile compared to their viral counterpart. Among these synthetic vectors are cationic polymers, branched dendrimers, cationic liposomes and cell-penetrating peptides (CPPs). The latter represent an assortment of fairly unrelated sequences essentially characterised by a high content of basic amino acids and a length of 10-30 residues. CPPs are capable of mediating the cellular uptake of hydrophilic macromolecules like peptides and nucleic acids (e.g. siRNAs, aptamers and antisense-oligonucleotides), which are internalised by cells at a very low rate when applied alone. Up to now, numerous sequences have been reported to show cell-penetrating properties and many of them have been used to successfully transport a variety of different cargos into mammalian cells. In recent years, it has become apparent that endocytosis is a major route of internalisation even though the mechanisms underlying the cellular translocation of CPPs are poorly understood and still subject to controversial discussions. In this review, we will summarise the latest developments in peptide-based cellular delivery of nucleic acid cargos. We will discuss different mechanisms of entry, the intracellular fate of the cargo, correlation studies of uptake versus biological activity of the cargo as well as technical problems and pitfalls. PMID:19325804

  4. Rational peptide design for functional materials via molecular self-assembly

    NASA Astrophysics Data System (ADS)

    Rajagopal, Karthikan

    Supra-molecular self-assembly of rationally designed peptides is a promising approach to construct functional materials. This thesis specifically focuses on hydrogels, an important class of materials with potential for applications in tissue engineering, drug delivery and micro-fluidic systems. The objective is to design short peptides that would specifically adopt a stimulus dependent conformation that is strongly amenable to self-assembly resulting in material formation. With this concept the rational design of a 20 amino acid peptide (MAX1) that folds into an amphiphilic beta-hairpin structure and then self-assembles to form a rigid hydrogel under alkaline conditions is presented. The molecular level conformation of MAX1 was characterized using circular dichroism and FTIR spectroscopies. The mesoscale structure of the hydrogel assessed using confocal and transmission electron micron microscopies and neutron scattering techniques shows that peptide self-assembly results in the formation of fibrils that are homogeneously 3 nm in diameter. The mechanical properties of the hydrogel probed using oscillatory rheology shows that MAX1 forms a stiff hydrogel. Since the self-assembly process is coupled to the intra-molecularly folded state of the peptide, stimulus responsiveness can be specifically engineered into the sequence by rational design. This was demonstrated in the design of peptides that form hydrogels in response to a specific stimulus such as temperature, pH or ionic strength. The significance of peptide design in the context of self-assembly and its relationship to the nanostructure was studied by designing a series of peptides derived from MAX1. Evolving from these studies is an understanding of the relationship between molecular level peptide structure and the nanoscale supra-molecular morphology. Based on this, it has been shown that alternate morphologies distinct from those observed with the gel forming peptides, such as non-twisting laminates or tube

  5. Design and Application of Antimicrobial Peptide Conjugates.

    PubMed

    Reinhardt, Andre; Neundorf, Ines

    2016-01-01

    Antimicrobial peptides (AMPs) are an interesting class of antibiotics characterized by their unique antibiotic activity and lower propensity for developing resistance compared to common antibiotics. They belong to the class of membrane-active peptides and usually act selectively against bacteria, fungi and protozoans. AMPs, but also peptide conjugates containing AMPs, have come more and more into the focus of research during the last few years. Within this article, recent work on AMP conjugates is reviewed. Different aspects will be highlighted as a combination of AMPs with antibiotics or organometallic compounds aiming to increase antibacterial activity or target selectivity, conjugation with photosensitizers for improving photodynamic therapy (PDT) or the attachment to particles, to name only a few. Owing to the enormous resonance of antimicrobial conjugates in the literature so far, this research topic seems to be very attractive to different scientific fields, like medicine, biology, biochemistry or chemistry. PMID:27187357

  6. Design and Application of Antimicrobial Peptide Conjugates

    PubMed Central

    Reinhardt, Andre; Neundorf, Ines

    2016-01-01

    Antimicrobial peptides (AMPs) are an interesting class of antibiotics characterized by their unique antibiotic activity and lower propensity for developing resistance compared to common antibiotics. They belong to the class of membrane-active peptides and usually act selectively against bacteria, fungi and protozoans. AMPs, but also peptide conjugates containing AMPs, have come more and more into the focus of research during the last few years. Within this article, recent work on AMP conjugates is reviewed. Different aspects will be highlighted as a combination of AMPs with antibiotics or organometallic compounds aiming to increase antibacterial activity or target selectivity, conjugation with photosensitizers for improving photodynamic therapy (PDT) or the attachment to particles, to name only a few. Owing to the enormous resonance of antimicrobial conjugates in the literature so far, this research topic seems to be very attractive to different scientific fields, like medicine, biology, biochemistry or chemistry. PMID:27187357

  7. Tuning Curvature and Stability of Monoolein Bilayers by Designer Lipid-Like Peptide Surfactants

    PubMed Central

    Yaghmur, Anan; Laggner, Peter; Zhang, Shuguang; Rappolt, Michael

    2007-01-01

    This study reports the effect of loading four different charged designer lipid-like short anionic and cationic peptide surfactants on the fully hydrated monoolein (MO)-based Pn3m phase (Q224). The studied peptide surfactants comprise seven amino acid residues, namely A6D, DA6, A6K, and KA6. D (aspartic acid) bears two negative charges, K (lysine) bears one positive charge, and A (alanine) constitutes the hydrophobic tail. To elucidate the impact of these peptide surfactants, the ternary MO/peptide/water system has been investigated using small-angle X-ray scattering (SAXS), within a certain range of peptide concentrations (R≤0.2) and temperatures (25 to 70°C). We demonstrate that the bilayer curvature and the stability are modulated by: i) the peptide/lipid molar ratio, ii) the peptide molecular structure (the degree of hydrophobicity, the type of the hydrophilic amino acid, and the headgroup location), and iii) the temperature. The anionic peptide surfactants, A6D and DA6, exhibit the strongest surface activity. At low peptide concentrations (R = 0.01), the Pn3m structure is still preserved, but its lattice increases due to the strong electrostatic repulsion between the negatively charged peptide molecules, which are incorporated into the interface. This means that the anionic peptides have the effect of enlarging the water channels and thus they serve to enhance the accommodation of positively charged water-soluble active molecules in the Pn3m phase. At higher peptide concentration (R = 0.10), the lipid bilayers are destabilized and the structural transition from the Pn3m to the inverted hexagonal phase (H2) is induced. For the cationic peptides, our study illustrates how even minor modifications, such as changing the location of the headgroup (A6K vs. KA6), affects significantly the peptide's effectiveness. Only KA6 displays a propensity to promote the formation of H2, which suggests that KA6 molecules have a higher degree of incorporation in the

  8. De Novo Design of a Tumor-Penetrating Peptide

    PubMed Central

    Alberici, Luca; Roth, Lise; Sugahara, Kazuki N.; Agemy, Lilach; Kotamraju, Venkata R.; Teesalu, Tambet; Bordignon, Claudio; Traversari, Catia; Rizzardi, Gian-Paolo; Ruoslahti, Erkki

    2012-01-01

    Poor penetration of anti-tumor drugs into the extravascular tumor tissue isoften a major factor limiting the efficacy of cancer treatments. Our group has recently described a strategy to enhance tumor penetration of chemotherapeutic drugs through use of iRGD peptide (CRGDK/RGPDC). This peptide comprises two sequence motifs: RGD, which binds to αvβ3/5 integrins on tumor endothelia and tumor cells and a cryptic CendR motif (R/KXXR/K-OH). Once integrin binding has brought iRGD to the tumor, the peptide is proteolytically cleaved to expose the cryptic CendR motif. The truncated peptide loses affinity for its primary receptor and binds to neuropilin-1, activating a tissue-penetration pathway that delivers the peptide along with attached or co-administered payload into the tumor mass. Here we describe the design of a new tumor-penetrating peptide based on the current knowledge of homing sequences and internalizing receptors. The tumor-homing motif in the new peptide is the NGR sequence, which binds to endothelial CD13. The NGR sequence was placed in the context of a CendR motif (RNGR), and this sequence was embedded in the iRGD framework. The resulting peptide (CRNGRGPDC, iNGR) homed to tumor vessels and penetrated into tumor tissue more effectively than the standard NGR peptide. iNGR induced greater tumor penetration of coupled nanoparticles and co-administered compounds than NGR. Doxorubicin given together with iNGR was significantly more efficacious than the drug alone. These results show that a tumor-specific, tissue-penetrating peptide can be constructed from known sequence elements. This principle may be useful in designing tissue-penetrating peptides for other diseases. PMID:23151901

  9. A designed buried salt bridge modulates heterodimerization of a membrane peptide.

    PubMed

    Shinde, Sandip; Binder, Jennifer K; Goyal, Bhupesh; Woodrum, B; De Munari, Sonia; Levitus, Marcia; Ghirlanda, Giovanna

    2014-11-01

    Specific helix-helix interactions underpin the correct assembly of multipass membrane proteins. Here, we show that a designed buried salt bridge mediates heterodimer formation of model transmembrane helical peptides in a pH-dependent manner. The model peptides bear side chains functionalized with either a carboxylic acid or a primary amine within a hydrophobic segment. The association behavior was monitored by Förster resonance energy transfer, revealing that heterodimer formation is maximized at a pH close to neutrality (pH 6.5), at which each peptide is found in a charged state. In contrast, heterodimerization is disfavored at low and high values of pH, because either the carboxylic acid or the primary amine is present in its neutral state, thus preventing the formation of a salt bridge. These findings provide a blueprint for the design and modulation of protein-protein interactions in membrane proteins. PMID:25250823

  10. Graphene Symmetry Amplified by Designed Peptide Self-Assembly.

    PubMed

    Mustata, Gina-Mirela; Kim, Yong Ho; Zhang, Jian; DeGrado, William F; Grigoryan, Gevorg; Wanunu, Meni

    2016-06-01

    We present a strategy for designed self-assembly of peptides into two-dimensional monolayer crystals on the surface of graphene and graphite. As predicted by computation, designed peptides assemble on the surface of graphene to form very long, parallel, in-register β-sheets, which we call β-tapes. Peptides extend perpendicularly to the long axis of each β-tape, defining its width, with hydrogen bonds running along the axis. Tapes align on the surface to create highly regular microdomains containing 4-nm pitch striations. Moreover, in agreement with calculations, the atomic structure of the underlying graphene dictates the arrangement of the β-tapes, as they orient along one of six directions defined by graphene's sixfold symmetry. A cationic-assembled peptide surface is shown here to strongly adhere to DNA, preferentially orienting the double helix along β-tape axes. This orientational preference is well anticipated from calculations, given the underlying peptide layer structure. These studies illustrate how designed peptides can amplify the Ångstrom-level atomic symmetry of a surface onto the micrometer scale, further imparting long-range directional order onto the next level of assembly. The remarkably stable nature of these assemblies under various environmental conditions suggests applications in enzymelike catalysis, biological interfaces for cellular recognition, and two-dimensional platforms for studying DNA-peptide interactions. PMID:27276268

  11. Formulation of a Peptide Nucleic Acid Based Nucleic Acid Delivery Construct

    PubMed Central

    Millili, Peter G.; Yin, Daniel H.; Fan, Haihong; Naik, Ulhas P.; Sullivan, Millicent O.

    2010-01-01

    Gene delivery biomaterials need to be designed to efficiently achieve nuclear delivery of plasmid DNA. Polycations have been used to package DNA and other nucleic acids within sub-micron sized particles, offering protection from shear-induced or enzymatic degradation. However, cytotoxicity issues coupled with limited in vivo transfection efficiencies minimize the effectiveness of this approach. In an effort to improve upon existing technologies aimed at delivering nucleic acids, an alternative approach to DNA packaging was explored. Peptide nucleic acids (PNAs) were used to directly functionalize DNA with poly(ethylene glycol) (PEG) chains that provide a steric layer and inhibit multimolecular aggregation during complexation. DNA prePEGylation by this strategy was predicted to enable the formation of more homogeneous and efficiently packaged polyplexes. In this work, DNA-PNA-peptide-PEG (DP3) conjugates were synthesized and self-assembled with 25 kDa poly(ethylenimine) (PEI). Complexes with small standard deviations and average diameters ranging from 30 – 50 nm were created, with minimal dependence of complex size on N:P ratio (PEI amines to DNA phosphates). Furthermore, PEI-DNA interactions were altered by the derivitization strategy, resulting in tighter compaction of the PEI-DP3 complexes in comparison with PEI-DNA complexes. Transfection experiments in Chinese Hamster Ovary (CHO) cells revealed comparable transfection efficiencies but reduced cytotoxicities of the PEI-DP3 complexes relative to PEI-DNA complexes. The enhanced cellular activities of the PEI-DP3 complexes were maintained following the removal of free PEI from the PEI-DP3 formulations, whereas the cellular activity of the conventional PEI-DNA formulations was reduced by free PEI removal. These findings suggest that DNA prePEGylation by the PNA-based strategy might provide a way to circumvent cytotoxicity and formulation issues related to the use of PEI for in vivo gene delivery. PMID:20131756

  12. Structure and Energetic Contributions of a Designed Modular Peptide-Binding Protein with Picomolar Affinity.

    PubMed

    Hansen, Simon; Tremmel, Dirk; Madhurantakam, Chaithanya; Reichen, Christian; Mittl, Peer R E; Plückthun, Andreas

    2016-03-16

    Natural armadillo repeat proteins (nArmRP) like importin-α or β-catenin bind their target peptides such that each repeat interacts with a dipeptide unit within the stretched target peptide. However, this modularity is imperfect and also restricted to short peptide stretches of usually four to six consecutive amino acids. Here we report the development and characterization of a regularized and truly modular peptide-specific binding protein, based on designed armadillo repeat proteins (dArmRP), binding to peptides of alternating lysine and arginine residues (KR)n. dArmRP were obtained from nArmRP through cycles of extensive protein engineering, which rendered them more uniform. This regularity is reflected in the consistent binding of dArmRP to (KR)-peptides, where affinities depend on the lengths of target peptides and the number of internal repeats in a very systematic manner, thus confirming the modularity of the interaction. This exponential dependency between affinity and recognition length suggests that each module adds a constant increment of binding energy to sequence-specific recognition. This relationship was confirmed by comprehensive mutagenesis studies that also reveal the importance of individual peptide side chains. The 1.83 Å resolution crystal structure of a dArmRP with five identical internal repeats in complex with the cognate (KR)5 peptide proves a modular binding mode, where each dipeptide is recognized by one internal repeat. The confirmation of this true modularity over longer peptide stretches lays the ground for the design of binders with different specificities and tailored affinities by the assembly of dipeptide-specific modules based on armadillo repeats. PMID:26878586

  13. Regulation of peptide YY homeostasis by gastric acid and gastrin.

    PubMed

    Gomez, G; Padilla, L; Udupi, V; Tarasova, N; Sundler, F; Townsend, C M; Thompson, J C; Greeley, G H

    1996-04-01

    Peptide YY (PYY) is a gut hormone localized primarily in the distal bowel. Because circulating PYY inhibits gastric acid secretion, we investigated the effects of gastric acid secretion and gastrin on gene expression and secretion of PYY. In conscious dogs, PYY release in response to oral food was inhibited (P < 0.05) by pharmacologic inhibition of gastric acid secretion (omeprazole, famotidine). In rats, omeprazole treatment resulted in a significant elevation in serum gastrin concentrations and a simultaneous decrease in PYY messenger RNA (mRNA) and peptide levels in the colon; administration of a gastrin receptor antagonist (L365, 260) prevented the inhibitory actions of omeprazole on colonic PYY mRNA levels. In athymic-nude mice, implantation of a human gastrinoma resulted in an elevation of serum gastrin concentrations and a concomitant depression of colonic PYY mRNA levels. We conclude that endogenous gastric acid secretion up-regulates PYY release and PYY mRNA expression. Circulating gastrin acts to down-regulate PYY release and PYY mRNA expression. This study provides evidence that foregut functions (i.e., gastric acid secretion and gastrin release) exert control over an antiacid signal (e.g. PYY release) emanating from the hindgut. PMID:8625912

  14. Design of nanostructures based on aromatic peptide amphiphiles.

    PubMed

    Fleming, Scott; Ulijn, Rein V

    2014-12-01

    Aromatic peptide amphiphiles are gaining popularity as building blocks for the bottom-up fabrication of nanomaterials, including gels. These materials combine the simplicity of small molecules with the versatility of peptides, with a range of applications proposed in biomedicine, nanotechnology, food science, cosmetics, etc. Despite their simplicity, a wide range of self-assembly behaviours have been described. Due to varying conditions and protocols used, care should be taken when attempting to directly compare results from the literature. In this review, we rationalise the structural features which govern the self-assembly of aromatic peptide amphiphiles by focusing on four segments, (i) the N-terminal aromatic component, (ii) linker segment, (iii) peptide sequence, and (iv) C-terminus. It is clear that the molecular structure of these components significantly influences the self-assembly process and resultant supramolecular architectures. A number of modes of assembly have been proposed, including parallel, antiparallel, and interlocked antiparallel stacking conformations. In addition, the co-assembly arrangements of aromatic peptide amphiphiles are reviewed. Overall, this review elucidates the structural trends and design rules that underpin the field of aromatic peptide amphiphile assembly, paving the way to a more rational design of nanomaterials based on aromatic peptide amphiphiles. PMID:25199102

  15. Design and synthesis of cationic antibacterial peptide based on Leucrocin I sequence, antibacterial peptide from crocodile (Crocodylus siamensis) white blood cell extracts.

    PubMed

    Yaraksa, Nualyai; Anunthawan, Thitiporn; Theansungnoen, Tinnakorn; Daduang, Sakda; Araki, Tomohiro; Dhiravisit, Apisak; Thammasirirak, Sompong

    2014-03-01

    Leucrocin I is an antibacterial peptide isolated from crocodile (Crocodylus siamensis) white blood cell extracts. Based on Leucrocin I sequence, cationic peptide, NY15, was designed, synthesized and evaluated for antibacterial activity against Bacillus sphaericus TISTR 678, Bacillus megaterium (clinical isolate), Vibrio cholerae (clinical isolate), Salmonella typhi (clinical isolate), Salmonella typhi ATCC 5784 and Escherichia coli 0157:H7. The efficacy of the peptide made from all L-amino acids was also compared with all D-amino acids. The peptide made from all D-amino acids was more active than the corresponding L-enantiomer. In our detailed study, the interaction between peptides and the cell membrane of Vibrio cholerae as part of their killing mechanism was studied by fluorescence and electron microscopy. The results show that the membrane was the target of action of the peptides. Finally, the cytotoxicity assays revealed that both L-NY15 and D-NY15 peptides are non-toxic to mammalian cells at bacteriolytic concentrations. PMID:24192554

  16. De Novo Designed Imaging Agents Based on Lanthanide Peptides Complexes.

    PubMed

    Peacock, A F A

    2016-01-01

    Herein are discussed a selection of lanthanide peptide/protein complexes in view of their potential applications as imaging agents, both in terms of luminescence detection and magnetic resonance imaging. Though this chapter covers a range of different peptides and protein, if focuses specifically on the opportunities afforded by the de novo design of coiled coils, miniature protein scaffolds, and the development on lanthanide-binding sites into these architectures. The requirements for lanthanide coordination and the challenges that need to be addressed when preparing lanthanide peptides with a view to their potential adoption as clinical imaging applications, will be highlighted. PMID:27586349

  17. Photosensitizing effect of cations on amino acids and peptides.

    PubMed

    Bogdanova, N P; Khenokh, M A

    1969-01-01

    In connection with a study of the chemical evolution of abiogenically synthesized organic compounds on primitive Earth and the physical conditions of other planets, this paper reports the experimental results obtained by the photolysis of solutions of aliphatic amino acids (glycine, alanine, valine, leucine, n. leucine) and peptides in the atmosphere of the air, N2, Ar and CO2 in the presence of the most simple photocatalyzers-cations of sulphates. The evidence shows that the photochemical conversion of NH2 acids depends on the content of the atmosphere. The decay of NH2-group is most active in air. N2 and Ar exert no significant influence on deamination, whereas in the atmosphere of CO2 the formation of ammonia in valine, for example, was only 29 per cent of its total amount during photolysis in the air. Cu2+ and Fe2+ catalyzed while Al3+ inhibited the ammonia excretion. The formation of acetaldehyde during alanine photolysis was actually independent from the atmosphere of N2 and was inhibited in Ar and CO2. Oxydative processes inducing the formation of glyoxalic acid and formaldehyde were sharply inhibited in Ar, N2 and CO2. Under the influence of ultraviolet light of the decay of NH2-acids is also accompanied by the formation of new NH2-acids. The photosensitizing effect of cations induces a rupture of -CO-NH-bonds in peptides and, provided heavy radiation doses, prevents the formation of new NH2-acids. The longer the dipeptide chain, the more significant the quantum yield of its decomposition. The photolysis of dipeptides, leading to their decay, does not necessarily induce a hydrolytic rupture of -CO-NH-bonds resulting in the formation of three amino acids. The results obtained permit approaching problems concerning the effect of the gas content of the atmosphere and various cations (photocatalyzers) on photolytic conversion of abiogenically synthesized and biogenically significant substances, amino acids for example, at the action of ultraviolet light. PMID

  18. Rational Design of MMP Degradable Peptide-Based Supramolecular Filaments

    PubMed Central

    2015-01-01

    One-dimensional nanostructures formed by self-assembly of small molecule peptides have been extensively explored for use as biomaterials in various biomedical contexts. However, unlike individual peptides that can be designed to be specifically degradable by enzymes/proteases of interest, their self-assembled nanostructures, particularly those rich in β-sheets, are generally resistant to enzymatic degradation because the specific cleavage sites are often embedded inside the nanostructures. We report here on the rational design of β-sheet rich supramolecular filaments that can specifically dissociate into less stable micellar assemblies and monomers upon treatment with matrix metalloproteases-2 (MMP-2). Through linkage of an oligoproline segment to an amyloid-derived peptide sequence, we first synthesized an amphiphilic peptide that can undergo a rapid morphological transition in response to pH variations. We then used MMP-2 specific peptide substrates as multivalent cross-linkers to covalently fix the amyloid-like filaments in the self-assembled state at pH 4.5. Our results show that the cross-linked filaments are stable at pH 7.5 but gradually break down into much shorter filaments upon cleavage of the peptidic cross-linkers by MMP-2. We believe that the reported work presents a new design platform for the creation of amyloid-like supramolecular filaments responsive to enzymatic degradation. PMID:24611531

  19. Designing Human m1 Muscarinic Receptor-Targeted Hydrophobic Eigenmode Matched Peptides as Functional Modulators

    PubMed Central

    Selz, Karen A.; Mandell, Arnold J.; Shlesinger, Michael F.; Arcuragi, Vani; Owens, Michael J.

    2004-01-01

    A new proprietary de novo peptide design technique generated ten 15-residue peptides targeting and containing the leading nontransmembrane hydrophobic autocorrelation wavelengths, “modes”, of the human m1 muscarinic cholinergic receptor, m1AChR. These modes were also shared by the m4AChR subtype (but not the m2, m3, or m5 subtypes) and the three-finger snake toxins that pseudoirreversibly bind m1AChR. The linear decomposition of the hydrophobically transformed m1AChR amino acid sequence yielded ordered eigenvectors of orthogonal hydrophobic variational patterns. The weighted sum of two eigenvectors formed the peptide design template. Amino acids were iteratively assigned to template positions randomly, within hydrophobic groups. One peptide demonstrated significant functional indirect agonist activity, and five produced significant positive allosteric modulation of atropine-reversible, direct-agonist-induced cellular activation in stably m1AChR-transfected Chinese hamster ovary cells, reflected in integrated extracellular acidification responses. The peptide positive allosteric ligands produced left-shifts and peptide concentration-response augmentation in integrated extracellular acidification response asymptotic sigmoidal functions and concentration-response behavior in Hill number indices of positive cooperativity. Peptide mode specificity was suggested by negative crossover experiments with human m2ACh and D2 dopamine receptors. Morlet wavelet transformation of the leading eigenvector-derived, m1AChR eigenfunctions locates seven hydrophobic transmembrane segments and suggests possible extracellular loop locations for the peptide-receptor mode-matched, modulatory hydrophobic aggregation sites. PMID:14990463

  20. Molecular design of specific metal-binding peptide sequences from protein fragments: theory and experiment.

    PubMed

    Kozísek, Milan; Svatos, Ales; Budesínský, Milos; Muck, Alexander; Bauer, Mikael C; Kotrba, Pavel; Ruml, Tomás; Havlas, Zdenek; Linse, Sara; Rulísek, Lubomír

    2008-01-01

    A novel strategy is presented for designing peptides with specific metal-ion chelation sites, based on linking computationally predicted ion-specific combinations of amino acid side chains coordinated at the vertices of the desired coordination polyhedron into a single polypeptide chain. With this aim, a series of computer programs have been written that 1) creates a structural combinatorial library containing Zi-(X)n-Zj sequences (n=0-14; Z: amino acid that binds the metal through the side chain; X: any amino acid) from the existing protein structures in the non-redundant Protein Data Bank; 2) merges these fragments into a single Z1-(X)n1 -Z2-(X)n2 -Z3-(X)n3 -...-Zj polypeptide chain; and 3) automatically performs two simple molecular mechanics calculations that make it possible to estimate the internal strain in the newly designed peptide. The application of this procedure for the most M2+-specific combinations of amino acid side chains (M: metal; see L. Rulísek, Z. Havlas J. Phys. Chem. B 2003, 107, 2376-2385) yielded several peptide sequences (with lengths of 6-20 amino acids) with the potential for specific binding with six metal ions (Co2+, Ni2+, Cu2+, Zn2+, Cd2+ and Hg2+). The gas-phase association constants of the studied metal ions with these de novo designed peptides were experimentally determined by MALDI mass spectrometry by using 3,4,5-trihydroxyacetophenone as a matrix, whereas the thermodynamic parameters of the metal-ion coordination in the condensed phase were measured by isothermal titration calorimetry (ITC), chelatometry and NMR spectroscopy methods. The data indicate that some of the computationally predicted peptides are potential M2+-specific metal-ion chelators. PMID:18633954

  1. Designing biomaterials exploiting beta-sheet forming peptides self-assembly

    NASA Astrophysics Data System (ADS)

    Saiani, Alberto

    2013-03-01

    The use of non-covalent self-assembly to construct materials has become a prominent strategy in material science offering practical routes for the construction of increasingly functional materials for a variety of applications ranging from electronic to biotechnology. A variety of molecular building blocks can be used for this purpose, one such block that has attracted considerable attention are de-novo designed peptides. The library of 20 natural amino acids offers the ability to play with the intrinsic properties of the peptide such as structure, hydrophobicity, charge and functionality allowing the design of materials with a wide range of properties. The beta-sheet motif is of particular interest as short peptides can be designed to form beta-sheet rich fibres that entangle and consequently form hydrogels. These hydrogels can be further functionalised using specific biological signals or drugs by synthesising functionalised peptides that are incorporated into the hydrogel network during the self-assembling process. This functionalisation approach is very attractive has it does not require any chemistry avoiding therefore the use of additional potentially toxic chemicals. It also offers the possibility to introduce multiple functionalities in a straightforward fashion. The hydrogels can also be made responsive through the use of enzymatic catalysis and/or conjugation with responsive polymers. In this presentation we will discuss the design opportunities offered by these peptides to create new functional biomaterials.

  2. Scanning of 16S Ribosomal RNA for Peptide Nucleic Acid Targets.

    PubMed

    Górska, Anna; Markowska-Zagrajek, Agnieszka; Równicki, Marcin; Trylska, Joanna

    2016-08-25

    We have designed a protocol and server to aid in the search for putative binding sites in 16S rRNA that could be targeted by peptide nucleic acid oligomers. Various features of 16S rRNA were considered to score its regions as potential targets for sequence-specific binding that could result in inhibition of ribosome function. Specifically, apart from the functional importance of a particular rRNA region, we calculated its accessibility, flexibility, energetics of strand invasion by an oligomer, as well as similarity to human rRNA. To determine 16S rRNA flexibility in the ribosome context, we performed all-atom molecular dynamics simulations of the 30S subunit in explicit solvent. We proposed a few 16S RNA target sites, and one of them was tested experimentally to verify inhibition of bacterial growth by a peptide nucleic acid oligomer. PMID:27105576

  3. Dissecting Electrostatic Contributions to Folding and Self-Assembly Using Designed Multicomponent Peptide Systems.

    PubMed

    Parmar, Avanish S; James, Jose K; Grisham, Daniel R; Pike, Douglas H; Nanda, Vikas

    2016-04-01

    We investigate formation of nano- to microscale peptide fibers and sheets where assembly requires association of two distinct collagen mimetic peptides (CMPs). The multicomponent nature of these designs allows the decoupling of amino acid contributions to peptide folding versus higher-order assembly. While both arginine and lysine containing CMP sequences can favor triple-helix folding, only arginine promotes rapid supramolecular assembly in each of the three two-component systems examined. Unlike lysine, the polyvalent guanidyl group of arginine is capable of both intra- and intermolecular contacts, promoting assembly. This is consistent with the supramolecular diversity of CMP morphologies observed throughout the literature. It also connects CMP self-assembly with a broad range of biomolecular interaction phenomena, providing general principles for modeling and design. PMID:26966815

  4. The nature of peptide interactions with acid end-group PLGAs and facile aqueous-based microencapsulation of therapeutic peptides

    PubMed Central

    Sophocleous, Andreas M.; Desai, Kashappa-Goud H.; Mazzara, J. Maxwell; Tong, Ling; Cheng, Ji-Xin; Olsen, Karl F.; Schwendeman, Steven P.

    2013-01-01

    An important poorly understood phenomenon in controlled-release depots involves the strong interaction between common cationic peptides and low Mw free acid end-group poly(lactic-co-glycolic acids) (PLGAs) used to achieve continuous peptide release kinetics. The kinetics of peptide sorption to PLGA was examined by incubating peptide solutions of 0.2-4 mM octreotide or leuprolide acetate salts in 0.1 M HEPES buffer, pH 7.4, with polymer particles or films at 4-37 °C for 24 h. The extent of absorption/loading of peptides in PLGA particles/films was assayed by two-phase extraction and amino acid analysis. Confocal Raman microspectroscopy and stimulated Raman scattering (SRS) and laser scanning confocal imaging techniques were used to examine peptide penetration in the polymer phase. The release of sorbed peptide from leuprolide-PLGA particles was evaluated both in vitro (PBST + 0.02% sodium azide, 37 °C) and in vivo (male Sprague-Dawley rats). We found that when the PLGA-COOH chains are sufficiently mobilized, therapeutic peptides not only bind at the surface, a common belief to date, but can also internalized and distributed throughout the polymer phase at physiological temperature forming a salt with low-molecular weight PLGA-COOH. Importantly, absorption of leuprolide into low MW PLGA-COOH particles yielded ~17 wt% leuprolide loading in the polymer (i.e., ~70% of PLGA-COOH acids occupied), and the absorbed peptide was released from the polymer for > 2 weeks in a controlled fashion in vitro and as indicated by sustained testosterone suppression in male Sprague-Dawley rats. This new approach, which bypasses the traditional encapsulation method and associated production cost, opens up the potential for facile production of low-cost controlled-release injectable depots for leuprolide and related peptides. PMID:24021356

  5. Design of non-aggregating variants of Aβ peptide

    SciTech Connect

    Caine, Joanne M.; Churches, Quentin; Waddington, Lynne; Nigro, Julie; Breheney, Kerry; Masters, Colin L.; Nuttall, Stewart D.; Streltsov, Victor A.

    2014-10-24

    Highlights: • Non-aggregating, non-toxic variants of Aβ peptide were designed using Aβ structure. • Mutations reduce aggregation by stabilising Aβ into small non-toxic oligomers. • Identification of these residues will assist the design of future therapeutic peptides. - Abstract: Self association of the amyloid-β (Aβ{sub 42}) peptide into oligomers, high molecular weight forms, fibrils and ultimately neuritic plaques, has been correlated with progressive cognitive decline in Alzheimer’s disease. Thus, insights into the drivers of the aggregation pathway have the capacity to significantly contribute to our understanding of disease mechanism. Functional assays and a three-dimensional crystal structure of the P3 amyloidogenic region 18–41 of Aβ were used to identify residues important in self-association and to design novel non-aggregating variants of the peptide. Biophysical studies (gel filtration, SDS–PAGE, dynamic light scattering, thioflavin T assay, and electron microscopy) demonstrate that in contrast to wild type Aβ these targeted mutations lose the ability to self-associate. Loss of aggregation also correlates with reduced neuronal toxicity. Our results highlight residues and regions of the Aβ peptide important for future targeting agents aimed at the amelioration of Alzheimer’s disease.

  6. Heteropoly acids triggered self-assembly of cationic peptides into photo- and electro-chromic gels.

    PubMed

    Li, Jingfang; Xu, Jing; Li, Xiaodong; Gao, Wenmei; Wang, Liyan; Wu, Lixin; Lee, Myongsoo; Li, Wen

    2016-07-01

    A series of cationic peptides with alternating lysines and hydrophobic residues were designed and synthesized. These kinds of short peptides with protonated lysines can complex with anionic heteropoly acids (HPAs) to form a stable gel in water/ethanol mixed solution. Circular dichroism spectroscopy showed that the short peptides adopted a mixed conformation (β-sheet and random-coil) within the gel matrix. Scanning and transmission electron microscopy revealed that the heteropoly acids, acting as nanosized cross-linkers, first initiated the self-assembly of the cationic peptides into spherical nanostructures. Then these nanospheres accumulated with each other through hydrogen bonds and hydrophobic interactions to form large sheet-like assemblies, which further interconnected with each other forming continuous 3D network structures. Fourier-transform infrared spectroscopy showed that the structural integrity of the HPAs was maintained during the gelation process. The resultant hybrid gels showed reversible photo- and elecrtro-chromic properties. X-ray photoelectron spectroscopy revealed that the hybrid gels, capable of persistent and reversible changes of their colour, are attributed to the intervalence charge-transfer transition of the HPAs. Reversible information writing and erasing were demonstrated through a repeated photo-lithograph or electric stimuli without significant loss of the gel performance. PMID:27240759

  7. Epitope prediction algorithms for peptide-based vaccine design.

    PubMed

    Florea, Liliana; Halldórsson, Bjarni; Kohlbacher, Oliver; Schwartz, Russell; Hoffman, Stephen; Istrail, Sorin

    2003-01-01

    Peptide-based vaccines, in which small peptides derived from target proteins (eptiopes) are used to provoke an immune reaction, have attracted considerable attention recently as a potential means both of treating infectious diseases and promoting the destruction of cancerous cells by a patient's own immune system. With the availability of large sequence databases and computers fast enough for rapid processing of large numbers of peptides, computer aided design of peptide-based vaccines has emerged as a promising approach to screening among billions of possible immune-active peptides to find those likely to provoke an immune response to a particular cell type. In this paper, we describe the development of three novel classes of methods for the prediction problem. We present a quadratic programming approach that can be trained on quantitative as well as qualitative data. The second method uses linear programming to counteract the fact that our training data contains mostly positive examples. The third class of methods uses sequence profiles obtained by clustering known epitopes to score candidate peptides. By integrating these methods, using a simple voting heuristic, we achieve improved accuracy over the state of the art. PMID:16826643

  8. Loligomers: design of de novo peptide-based intracellular vehicles.

    PubMed Central

    Sheldon, K; Liu, D; Ferguson, J; Gariépy, J

    1995-01-01

    Defined branched peptides (loligomers) incorporating cytoplasmic translocation signals, nuclear localization sequences, and fluorescent probes were designed and synthesized to demonstrate the feasibility and simplicity of creating novel classes of intracellular vehicles. Loligomers containing all the above signals were rapidly internalized by Chinese hamster ovary (CHO) cells and accumulated in their nucleus. At 4 degrees C, the interaction of peptide constructs with CHO cells was limited to membrane association. Loligomers entered cells at higher temperatures by adsorptive endocytosis. Inhibitors of ATP synthesis affected cytoplasmic import only weakly but abolished nuclear uptake. The peptide signals guided both cytoplasmic and nuclear localization events. The properties exhibited by loligomers suggest a strategy for the facile design of "guided" classes of intracellular agents. Images Fig. 3 PMID:7892224

  9. Light-Switchable Peptides with a Hemithioindigo Unit: Peptide Design, Photochromism, and Optical Spectroscopy.

    PubMed

    Kitzig, S; Thilemann, M; Cordes, T; Rück-Braun, Karola

    2016-05-01

    This Minireview focuses on the hemithioindigo photoswitch and its use for the reversible control of three-dimensional peptide structure and related biological functions. Both the general design aspects and biophysical properties of various hemithioindigo-based chromopeptides are summarized. Hemithioindigo undergoes reversible Z→E photoisomerization after absorption of visible light. The unique ultrafast switching mechanism of hemithioindigo combines picosecond isomerization kinetics with strong double-bond torsion after light absorption, making it the ideal tool for instantaneous modulation of biological structure. Various inhibitors and model peptides based on hemithioindigo are described that can directly regulate biological signaling or allow the fastest events in peptide folding to be studied. Finally, a diverse range of chromopeptides with photoswitchable β-hairpin structures based on azobenzenes, stilbenes, and hemithioindigo are compared to emphasize the unique properties of hemithioindigo. PMID:26789782

  10. De Novo Design of Skin-Penetrating Peptides for Enhanced Transdermal Delivery of Peptide Drugs.

    PubMed

    Menegatti, Stefano; Zakrewsky, Michael; Kumar, Sunny; De Oliveira, Joshua Sanchez; Muraski, John A; Mitragotri, Samir

    2016-03-01

    Skin-penetrating peptides (SPPs) are attracting increasing attention as a non-invasive strategy for transdermal delivery of therapeutics. The identification of SPP sequences, however, currently performed by experimental screening of peptide libraries, is very laborious. Recent studies have shown that, to be effective enhancers, SPPs must possess affinity for both skin keratin and the drug of interest. We therefore developed a computational process for generating and screening virtual libraries of disulfide-cyclic peptides against keratin and cyclosporine A (CsA) to identify SPPs capable of enhancing transdermal CsA delivery. The selected sequences were experimentally tested and found to bind both CsA and keratin, as determined by mass spectrometry and affinity chromatography, and enhance transdermal permeation of CsA. Four heptameric sequences that emerged as leading candidates (ACSATLQHSCG, ACSLTVNWNCG, ACTSTGRNACG, and ACSASTNHNCG) were tested and yielded CsA permeation on par with previously identified SPP SPACE (TM) . An octameric peptide (ACNAHQARSTCG) yielded significantly higher delivery of CsA compared to heptameric SPPs. The safety profile of the selected sequences was also validated by incubation with skin keratinocytes. This method thus represents an effective procedure for the de novo design of skin-penetrating peptides for the delivery of desired therapeutic or cosmetic agents. PMID:26799634

  11. Cyclolization of D-lysergic acid alkaloid peptides.

    PubMed

    Havemann, Judith; Vogel, Dominik; Loll, Bernhard; Keller, Ullrich

    2014-01-16

    The tripeptide chains of the ergopeptines, a class of pharmacologically important D-lysergic acid alkaloid peptides, are arranged in a unique bicyclic cyclol based on an amino-terminal α-hydroxyamino acid and a terminal orthostructure. D-lysergyl-tripeptides are assembled by the nonribosomal peptide synthetases LPS1 and LPS2 of the ergot fungus Claviceps purpurea and released as N-(D-lysergyl-aminoacyl)-lactams. We show total enzymatic synthesis of ergopeptines catalyzed by a Fe²⁺/2-ketoglutarate-dependent dioxygenase (EasH) in conjunction with LPS1/LPS2. Analysis of the reaction indicated that EasH introduces a hydroxyl group into N-(D-lysergyl-aminoacyl)-lactam at α-C of the aminoacyl residue followed by spontaneous condensation with the terminal lactam carbonyl group. Sequence analysis revealed that EasH belongs to the wide and diverse family of the phytanoyl coenzyme A hydroxylases. We provide a high-resolution crystal structure of EasH that is most similar to that of phytanoyl coenzyme A hydroxylase, PhyH, from human. PMID:24361048

  12. EPSA: A Novel Supercritical Fluid Chromatography Technique Enabling the Design of Permeable Cyclic Peptides

    PubMed Central

    2014-01-01

    Most peptides are generally insufficiently permeable to be used as oral drugs. Designing peptides with improved permeability without reliable permeability monitoring is a challenge. We have developed a supercritical fluid chromatography technique for peptides, termed EPSA, which is shown here to enable improved permeability design. Through assessing the exposed polarity of a peptide, this technique can be used as a permeability surrogate. PMID:25313332

  13. dPABBs: A Novel in silico Approach for Predicting and Designing Anti-biofilm Peptides

    PubMed Central

    Sharma, Arun; Gupta, Pooja; Kumar, Rakesh; Bhardwaj, Anshu

    2016-01-01

    Increasingly, biofilms are being recognised for their causative role in persistent infections (like cystic fibrosis, otitis media, diabetic foot ulcers) and nosocomial diseases (biofilm-infected vascular catheters, implants and prosthetics). Given the clinical relevance of biofilms and their recalcitrance to conventional antibiotics, it is imperative that alternative therapeutics are proactively sought. We have developed dPABBs, a web server that facilitates the prediction and design of anti-biofilm peptides. The six SVM and Weka models implemented on dPABBs were observed to identify anti-biofilm peptides on the basis of their whole amino acid composition, selected residue features and the positional preference of the residues (maximum accuracy, sensitivity, specificity and MCC of 95.24%, 92.50%, 97.73% and 0.91, respectively, on the training datasets). On the N-terminus, it was seen that either of the cationic polar residues, R and K, is present at all five positions in case of the anti-biofilm peptides, whereas in the QS peptides, the uncharged polar residue S is preponderant at the first (also anionic polar residues D, E), third and fifth positions. Positive predictions were also obtained for 29 FDA-approved peptide drugs and ten antimicrobial peptides in clinical development, indicating at their possible repurposing for anti-biofilm therapy. dPABBs is freely accessible on: http://ab-openlab.csir.res.in/abp/antibiofilm/. PMID:26912180

  14. dPABBs: A Novel in silico Approach for Predicting and Designing Anti-biofilm Peptides.

    PubMed

    Sharma, Arun; Gupta, Pooja; Kumar, Rakesh; Bhardwaj, Anshu

    2016-01-01

    Increasingly, biofilms are being recognised for their causative role in persistent infections (like cystic fibrosis, otitis media, diabetic foot ulcers) and nosocomial diseases (biofilm-infected vascular catheters, implants and prosthetics). Given the clinical relevance of biofilms and their recalcitrance to conventional antibiotics, it is imperative that alternative therapeutics are proactively sought. We have developed dPABBs, a web server that facilitates the prediction and design of anti-biofilm peptides. The six SVM and Weka models implemented on dPABBs were observed to identify anti-biofilm peptides on the basis of their whole amino acid composition, selected residue features and the positional preference of the residues (maximum accuracy, sensitivity, specificity and MCC of 95.24%, 92.50%, 97.73% and 0.91, respectively, on the training datasets). On the N-terminus, it was seen that either of the cationic polar residues, R and K, is present at all five positions in case of the anti-biofilm peptides, whereas in the QS peptides, the uncharged polar residue S is preponderant at the first (also anionic polar residues D, E), third and fifth positions. Positive predictions were also obtained for 29 FDA-approved peptide drugs and ten antimicrobial peptides in clinical development, indicating at their possible repurposing for anti-biofilm therapy. dPABBs is freely accessible on: http://ab-openlab.csir.res.in/abp/antibiofilm/. PMID:26912180

  15. Heavy-metal complexation by de novo peptide design.

    PubMed

    Farrer, Brian T; Pecoraro, Vincent L

    2002-11-01

    From poisoning caused by lead-based paint on domestic buildings to groundwater contamination by naturally occurring arsenic deposits in India, heavy-metal toxicity is a global health problem. Contaminated ground water and acute cases of heavy-metal poisoning are treated with chelators to remove the heavy metals from the contaminated site or person. This review discusses the effort to generate heavy-metal chelators through peptide de novo design. De novo design entails the design of a primary sequence that will precisely fold into a predetermined secondary and tertiary protein structure. The first-generation peptide chelator used to initiate this investigation is the three-stranded coild coil containing Cys. Cys provides a potential trigonal binding site with soft thiolate ligands, which has been proposed to provide specific interactions with heavy metals. This hypothesis derives from the observation that similar sites on natural proteins show selectivity for heavy metals over other essential metals, such as Zn or Mg. A description of two systems, the TRI series and the IZ-AC peptide, is given, highlighting the interaction of these peptides with Hg, Cd, As and Pb. Arguments are also presented for the potential use of three-helix bundles as a second-generation design. PMID:12478724

  16. Investigating the inclusion properties of aromatic amino acids complexing beta-cyclodextrins in model peptides.

    PubMed

    Caso, Jolanda Valentina; Russo, Luigi; Palmieri, Maddalena; Malgieri, Gaetano; Galdiero, Stefania; Falanga, Annarita; Isernia, Carla; Iacovino, Rosa

    2015-10-01

    Cyclodextrins are commonly used as complexing agents in biological, pharmaceutical, and industrial applications since they have an effect on protein thermal and proteolytic stability, refolding yields, solubility, and taste masking. β-cyclodextrins (β-CD), because of their cavity size are a perfectly suited complexing agent for many common guest moieties. In the case of peptide-cyclodextrin and protein-cyclodextrin host-guest complexes the aromatic amino acids are reported to be the principal responsible of the interaction. For these reasons, we have investigated the inclusion properties of nine designed tripeptides, obtained permuting the position of two L-alanines (Ala, A) with that of one L-tryptophan (Trp, W), L-phenylalanine (Phe, F), or L-tyrosine (Tyr, Y), respectively. Interestingly, the position of the aromatic side-chain in the sequence appears to modulate the β-CD:peptide binding constants, determined via UV-Vis and NMR spectroscopy, which in turn assumes values higher than those reported for the single amino acid. The tripeptides containing a tyrosine showed the highest binding constants, with the central position in the Ac-AYA-NH2 peptide becoming the most favorite for the interaction. A combined NMR and Molecular Docking approach permitted to build detailed complex models, highlighting the stabilizing interactions of the neighboring amino acids backbone atoms with the upper rim of the β-CD. PMID:25985927

  17. Ligand-Based Peptide Design and Combinatorial Peptide Libraries to Target G Protein-Coupled Receptors

    PubMed Central

    Gruber, Christian W.; Muttenthaler, Markus; Freissmuth, Michael

    2016-01-01

    G protein-coupled receptors (GPCRs) are considered to represent the most promising drug targets; it has been repeatedly said that a large fraction of the currently marketed drugs elicit their actions by binding to GPCRs (with cited numbers varying from 30–50%). Closer scrutiny, however, shows that only a modest fraction of (~60) GPCRs are, in fact, exploited as drug targets, only ~20 of which are peptide-binding receptors. The vast majority of receptors in the humane genome have not yet been explored as sites of action for drugs. Given the drugability of this receptor class, it appears that opportunities for drug discovery abound. In addition, GPCRs provide for binding sites other than the ligand binding sites (referred to as the “orthosteric site”). These additional sites include (i) binding sites for ligands (referred to as “allosteric ligands”) that modulate the affinity and efficacy of orthosteric ligands, (ii) the interaction surface that recruits G proteins and arrestins, (iii) the interaction sites of additional proteins (GIPs, GPCR interacting proteins that regulate G protein signaling or give rise to G protein-independent signals). These sites can also be targeted by peptides. Combinatorial and natural peptide libraries are therefore likely to play a major role in identifying new GPCR ligands at each of these sites. In particular the diverse natural peptide libraries such as the venom peptides from marine cone-snails and plant cyclotides have been established as a rich source of drug leads. High-throughput screening and combinatorial chemistry approaches allow for progressing from these starting points to potential drug candidates. This will be illustrated by focusing on the ligand-based drug design of oxytocin (OT) and vasopressin (AVP) receptor ligands using natural peptide leads as starting points. PMID:20687879

  18. De Novo Designed Synthetic Mimics of Antimicrobial Peptides

    PubMed Central

    Scott, Richard W.; DeGrado, William F.; Tew, Gregory N.

    2008-01-01

    Summary Antimicrobial peptides are small cationic amphiphiles that play an important role in the innate immune system. Given their broad specificity, they appear to be ideal therapeutic agents. As a result, over the last decade, there has been considerable interest in developing them as intravenously administered antibiotics. However, it has proven difficult to accomplish this goal with peptide-based structures. While it has been possible to solve some relatively simple problems such as susceptibility to proteolysis, more severe problems have included the expense of the materials, toxicity, limited efficacy, and limited tissue distribution. As a result, we developed small synthetic oligomers designed to adopt amphiphilic conformations and exhibit potent antimicrobial activity while being non-toxic to host cells. One class of these synthetic mimics of antimicrobial peptides (SMAMPs) is being developed as intravenous antibiotics PMID:18996193

  19. Di-heterometalation of thiol-functionalized peptide nucleic acids

    PubMed Central

    Joshi, Tanmaya; Patra, Malay; Spiccia, Leone; Gasser, Gilles

    2013-01-01

    As a proof-of-principle, two hetero-bimetallic PNA oligomers containing a ruthenium(II) polypyridyl and a cyclopentadienyl manganese tricarbonyl complex have been prepared by serial combination of solid-phase peptide coupling and in-solution thiol chemistry. Solid-phase N-terminus attachment of Ru(II)-polypyridyl carboxylic acid derivative, C1, onto the thiol-functionalized PNA backbone (H-a-a-g-t-c-t-g-c-linker-cys-NH2) has been performed by standard peptide coupling method. As two parallel approaches, the strong affinity of thiols for maleimide and haloacetyl group has been exploited for subsequent post-SPPS addition of cymantrene-based organometallic cores, C2 and C3. Michael-like addition and thioether ligation of thiol functionalized PNA1 (H-gly-a-a-g-t-c-t-g-c-linker-cys-NH2) and PNA2 (C1-a-a-g-t-c-t-g-c-linker-cys-NH2) to cymantrene maleimide and chloroacetyl derivatives, C2 and C3, respectively, has been performed. The synthesized ruthenium(II)-cymantrenyl PNA oligomers have been characterized by mass spectrometry (ESI-MS) and IR spectroscopy. The distinct Mn-CO vibrational IR stretches, between 1,924–2,074 cm−1, have been used as markers to confirm the presence of cymantrenyl units in the PNA sequences and the purity of the HPLC-purified PNA thioethers assessed using LC-MS. PMID:23422249

  20. Serum Stable Natural Peptides Designed by mRNA Display

    PubMed Central

    Howell, Shannon M.; Fiacco, Stephen V.; Takahashi, Terry T.; Jalali-Yazdi, Farzad; Millward, Steven W.; Hu, Biliang; Wang, Pin; Roberts, Richard W.

    2014-01-01

    Peptides constructed with the 20 natural amino acids are generally considered to have little therapeutic potential because they are unstable in the presence of proteases and peptidases. However, proteolysis cleavage can be idiosyncratic, and it is possible that natural analogues of functional sequences exist that are highly resistant to cleavage. Here, we explored this idea in the context of peptides that bind to the signaling protein Gαi1. To do this, we used a two-step in vitro selection process to simultaneously select for protease resistance while retaining function–first by degrading the starting library with protease (chymotrypsin), followed by positive selection for binding via mRNA display. Starting from a pool of functional sequences, these experiments revealed peptides with 100–400 fold increases in protease resistance compared to the parental library. Surprisingly, selection for chymotrypsin resistance also resulted in similarly improved stability in human serum (~100 fold). Mechanistically, the decreases in cleavage results from both a lower rate of cleavage (kcat) and a weaker interaction with the protease (Km). Overall, our results demonstrate that the hydrolytic stability of functional, natural peptide sequences can be improved by two orders of magnitude simply by optimizing the primary sequence. PMID:25234472

  1. High-throughput platform for design and screening of peptides as inhibitors of calcium oxalate monohydrate crystallization

    NASA Astrophysics Data System (ADS)

    Farmanesh, Sahar; Chung, Jihae; Chandra, Divya; Sosa, Ricardo D.; Karande, Pankaj; Rimer, Jeffrey D.

    2013-06-01

    Crystal growth modifiers present a versatile tool for controlling crystal shape and size. Our work described here focuses on the design and screening of short peptides as inhibitors of calcium oxalate monohydrate (COM) crystals using high-throughput approaches. We designed a small library of 13 peptides containing Ala and Asp amino acids arranged in varying sequences that mimic ubiquitous motifs in natural calcium-binding proteins. Peptides were screened using a quick assay to measure their efficacy for inhibiting COM crystallization. Our results show that subtle variations in the placement of Ala and Asp residues in the peptide sequence can have a profound effect on their inhibition potential. We were able to discover peptide sequences that inhibit COM crystallization more effectively than some of the well-known COM inhibitors, such as citrate. Our results also demonstrate that peptides can be engineered to bind to specific faces of COM crystals. Peptide sequences identified in this work are promising candidates for further development as therapies for biomineral-related diseases, such as kidney stone disease. Collectively, our work establishes new paradigms for the design, synthesis, and screening of peptides for controlling crystal habit with the potential to impact a variety of fields, including drug discovery, advanced materials, catalysis and separations.

  2. Targeting Multidrug-resistant Staphylococci with an anti-rpoA Peptide Nucleic Acid Conjugated to the HIV-1 TAT Cell Penetrating Peptide.

    PubMed

    Abushahba, Mostafa Fn; Mohammad, Haroon; Seleem, Mohamed N

    2016-01-01

    Staphylococcus aureus infections present a serious challenge to healthcare practitioners due to the emergence of resistance to numerous conventional antibiotics. Due to their unique mode of action, peptide nucleic acids are novel alternatives to traditional antibiotics to tackle the issue of bacterial multidrug resistance. In this study, we designed a peptide nucleic acid covalently conjugated to the HIV-TAT cell penetrating peptide (GRKKKRRQRRRYK) in order to target the RNA polymerase α subunit gene (rpoA) required for bacterial genes transcription. We explored the antimicrobial activity of the anti-rpoA construct (peptide nucleic acid-TAT) against methicillin-resistant S. aureus, vancomycin-intermediate S. aureus, vancomycin-resistant S. aureus, linezolid-resistant S. aureus, and methicillin-resistant S. epidermidis in pure culture, infected mammalian cell culture, and in an in vivo Caenorhabditis elegans infection model. The anti-rpoA construct led to a concentration-dependent inhibition of bacterial growth (at micromolar concentrations) in vitro and in both infected cell culture and in vivo in C. elegans. Moreover, rpoA gene silencing resulted in suppression of its message as well as reduced expression of two important methicillin-resistant S. aureus USA300 toxins (α-hemolysin and Panton-Valentine leukocidin). This study confirms that rpoA gene is a potential target for development of novel antisense therapeutics to treat infections caused by methicillin-resistant S. aureus. PMID:27434684

  3. 13-Helix folding of a β/γ-peptide manifold designed from a "minimal-constraint" blueprint.

    PubMed

    Grison, Claire M; Robin, Sylvie; Aitken, David J

    2016-06-14

    A bottom-up design rationale was adopted to devise β/γ-peptide foldamer manifolds which would adopt preferred 13-helix conformations, relying on minimal steric imposition brought by the constituent amino acid residues. In this way, a well-defined 13-helix conformer was revealed for short oligomers of trans-2-aminocyclobutanecarboxylic acid and γ(4)-amino acids in alternation, which gave good topological superposition upon an α-helix motif. PMID:27230541

  4. Preparation of ACE Inhibitory Peptides from Mytilus coruscus Hydrolysate Using Uniform Design

    PubMed Central

    Wu, Jin-Chao; Cheng, Jie; Shi, Xiao-lai

    2013-01-01

    The angiotensin-I-converting enzyme (ACE) inhibitory peptides from mussel, Mytilus coruscus, were investigated and the variable factors, protease concentration, hydrolysis time, pH, and temperature, were optimized using Uniform Design, a new statistical experimental method. The results proved that the hydrolysate of alkali proteases had high ACE-inhibitory activity, especially the alkali protease E1. Optimization by Uniform Design showed that the best hydrolysis conditions for preparation of ACE-inhibitory peptides from Mytilus coruscus were protease concentration of 36.0 U/mL, hydrolysis time of 2.7 hours, pH 8.2, and Temperature at 59.5°C, respectively. The verification experiments under optimum conditions showed that the ACE-inhibitory activity (91.3%) were agreed closely with the predicted activity of 90.7%. The amino acid composition analysis of Mytilus coruscus ACE-inhibitory peptides proved that it had high percent of lysine, leucine, glycine, aspartic acid, and glutamic acid. PMID:23484103

  5. Testing the limits of rational design by engineering pH sensitivity into membrane-active peptides.

    PubMed

    Wiedman, Gregory; Wimley, William C; Hristova, Kalina

    2015-04-01

    In this work, we sought to rationally design membrane-active peptides that are triggered by low pH to form macromolecular-sized pores in lipid bilayers. Such peptides could have broad utility in biotechnology and in nanomedicine as cancer therapeutics or drug delivery vehicles that promote release of macromolecules from endosomes. Our approach to rational design was to combine the properties of a pH-independent peptide, MelP5, which forms large pores allowing passage of macromolecules, with the properties of two pH-dependent membrane-active peptides, pHlip and GALA. We created two hybrid sequences, MelP5_Δ4 and MelP5_Δ6, by using the distribution of acidic residues on pHlip and GALA as a guide to insert acidic amino acids into the amphipathic helix of MelP5. We show that the new peptides bind to lipid bilayers and acquire secondary structure in a pH-dependent manner. The peptides also destabilize bilayers in a pH-dependent manner, such that lipid vesicles release the small molecules ANTS/DPX at low pH only. Thus, we were successful in designing pH-triggered pore-forming peptides. However, no macromolecular release was observed under any conditions. Therefore, we abolished the unique macromolecular poration properties of MelP5 by introducing pH sensitivity into its sequence. We conclude that the properties of pHlip, GALA, and MelP5 are additive, but only partially so. We propose that this lack of additivity is a limitation in the rational design of novel membrane-active peptides, and that high-throughput approaches to discovery will be critical for continued progress in the field. PMID:25572997

  6. Molecular Design, Structural Analysis and Antifungal Activity of Derivatives of Peptide CGA-N46.

    PubMed

    Li, Rui-Fang; Lu, Zhi-Fang; Sun, Ya-Nan; Chen, Shi-Hua; Yi, Yan-Jie; Zhang, Hui-Ru; Yang, Shuo-Ye; Yu, Guang-Hai; Huang, Liang; Li, Chao-Nan

    2016-09-01

    Chromogranin A (CGA)-N46, a derived peptide of human chromogranin A, has antifungal activity. To further research the active domain of CGA-N46, a series of derivatives were designed by successively deleting amino acid from both terminus of CGA-N46, and the amino acid sequence of each derivative was analyzed by bioinformatic software. Based on the predicted physicochemical properties of the peptides, including half-life time in mammalian reticulocytes (in vitro), yeast (in vivo) and E. coli (in vivo), instability index, aliphatic index and grand average of hydropathicity (GRAVY), the secondary structure, net charge, the distribution of hydrophobic residues and hydrophilic residues, the final derivatives CGA-N15, CGA-N16, CGA-N12 and CGA-N8 were synthesized by solid-phase peptide synthesis. The results of bioinformatic analysis showed that CGA-N46 and its derivatives were α-helix, neutral or weak positive charge, hydrophilic, and CGA-N12 and CGA-N8 were more stable than the other derivatives. The results of circular dichroism confirmed that CGA-N46 and its derived peptides displayed α-helical structure in an aqueous solution and 30 mM sodium dodecylsulfate, but α-helical contents decreased in hydrophobic lipid vesicles. CGA-N15, CGA-N16, CGA-N12 and CGA-N8 had higher antifungal activities than their mother peptide CGA-N46. Among of the derived peptides, CGA-N12 showed the least hemolytic activity. In conclusion, we have successfully identified the active domain of CGA-N46 with strong antifungal activity and weak hemolytic activity, which provides the possibility to develop a new class of antibiotics. PMID:27165480

  7. Biomimetic oligosaccharide and peptide surfactant polymers designed for cardiovascular biomaterials

    NASA Astrophysics Data System (ADS)

    Ruegsegger, Mark Andrew

    A common problem associated with cardiovascular devices is surface induced thrombosis initiated by the rapid, non-specific adsorption of plasma proteins onto the biomaterial surface. Control of the initial protein adsorption is crucial to achieve the desired longevity of the implanted biomaterial. The cell membrane glycocalyx acts as a non-thrombogenic interface through passive (dense oligosaccharide structures) and active (ligand/receptor interactions) mechanisms. This thesis is designed to investigate biomimicry of the cell glycocalyx to minimize non-specific protein adsorption and promote specific ligand/receptor interactions. Biomimetic macromolecules were designed through the molecular-scale engineering of polymer surfactants, utilizing a poly(vinyl amine) (PVAm) backbone to which hydrophilic (dextran, maltose, peptide) and hydrophobic alkyl (hexanoyl or hexanal) chains are simultaneously attached. The structure was controlled through the molar feed ratio of hydrophobic-to-hydrophilic groups, which also provided control of the solution and surface-active properties. To mimic passive properties, a series of oligomaltose surfactants were synthesized with increasing saccharide length (n = 2, 7, 15 where n is number of glucose units) to investigate the effect of coating height on protein adsorption. The surfactants were characterized by infra red (IR) and nuclear magnetic resonance (NMR) spectroscopies for structural properties and atomic force microscopy (AFM) and contact angle goniometry for surface activity. Protein adsorption under dynamic flow (5 dyn/cm2) was reduced by 85%--95% over the bare hydrophobic substrate; platelet adhesion dropped by ˜80% compared to glass. Peptide ligands were incorporated into the oligosaccharide surfactant to promote functional activity of the passive coating. The surfactants were synthesized to contain 0%, 25%, 50%, 75%, and 100% peptide ligand density and were stable on hydrophobic surfaces. The peptide surface density was

  8. Modular Design of Self-Assembling Peptide-Based Nanotubes.

    PubMed

    Burgess, Natasha C; Sharp, Thomas H; Thomas, Franziska; Wood, Christopher W; Thomson, Andrew R; Zaccai, Nathan R; Brady, R Leo; Serpell, Louise C; Woolfson, Derek N

    2015-08-26

    An ability to design peptide-based nanotubes (PNTs) rationally with defined and mutable internal channels would advance understanding of peptide self-assembly, and present new biomaterials for nanotechnology and medicine. PNTs have been made from Fmoc dipeptides, cyclic peptides, and lock-washer helical bundles. Here we show that blunt-ended α-helical barrels, that is, preassembled bundles of α-helices with central channels, can be used as building blocks for PNTs. This approach is general and systematic, and uses a set of de novo helical bundles as standards. One of these bundles, a hexameric α-helical barrel, assembles into highly ordered PNTs, for which we have determined a structure by combining cryo-transmission electron microscopy, X-ray fiber diffraction, and model building. The structure reveals that the overall symmetry of the peptide module plays a critical role in ripening and ordering of the supramolecular assembly. PNTs based on pentameric, hexameric, and heptameric α-helical barrels sequester hydrophobic dye within their lumens. PMID:26219086

  9. Cyclotides: macrocyclic peptides with applications in drug design and agriculture.

    PubMed

    Craik, David J; Mylne, Joshua S; Daly, Norelle L

    2010-01-01

    Cyclotides are disulfide-rich peptides from plants that are exceptionally stable as a result of their unique cyclic cystine knot structural motif. Their natural role is thought to be as plant defence agents, most notably against insect pests, but they also have potential applications in drug design and agriculture. This article identifies gaps in current knowledge on cyclotides and suggests future directions for research into this fascinating family of ultra-stable mini-proteins. PMID:19795188

  10. The paradox of conformational constraint in the design of Cbl(TKB)-binding peptides

    PubMed Central

    Kumar, Eric A.; Chen, Qianyi; Kizhake, Smitha; Kolar, Carol; Kang, Myungshim; Chang, Chia-en A.; Borgstahl, Gloria E. O.; Natarajan, Amarnath

    2013-01-01

    Solving the crystal structure of Cbl(TKB) in complex with a pentapeptide, pYTPEP, revealed that the PEP region adopted a poly-L-proline type II (PPII) helix. An unnatural amino acid termed a proline-templated glutamic acid (ptE) that constrained both the backbone and sidechain to the bound conformation was synthesized and incorporated into the pYTPXP peptide. We estimated imposing structural constraints onto the backbone and sidechain of the peptide and preorganize it to the bound conformation in solution will yield nearly an order of magnitude improvement in activity. NMR studies confirmed that the ptE-containing peptide adopts the PPII conformation, however, competitive binding studies showed an order of magnitude loss of activity. Given the emphasis that is placed on imposing structural constraints, we provide an example to support the contrary. These results point to conformational flexibility at the interface, which have implications in the design of potent Cbl(TKB)-binding peptides. PMID:23572190

  11. PH dependent adhesive peptides

    DOEpatents

    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.

  12. Fluorescent 2-Aminopyridine Nucleobases for Triplex-Forming Peptide Nucleic Acids.

    PubMed

    Cheruiyot, Samwel K; Rozners, Eriks

    2016-08-17

    Development of new fluorescent peptide nucleic acids (PNAs) is important for fundamental research and practical applications. The goal of this study was the design of fluorogenic nucleobases for incorporation in triplex-forming PNAs. The underlying design principle was the use of a protonation event that accompanied binding of a 2-aminopyridine (M) nucleobase to a G-C base pair as an on switch for a fluorescence signal. Two fluorogenic nucleobases, 3-(1-phenylethynyl)-M and phenylpyrrolo-M, were designed, synthesized and studied. The new M derivatives provided modest enhancement of fluorescence upon protonation but showed reduced RNA binding affinity and quenching of fluorescence signal upon triple-helix formation with cognate double-stranded RNA. Our study illustrates the principal challenges of design and provides guidelines for future improvement of fluorogenic PNA nucleobases. The 3-(1-phenylethynyl)-M may be used as a fluorescent nucleobase to study PNA-RNA triple-helix formation. PMID:27223320

  13. In Vitro and In Vivo Activities of Antimicrobial Peptides Developed Using an Amino Acid-Based Activity Prediction Method

    PubMed Central

    Wu, Xiaozhe; Wang, Zhenling; Li, Xiaolu; Fan, Yingzi; He, Gu; Wan, Yang; Yu, Chaoheng; Tang, Jianying; Li, Meng; Zhang, Xian; Zhang, Hailong; Xiang, Rong; Pan, Ying; Liu, Yan; Lu, Lian

    2014-01-01

    To design and discover new antimicrobial peptides (AMPs) with high levels of antimicrobial activity, a number of machine-learning methods and prediction methods have been developed. Here, we present a new prediction method that can identify novel AMPs that are highly similar in sequence to known peptides but offer improved antimicrobial activity along with lower host cytotoxicity. Using previously generated AMP amino acid substitution data, we developed an amino acid activity contribution matrix that contained an activity contribution value for each amino acid in each position of the model peptide. A series of AMPs were designed with this method. After evaluating the antimicrobial activities of these novel AMPs against both Gram-positive and Gram-negative bacterial strains, DP7 was chosen for further analysis. Compared to the parent peptide HH2, this novel AMP showed broad-spectrum, improved antimicrobial activity, and in a cytotoxicity assay it showed lower toxicity against human cells. The in vivo antimicrobial activity of DP7 was tested in a Staphylococcus aureus infection murine model. When inoculated and treated via intraperitoneal injection, DP7 reduced the bacterial load in the peritoneal lavage solution. Electron microscope imaging and the results indicated disruption of the S. aureus outer membrane by DP7. Our new prediction method can therefore be employed to identify AMPs possessing minor amino acid differences with improved antimicrobial activities, potentially increasing the therapeutic agents available to combat multidrug-resistant infections. PMID:24982064

  14. Design and characterization of the anion-sensitive coiled-coil peptide.

    PubMed Central

    Hoshino, M.; Yumoto, N.; Yoshikawa, S.; Goto, Y.

    1997-01-01

    As a model for analyzing the role of charge repulsion in proteins and its shielding by the solvent, we designed a peptide of 27 amino acid residues that formed a homodimeric coiled-coil. The interface between the coils consisted of hydrophobic Leu and Val residues, and 10 Lys residues per monomer were incorporated into the positions exposed to solvent. During the preparation of a disulfide-linked dimer in which the two peptides were linked in parallel by the two disulfide bonds located at the N and C terminals, a cyclic monomer with an intramolecular disulfide bond was also obtained. On the basis of CD and 1H-NMR, the conformational stabilities of these isomers and several reference peptides were examined. Whereas all these peptides were unfolded in the absence of salt at pH 4.7 and 20 degrees C, the addition of NaClO4 cooperatively stabilized the alpha-helical conformation. The crosslinking of the peptides by disulfide bonds significantly decreased the midpoint salt concentration of the transition. The 1H-NMR spectra in the presence of NaClO4 suggested that, whereas the disulfide-bonded dimer assumed a native-like conformation, the cyclic monomer assumed a molten globule-like conformation with disordered side chains. However, the cyclic monomer exhibited cooperative transitions against temperature and Gdn-HCl that were only slightly less cooperative than those of the disulfide-bonded parallel dimer. These results indicate that the charge repulsion critically destabilizes the native-like state as well as the molten globule-like state, and that the solvent-dependent charge repulsion may be useful for controlling the conformation of designed peptides. PMID:9232640

  15. Designer interface peptide grafts target estrogen receptor alpha dimerization.

    PubMed

    Chakraborty, S; Asare, B K; Biswas, P K; Rajnarayanan, R V

    2016-09-01

    The nuclear transcription factor estrogen receptor alpha (ERα), triggered by its cognate ligand estrogen, regulates a variety of cellular signaling events. ERα is expressed in 70% of breast cancers and is a widely validated target for anti-breast cancer drug discovery. Administration of anti-estrogen to block estrogen receptor activation is still a viable anti-breast cancer treatment option but anti-estrogen resistance has been a significant bottle-neck. Dimerization of estrogen receptor is required for ER activation. Blocking ERα dimerization is therefore a complementary and alternative strategy to combat anti-estrogen resistance. Dimer interface peptide "I-box" derived from ER residues 503-518 specifically blocks ER dimerization. Recently using a comprehensive molecular simulation we studied the interaction dynamics of ERα LBDs in a homo-dimer. Based on this study, we identified three interface recognition peptide motifs LDKITDT (ERα residues 479-485), LQQQHQRLAQ (residues 497-506), and LSHIRHMSNK (residues 511-520) and reported the suitability of using LQQQHQRLAQ (ER 497-506) as a template to design inhibitors of ERα dimerization. Stability and self-aggregation of peptide based therapeutics poses a significant bottle-neck to proceed further. In this study utilizing peptide grafted to preserve their pharmacophoric recognition motif and assessed their stability and potential to block ERα mediated activity in silico and in vitro. The Grafted peptides blocked ERα mediated cell proliferation and viability of breast cancer cells but did not alter their apoptotic fate. We believe the structural clues identified in this study can be used to identify novel peptidometics and small molecules that specifically target ER dimer interface generating a new breed of anti-cancer agents. PMID:27462021

  16. Rationale-Based, De Novo Design of Dehydrophenylalanine-Containing Antibiotic Peptides and Systematic Modification in Sequence for Enhanced Potency▿

    PubMed Central

    Pathak, Sarika; Chauhan, Virander Singh

    2011-01-01

    Increased microbial drug resistance has generated a global requirement for new anti-infective agents. As part of an effort to develop new, low-molecular-mass peptide antibiotics, we used a rationale-based minimalist approach to design short, nonhemolytic, potent, and broad-spectrum antibiotic peptides with increased serum stability. These peptides were designed to attain an amphipathic structure in helical conformations. VS1 was used as the lead compound, and its properties were compared with three series of derivates obtained by (i) N-terminal amino acid addition, (ii) systematic Trp substitution, and (iii) peptide dendrimerization. The Trp substitution approach underlined the optimized sequence of VS2 in terms of potency, faster membrane permeation, and cost-effectiveness. VS2 (a variant of VS1 with two Trp substitutions) was found to exhibit good antimicrobial activity against both the Gram-negative Escherichia coli and the Gram-positive bacterium Staphylococcus aureus. It was also found to have noncytolytic activity and the ability to permeate and depolarize the bacterial membrane. Lysis of the bacterial cell wall and inner membrane by the peptide was confirmed by transmission electron microscopy. A combination of small size, the presence of unnatural amino acids, high antimicrobial activity, insignificant hemolysis, and proteolytic resistance provides fundamental information for the de novo design of an antimicrobial peptide useful for the management of infectious disease. PMID:21321136

  17. 2D-Qsar for 450 types of amino acid induction peptides with a novel substructure pair descriptor having wider scope

    PubMed Central

    2011-01-01

    Background Quantitative structure-activity relationships (QSAR) analysis of peptides is helpful for designing various types of drugs such as kinase inhibitor or antigen. Capturing various properties of peptides is essential for analyzing two-dimensional QSAR. A descriptor of peptides is an important element for capturing properties. The atom pair holographic (APH) code is designed for the description of peptides and it represents peptides as the combination of thirty-six types of key atoms and their intermediate binding between two key atoms. Results The substructure pair descriptor (SPAD) represents peptides as the combination of forty-nine types of key substructures and the sequence of amino acid residues between two substructures. The size of the key substructures is larger and the length of the sequence is longer than traditional descriptors. Similarity searches on C5a inhibitor data set and kinase inhibitor data set showed that order of inhibitors become three times higher by representing peptides with SPAD, respectively. Comparing scope of each descriptor shows that SPAD captures different properties from APH. Conclusion QSAR/QSPR for peptides is helpful for designing various types of drugs such as kinase inhibitor and antigen. SPAD is a novel and powerful descriptor for various types of peptides. Accuracy of QSAR/QSPR becomes higher by describing peptides with SPAD. PMID:22047717

  18. Forcefield_NCAA: Ab Initio Charge Parameters to Aid in the Discovery and Design of Therapeutic Proteins and Peptides with Unnatural Amino Acids and Their Application to Complement Inhibitors of the Compstatin Family

    PubMed Central

    2015-01-01

    We describe the development and testing of ab initio derived, AMBER ff03 compatible charge parameters for a large library of 147 noncanonical amino acids including β- and N-methylated amino acids for use in applications such as protein structure prediction and de novo protein design. The charge parameter derivation was performed using the RESP fitting approach. Studies were performed assessing the suitability of the derived charge parameters in discriminating the activity/inactivity between 63 analogs of the complement inhibitor Compstatin on the basis of previously published experimental IC50 data and a screening procedure involving short simulations and binding free energy calculations. We found that both the approximate binding affinity (K*) and the binding free energy calculated through MM-GBSA are capable of discriminating between active and inactive Compstatin analogs, with MM-GBSA performing significantly better. Key interactions between the most potent Compstatin analog that contains a noncanonical amino acid are presented and compared to the most potent analog containing only natural amino acids and native Compstatin. We make the derived parameters and an associated web interface that is capable of performing modifications on proteins using Forcefield_NCAA and outputting AMBER-ready topology and parameter files freely available for academic use at http://selene.princeton.edu/FFNCAA. The forcefield allows one to incorporate these customized amino acids into design applications with control over size, van der Waals, and electrostatic interactions. PMID:24932669

  19. Forcefield_NCAA: ab initio charge parameters to aid in the discovery and design of therapeutic proteins and peptides with unnatural amino acids and their application to complement inhibitors of the compstatin family.

    PubMed

    Khoury, George A; Smadbeck, James; Tamamis, Phanourios; Vandris, Andrew C; Kieslich, Chris A; Floudas, Christodoulos A

    2014-12-19

    We describe the development and testing of ab initio derived, AMBER ff03 compatible charge parameters for a large library of 147 noncanonical amino acids including β- and N-methylated amino acids for use in applications such as protein structure prediction and de novo protein design. The charge parameter derivation was performed using the RESP fitting approach. Studies were performed assessing the suitability of the derived charge parameters in discriminating the activity/inactivity between 63 analogs of the complement inhibitor Compstatin on the basis of previously published experimental IC50 data and a screening procedure involving short simulations and binding free energy calculations. We found that both the approximate binding affinity (K*) and the binding free energy calculated through MM-GBSA are capable of discriminating between active and inactive Compstatin analogs, with MM-GBSA performing significantly better. Key interactions between the most potent Compstatin analog that contains a noncanonical amino acid are presented and compared to the most potent analog containing only natural amino acids and native Compstatin. We make the derived parameters and an associated web interface that is capable of performing modifications on proteins using Forcefield_NCAA and outputting AMBER-ready topology and parameter files freely available for academic use at http://selene.princeton.edu/FFNCAA . The forcefield allows one to incorporate these customized amino acids into design applications with control over size, van der Waals, and electrostatic interactions. PMID:24932669

  20. Antitumor Effects of EGFR Antisense Guanidine-Based Peptide Nucleic Acids in Cancer Models

    PubMed Central

    Thomas, Sufi M.; Sahu, Bichismita; Rapireddy, Srinivas; Bahal, Raman; Wheeler, Sarah E.; Procopio, Eva M.; Kim, Joseph; Joyce, Sonali C.; Contrucci, Sarah; Wang, Yun; Chiosea, Simion I.; Lathrop, Kira L.; Watkins, Simon; Grandis, Jennifer R.; Armitage, Bruce A.; Ly, Danith H.

    2013-01-01

    Peptide nucleic acids have emerged over the past two decades as a promising class of nucleic acid mimics because of their strong binding affinity and sequence selectivity toward DNA and RNA, and resistance to enzymatic degradation by proteases and nucleases. While they have been shown to be effective in regulation of gene expression in vitro, and to a small extent in vivo, their full potential for molecular therapy has not yet been fully realized due to poor cellular uptake. Herein, we report the development of cell-permeable, guanidine-based peptide nucleic acids targeting the epidermal growth factor receptor (EGFR) in preclinical models as therapeutic modality for head and neck squamous cell carcinoma (HNSCC) and nonsmall cell lung cancer (NSCLC). A GPNA oligomer, 16 nucleotides in length, designed to bind to EGFR gene transcript elicited potent antisense effects in HNSCC and NSCLC cells in preclinical models. When administered intraperitoneally in mice, EGFRAS-GPNA was taken-up by several tissues including the xenograft tumor. Systemic administration of EGFRAS-GPNA induced antitumor effects in HNSCC xenografts, with similar efficacies as the FDA-approved EGFR inhibitors: cetuximab and erlotinib. In addition to targeting wild-type EGFR, EGFRAS-GPNA is effective against the constitutively active EGFR vIII mutant implicated in cetuximab resistance. Our data reveals that GPNA is just as effective as a molecular platform for treating cetuximab resistant cells, demonstrating its utility in the treatment of cancer. PMID:23113581

  1. Amino acid sequence of a vitamin K-dependent Ca2+-binding peptide from bovine prothrombin.

    PubMed

    Howard, J B; Fausch, M D

    1975-08-10

    The amino acid sequence of a 31-residue peptide from bovine prothrombin has been determined. This peptide has been shown to contain the vitamin K-dependent modification required for Ca2+ binding (Nelsestuen, G. L., and Suttie, J. W. (1973) Proc. Natl. Acad. Sci. U. S. A. 70, 3366-3370) and the modified amino acid, gamma-carboxyglutamic acid (Nelsestuen, G. L., Zytkovicz, T., and Howard, J. B. (1974) J. Biol. Chem. 249, 6347-6350). The peptide was shown to correspond to residues 12 to 42 of prothrombin. PMID:807581

  2. Harnessing natures ability to control metal ion coordination geometry using de novo designed peptides

    PubMed Central

    Peacock, Anna F. A.; Iranzo, Olga; Pecoraro, Vincent L.

    2011-01-01

    Advances in protein chemistry and molecular and structural biology have empowered modern chemists to build complex biological architectures using a “first principles” approach, which is known as de novo protein design. In this Perspective we demonstrate how simple three-stranded α-helical constructs can be prepared by the sole consideration of the primary amino acid sequence of a peptide. With these well defined systems, we then demonstrate that metal binding cavities can be carved out of the hydrophobic cores of these aggregates in order to bind metal ions such as cadmium with well defined coordination geometries. Examples will be given of homoleptic CdS3 complexes, CdS3O sites and proteins which contain equilibrium mixtures of these two species. We will provide a description of a strategy that allows us to build heterochromic peptides (small proteins that complex two metals in nearly identical environments but which result in different physical properties and allow for metal site selectivity). We conclude with a new class of designed peptides, diastereopeptides, which can exploit changes in amino acid chirality to control metal ion coordination number and lead to an alternative path towards heterochromic systems. The constructs described herein represent the initial steps of preparing protein structures that may simultaneous contain structural and catalytic metal binding centers. These studies inform the community on a developing field, which promises new opportunities for the study of bioinorganic chemistry. PMID:19290357

  3. Native like helices in a specially designed β peptide in the gas phase.

    PubMed

    Schubert, Franziska; Pagel, Kevin; Rossi, Mariana; Warnke, Stephan; Salwiczek, Mario; Koksch, Beate; von Helden, Gert; Blum, Volker; Baldauf, Carsten; Scheffler, Matthias

    2015-02-21

    In the natural peptides, helices are stabilized by hydrogen bonds that point backward along the sequence direction. Until now, there is only little evidence for the existence of analogous structures in oligomers of conformationally unrestricted β amino acids. We specifically designed the β peptide Ac-(β(2)hAla)6-LysH(+) to form native like helical structures in the gas phase. The design follows the known properties of the peptide Ac-Ala6-LysH(+) that forms a α helix in isolation. We perform ion-mobility mass-spectrometry and vibrational spectroscopy in the gas phase, combined with state-of-the-art density-functional theory simulations of these molecular systems in order to characterize their structure. We can show that the straightforward exchange of alanine residues for the homologous β amino acids generates a system that is generally capable of adopting native like helices with backward oriented H-bonds. By pushing the limits of theory and experiments, we show that one cannot assign a single preferred structure type due to the densely populated energy landscape and present an interpretation of the data that suggests an equilibrium of three helical structures. PMID:25611682

  4. Peptides Composed of Alternating L- and D-Amino Acids Inhibit Amyloidogenesis in Three Distinct Amyloid Systems Independent of Sequence.

    PubMed

    Kellock, Jackson; Hopping, Gene; Caughey, Byron; Daggett, Valerie

    2016-06-01

    There is now substantial evidence that soluble oligomers are primary toxic agents in amyloid diseases. The development of an antibody recognizing the toxic soluble oligomeric forms of different and unrelated amyloid species suggests a common conformational intermediate during amyloidogenesis. We previously observed a common occurrence of a novel secondary structure element, which we call α-sheet, in molecular dynamics (MD) simulations of various amyloidogenic proteins, and we hypothesized that the toxic conformer is composed of α-sheet structure. As such, α-sheet may represent a conformational signature of the misfolded intermediates of amyloidogenesis and a potential unique binding target for peptide inhibitors. Recently, we reported the design and characterization of a novel hairpin peptide (α1 or AP90) that adopts stable α-sheet structure and inhibits the aggregation of the β-Amyloid Peptide Aβ42 and transthyretin. AP90 is a 23-residue hairpin peptide featuring alternating D- and L-amino acids with favorable conformational propensities for α-sheet formation, and a designed turn. For this study, we reverse engineered AP90 to identify which of its design features is most responsible for conferring α-sheet stability and inhibitory activity. We present experimental characterization (CD and FTIR) of seven peptides designed to accomplish this. In addition, we measured their ability to inhibit aggregation in three unrelated amyloid species: Aβ42, transthyretin, and human islet amylin polypeptide. We found that a hairpin peptide featuring alternating L- and D-amino acids, independent of sequence, is sufficient for conferring α-sheet structure and inhibition of aggregation. Additionally, we show a correlation between α-sheet structural stability and inhibitory activity. PMID:27012425

  5. Supramolecular control of self-assembling terthiophene-peptide conjugates through the amino acid side chain

    SciTech Connect

    Lehrman, Jessica A.; Cui, Honggang; Tsai, Wei-Wen; Moyer, Tyson J.; Stupp, Samuel I.

    2013-07-30

    The self-assembly of oligothiophene–peptide conjugates can be directed through the systematic variation of the peptide sequence into different nanostructures, including flat spicules, nanotubes, spiral sheets, and giant, flat sheets. Furthermore, the assembly of these molecules is not controlled by steric interactions between the amino acid side chains.

  6. pH-Dependent membrane lysis by using melittin-inspired designed peptides.

    PubMed

    Kashiwada, A; Mizuno, M; Hashimoto, J

    2016-07-14

    We developed a membrane-lytic peptide (LP) having 26 amino acid residues composed of a helix-promoting hydrophobic segment (Leu-Ala repetitive sequence) and a cationic segment from melittin. In the presence of liposomes, LP interacts with liposomal surfaces to form a hydrophobic helix in the lipid bilayer in a wide pH range. In order to provide LP with a weakly acidic (endosomal) pH-controlled membrane-lytic activity, we have designed an LPE peptide series (a typical peptide, LPE3-1) with a hydrophobic segment in which Leu (L) residues are replaced by acidic Glu (E) residues. To analyze the pH-selective membrane-lytic activity of the designed peptides, both calcein leakage and membrane accessibility assays were performed. In the case of membrane disruption induced by the active pore formation, the incorporated calcein would leak from the liposomes and simultaneously the aqueous solution in the membrane surrounding would be accessible to the liposome interior at pH 5.0. The assays in the presence of LPE3-1 indicated no significant leakage or accessibility at pH 7.4, but a typical leakage and some accessibility to liposomes were positively observed at pH 5.0. In order to estimate whether the weakly acidic pH-controlled lytic activity is due to a secondary structural change of the hydrophobic segment of LPE3-1 in the liposome membrane, we have measured circular dichroism spectra. In the presence of liposomes, the minimum showing the characteristic helical structure was observed at 222 nm only under weakly acidic conditions. This pH dependence is in good agreement with the results from the leakage and accessibility assays. The pH-dependent membrane disruption properties of LPE3-1 may open a new avenue to gain insight into the interaction between peptides and lipids for the development of efficient drug/gene delivery systems. PMID:27270896

  7. Design and Synthesis of Near-Infrared Peptide for in Vivo Molecular Imaging of HER2.

    PubMed

    Joshi, Bishnu P; Zhou, Juan; Pant, Asha; Duan, Xiyu; Zhou, Quan; Kuick, Rork; Owens, Scott R; Appelman, Henry; Wang, Thomas D

    2016-02-17

    We report the development, characterization, and validation of a peptide specific for the extracellular domain of HER2. This probe chemistry was developed for molecular imaging by using a structural model to select an optimal combination of amino acids that maximize the likelihood for unique hydrophobic and hydrophilic interactions with HER2 domain 3. The sequence KSPNPRF was identified and conjugated with either FITC or Cy5.5 via a GGGSK linker using Fmoc-mediated solid-phase synthesis to demonstrate flexibility for this chemical structure to be labeled with different fluorophores. A scrambled sequence was developed for control by altering the conformationally rigid spacer and moving both hydrophobic and hydrophilic amino acids on the C-terminus. We validated peptide specificity for HER2 in knockdown and competition experiments using human colorectal cancer cells in vitro, and measured a binding affinity of kd = 21 nM and time constant of k = 0.14 min(-1) (7.14 min). We used this peptide with either topical or intravenous administration in a preclinical model of colorectal cancer to demonstrate specific uptake in spontaneous adenomas and to show feasibility for real time in vivo imaging with near-infrared fluorescence. We used this peptide in immunofluorescence studies of human proximal colon specimens to evaluate specificity for sessile serrated and sporadic adenomas. Improved visualization can be used endoscopically to guide tissue biopsy and detect premalignant lesions that would otherwise be missed. Our peptide design for specificity to HER2 is promising for clinical translation in molecular imaging methods for early cancer detection. PMID:26709709

  8. Peptides inhibitors of acid-sensing ion channels.

    PubMed

    Diochot, S; Salinas, M; Baron, A; Escoubas, P; Lazdunski, M

    2007-02-01

    Acid-sensing ion channels (ASICs) channels are proton-gated cationic channels mainly expressed in central and peripheric nervous system and related to the epithelial amiloride-sensitive Na(+) channels and to the degenerin family of ion channels. ASICs comprise four proteins forming functional channel subunits (ASIC1a, ASIC1b, ASIC2a, and ASIC3) and two proteins (ASIC2b and ASIC4) without yet known activators. Functional channels are activated by external pH variations ranging from pH(0.5) 6.8 to 4.0 and currents are characterized by either rapid kinetics of inactivation (ASIC1a, ASIC1b, ASIC3) or slow kinetics of inactivation (ASIC2a) and sometimes the presence of a plateau phase (ASIC3). ASIC1a and ASIC3, which are expressed in nociceptive neurons, have been implicated in inflammation and knockout mice studies support the role of ASIC3 in various pain processes. ASIC1a seems more related to synaptic plasticity, memory, learning and fear conditioning in the CNS. ASIC2a contributes to hearing in the cochlea, sour taste sensation, and visual transduction in the retina. The pharmacology of ASICs is limited to rather nonselective drugs such as amiloride, nonsteroid anti-inflammatory drugs, and neuropeptides. Recently, two peptides, PcTx1 and APETx2, isolated from a spider and a sea anemone, have been characterized as selective and high-affinity inhibitors for ASIC1a and ASIC3 channels, respectively. PcTx1 inhibits ASIC1a homomers with an affinity of 0.7 nM (IC(50)) without any effect on ASIC1a containing heteromers and thus helped to characterize ASIC1a homomeric channels in peripheric and central neurons. PcTx1 acts as a gating modifier since it shifts the channel from the resting to an inactivated state by increasing its affinity for H(+). APETx2 is less selective since it inhibits several ASIC3-containing channels (IC(50) from 63 nM to 2 microM) and to date its mode of action is unknown. Nevertheless, APETx2 structure is related to other sea anemone peptides, which

  9. Amino acid and peptide absorption from partial digests of proteins in isolated rat small intestine.

    PubMed Central

    Gardner, M L

    1978-01-01

    1. Absorption of each of sixteen amino acids, free and peptide-bound, has been measured in isolated rat small intestine perfused with five partial digests of proteins. 2. At low concentrations net absorption of each amino acid was proportional to its luminal concentration and independent of the nature of the amino acid. 3. A series of first-order multiple regressions was found to describe well the characteristics of absorption. 4. Rate constants for disappearance of free and peptide-bound amino acids from the lumen were closely similar. However, substantial back-flux occurred of amino acids derived from peptide hydrolysis. Hence 60-70% of the amino-N entering the serosal tissue fluid probably had left the lumen as free amino acids. 5. Intact peptides crossed the mucosa during absorption from a soy bean hydrolysate and in substantial quantities during absorption from one casein digest but not from another. With other hydrolysates there was no evidence for passage of peptides to the serosa. 6. In several cases there was a serious discrepancy between the amount of amino-N absorbed from the lumen and the amount accounted for as peptide or free amino acid in the serosal secretion. 7. The characteristics of absorption were similar (apart from the exceptions in 5 above) for all the digests studied except for soy bean hydrolysate. PMID:731590

  10. The design of peptide-based substrates for the cdc2 protein kinase.

    PubMed Central

    Srinivasan, J; Koszelak, M; Mendelow, M; Kwon, Y G; Lawrence, D S

    1995-01-01

    The substrate sequence specificity of the cdc2 protein kinase from Pisaster ochraceus has been evaluated. The peptide, Ac-Ser-Pro-Gly-Arg-Arg-Arg-Arg-Lys-amide, serves as an efficient cdc2 kinase substrate with a Km of 1.50 +/- 0.04 microM and a Vmax. of 12.00 +/- 0.18 mumol/min per mg. The amino acid sequence of this peptide is not based on any sequence in a known protein substrate of the cyclin-dependent kinase, but rather was designed from structural attributes that appear to be important in the majority of cdc2 substrates. The cyclin-dependent enzyme is remarkably indiscriminate in its ability to recognize and phosphorylate peptides that contain an assortment of structurally diverse residues at the P-2, P-1 and P+2 positions. However, peptides that contain a free N-terminal serine or lack an arginine at the P+4 position are relatively poor substrates. These aspects of the substrate specificity of the cdc2 protein kinase are compared and contrasted with the previously reported substrate specificity of a cdc2-like protein kinase from bovine brain [Beaudette, Lew and Wang (1993) J. Biol. Chem. 268, 20825-20830]. PMID:7639712

  11. Quantum Chemical Analysis of MHC-Peptide Interactions for Vaccine Design

    PubMed Central

    Agudelo, W.A; Patarroyo, M.E

    2010-01-01

    The development of an adequate immune response against pathogens is mediated by molecular interactions between different cell types. Among them, binding of antigenic peptides to the Major Histocompatibility Complex (MHC) molecule expressed on the membrane of antigen presenting cells (APCs), and their subsequent recognition by the T cell receptor have been demonstrated to be crucial for developing an adequate immune response. The present review compiles computational quantum chemistry studies about the electrostatic potential variations induced on the MHC binding region by peptide’s amino acids, carried out with the aim of describing MHC–peptide binding interactions. The global idea is that the electrostatic potential can be represented in terms of a series expansion (charge, dipole, quadrupole, hexadecapole, etc.) whose three first terms provide a good local approximation to the molecular electrostatic ‘landscape’ and to the variations induced on such landscape by targeted modifications on the residues of the antigenic peptide. Studies carried out in four MHC class II human allele molecules, which are the most representative alleles of their corresponding haplotypes, showed that each of these molecules have conserved as well as specific electrostatic characteristics, which can be correlated at a good extent with the peptide binding profiles reported experimentally for these molecules. The information provided by such characteristics would help increase our knowledge about antigen binding and presentation, and could ultimately contribute to developing a logical and rational methodology for designing chemically synthesized, multi-antigenic, subunit-based vaccines, through the application of quantum chemistry methods. PMID:20394575

  12. Aqueous Synthesis of Peptide Thioesters from Amino Acids and a Thiol Using 1,1‧-Carbonyldiimidazole

    NASA Astrophysics Data System (ADS)

    Weber, Arthur L.

    2005-10-01

    A new method was developed for the synthesis of peptide thioesters from free amino acids and thiols in water. This one-pot simple method involves two steps: (1) activation in water of an amino acid presumably as its N-carboxyanhydride (NCA) using 1,1‧-carbonyldiimidazole (CDI), and (2) subsequent condensation of the activated amino acid-NCA in the presence of a thiol. With this method citrulline peptide thioesters containing up to 10 amino acid residues were prepared in a single reaction. This aqueous synthetic method provides a simple way to prepare peptide thioesters for studies of peptide replication involving ligation of peptide thioesters on peptide templates. The relevance of peptide replication to the origin-of-life process is supported by previous studies showing that amino acid thioesters (peptide thioester precursors) can be synthesized under prebiotic conditions by reaction of small sugars with ammonia and a thiol.

  13. Cyclic Sulfamidate Enabled Syntheses of Amino Acids, Peptides, Carbohydrates, and Natural Products

    EPA Science Inventory

    This article reviews the emergence of cyclic sulfamidates as versatile intermediatesfor the synthesis of unnatural amino acids, chalcogen peptides, modified sugars, drugs and drug candidates, and important natural products.

  14. Tetrazine-Containing Amino Acid for Peptide Modification and Live Cell Labeling

    PubMed Central

    Ni, Zhongqiu; Zhou, Lanxia; Li, Xu; Zhang, Jing; Dong, Shouliang

    2015-01-01

    A novel amino acid derivative 3-(4-(1, 2, 4, 5-tetrazine-3-yl) phenyl)-2-aminopropanoic acid was synthesized in this study. The compound possessed better water-solubility and was synthesized more easily compared with the well-known and commercially available 3-(p-benzylamino)-1, 2, 4, 5-tetrazine. Tetrazine-containing amino acid showed excellent stability in biological media and might be used for cancer cell labeling. Moreover, the compound remained relatively stable in 50% TFA/DCM with little decomposition after prolonged exposure at room temperature. The compound could be utilized as phenylalanine or tyrosine analogue in peptide modification, and the tetrazine-containing peptide demonstrated more significant biological activity than that of the parent peptide. The combination of tetrazine group and amino acid offered broad development prospects of the bioorthogonal labeling and peptide synthesis. PMID:26536589

  15. Stimulation of Lysine Decarboxylase Production in Escherichia coli by Amino Acids and Peptides1

    PubMed Central

    Cascieri, T.; Mallette, M. F.

    1973-01-01

    A commercial hydrolysate of casein stimulated production of lysine decarboxylase (EC 4.1.1.18) by Escherichia coli B. Cellulose and gel chromatography of this hydrolysate yielded peptides which were variably effective in this stimulation. Replacement of individual, stimulatory peptides by equivalent amino acids duplicated the enzyme levels attained with those peptides. There was no indication of specific stimulation by any peptide. The peptides were probably taken up by the oligopeptide transport system of E. coli and hydrolyzed intracellularly by peptidases to their constituent amino acids for use in enzyme synthesis. Single omission of amino acids from mixtures was used to screen them for their relative lysine decarboxylase stimulating abilities. Over 100 different mixtures were evaluated in establishing the total amino acid requirements for maximal synthesis of lysine decarboxylase by E. coli B. A mixture containing all of the common amino acids except glutamic acid, aspartic acid, and alanine increased lysine decarboxylase threefold over an equivalent weight of casein hydrolysate. The nine most stimulatory amino acids were methionine, arginine, cystine, leucine, isoleucine, glutamine, threonine, tyrosine, and asparagine. Methionine and arginine quantitatively were the most important. A mixture of these nine was 87% as effective as the complete mixture. Several amino acids were inhibitory at moderate concentrations, and alanine (2.53 mM) was the most effective. Added pyridoxine increased lysine decarboxylase activity 30%, whereas other B vitamins and cyclic adenosine 5′-monophosphate had no effect. PMID:4588201

  16. Design of Peptide Immunotherapies for MHC Class-II-Associated Autoimmune Disorders

    PubMed Central

    2013-01-01

    Autoimmune disorders, that occur when autoreactive immune cells are induced to activate their responses against self-tissues, affect one percent of the world population and represent one of the top 10 leading causes of death. The major histocompatibility complex (MHC) is a principal susceptibility locus for many human autoimmune diseases, in which self-tissue antigens providing targets for pathogenic lymphocytes are bound to HLA molecules encoded by disease-associated alleles. In spite of the attempts to design strategies for inhibition of antigen presentation targeting the MHC-peptide/TCR complex via generation of blocking antibodies, altered peptide ligands (APL), or inhibitors of costimulatory molecules, potent therapies with minimal side effects have yet to be developed. Copaxone (glatiramer acetate, GA) is a random synthetic amino acid copolymer that reduces the relapse rate by about 30% in relapsing-remitting multiple sclerosis (MS) patients. Based on the elucidated binding motifs of Copaxone and of the anchor residues of the immunogenic myelin basic protein (MBP) peptide to HLA-DR molecules, novel copolymers have been designed and proved to be more effective in suppressing MS-like disease in mice. In this report, we describe the rationale for design of second-generation synthetic random copolymers as candidate drugs for a number of MHC class-II-associated autoimmune disorders. PMID:24324511

  17. Design of Catalytic Peptides and Proteins Through Rational and Combinatorial Approaches.

    PubMed

    Maeda, Yoshiaki; Makhlynets, Olga V; Matsui, Hiroshi; Korendovych, Ivan V

    2016-07-11

    This review focuses on recent progress in noncomputational methods to introduce catalytic function into proteins, peptides, and peptide assemblies. We discuss various approaches to creating catalytic activity and classification of noncomputational methods into rational and combinatorial classes. The section on rational design covers recent progress in the development of short peptides and oligomeric peptide assemblies for various natural and unnatural reactions. The section on combinatorial design describes recent advances in the discovery of catalytic peptides. We present the future prospects of these and other new approaches in a broader context, including implications for functional material design. PMID:27022702

  18. Efficacy of peptide nucleic acid and selected conjugates against specific cellular pathologies of amyotrophic lateral sclerosis.

    PubMed

    Browne, Elisse C; Parakh, Sonam; Duncan, Luke F; Langford, Steven J; Atkin, Julie D; Abbott, Belinda M

    2016-04-01

    Cellular studies have been undertaken on a nonamer peptide nucleic acid (PNA) sequence, which binds to mRNA encoding superoxide dismutase 1, and a series of peptide nucleic acids conjugated to synthetic lipophilic vitamin analogs including a recently prepared menadione (vitamin K) analog. Reduction of both mutant superoxide dismutase 1 inclusion formation and endoplasmic reticulum stress, two of the key cellular pathological hallmarks in amyotrophic lateral sclerosis, by two of the prepared PNA oligomers is reported for the first time. PMID:26935939

  19. Exploring the sequence space for (tri-)peptide self-assembly to design and discover new hydrogels

    NASA Astrophysics Data System (ADS)

    Frederix, Pim W. J. M.; Scott, Gary G.; Abul-Haija, Yousef M.; Kalafatovic, Daniela; Pappas, Charalampos G.; Javid, Nadeem; Hunt, Neil T.; Ulijn, Rein V.; Tuttle, Tell

    2015-01-01

    Peptides that self-assemble into nanostructures are of tremendous interest for biological, medical, photonic and nanotechnological applications. The enormous sequence space that is available from 20 amino acids probably harbours many interesting candidates, but it is currently not possible to predict supramolecular behaviour from sequence alone. Here, we demonstrate computational tools to screen for the aqueous self-assembly propensity in all of the 8,000 possible tripeptides and evaluate these by comparison with known examples. We applied filters to select for candidates that simultaneously optimize the apparently contradicting requirements of aggregation propensity and hydrophilicity, which resulted in a set of design rules for self-assembling sequences. A number of peptides were subsequently synthesized and characterized, including the first reported tripeptides that are able to form a hydrogel at neutral pH. These tools, which enable the peptide sequence space to be searched for supramolecular properties, enable minimalistic peptide nanotechnology to deliver on its promise.

  20. Proteins, Peptides and Amino Acids: Role in Infant Nutrition.

    PubMed

    Nutten, Sophie

    2016-01-01

    Proteins are polymers composed of 30 or more amino acids; some of them are essential dietary components, since they are not synthetized by human metabolic processes. They are crucial for healthy growth and development and influence major functions of the body. The infant's first year is a critical time of rapid growth and development, which must be supported by a high rate of protein synthesis. Breast milk, as a single specific food source in the first months of life, is providing the total protein and essential amino acids required. Infant formulas have been designed for infants who cannot be breastfed. They should be similar to breast milk in their composition and their functional outcomes, insuring appropriate growth, optimal development, maturation of the immune system, easy digestion and healthy metabolic programming. By modifying their protein components, specific infant formulas have also been developed for specific needs. For example, partially hydrolyzed (prevention of atopic dermatitis) and extensively hydrolyzed or amino-acid-based infant formulas (reduction in allergy symptoms) have been designed for the management of cow's milk protein allergy. In conclusion, proteins provided via breast milk or infant formula are essential components of the infant's diet; therefore, the specific quality, quantity and conformation of proteins are of utmost importance for healthy growth and development. PMID:27336588

  1. Site-Selective Binding of Nanoparticles to Double-Stranded DNA via Peptide Nucleic Acid "Invasion"

    SciTech Connect

    Stadler, A.L.; van der Lelie, D.; Sun, D.; Maye, M. M.; Gang, O.

    2011-04-01

    We demonstrate a novel method for by-design placement of nano-objects along double-stranded (ds) DNA. A molecular intercalator, designed as a peptide nucleic acid (PNA)-DNA chimera, is able to invade dsDNA at the PNA-side due to the hybridization specificity between PNA and one of the duplex strands. At the same time, the single-stranded (ss) DNA tail of the chimera, allows for anchoring of nano-objects that have been functionalized with complementary ssDNA. The developed method is applied for interparticle attachment and for the fabrication of particle clusters using a dsDNA template. This method significantly broadens the molecular toolbox for constructing nanoscale systems by including the most conventional not yet utilized DNA motif, double helix DNA.

  2. Plasmid DNA delivery by arginine-rich cell-penetrating peptides containing unnatural amino acids.

    PubMed

    Kato, Takuma; Yamashita, Hiroko; Misawa, Takashi; Nishida, Koyo; Kurihara, Masaaki; Tanaka, Masakazu; Demizu, Yosuke; Oba, Makoto

    2016-06-15

    Cell-penetrating peptides (CPPs) have been developed as drug, protein, and gene delivery tools. In the present study, arginine (Arg)-rich CPPs containing unnatural amino acids were designed to deliver plasmid DNA (pDNA). The transfection ability of one of the Arg-rich CPPs examined here was more effective than that of the Arg nonapeptide, which is the most frequently used CPP. The transfection efficiencies of Arg-rich CPPs increased with longer post-incubation times and were significantly higher at 48-h and 72-h post-incubation than that of the commercially available transfection reagent TurboFect. These Arg-rich CPPs were complexed with pDNA for a long time in cells and effectively escaped from the late endosomes/lysosomes into the cytoplasm. These results will be helpful for designing novel CPPs for pDNA delivery. PMID:27132868

  3. Potent Antibacterial Antisense Peptide–Peptide Nucleic Acid Conjugates Against Pseudomonas aeruginosa

    PubMed Central

    Ghosal, Anubrata

    2012-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen causing severe infections in hospital settings, especially with immune compromised patients, and the increasing prevalence of multidrug resistant strains urges search for new drugs with novel mechanisms of action. In this study we introduce antisense peptide–peptide nucleic acid (PNA) conjugates as antibacterial agents against P. aeruginosa. We have designed and optimized antisense peptide–PNA conjugates targeting the translation initiation region of the ftsZ gene (an essential bacterial gene involved in cell division) or the acpP gene (an essential bacterial gene involved in fatty acid synthesis) of P. aeruginosa (PA01) and characterized these compounds according to their antimicrobial activity and mode of action. Four antisense PNA oligomers conjugated to the H-(R-Ahx-R)4-Ahx-βala or the H-(R-Ahx)6-βala peptide exhibited complete growth inhibition of P. aeruginosa strains PA01, PA14, and LESB58 at 1–2 μM concentrations without any indication of bacterial membrane disruption (even at 20 μM), and resulted in specific reduction of the targeted mRNA levels. One of the four compounds showed clear bactericidal activity while the other significantly reduced bacterial survival. These results open the possibility of development of antisense antibacterials for treatment of Pseudomonas infections. PMID:23030590

  4. Installing amino acids and peptides on N-heterocycles under visible-light assistance

    PubMed Central

    Jin, Yunhe; Jiang, Min; Wang, Hui; Fu, Hua

    2016-01-01

    Readily available natural α-amino acids are one of nature’s most attractive and versatile building blocks in synthesis of natural products and biomolecules. Peptides and N-heterocycles exhibit various biological and pharmaceutical functions. Conjugation of amino acids or peptides with N-heterocycles provides boundless potentiality for screening and discovery of diverse biologically active molecules. However, it is a great challenge to install amino acids or peptides on N-heterocycles through formation of carbon-carbon bonds under mild conditions. In this article, eighteen N-protected α-amino acids and three peptides were well assembled on phenanthridine derivatives via couplings of N-protected α-amino acid and peptide active esters with substituted 2-isocyanobiphenyls at room temperature under visible-light assistance. Furthermore, N-Boc-proline residue was successfully conjugated with oxindole derivatives using similar procedures. The simple protocol, mild reaction conditions, fast reaction, and high efficiency of this method make it an important strategy for synthesis of diverse molecules containing amino acid and peptide fragments. PMID:26830014

  5. Installing amino acids and peptides on N-heterocycles under visible-light assistance.

    PubMed

    Jin, Yunhe; Jiang, Min; Wang, Hui; Fu, Hua

    2016-01-01

    Readily available natural α-amino acids are one of nature's most attractive and versatile building blocks in synthesis of natural products and biomolecules. Peptides and N-heterocycles exhibit various biological and pharmaceutical functions. Conjugation of amino acids or peptides with N-heterocycles provides boundless potentiality for screening and discovery of diverse biologically active molecules. However, it is a great challenge to install amino acids or peptides on N-heterocycles through formation of carbon-carbon bonds under mild conditions. In this article, eighteen N-protected α-amino acids and three peptides were well assembled on phenanthridine derivatives via couplings of N-protected α-amino acid and peptide active esters with substituted 2-isocyanobiphenyls at room temperature under visible-light assistance. Furthermore, N-Boc-proline residue was successfully conjugated with oxindole derivatives using similar procedures. The simple protocol, mild reaction conditions, fast reaction, and high efficiency of this method make it an important strategy for synthesis of diverse molecules containing amino acid and peptide fragments. PMID:26830014

  6. Rapid complexing of oxoacylglycerols with amino acids, peptides and aminophospholipids.

    PubMed

    Kurvinen, J P; Kuksis, A; Ravandi, A; Sjövall, O; Kallio, H

    1999-03-01

    We prepared model Schiff bases from 2-[9-oxo]nonanoyl glycerol (2-MAG-ALD) and various amino compounds. 2-MAG-ALD was obtained by pancreatic lipase hydrolysis of trioleoyl glycerol and reductive ozonolysis of the resulting 2-monooleoyl glycerol. The reaction products were purified by thin-layer chromatography. Schiff bases were synthesized in greater than 50% yield by reacting 2-MAG-ALD with twofold molar excess of valine, Nalpha-acetyl-L-lysine methyl ester and the tripeptides glycyl-glycyl-glycine, glycyl-glycyl-histidine, and glycyl-histidyl-lysine in aqueous methanol and with 1-palmitoyl-2-stearoyl glycerophosphoethanolamine (PE) in chloroform/methanol for 16 h at room temperature. Prior to analysis the bases were reduced with sodium cyanoborohydride in methanol for 30 min at 4 degrees C. Reaction products were analyzed by high-performance liquid chromatography/electrospray ionization/mass spectrometry (HPLC/ESI/MS). Reduced Schiff bases of 2-MAG-ALD with PE and amino acids were analyzed by normal-phase HPLC/ESI/MS and those with peptides by reversed-phase HPLC/ESI/MS. Single adducts were obtained in all cases and both the alpha-amino group of valine and the epsilon-amino group of Nalpha-acetyl-L-lysine methyl ester were reactive. Molecular ions of reaction products were the only detected ions in the negative ionization mode, whereas in the positive ion mode sodiated molecular ions were also detected. The present study suggests that 2-MAG-ALD may form Schiff base adducts with amino compounds in other aqueous media, such as the intestinal lumen and in the hydrophobic environment of cell membranes. PMID:10230725

  7. L-amino acid ligase from Pseudomonas syringae producing tabtoxin can be used for enzymatic synthesis of various functional peptides.

    PubMed

    Arai, Toshinobu; Arimura, Yasuhiro; Ishikura, Shun; Kino, Kuniki

    2013-08-01

    Functional peptides are expected to be beneficial compounds that improve our quality of life. To address the growing need for functional peptides, we have examined peptide synthesis by using microbial enzymes. l-Amino acid ligase (Lal) catalyzes the condensation of unprotected amino acids in an ATP-dependent manner and is applicable to fermentative production. Hence, Lal is a promising enzyme to achieve cost-effective synthesis. To obtain a Lal with novel substrate specificity, we focused on the putative Lal involved in the biosynthesis of the dipeptidic phytotoxin designated tabtoxin. The tabS gene was cloned from Pseudomonas syringae NBRC14081 and overexpressed in Escherichia coli cells. The recombinant TabS protein produced showed the broadest substrate specificity of any known Lal; it detected 136 of 231 combinations of amino acid substrates when dipeptide synthesis was examined. In addition, some new substrate specificities were identified and unusual amino acids, e.g., l-pipecolic acid, hydroxy-l-proline, and β-alanine, were found to be acceptable substrates. Furthermore, kinetic analysis and monitoring of the reactions over a short time revealed that TabS showed distinct substrate selectivity at the N and C termini, which made it possible to specifically synthesize a peptide without by-products such as homopeptides and heteropeptides with the reverse sequence. TabS specifically synthesized the following functional peptides, including their precursors: l-arginyl-l-phenylalanine (antihypertensive effect; yield, 62%), l-leucyl-l-isoleucine (antidepressive effect; yield, 77%), l-glutaminyl-l-tryptophan (precursor of l-glutamyl-l-tryptophan, which has antiangiogenic activity; yield, 54%), l-leucyl-l-serine (enhances saltiness; yield, 83%), and l-glutaminyl-l-threonine (precursor of l-glutamyl-l-threonine, which enhances saltiness; yield, 96%). Furthermore, our results also provide new insights into tabtoxin biosynthesis. PMID:23770908

  8. The impact of α-hydrazino acids embedded in short fluorescent peptides on peptide interactions with DNA and RNA.

    PubMed

    Suć, Josipa; Tumir, Lidija-Marija; Glavaš-Obrovac, Ljubica; Jukić, Marijana; Piantanida, Ivo; Jerić, Ivanka

    2016-06-01

    A series of novel hydrazino-based peptidomimetics and analogues comprising N-terminal lysine and C-terminal phenanthridinyl-l-alanine were prepared. The presented results demonstrate the up to now unknown possibility to finely modulate peptide interactions with DNA/RNA by α-hydrazino group insertion and how the different positioning of two α-hydrazino groups in peptides controls binding to various double stranded and single stranded DNA and RNA. All peptidomimetics bind with 1-10 micromolar affinity to ds-DNA/RNA, whereby the binding mode is a combination of electrostatic interactions and hydrophobic interactions within DNA/RNA grooves. Insertion of the α-hydrazino group into the peptide systematically decreased its fluorimetric response to DNA/RNA binding in the order: mono-hydrazino < alternating-hydrazino < sequential-hydrazino group. Binding studies of ss-polynucleotides suggest intercalation of phenanthridine between polynucleotide bases, whereby affinity and fluorimetric response decrease with the number of α-hydrazino groups in the peptide sequence. Particularly interesting was the interaction of two sequential α-hydrazino acids-peptidomimetic with poly rG, characterised by a specific strong increase of CD bands, while all other peptide/ssRNA combinations gave only a CD-band decrease. All mentioned interactions could also be reversibly controlled by adjusting the pH, due to the protonation of the fluorophore. PMID:27161341

  9. The world of beta- and gamma-peptides comprised of homologated proteinogenic amino acids and other components.

    PubMed

    Seebach, Dieter; Beck, Albert K; Bierbaum, Daniel J

    2004-08-01

    helices (10/12-, 12-, and 14-helix) if we include oligomers of trans-2-aminocyclopentanecarboxylic acid, for example - the structures are already observable with chains made up of only four components, and, having now undergone a learning process, we are able to construct them by design. The structures of the shorter beta-peptides can also be reliably determined by molecular-dynamics calculations (in solution; GROMOS program package). Unlike in the case of the natural helices, these compounds' folding into secondary structures is not cooperative. In beta- and gamma-peptides, it is possible to introduce heteroatom substituents (such as halogen or OH) onto the backbones or to incorporate heteroatoms (NH, O) directly into the chain, and, thanks to this, it has been possible to study effects unobservable in the world of the alpha-peptides. Tests with proteolytic enzymes of all types (from mammals, microorganisms, yeasts) and in vivo examination (mice, rats, insects, plants) showed beta- and gamma-peptides to be completely stable towards proteolysis and, as demonstrated for two beta-peptides, extraordinarily stable towards metabolism, even when bearing functionalized side chains (such as those of Thr, Tyr, Trp, Lys, or Arg). The beta-peptides so far examined also normally display no or only very weak cytotoxic, antiproliferative, antimicrobial, hemolytic, immunogenic, or inflammatory properties either in cell cultures or in vivo. Even biological degradation by microbial colonies of the types found in sewage-treatment plants or in soil is very slow. That there are indeed interactions of beta- and gamma-peptides with biological systems, however, can be seen in the following findings: i) organ-specific distribution takes place after intravenous (i.v.) administration in rats, ii) transport through the intestines of rodents has been observed, iii) beta-peptides with positively charged side chains (Arg and Lys) settle on cell surfaces, are able to enter into mammalian cells

  10. New Compstatin Peptides Containing N-Terminal Extensions and Non-Natural Amino Acids Exhibit Potent Complement Inhibition and Improved Solubility Characteristics

    PubMed Central

    2015-01-01

    Compstatin peptides are complement inhibitors that bind and inhibit cleavage of complement C3. Peptide binding is enhanced by hydrophobic interactions; however, poor solubility promotes aggregation in aqueous environments. We have designed new compstatin peptides derived from the W4A9 sequence (Ac-ICVWQDWGAHRCT-NH2, cyclized between C2 and C12), based on structural, computational, and experimental studies. Furthermore, we developed and utilized a computational framework for the design of peptides containing non-natural amino acids. These new compstatin peptides contain polar N-terminal extensions and non-natural amino acid substitutions at positions 4 and 9. Peptides with α-modified non-natural alanine analogs at position 9, as well as peptides containing only N-terminal polar extensions, exhibited similar activity compared to W4A9, as quantified via ELISA, hemolytic, and cell-based assays, and showed improved solubility, as measured by UV absorbance and reverse-phase HPLC experiments. Because of their potency and solubility, these peptides are promising candidates for therapeutic development in numerous complement-mediated diseases. PMID:25494040

  11. Enzyme reaction engineering: design of peptide synthesis by stabilized trypsin.

    PubMed

    Blanco, R M; Alvaro, G; Guisán, J M

    1991-07-01

    By using very active and very stable trypsin agarose derivatives, we have optimized the design of the synthesis of a model dipeptide, benzoylarginine leucinamide, by two different strategies: (i) kinetically controlled synthesis (KCS), by using benzoyl arginine ethyl ester and leucinamide as substrates, and (ii) thermodynamically controlled synthesis (TCS), by using benzoyl arginine and leucinamide as substrates. In each strategy, we have studied the integrated effect of a number of variables that define the reaction medium on different parameters of industrial interest, e.g. time course of peptide synthesis, higher synthetic yields, and stability of the catalyst, as well as aminolysis/hydrolysis ratios and rate of peptide hydrolysis in the case of KCS. Both synthetic approaches were carried out in monophasic water or water-organic cosolvent systems. We have mainly tested a number of variables, e.g. temperature, polarity of the reaction medium (presence of cosolvents, presence of ammonium sulfate), and exact structure of the trypsin derivatives. Optimal experimental conditions for these synthetic approaches were established in order to simultaneously obtain good values for all industrial parameters. The use of previously stabilized trypsin derivatives greatly improves the design of these synthetic approaches (e.g. by using drastic experimental conditions: 1 M ammonium sulfate (KCS) or 90% organic cosolvents (TCS]. In these conditions, our derivatives preserve more than 95% of activity after 2 months and we have been able to reach synthetic productivities of 180 (KCS) and 1 (TCS) tons of dipeptide per year per liter of catalyst. PMID:1367640

  12. Formation of Amino Acid Thioesters for Prebiotic Peptide Synthesis: Catalysis By Amino Acid Products

    NASA Technical Reports Server (NTRS)

    Weber, Arthur L.; DeVincenzi, Donald L. (Technical Monitor)

    1999-01-01

    The origin of life can be described as a series of events in which a prebiotic chemical process came increasingly under the control of its catalytic products. In our search for this prebiotic process that yielded catalytic takeover products (such as polypeptides), we have been investigating a reaction system that generates peptide-forming amino acid thioesters from formaldehyde, glycolaldehyde, and ammonia in the presence of thiols. As shown below, this model process begins by aldol condensation of formaldehyde and glycolaldehyde to give trioses and releases. These sugars then undergo beta-dehydration yielding their respective alpha-ketoaldehydes. Addition of ammonia to the alpha-ketoaldehydes yields imines which can either: (a) rearrange in the presence of thesis to give amino acid thioesters or (be react with another molecule of aldehyde to give imidazoles. This 'one-pot' reaction system operates under mild aqueous conditions, and like modem amino acid biosynthesis, uses sugar intermediates which are converted to products by energy-yielding redox reactions. Recently, we discovered that amino acids, such as the alanine reaction product, catalyze the first and second steps of the process. In the presence of ammonia the process also generates other synthetically useful products, like the important biochemical -- pyruvic acid.

  13. Rational Design of a Carrier Protein for the Production of Recombinant Toxic Peptides in Escherichia coli.

    PubMed

    Pane, Katia; Durante, Lorenzo; Pizzo, Elio; Varcamonti, Mario; Zanfardino, Anna; Sgambati, Valeria; Di Maro, Antimo; Carpentieri, Andrea; Izzo, Viviana; Di Donato, Alberto; Cafaro, Valeria; Notomista, Eugenio

    2016-01-01

    Commercial uses of bioactive peptides require low cost, effective methods for their production. We developed a new carrier protein for high yield production of recombinant peptides in Escherichia coli very well suited for the production of toxic peptides like antimicrobial peptides. GKY20, a short antimicrobial peptide derived from the C-terminus of human thrombin, was fused to the C-terminus of Onconase, a small ribonuclease (104 amino acids), which efficiently drove the peptide into inclusion bodies with very high expression levels (about 200-250 mg/L). After purification of the fusion protein by immobilized metal ion affinity chromatography, peptide was obtained by chemical cleavage in diluted acetic acid of an acid labile Asp-Pro sequence with more than 95% efficiency. To improve peptide purification, Onconase was mutated to eliminate all acid labile sequences thus reducing the release of unwanted peptides during the acid cleavage. Mutations were chosen to preserve the differential solubility of Onconase as function of pH, which allows its selective precipitation at neutral pH after the cleavage. The improved carrier allowed the production of 15-18 mg of recombinant peptide per liter of culture with 96-98% purity without the need of further chromatographic steps after the acid cleavage. The antimicrobial activity of the recombinant peptide, with an additional proline at the N-terminus, was tested on Gram-negative and Gram-positive strains and was found to be identical to that measured for synthetic GKY20. This finding suggests that N-terminal proline residue does not change the antimicrobial properties of recombinant (P)GKY20. The improved carrier, which does not contain cysteine and methionine residues, Asp-Pro and Asn-Gly sequences, is well suited for the production of peptides using any of the most popular chemical cleavage methods. PMID:26808536

  14. Rational Design of a Carrier Protein for the Production of Recombinant Toxic Peptides in Escherichia coli

    PubMed Central

    Pizzo, Elio; Varcamonti, Mario; Zanfardino, Anna; Sgambati, Valeria; Di Maro, Antimo; Carpentieri, Andrea; Izzo, Viviana; Di Donato, Alberto; Cafaro, Valeria; Notomista, Eugenio

    2016-01-01

    Commercial uses of bioactive peptides require low cost, effective methods for their production. We developed a new carrier protein for high yield production of recombinant peptides in Escherichia coli very well suited for the production of toxic peptides like antimicrobial peptides. GKY20, a short antimicrobial peptide derived from the C-terminus of human thrombin, was fused to the C-terminus of Onconase, a small ribonuclease (104 amino acids), which efficiently drove the peptide into inclusion bodies with very high expression levels (about 200–250 mg/L). After purification of the fusion protein by immobilized metal ion affinity chromatography, peptide was obtained by chemical cleavage in diluted acetic acid of an acid labile Asp-Pro sequence with more than 95% efficiency. To improve peptide purification, Onconase was mutated to eliminate all acid labile sequences thus reducing the release of unwanted peptides during the acid cleavage. Mutations were chosen to preserve the differential solubility of Onconase as function of pH, which allows its selective precipitation at neutral pH after the cleavage. The improved carrier allowed the production of 15–18 mg of recombinant peptide per liter of culture with 96–98% purity without the need of further chromatographic steps after the acid cleavage. The antimicrobial activity of the recombinant peptide, with an additional proline at the N-terminus, was tested on Gram-negative and Gram-positive strains and was found to be identical to that measured for synthetic GKY20. This finding suggests that N-terminal proline residue does not change the antimicrobial properties of recombinant (P)GKY20. The improved carrier, which does not contain cysteine and methionine residues, Asp-Pro and Asn-Gly sequences, is well suited for the production of peptides using any of the most popular chemical cleavage methods. PMID:26808536

  15. Design and Evaluation of Antimalarial Peptides Derived from Prediction of Short Linear Motifs in Proteins Related to Erythrocyte Invasion

    PubMed Central

    Bianchin, Alessandra; Bell, Angus; Chubb, Anthony J.; Doolan, Nathalie; Leneghan, Darren; Stavropoulos, Ilias; Shields, Denis C.; Mooney, Catherine

    2015-01-01

    The purpose of this study was to investigate the blood stage of the malaria causing parasite, Plasmodium falciparum, to predict potential protein interactions between the parasite merozoite and the host erythrocyte and design peptides that could interrupt these predicted interactions. We screened the P. falciparum and human proteomes for computationally predicted short linear motifs (SLiMs) in cytoplasmic portions of transmembrane proteins that could play roles in the invasion of the erythrocyte by the merozoite, an essential step in malarial pathogenesis. We tested thirteen peptides predicted to contain SLiMs, twelve of them palmitoylated to enhance membrane targeting, and found three that blocked parasite growth in culture by inhibiting the initiation of new infections in erythrocytes. Scrambled peptides for two of the most promising peptides suggested that their activity may be reflective of amino acid properties, in particular, positive charge. However, one peptide showed effects which were stronger than those of scrambled peptides. This was derived from human red blood cell glycophorin-B. We concluded that proteome-wide computational screening of the intracellular regions of both host and pathogen adhesion proteins provides potential lead peptides for the development of anti-malarial compounds. PMID:26039561

  16. Design, synthesis and DNA interactions of a chimera between a platinum complex and an IHF mimicking peptide.

    PubMed

    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. PMID:26477860

  17. Molecular mechanics and dynamics studies on the interaction of gallic acid with collagen-like peptides

    NASA Astrophysics Data System (ADS)

    Madhan, B.; Thanikaivelan, P.; Subramanian, V.; Raghava Rao, J.; Unni Nair, Balachandran; Ramasami, T.

    2001-10-01

    Molecular modelling approaches have been used to understand the interaction of collagen-like peptides with gallic acid, which mimic vegetable tanning processes involved in protein stabilization. Several interaction sites have been identified and the binding energies of the complexes have been calculated. The calculated binding energies for various geometries are in the range 6-13 kcal/mol. It is found that some complexes exhibit hydrogen bonding, and electrostatic interaction plays a dominant role in the stabilization of the peptide by gallic acid. The π-OH type of interaction is also observed in the peptide stabilization. Molecular dynamics (MD) simulation for 600 ps revealed the possibility of hydrogen bonding between the collagen-like peptide and gallic acid.

  18. Relationship between cadmium, zinc, Cd-peptide, and organic acid in tobacco suspension cells

    SciTech Connect

    Krotz, R.M.; Evangelou, B.P.; Wagner, G.J. )

    1989-10-01

    Responses of tobacco (Nicotiana tabacum) suspension cells to Cd and Zn were studied in the presence and absence of ligand of Cd-peptide in order to understand the role of this peptide versus other mechanisms in Cd and Zn accumulation and accommodation in plants. With 45 micromolar Cd and 300 micromolar Zn (non-growth-inhibiting levels), metals appeared rapidly within cells, and intracellular Cd and Zn reached medium concentrations after 6 to 10 hours. Cd-peptide was observed in response to Cd after 2 hours, but this form only accounted for {approximately}30% of soluble Cd after 24 hours. Peptide was not observed in cells exposed to 300 micromolar Zn for up to 7 days. Organic acid-to-metal stoichiometry indicated that endogenous organic acid content of cells was more than sufficient to complex absorbed metals and no evidence was found for stimulation of organic acid biosynthesis by Cd or Zn. Metal-complexing potential of organic acids for Cd and Zn versus endogenous cations is discussed as is vacuolar-extravacuolar distribution of metals. The absence of Cd-peptide does not limit Cd-accumulation in the system studied. Results suggest that tobacco suspension cells accommodte the presence of non-growth-inhibiting and growth-inhibiting levels of Cd and Zn by sequestration in the vacuole as complexes with endogenous organic acids and that this may be a principal means for accommodation of Cd as well as Zn in the presence and absence of Cd-peptide.

  19. Endogenous flow of amino acids in the avian ileum as influenced by increasing dietary peptide concentrations.

    PubMed

    Ravindran, Velmurugu; Morel, Patrick C H; Rutherfurd, Shane M; Thomas, Donald V

    2009-03-01

    The aim of the present study was to establish whether feeding broiler chickens with diets containing increasing dietary peptide concentrations would cause increases in ileal endogenous amino acid flow. The flow of N and most amino acids increased quadratically (P < 0.05 to 0.001) with increasing dietary concentrations of peptides. The exceptions were the flow of threonine, serine, glycine, tyrosine and cystine, which increased linearly (P < 0.001) with dietary peptide levels. Another notable exception to the general trend was the flow of proline, which was significantly higher (P < 0.01) in birds fed the protein-free diet. The amino acid profile of endogenous protein, expressed as proportion of crude protein, indicated that the ratios of threonine, glutamic acid, proline, glycine, leucine, histidine, arginine and cystine were influenced (P < 0.05) with increasing dietary peptide concentrations. In general, compared with the protein-free diet, the ratios of threonine and arginine in endogenous protein were lower (P < 0.05) and those of glutamic acid, glycine and histidine were greater (P < 0.05) in diets with high concentrations of peptides. The ratio of proline was found to decrease (P < 0.05) with increasing dietary peptide concentrations. These changes in the amino acid profile of endogenous protein are probably reflective of changes in the output of one or more of the components of endogenous protein. Overall, the present results demonstrated that increasing dietary peptide concentrations increased the flow of endogenous amino acid flow at the terminal ileum of broiler chickens in a dose-dependent manner and also caused changes in the composition of endogenous protein. The observed changes in endogenous amino flow will influence the maintenance requirements for amino acids and also have implications for the calculation of true digestibility coefficient of feedstuffs. PMID:18662428

  20. A toy model of prebiotic peptide evolution: the possible role of relative amino acid abundances.

    PubMed

    Polanco, Carlos; Buhse, Thomas; Samaniego, José Lino; Castañón González, Jorge Alberto

    2013-01-01

    This paper presents a mathematical-computational toy model based on the assumed dynamic principles of prebiotic peptide evolution. Starting from a pool of amino acid monomers, the model describes in a generalized manner the generation of peptides and their sequential information. The model integrates the intrinsic and dynamic key elements of the initiation of biopolymerization, such as the relative amino acid abundances and polarities, as well as the oligomer reversibility, i.e. fragmentation and recombination, and peptide self-replication. Our modeling results suggest that the relative amino acid abundances, as indicated by Miller-Urey type electric discharge experiments, played a principal role in the early sequential information of peptide profiles. Moreover, the computed profiles display an astonishing similarity to peptide profiles observed in so-called biological common ancestors found in the following three microorganisms; E. coli, M. jannaschii, and S. cereviasiae. The prebiotic peptide fingerprint was obtained by the so-called polarity index method that was earlier reported as a tool for the identification of cationic amphipathic antibacterial short peptides. PMID:23741717

  1. Analysis of Endogenous D-Amino Acid-Containing Peptides in Metazoa

    PubMed Central

    Bai, Lu; Sheeley, Sarah; Sweedler, Jonathan V.

    2010-01-01

    Peptides are chiral molecules with their structure determined by the composition and configuration of their amino acid building blocks. The naturally occurring amino acids, except glycine, possess two chiral forms. This allows the formation of multiple peptide diastereomers that have the same sequence. Although living organisms use L-amino acids to make proteins, a group of D-amino acid-containing peptides (DAACPs) has been discovered in animals that have at least one of their residues isomerized to the D-form via an enzyme-catalyzed process. In many cases, the biological functions of these peptides are enhanced due to this structural conversion. These DAACPs are different from those known to occur in bacterial cell wall and antibiotic peptides, the latter of which are synthesized in a ribosome-independent manner. DAACPs have now also been identified in a number of distinct groups throughout the Metazoa. Their serendipitous discovery has often resulted from discrepancies observed in bioassays or in chromatographic behavior between natural peptide fractions and peptides synthesized according to a presumed all-L sequence. Because this L-to-D post-translational modification is subtle and not detectable by most sequence determination approaches, it is reasonable to suspect that many studies have overlooked this change; accordingly, DAACPs may be more prevalent than currently thought. Although diastereomer separation techniques developed with synthetic peptides in recent years have greatly aided in the discovery of natural DAACPs, there is a need for new, more robust methods for naturally complex samples. In this review, a brief history of DAACPs in animals is presented, followed by discussion of a variety of analytical methods that have been used for diastereomeric separation and detection of peptides. PMID:20490347

  2. The Prebiotic Synthesis of Ethylenediamine Monoacetic Acid, The Repeating Unit of Peptide Nucleic Acids

    NASA Technical Reports Server (NTRS)

    Nelson, Kevin E.; Miller, Stanley L.

    1992-01-01

    The polymerization of ribonucleic acids or their precursors constitutes an important event in prebiotic chemistry. The various problems using ribonucleotides to make RNA suggest that there may have been a precursor. An attractive possibility are the peptide nucleic acids (PNA). PNAs are nucleotide analogs that make use of a polymer of ethylenediamine monoacetic acid (EDMA or 2-amninoethyl glycine) with the bases attached by an acetic acid. EDMA is an especially attractive alternative to the ribose phosphate or deoxyribose phosphate backbone because it contains no chiral centers and is potentially prebiotic, but there is no reported prebiotic synthesis. We have synthesized both EDMA and ethylenediamine diacetic acid (EDDA) from the prebiotic compounds ethylenediamine, formaldehyde, and hydrogen cyanide. The yields of EDMA range from 11 to 79% along with some sEDDA and uEDDA. These reactions work with concentrations of 10(exp -1)M and as low as 10(exp -4)M, and the reaction is likely to be effective at even lower concentrations. Ethylenediamine is a likely prebiotic compound, but it has not yet been demonstrated, although compounds such as ethanolamine and cysteamine have been proven to be prebiotic. Under neutral pH and heating at l00 C, EDMA is converted to the lactam, monoketopiperazine (MKP). The cyclization occurs and has an approximate ratio of MKP/EDMA = 3 at equilibrium. We have measured the solubilities of EDMA center dot H20 as 6.4 m, EDMA center dot HCl center dot H20 as 13.7 m, and EDMA center dot 2HCl center dot H20 as 3.4 m. These syntheses together with the high solubility of EDMA suggest that EDMA would concentrate in drying lagoons and might efficiently form polymers. Given the instability of ribose and the poor polymerizability of nucleotides, the prebiotic presence of EDMA and the possibility of its polymerization raises the possibility that PNAs are the progenitors of present day nucleic acids. A pre-RNA world may have existed in which PNAs or

  3. New peptide deformylase inhibitors design, synthesis and pharmacokinetic assessment.

    PubMed

    Lv, Fengping; Chen, Chen; Tang, Yang; Wei, Jianhai; Zhu, Tong; Hu, Wenhao

    2016-08-01

    The docking approach for the screening of designed small molecule ligands, led to the identification of a critical arginine residue in peptide deformylase for spiro cyclopropyl PDF inhibitor's extra hydrophobic binding, providing us a useful tool for searching more efficient PDF inhibitors to fight for horrifying antibiotics resistance. Further synthetic modification was undertaken to optimize the potency of amide compounds. To lower metabolic susceptibility and in turn reduce unwanted metabolic toxicity that was observed clinically, while retaining desired antibacterial activity, the use of azoles as amide bioisosteres had also been investigated. After the completion of chemical synthesis, all the compounds were evaluated through in vitro antibacterial activity assay, some of which were further subject to in vivo rat pharmacokinetic assessment. Those findings in this letter showed that spiro cyclopropyl proline N-formyl hydroxylamines, and especially the bioisosteric azoles, can represent a promising class of PDF inhibitors. PMID:27293070

  4. A method for the 32P labeling of peptides or peptide nucleic acid oligomers

    NASA Technical Reports Server (NTRS)

    Kozlov, I. A.; Nielsen, P. E.; Orgel, L. E.; Bada, J. L. (Principal Investigator)

    1998-01-01

    A novel approach to the radioactive labeling of peptides and PNA oligomers is described. It is based on the conjugation of a deoxynucleoside 3'-phosphate with the terminal amine of the substrate, followed by phosphorylation of the 5'-hydroxyl group of the nucleotide using T4 polynucleotide kinase and [gamma-32P]ATP.

  5. Nucleic Acid-Peptide Complex Phase Controlled by DNA Hybridization

    NASA Astrophysics Data System (ADS)

    Vieregg, Jeffrey; Lueckheide, Michael; Leon, Lorraine; Marciel, Amanda; Tirrell, Matthew

    When polyanions and polycations are mixed, counterion release drives formation of polymer-rich complexes that can either be solid (precipitates) or liquid (coacervates) depending on the properties of the polyelectrolytes. These complexes are important in many fields, from encapsulation of industrial polymers to membrane-free segregation of biomolecules such as nucleic acids and proteins. Condensation of long double-stranded DNA has been studied for several decades, but comparatively little attention has been paid to the polyelectrolyte behavior of oligonucleotides. We report here studies of DNA oligonucleotides (10 - 88 nt) complexed with polylysine (10 - 100 aa). Unexpectedly, we find that the phase of the resulting complexes is controlled by the hybridization state of the nucleic acid, with double-stranded DNA forming precipitates and single-stranded DNA forming coacervates. Stability increases with polyelectrolyte length and decreases with solution salt concentration, with complexes of the longer double-stranded polymers undergoing precipitate/coacervate/soluble transitions as ionic strength is increased. Mixing coacervates formed by complementary single-stranded oligonucleotides results in precipitate formation, raising the possibility of stimulus-responsive material design.

  6. Structural Dynamic of a Self-Assembling Peptide d-EAK16 Made of Only D-Amino Acids

    PubMed Central

    Luo, Zhongli; Zhao, Xiaojun; Zhang, Shuguang

    2008-01-01

    We here report systematic study of structural dynamics of a 16-residue self-assembling peptide d-EAK16 made of only D-amino acids. We compare these results with its chiral counterpart L-form, l-EAK16. Circular dichroism was used to follow the structural dynamics under various temperature and pH conditions. At 25°C the d-EAK16 peptide displayed a typical beta-sheet spectrum. Upon increasing the temperature above 70°C, there was a spectrum shift as the 218 nm valley widens toward 210 nm. Above 80°C, the d-EAK16 peptide transformed into a typical alpha-helix CD spectrum without going through a detectable random-coil intermediate. When increasing the temperature from 4°C to 110°C then cooling back from 110°C to 4°C, there was a hysteresis: the secondary structure from beta-sheet to alpha-helix and then from alpha-helix to beta-sheet occurred. d-EAK16 formed an alpha-helical conformation at pH0.76 and pH12 but formed a beta-sheet at neutral pH. The effects of various pH conditions, ionic strength and denaturing agents were also noted. Since D-form peptides are resistant to natural enzyme degradation, such drastic structural changes may be exploited for fabricating molecular sensors to detect minute environmental changes. This provides insight into the behaviors of self-assembling peptides made of D-amino acids and points the way to designing new peptide materials for biomedical engineering and nanobiotechnology. PMID:18509542

  7. Site-Specific Characterization of d-Amino Acid Containing Peptide Epimers by Ion Mobility Spectrometry

    PubMed Central

    2013-01-01

    Traditionally, the d-amino acid containing peptide (DAACP) candidate can be discovered by observing the differences of biological activity and chromatographic retention time between the synthetic peptides and naturally occurring peptides. However, it is difficult to determine the exact position of d-amino acid in the DAACP candidates. Herein, we developed a novel site-specific strategy to rapidly and precisely localize d-amino acids in peptides by ion mobility spectrometry (IMS) analysis of mass spectrometry (MS)-generated epimeric fragment ions. Briefly, the d/l-peptide epimers were separated by online reversed-phase liquid chromatography and fragmented by collision-induced dissociation (CID), followed by IMS analysis. The epimeric fragment ions resulting from d/l-peptide epimers exhibit conformational differences, thus showing different mobilities in IMS. The arrival time shift between the epimeric fragment ions was used as criteria to localize the d-amino acid substitution. The utility of this strategy was demonstrated by analysis of peptide epimers with different molecular sizes, [d-Trp]-melanocyte-stimulating hormone, [d-Ala]-deltorphin, [d-Phe]-achatin-I, and their counterparts that contain all-l amino acids. Furthermore, the crustacean hyperglycemia hormones (CHHs, 8.5 kDa) were isolated from the American lobster Homarus americanus and identified by integration of MS-based bottom-up and top-down sequencing approaches. The IMS data acquired using our novel site-specific strategy localized the site of isomerization of l- to d-Phe at the third residue of the CHHs from the N-terminus. Collectively, this study demonstrates a new method for discovery of DAACPs using IMS technique with the ability to localize d-amino acid residues. PMID:24328107

  8. Targeting lipopolyplexes using bifunctional peptides incorporating hydrophobic spacer amino acids: synthesis, transfection, and biophysical studies.

    PubMed

    Pilkington-Miksa, Michael A; Writer, Michele J; Sarkar, Supti; Meng, Qing-Hai; Barker, Suzie E; Shamlou, Parviz Ayazi; Hailes, Helen C; Hart, Stephen L; Tabor, Alethea B

    2007-01-01

    We have developed efficient synthetic routes to two hydrophobic amino acids, suitably protected for solid-phase peptide synthesis, and have successfully synthesized peptides containing these or other hydrophobic amino acids as spacers between a Lys16 moiety and an integrin-targeting motif. These peptides have in turn been used to formulate a range of lipopolyplex vectors with Lipofectin and plasmid DNA. The transfection efficiencies of these vectors and their aggregation behavior in buffers and in serum have been studied. We have shown that vectors containing peptides incorporating long linkers that are entirely hydrophobic are less efficient transfection agents. However, linkers of equivalent length that are in part hydrophobic show improved transfection properties, which is probably due to the improved accessibility of the integrin-binding motif. PMID:17915956

  9. Incorporation of N-amidino-pyroglutamic acid into peptides using intramolecular cyclization of alpha-guanidinoglutaric acid.

    PubMed

    Burov, Sergey; Moskalenko, Yulia; Dorosh, Marina; Shkarubskaya, Zoya; Panarin, Evgeny

    2009-11-01

    N-terminal modification of peptides by unnatural amino acids significantly affects their enzymatic stability, conformational properties and biological activity. Application of N-amidino-amino acids, positively charged under physiological conditions, can change peptide conformation and its affinity to the corresponding receptor. In this article, we describe synthesis of short peptides, containing a new building block-N-amidino-pyroglutamic acid. Although direct guanidinylation of pyroglutamic acid and oxidation of N-amidino-proline using RuO(4) did not produce positive results, N-amidino-Glp-Phe-OH was synthesized on Wang polymer by cyclization of alpha-guanidinoglutaric acid residue. In the course of synthesis, it was found that literature procedure of selective Boc deprotection using TMSOTf/TEA reagent is accompanied by concomitant side reaction of triethylamine alkylation by polymer linker fragment. It should be mentioned that independently from cyclization time and coupling agent (DIC or HCTU), the lactam formation was incomplete. Separation of the cyclic product from the linear precursor was achieved by HPLC in ammonium formate buffer at pH 6. HPLC analysis showed N-amidino-Glp-Phe-OH stability at acidic and physiological pH and fast ring opening in water solution at pH 9. The suggested method of N-amidino-Glp residue formation can be applied in the case of short peptide chains, whereas synthesis of longer ones will require fragment condensation approach. PMID:19739127

  10. A High-Adhesive Lysine-Cyclic RGD Peptide Designed for Selective Cell Retention Technology.

    PubMed

    Luo, Keyu; Mei, Tieniu; Li, Zhiqiang; Deng, Moyuan; Zhang, Zehua; Hou, Tianyong; Dong, Shiwu; Xie, Zhao; Xu, Jianzhong; Luo, Fei

    2016-06-01

    Cell adhesion is an important property of biomaterials used in selective cell retention (SCR) technology, which fabricates bone grafts rapidly in clinical settings. This could be improved by physical and biologic manipulations. To facilitate retention of the cells on the scaffold, especially osteoprogenitors from bone marrow in the convenient SCR procedure, a lysine-cyclic RGD (LcRGD) peptide was here designed to coordinate positively charged amino acids and the RGD sequence to enhance the adhesion performance of the scaffold. Demineralized bone matrix (DBM) is an important therapeutic resource, but its cell adhesion ability and osteoinductive capacity are low because of its processing. These capabilities can be increased to enhance the performance of DBM when used in SCR technology. Here, LcRGD peptide was used to modify DBM and produce a DBM/LcRGD composite. This composite exhibited enhanced adhesion performance on cultured human bone marrow-derived mesenchymal stem cells and retained more osteoprogenitors from bone marrow than other materials did. The DBM/LcRGD composite displayed a preferable osteoinduction in vitro and osteogenic capacity in vivo. Thus, LcRGD peptide as a commendable modifier of DBM applied in SCR technology can improve bone transplantation. PMID:27154386

  11. Sugar amino acids and their uses in designing bioactive molecules.

    PubMed

    Chakraborty, Tushar K; Ghosh, Subhash; Jayaprakash, Sarva

    2002-02-01

    In search of new molecular entities for discovering new drugs and materials, organic chemists are looking for innovative approaches that try to imitate nature in assembling quickly large number of distinct and diverse molecular structures from 'nature-like' and yet unnatural designer building blocks using combinatorial approach. The main objective in developing such libraries is to mimic the diversities displayed in structures and properties of natural products. The unnatural building blocks used in these assemblies are carefully designed to manifest the structural diversities of the monomeric units used by nature like amino acids, carbohydrates and nucleosides to build its arsenal. Compounds made of such unnatural building blocks are also expected to be more stable toward proteolytic cleavage in physiological systems than their natural counterparts. Sugar amino acids constitute an important class of such polyfunctional scaffolds where the carboxyl, amino and hydroxyl termini provide an excellent opportunity to organic chemists to create structural diversities akin to nature's molecular arsenal. Recent advances in the area of combinatorial chemistry give an unprecedented technological support for rapid compilations of sugar amino acid-based libraries exploiting the diversities of carbohydrate molecules and well-developed solid-phase peptide synthesis methods. This review describes the development of sugar amino acids as a novel class of peptidomimetic building blocks and their applications in creating large number of structurally diverse peptide-based molecules many of which display interesting three-dimensional structures as well as useful biological properties. PMID:11945118

  12. Interaction of linear cationic peptides with phospholipid membranes and polymers of sialic acid.

    PubMed

    Kuznetsov, A S; Dubovskii, P V; Vorontsova, O V; Feofanov, A V; Efremov, R G

    2014-05-01

    Polysialic acid (PSA) is a natural anionic polymer typically occurring on the outer surface of cell membranes. PSA is involved in cell signaling and intermolecular interactions with proteins and peptides. The antimicrobial potential of peptides is usually evaluated in model membranes consisting of lipid bilayers but devoid of either PSA or its analogs. The goal of this work was to investigate the possible effect of PSA on the structure of melittin (Mlt) and latarcins Ltc1K, Ltc2a, and the activity of these peptides with respect to model membranes. These peptides are linear cationic ones derived from the venom of bee (Mlt) and spider (both latarcins). The length of each of the peptides is 26 amino acid residues, and they all have antimicrobial activity. However, they differ with respect to conformational mobility, hydrophobic characteristics, and overall charge. In this work, using circular dichroism spectroscopy, we show that the peptides adopt an α-helical conformation upon interaction with either PSA or phospholipid liposomes formed of either zwitterionic or anionic phospholipids or their mixtures. The extent of helicity depends on the amino acid sequence and properties of the medium. Based on small angle X-ray scattering data and the analysis of the fluorescence spectrum of the Trp residue in Mlt, we conclude that the peptide forms an oligomeric complex consisting of α-helical Mlt and several PSA molecules. Both latarcins, unlike Mlt, the most hydrophobic of the peptides, interact weakly with zwitterionic liposomes. However, they bind anionic liposomes or those composed of anionic/zwitterionic lipid mixtures. Latarcin Ltc1K forms associates on liposomes composed of zwitterionic/anionic lipid mixture. The structure of the peptide associates is either disordered or of β-sheet conformation. In all other cases the studied peptides adopt predominately α-helical conformation. In addition, we demonstrate that PSA inhibits membranolytic activity of Mlt and latarcin

  13. From amino acid sequence to bioactivity: The biomedical potential of antitumor peptides.

    PubMed

    Blanco-Míguez, Aitor; Gutiérrez-Jácome, Alberto; Pérez-Pérez, Martín; Pérez-Rodríguez, Gael; Catalán-García, Sandra; Fdez-Riverola, Florentino; Lourenço, Anália; Sánchez, Borja

    2016-06-01

    Chemoprevention is the use of natural and/or synthetic substances to block, reverse, or retard the process of carcinogenesis. In this field, the use of antitumor peptides is of interest as, (i) these molecules are small in size, (ii) they show good cell diffusion and permeability, (iii) they affect one or more specific molecular pathways involved in carcinogenesis, and (iv) they are not usually genotoxic. We have checked the Web of Science Database (23/11/2015) in order to collect papers reporting on bioactive peptide (1691 registers), which was further filtered searching terms such as "antiproliferative," "antitumoral," or "apoptosis" among others. Works reporting the amino acid sequence of an antiproliferative peptide were kept (60 registers), and this was complemented with the peptides included in CancerPPD, an extensive resource for antiproliferative peptides and proteins. Peptides were grouped according to one of the following mechanism of action: inhibition of cell migration, inhibition of tumor angiogenesis, antioxidative mechanisms, inhibition of gene transcription/cell proliferation, induction of apoptosis, disorganization of tubulin structure, cytotoxicity, or unknown mechanisms. The main mechanisms of action of those antiproliferative peptides with known amino acid sequences are presented and finally, their potential clinical usefulness and future challenges on their application is discussed. PMID:27010507

  14. Biomimetic vaterite formation at surfaces structurally templated by oligo(glutamic acid) peptides.

    PubMed

    Lu, Hao; Hood, Matthew A; Mauri, Sergio; Baio, Joe E; Bonn, Mischa; Muñoz-Espí, Rafael; Weidner, Tobias

    2015-11-14

    Previous studies have reported that the metastable vaterite phase of calcium carbonate can be stabilized in solution by acidic additives. Here we demonstrate that vaterite can also be stabilized directly at surfaces by engineered peptides. Our data show that the mineralisation occurs in a 'self-templating' process where calcium ions restructure the peptide backbone, which in turn allows for effective vaterite precipitation. PMID:26376942

  15. Self-assembly pathway of peptide nanotubes formed by a glutamatic acid-based bolaamphiphile.

    PubMed

    da Silva, Emerson Rodrigo; Alves, Wendel Andrade; Castelletto, Valeria; Reza, Mehedi; Ruokolainen, Janne; Hussain, Rohanah; Hamley, Ian William

    2015-07-25

    The self-assembly of peptide nanotubes formed by an L-glutamic acid-based bolaamphiphile is shown to proceed via a remarkable mechanism where the peptide conformation changes from β-sheet to unordered. The kinetics of this process are elucidated via X-ray scattering and UV circular dichroism methods. The reverse transition from "unordered" to β-sheet structures is triggered by UV radiation. PMID:26094619

  16. Predicting Three-Dimensional Conformations of Peptides Constructed of Only Glycine, Alanine, Aspartic Acid, and Valine

    NASA Astrophysics Data System (ADS)

    Oda, Akifumi; Fukuyoshi, Shuichi

    2015-06-01

    The GADV hypothesis is a form of the protein world hypothesis, which suggests that life originated from proteins (Lacey et al. 1999; Ikehara 2002; Andras 2006). In the GADV hypothesis, life is thought to have originated from primitive proteins constructed of only glycine, alanine, aspartic acid, and valine ([GADV]-proteins). In this study, the three-dimensional (3D) conformations of randomly generated short [GADV]-peptides were computationally investigated using replica-exchange molecular dynamics (REMD) simulations (Sugita and Okamoto 1999). Because the peptides used in this study consisted of only 20 residues each, they could not form certain 3D structures. However, the conformational tendencies of the peptides were elucidated by analyzing the conformational ensembles generated by REMD simulations. The results indicate that secondary structures can be formed in several randomly generated [GADV]-peptides. A long helical structure was found in one of the hydrophobic peptides, supporting the conjecture of the GADV hypothesis that many peptides aggregated to form peptide multimers with enzymatic activity in the primordial soup. In addition, these results indicate that REMD simulations can be used for the structural investigation of short peptides.

  17. Efficient 18F-Labeling of Large 37-Amino Acid pHLIP Peptide Analogues and their Biological Evaluation

    PubMed Central

    Daumar, Pierre; Wanger-Baumann, Cindy A.; Pillarsetty, NagaVaraKishore; Fabrizio, Laura; Carlin, Sean D.; Andreev, Oleg A.; Reshetnyak, Yana K.; Lewis, Jason S.

    2012-01-01

    Solid tumors often develop an acidic microenvironment, which plays a critical role in tumor progression and is associated with increased level of invasion and metastasis. The 37-residue pH (low) insertion peptide (pHLIP®) is under study as an imaging platform because of its unique ability to insert into cell membranes at a low extracellular pH (pHe<7). Labeling of peptides with [18F]-fluorine is usually performed via prosthetic groups using chemoselective coupling reactions. One of the most successful procedures involves the alkyne-azide copper(I) catalyzed cycloaddition (CuAAC). However, none of the known “click” methods have been applied to peptides as large as pHLIP. We designed a novel prosthetic group and extended the use of the CuAAC “click chemistry” for the simple and efficient 18F-labeling of large peptides. For the evaluation of this labeling approach, a D-amino acid analogue of WT-pHLIP and a L-amino acid control peptide K-pHLIP, both functionalized at the N-terminus with 6-azidohexanoic acid, were used. The novel 6-[18F]fluoro-2-ethynylpyridine prosthetic group, was obtained via nucleophilic substitution on the corresponding bromo-precursor after 10 min at 130 °C with a radiochemical yield of 27.5 ± 6.6% (decay corrected) with high radiochemical purity ≥ 98%. The subsequent CuI catalyzed “click” reaction with the azido functionalized pHLIP peptides was quantitative within 5 min at 70 °C in a mixture of water and ethanol using Cu-acetate and sodium L-ascorbate. [18F]-D-WT-pHLIP and [18F]-L-K-pHLIP were obtained with total radiochemical yields of 5–20% after HPLC purification. The total reaction time was only 85 min including formulation. In vitro stability tests revealed high stability of the [18F]-D-WT-pHLIP in human and mouse plasma after 120 min, with the parent tracer remaining intact at 65 and 85%, respectively. PET imaging and biodistribution studies in LNCaP and PC-3 xenografted mice with the [18F]-D-WT-pHLIP and the negative

  18. Disrupting Protein Expression with Peptide Nucleic Acids Reduces Infection by Obligate Intracellular Rickettsia

    PubMed Central

    Pelc, Rebecca S.; McClure, Jennifer C.; Kaur, Simran J.; Sears, Khandra T.; Rahman, M. Sayeedur; Ceraul, Shane M.

    2015-01-01

    Peptide Nucleic Acids (PNAs) are single-stranded synthetic nucleic acids with a pseudopeptide backbone in lieu of the phosphodiester linked sugar and phosphate found in traditional oligos. PNA designed complementary to the bacterial Shine-Dalgarno or start codon regions of mRNA disrupts translation resulting in the transient reduction in protein expression. This study examines the use of PNA technology to interrupt protein expression in obligate intracellular Rickettsia sp. Their historically intractable genetic system limits characterization of protein function. We designed PNA targeting mRNA for rOmpB from Rickettsia typhi and rickA from Rickettsia montanensis, ubiquitous factors important for infection. Using an in vitro translation system and competitive binding assays, we determined that our PNAs bind target regions. Electroporation of R. typhi and R. montanensis with PNA specific to rOmpB and rickA, respectively, reduced the bacteria’s ability to infect host cells. These studies open the possibility of using PNA to suppress protein synthesis in obligate intracellular bacteria. PMID:25781160

  19. De Novo Design and Experimental Characterization of Ultrashort Self-Associating Peptides

    PubMed Central

    Xue, Bo; Robinson, Robert C.; Hauser, Charlotte A. E.; Floudas, Christodoulos A.

    2014-01-01

    -associating tripeptides. We expect that this enhanced multimeric de novo peptide design framework will find future application in creating novel self-associating peptides based on unnatural amino acids, and inhibitor peptides of detrimental self-aggregating biological proteins. PMID:25010703

  20. Simultaneous Inhibition of Key Growth Pathways in Melanoma Cells and Tumor Regression by a Designed Bidentate Constrained Helical Peptide

    PubMed Central

    Dhar, Amlanjyoti; Mallick, Shampa; Ghosh, Piya; Maiti, Atanu; Ahmed, Israr; Bhattacharyya, Seemana; Mandal, Tapashi; Manna, Asit; Roy, Koushik; Singh, Sandeep; Nayak, Dipak Kumar; Wilder, Paul T.; Markowitz, Joseph; Weber, David J.; Ghosh, Mrinal K.; Chattopadhyay, Samit; Guha, Rajdeep; Konar, Aditya; Bandyopadhyay, Santu; Roy, Siddhartha

    2014-01-01

    Protein-protein interactions are part of a large number of signaling networks and potential targets for drug development. However, discovering molecules that can specifically inhibit such interactions is a major challenge. S100B, a calcium-regulated protein, plays a crucial role in the proliferation of melanoma cells through protein-protein interactions. In this article, we report the design and development of a bidentate conformationally constrained peptide against dimeric S100B based on a natural tight binding peptide, TRTK-12. The helical conformation of the peptide was constrained by substitution of α-amino isobutyric acid----an amino acid having high helical propensity----in positions which do not interact with S100B. A branched bidentate version of the peptide, bound to S100B tightly with a dissociation constant of 8 nM. When conjugated to a cell penetrating peptide, it caused growth inhibition and rapid apoptosis in melanoma cells. The molecule exerts anti-proliferative action through simultaneous inhibition of key growth pathways including reactivation of wild-type p53 and inhibition of Akt and STAT-3 phosphorylation. The apoptosis induced by the bidentate constrained helix is caused by direct migration of p53 to mitochondria. At moderate intravenous dose, the peptide completely inhibits melanoma growth in a mouse model without any significant observable toxicity. The specificity was shown by lack of ability of a double mutant peptide to cause tumor regression at the same dose level. The methodology described here for direct protein-protein interaction inhibition may be effective for rapid development of inhibitors against relatively weak protein-protein interactions for de novo drug development. PMID:24839139

  1. Structure-based Design of Peptides with High Affinity and Specificity to HER2 Positive Tumors

    PubMed Central

    Geng, Lingling; Wang, Zihua; Yang, Xiaoliang; Li, Dan; Lian, Wenxi; Xiang, Zhichu; Wang, Weizhi; Bu, Xiangli; Lai, Wenjia; Hu, Zhiyuan; Fang, Qiaojun

    2015-01-01

    To identify peptides with high affinity and specificity against human epidermal growth factor receptor 2 (HER2), a series of peptides were designed based on the structure of HER2 and its Z(HER2:342) affibody. By using a combination protocol of molecular dynamics modeling, MM/GBSA binding free energy calculations, and binding free energy decomposition analysis, two novel peptides with 27 residues, pep27 and pep27-24M, were successfully obtained. Immunocytochemistry and flow cytometry analysis verified that both peptides can specifically bind to the extracellular domain of HER2 protein at cellular level. The Surface Plasmon Resonance imaging (SPRi) analysis showed that dissociation constants (KD) of these two peptides were around 300 nmol/L. Furthermore, fluorescence imaging of peptides against nude mice xenografted with SKBR3 cells indicated that both peptides have strong affinity and high specificity to HER2 positive tumors. PMID:26284145

  2. Structure-based Design of Peptides with High Affinity and Specificity to HER2 Positive Tumors.

    PubMed

    Geng, Lingling; Wang, Zihua; Yang, Xiaoliang; Li, Dan; Lian, Wenxi; Xiang, Zhichu; Wang, Weizhi; Bu, Xiangli; Lai, Wenjia; Hu, Zhiyuan; Fang, Qiaojun

    2015-01-01

    To identify peptides with high affinity and specificity against human epidermal growth factor receptor 2 (HER2), a series of peptides were designed based on the structure of HER2 and its Z(HER2:342) affibody. By using a combination protocol of molecular dynamics modeling, MM/GBSA binding free energy calculations, and binding free energy decomposition analysis, two novel peptides with 27 residues, pep27 and pep27-24M, were successfully obtained. Immunocytochemistry and flow cytometry analysis verified that both peptides can specifically bind to the extracellular domain of HER2 protein at cellular level. The Surface Plasmon Resonance imaging (SPRi) analysis showed that dissociation constants (K D) of these two peptides were around 300 nmol/L. Furthermore, fluorescence imaging of peptides against nude mice xenografted with SKBR3 cells indicated that both peptides have strong affinity and high specificity to HER2 positive tumors. PMID:26284145

  3. Inorganic binding peptides designed by phage display techniques for biotechnology applications

    NASA Astrophysics Data System (ADS)

    Liao, Chih-Wei

    Biomacromolecules play an important role in the control of hard tissue structure and function via specific molecular recognition interactions between proteins of the matrix and inorganic species of the biomineral phase. During the construction of the tissue, biomacromolecules are usually folded into a certain comformation, analogous to a "lock" for fitting with other proteins or smaller molecules as a "key". Currently, the rational design of molecular recognition in biomacro-molecules is still hard to accomplish because the protein conformation is too complex to precisely predict based on the existing conformational information of proteins found in biological systems. In the past two decades, the combinatorial approach (e.g. phage display techniques) has been used to select short binding peptides with molecular recognition to an inorganic target material without a prior knowledge of the amino acid sequence required for the specific binding. The technique has been referred to as "biopanning" because bacteriophages are used to "screen" for peptides that exhibit strong binding to a target material of interest. In this study, two diverse applications were chosen to demonstrate the utility of the biopanning approach. In one project, phage display techniques were used to pan for Indium Zinc Oxide (InZnO) binding peptides to serve as linkers between transducer devices and biosensing elements for demonstration of the feasibility of reversibly electro-activated biosensors. The amorphous InZnO, with its homogeneous surface, led to three consensus peptide sequences, AGFPNSTHSSNL, SHAPDSTWFALF, and TNSSSQFVVAIP. In addition, it was demonstrated that some selected phage clones of the InZnO binding peptides were able to be released from the InZnO surface after applying a voltage of 1400 mV on an electro-activated releasing device. In the second project, phage display techniques were used to select phage clones that bind specifically to francolite mineral in order to achieve

  4. [Amino acid composition and peptide maps of udder and serum albumins in lactating and nonlactating cows].

    PubMed

    Lagodiuk, P Z; Klos, Iu S; Charkin, V A; Kisil', I O

    1983-01-01

    Amino acids and peptides of albumin hydrolyzates from the mammary gland and blood serum were studied for lactating and nonlactating (dry, pregnant 1-4.5 and 4.5-9 months) black-and-white cows. Most pronounced difference between the content of certain amino acids of the mammary gland and blood serum albumins are established for lactating cows and least pronounced for nonlactating dry cows. Dactylography detected 55-57 fragments of products resulted from trypsin hydrolysis of the mammary gland and blood serum albumins of the animals under study. Differences are found in the content and mobility of certain peptides. PMID:6829076

  5. Design of protease-resistant peptide ligands for the purification of antibodies from human plasma.

    PubMed

    Menegatti, Stefano; Bobay, Benjamin G; Ward, Kevin L; Islam, Tuhidul; Kish, William S; Naik, Amith D; Carbonell, Ruben G

    2016-05-01

    A strategy is presented for developing variants of peptide ligands with enhanced biochemical stability for the purification of antibodies from animal sera. Antibody-binding sequences HWRGWV, HYFKFD, and HFRRHL, previously discovered by our group, were modified with non-natural amino acids to gain resistance to proteolysis, while maintaining target affinity and selectivity. As trypsin and α-chymotrypsin were chosen as models of natural proteolytic enzymes, the basic (arginine and lysine) and aromatic (tryptophan, phenylalanine, and tyrosine) amino acids were replaced with non-natural analogs. Using the docking software HADDOCK, a virtual library of peptide variants was designed and screened in-silico against the known HWRGWV binding site on the pFc fragment of IgG. A pool of selected sequences with the highest predicted free energy of binding was synthesized on chromatographic resin, and the resulting adsorbents were tested for IgG binding and resistance to proteases. The ligand variants exhibited binding capacities and specificities comparable to the original sequences, yet with much higher proteolytic resistances. The sequences HWMetCitGWMetV and HFMetCitCitHL was used for purifying polyclonal IgG from IgG-rich fractions of human plasma, with yields and purity above 90%. Notably, due to electrical neutrality, the variant showed higher selectivity than the original sequence. Binding isotherms were also constructed, which confirmed the docking predictions. This method represents a general strategy for enhancing the biochemical stability as well as the affinity and selectivity of natural or synthetic peptide ligands for bioseparations. PMID:27072524

  6. Extracellular matrix-like surfactant polymers containing arginine-glycine-aspartic acid (RGD) peptides.

    PubMed

    Anderson, Eric H; Ruegsegger, Mark A; Murugesan, Gurunathan; Kottke-Marchant, Kandice; Marchant, Roger E

    2004-08-01

    We report on a novel series of biomimetic polymers exhibiting interfacial properties similar to the extracellular matrix. A series of well-defined surfactant polymers were synthesized by simultaneously incorporating arginine-glycine-aspartic acid (RGD) peptide, dextran oligosaccharide, and hexyl ligands with controlled feed ratios onto a poly(vinyl amine) (PVAm) backbone. The peptide sequence was H-GSSSGRGDSPA-NH(2) (Pep) having a hydrophilic extender at the amino terminus and capped carboxy terminus. The peptide-to-dextran (Pep:Dex) ratios were varied to create surfactants having 0, 25, 50, 75, and 100 mol-% peptide relative to dextran. The surfactants were characterized by IR, NMR and atomic force microscopy (AFM) for composition and surface active properties. AFM confirmed full surface coverage of PVAm(Pep)(100%) on graphite, and supported the mechanism of interdigitation of hexyl ligands between surfactant molecules within a specified range of hexyl chain densities. the attachment and growth of human pulmonary artery endothelial cells on the PVAm(Pep)(100%) surface was identical to the fibronectin positive control. Cell adhesion decreased dramatically with decreasing peptide density on the surfactant polymers. Molecular model of a peptide surfactant polymer, consisting of poly(vinyl amine) backbone with peptide, dextran oligosaccharide and hexyl branches coupled to the polymer chain. PMID:15468270

  7. De novo design and characterization of an apolar helical hairpin peptide at atomic resolution: Compaction mediated by weak interactions

    PubMed Central

    Ramagopal, Udupi A.; Ramakumar, Suryanarayanarao; Sahal, Dinkar; Chauhan, Virander S.

    2001-01-01

    Design of helical super secondary structural motifs is expected to provide important scaffolds to incorporate functional sites, thus allowing the engineering of novel miniproteins with function. An α,β-dehydrophenylalanine containing 21-residue apolar peptide was designed to mimic the helical hairpin motif by using a simple geometrical design strategy. The synthetic peptide folds into the desired structure as assessed crystallographically at 1.0-Å resolution. The two helices of the helical-hairpin motif, connected by a flexible (Gly)4 linker, are docked to each other by the concerted influence of weak interactions. The folding of the peptide without binary patterning of amino acids, disulfide bonds, or metal ions is a remarkable observation. The results demonstrate that preferred interactions among the hydrophobic residues selectively discriminate their putative partners in space, leading to the unique folding of the peptide, also a hallmark of the unique folding of hydrophobic core in globular proteins. We demonstrate here the engineering of molecules by using weak interactions pointing to their possible further exploitation in the de novo design of protein super secondary structural elements. PMID:11158562

  8. Peptide Synthesis through Cell-Free Expression of Fusion Proteins Incorporating Modified Amino Acids as Latent Cleavage Sites for Peptide Release.

    PubMed

    Liutkus, Mantas; Fraser, Samuel A; Caron, Karine; Stigers, Dannon J; Easton, Christopher J

    2016-05-17

    Chlorinated analogues of Leu and Ile are incorporated during cell-free expression of peptides fused to protein, by exploiting the promiscuity of the natural biosynthetic machinery. They then act as sites for clean and efficient release of the peptides simply by brief heat treatment. Dehydro analogues of Leu and Ile are similarly incorporated as latent sites for peptide release through treatment with iodine under cold conditions. These protocols complement enzyme-catalyzed methods and have been used to prepare calcitonin, gastrin-releasing peptide, cholecystokinin-7, and prolactin-releasing peptide prohormones, as well as analogues substituted with unusual amino acids, thus illustrating their practical utility as alternatives to more traditional chemical peptide synthesis. PMID:26918308

  9. THE AMPHOTERIC PROPERTIES OF SOME AMINO-ACIDS AND PEPTIDES.

    PubMed

    Eckweiler, H; Noyes, H M; Falk, K G

    1921-01-20

    The titration curves of solutions of glycine, alanine, alpha-ammo-butyric acid, leucine, glycyl-glycine, alanyl-glycine, alanyl-alanine, acetone, acetamide, urea, acetic acid, and aceturic acid were determined and some of the relations as dependent upon the chemical structures discussed. The isoelectric points of some of the amphoteric electrolytes were found experimentally. The definition of isoelectric point, its theoretical significance, and method of calculation were considered in some detail. PMID:19871865

  10. Hybrid polymeric hydrogels via peptide nucleic acid (PNA)/DNA complexation.

    PubMed

    Chu, Te-Wei; Feng, Jiayue; Yang, Jiyuan; Kopeček, Jindřich

    2015-12-28

    This work presents a new concept in hybrid hydrogel design. Synthetic water-soluble N-(2-hydroxypropyl)methacrylamide (HPMA) polymers grafted with multiple peptide nucleic acids (PNAs) are crosslinked upon addition of the linker DNA. The self-assembly is mediated by the PNA-DNA complexation, which results in the formation of hydrophilic polymer networks. We show that the hydrogels can be produced through two different types of complexations. Type I hydrogel is formed via the PNA/DNA double-helix hybridization. Type II hydrogel utilizes a unique "P-form" oligonucleotide triple-helix that comprises two PNA sequences and one DNA. Microrheology studies confirm the respective gelation processes and disclose a higher critical gelation concentration for the type I gel when compared to the type II design. Scanning electron microscopy reveals the interconnected microporous structure of both types of hydrogels. Type I double-helix hydrogel exhibits larger pore sizes than type II triple-helix gel. The latter apparently contains denser structure and displays greater elasticity as well. The designed hybrid hydrogels have potential as novel biomaterials for pharmaceutical and biomedical applications. PMID:26394062

  11. Characterization of bioactive RGD peptide immobilized onto poly(acrylic acid) thin films by plasma polymerization

    NASA Astrophysics Data System (ADS)

    Seo, Hyun Suk; Ko, Yeong Mu; Shim, Jae Won; Lim, Yun Kyong; Kook, Joong-Ki; Cho, Dong-Lyun; Kim, Byung Hoon

    2010-11-01

    Plasma surface modification can be used to improve the surface properties of commercial pure Ti by creating functional groups to produce bioactive materials with different surface topography. In this study, a titanium surface was modified with acrylic acid (AA) using a plasma treatment and immobilized with bioactive arginine-glycine-aspartic acid (RGD) peptide, which may accelerate the tissue integration of bone implants. Both terminals containing the -NH2 of RGD peptide sequence and -COOH of poly(acrylic acid) (PAA) thin film were combined with a covalent bond in the presence of 1-ethyl-3-3-dimethylaminopropyl carbodiimide (EDC). The chemical structure and morphology of AA film and RGD immobilized surface were investigated by X-ray photoelectron spectroscopy (XPS), Fourier transform infrared (FT-IR), atomic force microscopy (AFM), and scanning electron microscopy (SEM). All chemical analysis showed full coverage of the Ti substrate with the PAA thin film containing COOH groups and the RGD peptide. The MC3T3-E1 cells were cultured on each specimen, and the cell alkaline phosphatase (ALP) activity were examined. The surface-immobilized RGD peptide has a significantly increased the ALP activity of MC3T3-E1 cells. These results suggest that the RGD peptide immobilization on the titanium surface has an effect on osteoblastic differentiation of MC3T3-E1 cells and potential use in osteo-conductive bone implants.

  12. Lactobacillus gasseri requires peptides, not proteins or free amino acids, for growth in milk.

    PubMed

    Arakawa, K; Matsunaga, K; Takihiro, S; Moritoki, A; Ryuto, S; Kawai, Y; Masuda, T; Miyamoto, T

    2015-03-01

    Lactobacillus gasseri is a widespread commensal lactic acid bacterium inhabiting human mucosal niches and has many beneficial effects as a probiotic. However, L. gasseri is difficult to grow in milk, which hurts usability for the food industry. It had been previously reported that supplementation with yeast extract or proteose peptone, including peptides, enables L. gasseri to grow well in milk. In this study, our objective was to confirm peptide requirement of L. gasseri and evaluate efficacy of peptide release by enzymatic proteolysis on growth of L. gassei in milk. Three strains of L. gasseri did not grow well in modified DeMan, Rogosa, Sharpe broth without any nitrogen sources (MRS-N), but addition of a casein-derived peptide mixture, tryptone, promoted growth. In contrast, little effect was observed after adding casein or a casein-derived amino acid mixture, casamino acids. These results indicate that L. gasseri requires peptides, not proteins or free amino acids, among milk-derived nitrogen sources for growth. Lactobacillus gasseri JCM 1131T hardly had growth capacity in 6 kinds of milk-based media: bovine milk, human milk, skim milk, cheese whey, modified MRS-N (MRSL-N) supplemented with acid whey, and MRSL-N supplemented with casein. Moreover, treatment with digestive proteases, particularly pepsin, to release peptides made it grow well in each milk-based medium. The pepsin treatment was the most effective for growth of strain JCM 1131T in skim milk among the tested food-grade proteases such as trypsin, α-chymotrypsin, calf rennet, ficin, bromelain, and papain. As well as strain JCM 1131T, pepsinolysis of milk improved growth of other L. gasseri strains and some strains of enteric lactobacilli such as Lactobacillus crispatus, Lactobacillus gallinarum, Lactobacillus johnsonii, and Lactobacillus reuteri. These results suggest that some relatives of L. gasseri also use peptides as desirable nitrogen sources, and that milk may be a good supplier of nutritious

  13. Remote Enantioselection Transmitted by an Achiral Peptide Nucleic Acid Backbone

    NASA Technical Reports Server (NTRS)

    Kozlov, Igor A.; Orgel, Leslie E.; Nielsen, Peter E.

    2000-01-01

    short homochiral segment of DNA into a PNA helix could have guaranteed that the next short segment of DNA to be incorporated would have the same handedness as the first. Once two segments of the same handedness were present, the probability that a third segment would have the same handedness would increase, and so on. Evolution could then slowly dilute out the PNA part. This scenario would ultimately allow the formation of a chiral oligonucleotide by processes that are largely resistant to enantiomeric crossinhibition. It is important to note that the ligation of homochiral dinucleotides on a nucleic acid template would probably be at least as enantiospecific as the reaction that we have studied. The disadvantage of using chiral monomers as components of a replicating system arises from the difficulty of generating a first long homochiral template from a racemic mixture of monomers, although results of experiments designed to overcome this difficulty by employing homochiral tetramers have been reported.l l The probability of obtaining a homochiral n-mer from achiral substrates is approximately 1P-I if the nontemplate-directed extension of the primer is not enantioselective. Hence, it would be very hard to get started with a homochiral 40-mer, for example. No such difficulty exists in a scenario that originates with an achiral genetic material and in which the incorporation of very few chiral monomers in this achiral background gradually progresses towards homochirality. It seems possible that some PNA sequences could act as catalysts, analogous to ribozymes, even though PNA lacks clear metal binding sites. Although such catalysts could not be enantioselective, the incorporation of as few as two chiral nucleotides could then impose chiral specificity on the system. Furthermore, such patch chimeras could help to bridge the gap in catalytic potential between PNA and RNA, while guaranteeing enantioselectivity.

  14. Design of cocktail peptide vaccine against Cytomegalovirus infection

    PubMed Central

    Tabaei, Samira; Mashkani, Baratali; Esmaili, Arezoo; Karimi, Reza; Jamehdar, Saeid Amel

    2016-01-01

    Objective(s): Human Cytomegalovirus (HCMV) remains a major morbidity and mortality cause in immuno suppressed patients. Therefore, significant effort has been made towards the development of a vaccine. In this study, the expression of the pp65 and gB fusion peptides and Fc domain of mouse IgG2a as a novel delivery system for selective uptake of antigens by antigen-presenting cells (APCs) in Pichia pastoris yeast system were studied. Materials and Method: In this study, four immune dominant sequences in pp65 protein and 3 immuno dominant sequences in gB protein were selected according to literature review. Peptide linker -GGGGS- was used for construction of fusion peptide. This fusion peptide was cloned in the pPICZαA expression vector and transfected into P. pastoris host cells. Results: Dot blot and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) techniques showed that a high level of pp65-gB-Fc fusion peptide was expressed. Conclusion: This CMV pp65-gB-Fc fusion peptide could be a promising candidate for the development of a novel peptide vaccine. PMID:27279990

  15. Biomimetic design of affinity peptide ligand for capsomere of virus-like particle.

    PubMed

    Li, Yanying; Liu, Xiaodan; Dong, Xiaoyan; Zhang, Lin; Sun, Yan

    2014-07-22

    Virus-like particle (VLP) of murine polyomavirus (MPV) is a T = 7d icosahedral capsid that self-assembles from 72 capsomeres (Caps), each of which is a pentamer of major coat protein VP1. VLP has great potential in vaccinology, gene therapy, drug delivery, and materials science. However, its application is hindered by high cost downstream processes, leading to an urgent demand of a highly efficient affinity ligand for the separation and purification of Cap by affinity chromatography. Herein a biomimetic design strategy of an affinity peptide ligand of Cap has been developed on the basis of the binding structure of the C-terminus of minor coat protein (VP2-C) on the inner surface of Cap. The molecular interactions between VP2-C and Cap were first examined using all-atom molecular dynamics (MD) simulations coupled with the molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) method, where V283, P285, D286, W287, L289, and Y296 of VP2-C were identified as the hot spots. An affinity peptide library (DWXLXLXY, X denotes arbitrary amino acids except cysteine) was then constructed for virtual screening sequently by docking with AUTODOCK VINA, binding structure comparison, and final docking with ROSETTA FlexPepDock. Ten peptide candidates were selected and further confirmed by MD simulations and MM/PBSA, where DWDLRLLY was found to have the highest affinity to Cap. In DWDLRLLY, six residues are favorable for the binding, including W2, L4, L6 and Y8 inheriting from VP2-C, and R5 and L7 selected in the virtual screening. This confirms the high efficiency and accuracy of the biomimetic design strategy. DWDLRLLY was then experimentally validated by a one-step purification of Cap from crude cell lysate using affinity chromatography with the octapeptide immobilized on Sepharose gel. The purified Caps were observed to self-assemble into VLP with consistent structure of authentic MPV. PMID:24976378

  16. p-Nitromandelic acid as a highly acid-stable safety-catch linker for solid-phase synthesis of peptide and depsipeptide acids.

    PubMed

    Isidro-Llobet, Albert; Alvarez, Mercedes; Burger, Klaus; Spengler, Jan; Albericio, Fernando

    2007-04-12

    [reaction: see text] p-Nitromandelic acid as a safety-catch linker for Boc/Bzl-SPPS of base-labile compounds like peptides and depsipeptides is described. This linker permits acidic removal of side-chain protection groups from the resin. For cleavage from the solid support, the p-nitro group was reduced with tin(II) chloride. After washing off the reducing agents, the (depsi)peptide acids with or without the side-chain protection schemes were obtained by microwave irradiation at 50 degrees C with 5% TFA in dioxane. PMID:17367151

  17. Small acidic peptides from wheat germ chromatin. I. Isolation and biochemical characterization.

    PubMed

    Mancinelli, L; Castigli, E; Qualadrucci, P; Gianfranceschi, G L; Bramucci, M; Miano, A; Amici, D

    1992-01-01

    RNA synthesis in cell and cell-free systems is inhibited by a family of acidic, low molecular weight chromatin peptides (CPs). These peptides were extracted from deproteinized DNA of prokaryotic and eukaryotic cells, but the low yield of purified material by this procedure hinders efforts aimed at understanding their action mechanism in gene regulation. In this report we describe two purification methods of CPs from an easily available source, wheat germ. A comparison is made between the method starting from deproteinized DNA and the method from purified chromatin. The biological effects (inhibition of L1210 cell growth and DNA in vitro transcription) of CPs from wheat germ together with their chemical characteristics (molecular weight, amino acid composition and presence of phosphoserine) show strong homology with those of CPs from other sources. These results suggest a possible role of these chromatin peptides in controlling gene expression. PMID:1508994

  18. Synthesis of peptides from amino acids and ATP with lysine-rich proteinoid

    NASA Technical Reports Server (NTRS)

    Nakashima, T.; Fox, S. W.

    1980-01-01

    The paper examines the synthesis of peptides from aminoacids and ATP with a lysine-rich protenoid. The latter in aqueous solution catalyzes the formation of peptides from free amino acids and ATP; this catalytic activity is not found in acidic protenoids, even though the latter contain a basic aminoacid. The pH optimum for the synthesis is about 11, but it is appreciable below 8 and above 13. Temperature data indicate an optimum at 20 C or above, with little increase in rate up to 60 C. Pyrophosphate can be used instead of ATP, but the yields are lower. The ATP-aided syntheses of peptides in aqueous solution occur with several types of proteinous aminoacids.

  19. Role of the transmembrane domain in SNARE protein mediated membrane fusion: peptide nucleic acid/peptide model systems.

    PubMed

    Wehland, Jan-Dirk; Lygina, Antonina S; Kumar, Pawan; Guha, Samit; Hubrich, Barbara E; Jahn, Reinhard; Diederichsen, Ulf

    2016-08-16

    Fusion of synaptic vesicles with the presynaptic plasma membrane is mediated by Soluble NSF (N-ethylmaleimide-sensitive factor) Attachment Protein Receptor proteins also known as SNAREs. The backbone of this essential process is the assembly of SNAREs from opposite membranes into tight four helix bundles forcing membranes in close proximity. With model systems resembling SNAREs with reduced complexity we aim to understand how these proteins work at the molecular level. Here, peptide nucleic acids (PNAs) are used as excellent candidates for mimicking the SNARE recognition motif by forming well-characterized duplex structures. Hybridization between complementary PNA strands anchored in liposomes through native transmembrane domains (TMDs) induces the merger of the outer leaflets of the participating vesicles but not of the inner leaflets. A series of PNA/peptide hybrids differing in the length of TMDs and charges at the C-terminal end is presented. Interestingly, mixing of both outer and inner leaflets is seen for TMDs containing an amide in place of the natural carboxylic acid at the C-terminal end. Charged side chains at the C-terminal end of the TMDs are shown to have a negative impact on the mixing of liposomes. The length of the TMDs is vital for fusion as with the use of shortened TMDs, fusion was completely prevented. PMID:27345759

  20. Recognition Dynamics of p53 and MDM2: Implications for Peptide Design.

    PubMed

    ElSawy, Karim M; Lane, David P; Verma, Chandra S; Caves, Leo S D

    2016-01-21

    Peptides that inhibit MDM2 and attenuate MDM2-p53 interactions, thus activating p53, are currently being pursued as anticancer drug leads for tumors harboring wild type p53. The thermodynamic determinants of peptide-MDM2 interactions have been extensively studied. However, a detailed understanding of the dynamics that underlie these interactions is largely missing. In this study, we explore the kinetics of the binding of a set of peptides using Brownian dynamics simulations. We systematically investigate the effect of peptide C-terminal substitutions (Ser, Ala, Asn, Pro) of a Q16ETFSDLWKLLP27 p53-based peptide and a M1PRFMDYWEGLN12 12/1 phage-derived peptide on their interaction dynamics with MDM2. The substitutions modulate peptide residence times around the MDM2 protein. In particular, the highest affinity peptide, Q16ETFSDLWKLLS27, has the longest residence time (t ∼ 25 μs) around MDM2, suggesting its potentially important contribution to binding affinity. The binding of the p53-based peptides appears to be kinetically driven while that of the phage-derived series appears to be thermodynamically driven. The phage-derived peptides were found to adopt distinctly different modes of interaction with the MDM2 protein compared to their p53-based counterparts. The p53-based peptides approach the N-terminal region of the MDM2 protein with the peptide C-terminal end oriented toward the protein, while the M1PRFMDYWEGLN12-based peptides adopt the reverse orientation. To probe the determinants of this switch in orientation, a designed mutant of the phage-derived peptide, R3E (M1PEFMDYWEGLN12), was simulated and found to adopt the orientation adopted by the p53-based peptides and also to result in almost a 5-fold increase in the peptide residence time (∼120 μs) relative to the p53-based peptides. On this basis, we suggest that the R3E mutant phage-derived peptide has a higher affinity for MDM2 than the p53-based peptides and would therefore, competitively inhibit MDM2

  1. 2-Chlorotrityl chloride resin. Studies on anchoring of Fmoc-amino acids and peptide cleavage.

    PubMed

    Barlos, K; Chatzi, O; Gatos, D; Stavropoulos, G

    1991-06-01

    The esterification of 2-chlorotrityl chloride resin with Fmoc-amino acids in the presence of DIEA is studied under various conditions. High esterification yields are obtained using 0.6 equiv. Fmoc-amino acid/mmol resin in DCM or DCE, in 25 min, at room temperature. The reaction proceeds without by product formation even in the case of Fmoc-Asn and Fmoc-Gln. The quantitative and easy cleavage of amino acids and peptides from 2-chlorotrityl resin, by using AcOH/TFE/DCM mixtures, is accomplished within 15-60 min at room temperature, while t-butyl type protecting groups remain unaffected. Under these exceptionally mild conditions 2-chlorotrityl cations generated during the cleavage of amino acids and peptides from resin do not attack the nucleophilic side chains of Trp, Met, and Tyr. PMID:1917309

  2. Binding of small basic peptides to membranes containing acidic lipids: theoretical models and experimental results.

    PubMed Central

    Ben-Tal, N; Honig, B; Peitzsch, R M; Denisov, G; McLaughlin, S

    1996-01-01

    We measured directly the binding of Lys3, Lys5, and Lys7 to vesicles containing acidic phospholipids. When the vesicles contain 33% acidic lipids and the aqueous solution contains 100 mM monovalent salt, the standard Gibbs free energy for the binding of these peptides is 3, 5, and 7 kcal/mol, respectively. The binding energies decrease as the mol% of acidic lipids in the membrane decreases and/or as the salt concentration increases. Several lines of evidence suggest that these hydrophilic peptides do not penetrate the polar headgroup region of the membrane and that the binding is mainly due to electrostatic interactions. To calculate the binding energies from classical electrostatics, we applied the nonlinear Poisson-Boltzmann equation to atomic models of the phospholipid bilayers and the basic peptides in aqueous solution. The electrostatic free energy of interaction, which arises from both a long-range coulombic attraction between the positively charged peptide and the negatively charged lipid bilayer, and a short-range Born or image charge repulsion, is a minimum when approximately 2.5 A (i.e., one layer of water) exists between the van der Waals surfaces of the peptide and the lipid bilayer. The calculated molar association constants, K, agree well with the measured values: K is typically about 10-fold smaller than the experimental value (i.e., a difference of about 1.5 kcal/mol in the free energy of binding). The predicted dependence of K (or the binding free energies) on the ionic strength of the solution, the mol% of acidic lipids in the membrane, and the number of basic residues in the peptide agree very well with the experimental measurements. These calculations are relevant to the membrane binding of a number of important proteins that contain clusters of basic residues. Images FIGURE 2 FIGURE 3 PMID:8842196

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

    NASA Astrophysics Data System (ADS)

    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.

  4. Designing photoswitchable peptides using the AsLOV2 domain

    PubMed Central

    Lungu, Oana I.; Hallett, Ryan A.; Choi, Eun Jung; Aiken, Mary J.; Hahn, Klaus M.; Kuhlman, Brian

    2012-01-01

    Summary Photocontrol of functional peptides is a powerful tool for spatial and temporal control of cell signaling events. We show that the genetically encoded light-sensitive LOV2 domain of Avena Sativa phototropin 1 (AsLOV2) can be used to reversibly photomodulate the affinity of peptides for their binding partners. Sequence analysis and molecular modeling were used to embed two peptides into the Ja helix of the AsLOV2 domain while maintaining AsLOV2 structure in the dark, but allowing for binding to effector proteins when the Jα helix unfolds in the light. Caged versions of the ipaA and SsrA peptides, LOV-ipaA and LOV-SsrA, bind their targets with 49-fold and 8-fold enhanced affinity in the light, respectively. These switches can be used as general tools for light dependent co-localization, which we demonstrate with photoactivable gene transcription in yeast. PMID:22520757

  5. Phytochemicals that modulate amino acid and peptide catabolism by caprine rumen microbes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Microbe-derived ionophores and macrolide antibiotics are often added to ruminant diets, and growth promotion and feed efficiency are among the benefits. One mechanism is inhibition of microbes that catabolize amino acids or peptides and produce ammonia. Plants also produce antimicrobial ...

  6. Spectral Transition in Bio-Inspired Self-Assembled Peptide Nucleic Acid Photonic Crystals.

    PubMed

    Berger, Or; Yoskovitz, Eyal; Adler-Abramovich, Lihi; Gazit, Ehud

    2016-03-01

    The self-assembly of guanine-based peptide nucleic acid monomers into photonic crystals is described. A highly reflective lattice of guanine nanocrystals is found in the skin and ocular tissues of different species providing vivid structural colors. The fabricated guanine-based supramolecular structures respond to changes in osmolarity similar to the active spectral change mechanism employed by chameleons. PMID:26779770

  7. One-pot nanoparticulation of potentially bioactive peptides and gallic acid encapsulation.

    PubMed

    Nourbakhsh, Himan; Madadlou, Ashkan; Emam-Djomeh, Zahra; Wang, Yi-Cheng; Gunasekaran, Sundaram

    2016-11-01

    Whey protein isolate was hydrolyzed to an in vitro antioxidative hydrolysate, followed by transglutaminase-induced cross-linking and microemulsification in an oil phase. The obtained microemulsion was then dispersed in a gallic acid-rich model wastewater which caused gallic acid transportation into internal nanodroplets. Whey peptides were consequently gelled, yielding nanoparticles. Electrophoresis showed that β-lactoglobulin and low molecular weight peptides were cross-linked by transglutaminase. Protein hydrolysis and subsequent enzymatic cross-linking increased the ζ-potential value. Microscopic investigation indicated that most particles were non-spherical. Non-cross-linked and cross-linked peptides underwent a form of heat-triggered self-assembly in the dry state, while nanoparticles did not show such behavior. Peptide crystallites size was increased by cross-linking and acid-induced particle formation. The latter also caused a reduction in intensity of C-H stretching and C-N bending peaks in infra-red spectrum. Gallic acid release from particles to simulated gastrointestinal fluids was through diffusion from swollen particles, and reached almost 70% release. PMID:27211653

  8. Peptide nanofibers modified with a protein by using designed anchor molecules bearing hydrophobic and functional moieties.

    PubMed

    Miyachi, Ayaka; Takahashi, Tsuyoshi; Matsumura, Sachiko; Mihara, Hisakazu

    2010-06-11

    Self-assembly of peptides and proteins is a key feature of biological functions. Short amphiphilic peptides designed with a beta-sheet structure can form sophisticated nanofiber structures, and the fibers are available as nanomaterials for arranging biomolecules. Peptide FI (H-PKFKIIEFEP-OH) self-assembles into nanofibers with a coiled fine structure, as reported in our previous work. We have constructed anchor molecules that have both a binding moiety for the fiber structure and a functional unit capable of capturing target molecules, with the purpose of arranging proteins on the designed peptide nanofibers. Designed anchors containing an alkyl chain as a binding unit and biotin as a functional moiety were found to bind to peptide fibers FI and F2i (H-ALEAKFAAFEAKLA-NH(2)). The surface-exposed biotin moiety on the fibers could capture an anti-biotin antibody. Moreover, hydrophobic dipeptide anchor units composed of iminodiacetate connected to Phe-Phe or Ile-Ile and a peptide composed of six histidine residues connected to biotin could also connect FI peptide fibers to the anti-biotin antibody through the chelation of Ni(2+) ions. This strategy of using designed anchors opens a novel approach to constructing nanoscale protein arrays on peptide nanomaterials. PMID:20419712

  9. Designed amphiphilic peptide forms stable nanoweb, slowly releases encapsulated hydrophobic drug, and accelerates animal hemostasis

    PubMed Central

    Ruan, Liping; Zhang, Hangyu; Luo, Hanlin; Liu, Jingping; Tang, Fushan; Shi, Ying-Kang; Zhao, Xiaojun

    2009-01-01

    How do you design a peptide building block to make 2-dimentional nanowebs and 3-dimensional fibrous mats? This question has not been addressed with peptide self-assembling nanomaterials. This article describes a designed 9-residue peptide, N-Pro-Ser-Phe-Cys-Phe-Lys-Phe-Glu-Pro-C, which creates a strong fishnet-like nanostructure depending on the peptide concentrations and mechanical disruptions. This peptide is intramolecularly amphiphilic because of a single pair of ionic residues, Lys and Glu, at one end and nonionic residues, Phe, Cys, and Phe, at the other end. Circular dichroism and Fourier transform infrared spectroscopy analysis demonstrated that this peptide adopts stable β-turn and β-sheet structures and self-assembles into hierarchically arranged supramolecular aggregates in a concentration-dependent fashion, demonstrated by atomic force microscopy and electron microscopy. At high concentrations, the peptide dominantly self-assembled into globular aggregates that were extensively connected with each other to form “beads-on-a-thread” type nanofibers. These long nanofibers were extensively branched and overlapped to form a self-healing peptide hydrogel consisting of >99% water. This peptide can encapsulate the hydrophobic model drug pyrene and slowly release pyrene from coated microcrystals to liposomes. It can effectively stop animal bleeding within 30 s. We proposed a plausible model to interpret the intramolecular amphiphilic self-assembly process and suggest its importance for the future development of new biomaterials for drug delivery and regenerative medicine. PMID:19289834

  10. An effective conjugation strategy for designing short peptide-based HIV-1 fusion inhibitors.

    PubMed

    Liang, Guodong; Wang, Huixin; Chong, Huihui; Cheng, Siqi; Jiang, Xifeng; He, Yuxian; Wang, Chao; Liu, Keliang

    2016-08-16

    Lengthy peptides corresponding to the C-terminal heptad repeat (C-peptides) of human immunodeficiency virus type 1 (HIV-1) gp41 are potent inhibitors against virus-cell fusion. Designing short C-peptide-based HIV-1 fusion inhibitors could potentially redress the physicochemical and technical liabilities of a long-peptide therapeutic. However, designing such inhibitors with high potency has been challenging. We generated a conjugated architecture by incorporating small-molecule inhibitors of gp41 into the N-terminus of a panel of truncated C-peptides. Among these small molecule-capped short peptides, the 26-residue peptide Indole-T26 inhibited HIV-1 Env-mediated cell-cell fusion and viral replication at low nanomolar levels, reaching the potency of the only clinically used 36-residue peptide T20 (enfuvirtide). Collectively, our work opens up a new avenue for developing short peptide-based HIV-1 fusion inhibitors, and may have broad applicability to the development of modulators of other class I fusion proteins. PMID:27454320

  11. Variant fatty acid-like molecules Conjugation, novel approaches for extending the stability of therapeutic peptides

    PubMed Central

    Li, Ying; Wang, Yuli; Wei, Qunchao; Zheng, Xuemin; Tang, Lida; Kong, Dexin; Gong, Min

    2015-01-01

    The multiple physiological properties of glucagon-like peptide-1 (GLP-1) make it a promising drug candidate for the treatment of type 2 diabetes. However, the in vivo half-life of GLP-1 is short due to rapid degradation by dipeptidyl peptidase-IV (DPP-IV) and renal clearance. The poor stability of GLP-1 has significantly limited its clinical utility; however, many studies are focused on extending its stability. Fatty acid conjugation is a traditional approach for extending the stability of therapeutic peptides because of the high binding affinity of human serum albumin for fatty acids. However, the conjugate requires a complex synthetic approach, usually involving Lys and occasionally involving a linker. In the current study, we conjugated the GLP-1 molecule with fatty acid derivatives to simplify the synthesis steps. Human serum albumin binding assays indicated that the retained carboxyl groups of the fatty acids helped maintain a tight affinity to HSA. The conjugation of fatty acid-like molecules improved the stability and increased the binding affinity of GLP-1 to HSA. The use of fatty acid-like molecules as conjugating components allowed variant conjugation positions and freed carboxyl groups for other potential uses. This may be a novel, long-acting strategy for the development of therapeutic peptides. PMID:26658631

  12. Variant fatty acid-like molecules Conjugation, novel approaches for extending the stability of therapeutic peptides.

    PubMed

    Li, Ying; Wang, Yuli; Wei, Qunchao; Zheng, Xuemin; Tang, Lida; Kong, Dexin; Gong, Min

    2015-01-01

    The multiple physiological properties of glucagon-like peptide-1 (GLP-1) make it a promising drug candidate for the treatment of type 2 diabetes. However, the in vivo half-life of GLP-1 is short due to rapid degradation by dipeptidyl peptidase-IV (DPP-IV) and renal clearance. The poor stability of GLP-1 has significantly limited its clinical utility; however, many studies are focused on extending its stability. Fatty acid conjugation is a traditional approach for extending the stability of therapeutic peptides because of the high binding affinity of human serum albumin for fatty acids. However, the conjugate requires a complex synthetic approach, usually involving Lys and occasionally involving a linker. In the current study, we conjugated the GLP-1 molecule with fatty acid derivatives to simplify the synthesis steps. Human serum albumin binding assays indicated that the retained carboxyl groups of the fatty acids helped maintain a tight affinity to HSA. The conjugation of fatty acid-like molecules improved the stability and increased the binding affinity of GLP-1 to HSA. The use of fatty acid-like molecules as conjugating components allowed variant conjugation positions and freed carboxyl groups for other potential uses. This may be a novel, long-acting strategy for the development of therapeutic peptides. PMID:26658631

  13. In vitro properties of designed antimicrobial peptides that exhibit potent antipneumococcal activity and produces synergism in combination with penicillin

    PubMed Central

    Le, Cheng-Foh; Yusof, Mohd Yasim Mohd; Hassan, Hamimah; Sekaran, Shamala Devi

    2015-01-01

    Antimicrobial peptides (AMPs) represent a promising class of novel antimicrobial agents owing to their potent antimicrobial activity. In this study, two lead peptides from unrelated classes of AMPs were systematically hybridized into a series of five hybrid peptides (DM1- DM5) with conserved N- and C-termini. This approach allows sequence bridging of two highly dissimilar AMPs and enables sequence-activity relationship be detailed down to single amino acid level. Presence of specific amino acids and physicochemical properties were used to describe the antipneumococcal activity of these hybrids. Results obtained suggested that cell wall and/or membrane targeting could be the principal mechanism exerted by the hybrids leading to microbial cell killing. Moreover, the pneumocidal rate was greater than penicillin (PEN). Combination treatment with both DMs and PEN produced synergism. The hybrids were also broad spectrum against multiple common clinical bacteria. Sequence analysis showed that presence of specific residues has a major role in affecting the antimicrobial and cell toxicity of the hybrids than physicochemical properties. Future studies should continue to investigate the mechanisms of actions, in vivo therapeutic potential, and improve rational peptide design based on the current strategy. PMID:25985150

  14. Computational de novo design of a self-assembling peptide with predefined structure.

    PubMed

    Kaltofen, Sabine; Li, Chenge; Huang, Po-Ssu; Serpell, Louise C; Barth, Andreas; André, Ingemar

    2015-01-30

    Protein and peptide self-assembly is a powerful design principle for engineering of new biomolecules. More sophisticated biomaterials could be built if both the structure of the overall assembly and that of the self-assembling building block could be controlled. To approach this problem, we developed a computational design protocol to enable de novo design of self-assembling peptides with predefined structure. The protocol was used to design a peptide building block with a βαβ fold that self-assembles into fibrillar structures. The peptide associates into a double β-sheet structure with tightly packed α-helices decorating the exterior of the fibrils. Using circular dichroism, Fourier transform infrared spectroscopy, electron microscopy and X-ray fiber diffraction, we demonstrate that the peptide adopts the designed conformation. The results demonstrate that computational protein design can be used to engineer protein and peptide assemblies with predefined three-dimensional structures, which can serve as scaffolds for the development of functional biomaterials. Rationally designed proteins and peptides could also be used to investigate the subtle energetic and entropic tradeoffs in natural self-assembly processes and the relation between assembly structure and assembly mechanism. We demonstrate that the de novo designed peptide self-assembles with a mechanism that is more complicated than expected, in a process where small changes in solution conditions can lead to significant differences in assembly properties and conformation. These results highlight that formation of structured protein/peptide assemblies is often dependent on the formation of weak but highly precise intermolecular interactions. PMID:25498388

  15. Laser ion beam photodissociation studies of model amino acids and peptides

    SciTech Connect

    Techlenburg, R.E. Jr.; Miller, M.N.; Russell, D.H. )

    1989-02-15

    Visible (458-514.5 nm) and uv (333-385 nm) photodissociation of the (M + H){sup +} ions of dinitrophenyl (DNP) derivatized amino acids and peptides is reported. Photoexcitation of the DNP peptides by a visible proton results in fragmentation of the peptide chain with little fragmentation within the chromophore. Conversely, uv photoexcitation of the DNP peptides results in fragmentation of the chromophore as well as the peptide chain, but loss of NO or NO{sub 2} (within the chromophore) often dominates the photofragment ion spectrum. These results are rationalized with particular emphasis on energy-selective dissociation channels of large ionic systems. DNP-leucine and DNP-isoleucine (M + H){sup +} can be differentiated on the basis of photodissociation reactions which yield distonic radical cations. The rate of dissociation of photoexcited ions of DNP peptides is shown to decrease with increasing molecular weight (degrees of freedom). Lastly, comparisons between photodissociation and collision-induced dissociation as a structural probe are presented. 55 refs., 8 figs., 3 tabs.

  16. Effect of acid predissolution on fibril size and fibril flexibility of synthetic beta-amyloid peptide.

    PubMed Central

    Shen, C L; Fitzgerald, M C; Murphy, R M

    1994-01-01

    beta-amyloid peptide (A beta) is the major protein component of senile plaques and cerebrovascular amyloid deposits in Alzheimer's patients. Several researchers have demonstrated that A beta is neurotoxic in in vitro and in vivo systems. Peptide aggregation state and/or conformation might play a significant role in determining the toxicity of the peptide. The size and flexibility of fibrils formed from the synthetic peptide beta (1-39), corresponding to the first 39 residues of A beta, were determined. Samples were prepared either directly from lyophilized peptide or diluted from a 10 mg/ml stock solution in 0.1% trifluoroacetic acid (TFA). All samples had a final peptide concentration of 0.5 mg/ml, a final pH of 7.4, and a final NaCl concentration of 0.14 M. The molecular weight and linear density of the fibrils increased with increasing pre-incubation time in TFA, based on static light scattering measurements. Analysis of the angular dependence of the intensity of scattered light indicated that the fibrils were semi-flexible chains and that the fibril flexibility decreased with increasing pre-incubation time in TFA. There was a concomitant change in phase behavior from precipitation to gelation with the decrease in fibril flexibility. Images FIGURE 3 PMID:7811938

  17. Novel amino acids: synthesis of furoxan and sydnonimine containing amino acids and peptides as potential nitric oxide releasing motifs.

    PubMed

    Nortcliffe, Andrew; Botting, Nigel P; O'Hagan, David

    2013-07-28

    The incorporation of furoxan and sydnonimine ring systems into amino acid side chains is demonstrated with the preparation of four novel amino acids which carry these nitric oxide-releasing motifs. N-((4-Nitrophenoxy)carbonyl)-3-phenylsydnonimine 9 and bis(phenylsulfonyl)furoxan 10 are the key intermediates for introducing the heterocycle side chains onto appropriate amine and alcohol functionalities respectively. Furoxan 5 and 7 both displayed NO release based on determination of nitrite production. Orthogonal amino acid protecting group strategies were deployed to demonstrate that the amino acids could be incorporated into peptide frameworks. By way of demonstration the amino acids were placed centrally into several tripeptide motifs. Griess test assays showed that these amino acids released NO in the presence of γ-glutathione (GST). PMID:23753002

  18. Overcoming the Refractory Expression of Secreted Recombinant Proteins in Mammalian Cells through Modification of the Signal Peptide and Adjacent Amino Acids

    PubMed Central

    Güler-Gane, Gülin; Kidd, Sara; Sridharan, Sudharsan; Vaughan, Tristan J.; Wilkinson, Trevor C. I.

    2016-01-01

    The expression and subsequent purification of mammalian recombinant proteins is of critical importance to many areas of biological science. To maintain the appropriate tertiary structure and post-translational modifications of such proteins, transient mammalian expression systems are often adopted. The successful utilisation of these systems is, however, not always forthcoming and some recombinant proteins prove refractory to expression in mammalian hosts. In this study we focussed on the role of different N-terminal signal peptides and residues immediately downstream, in influencing the level of secreted recombinant protein obtained from suspension HEK293 cells. Using secreted alkaline phosphatase (SEAP) as a model protein, we identified that the +1/+2 downstream residues flanking a heterologous signal peptide significantly affect secreted levels. By incorporating these findings we conducted a comparison of different signal peptide sequences and identified the most productive as secrecon, a computationally-designed sequence. Importantly, in the context of the secrecon signal peptide and SEAP, we also demonstrated a clear preference for specific amino acid residues at the +1 position (e.g. alanine), and a detrimental effect of others (cysteine, proline, tyrosine and glutamine). When proteins that naturally contain these “undesirable” residues at the +1 position were expressed with their native signal peptide, the heterologous secrecon signal peptide, or secrecon with an additional alanine at the +1 or +1 and +2 position, the level of expression differed significantly and in an unpredictable manner. For each protein, however, at least one of the panel of signal peptide/adjacent amino acid combinations enabled successful recombinant expression. In this study, we highlight the important interplay between a signal peptide and its adjacent amino acids in enabling protein expression, and we describe a strategy that could enable recombinant proteins that have so far

  19. Overcoming the Refractory Expression of Secreted Recombinant Proteins in Mammalian Cells through Modification of the Signal Peptide and Adjacent Amino Acids.

    PubMed

    Güler-Gane, Gülin; Kidd, Sara; Sridharan, Sudharsan; Vaughan, Tristan J; Wilkinson, Trevor C I; Tigue, Natalie J

    2016-01-01

    The expression and subsequent purification of mammalian recombinant proteins is of critical importance to many areas of biological science. To maintain the appropriate tertiary structure and post-translational modifications of such proteins, transient mammalian expression systems are often adopted. The successful utilisation of these systems is, however, not always forthcoming and some recombinant proteins prove refractory to expression in mammalian hosts. In this study we focussed on the role of different N-terminal signal peptides and residues immediately downstream, in influencing the level of secreted recombinant protein obtained from suspension HEK293 cells. Using secreted alkaline phosphatase (SEAP) as a model protein, we identified that the +1/+2 downstream residues flanking a heterologous signal peptide significantly affect secreted levels. By incorporating these findings we conducted a comparison of different signal peptide sequences and identified the most productive as secrecon, a computationally-designed sequence. Importantly, in the context of the secrecon signal peptide and SEAP, we also demonstrated a clear preference for specific amino acid residues at the +1 position (e.g. alanine), and a detrimental effect of others (cysteine, proline, tyrosine and glutamine). When proteins that naturally contain these "undesirable" residues at the +1 position were expressed with their native signal peptide, the heterologous secrecon signal peptide, or secrecon with an additional alanine at the +1 or +1 and +2 position, the level of expression differed significantly and in an unpredictable manner. For each protein, however, at least one of the panel of signal peptide/adjacent amino acid combinations enabled successful recombinant expression. In this study, we highlight the important interplay between a signal peptide and its adjacent amino acids in enabling protein expression, and we describe a strategy that could enable recombinant proteins that have so far

  20. A recipe for designing water-soluble, beta-sheet-forming peptides.

    PubMed Central

    Mayo, K. H.; Ilyina, E.; Park, H.

    1996-01-01

    Based on observations of solubility and folding properties of peptide 33-mers derived from the beta-sheet domains of platelet factor-4 (PF4), interleukin-8 (IL-8), and growth related protein (Gro-alpha), as well as other beta-sheet-forming peptides, general guidelines have been developed to aid in the design of water soluble, self-association-induced beta-sheet-forming peptides. CD, 1H-NMR, and pulsed field gradient NMR self-diffusion measurements have been used to assess the degree of folding and state of aggregation. PF4 peptide forms native-like beta-sheet tetramers and is sparingly soluble above pH 6. IL-8 peptide is insoluble between pH 4.5 and pH 7.5, yet forms stable, native-like beta-sheet dimers at higher pH. Gro-alpha peptide is soluble at all pH values, yet displays no discernable beta-sheet structure even when diffusion data indicate dimer-tetramer aggregation. A recipe used in the de novo design of water-soluble beta-sheet-forming peptides calls for the peptide to contain 40-50% hydrophobic residues, usually aliphatic ones (I, L, V, A, M) (appropriately paired and mostly but not always alternating with polar residues in the sheet sequence), a positively charged (K, R) to negatively charged (E, D) residue ratio between 4/2 and 6/2, and a noncharged polar residue (N, Q, T, S) composition of about 20% or less. Results on four de novo designed, 33-residue peptides are presented supporting this approach. Under near physiologic conditions, all four peptides are soluble, form beta-sheet structures to varying degrees, and self-associate. One peptide folds as a stable, compact beta-sheet tetramer, whereas the others are transient beta-sheet-containing aggregates. PMID:8819163

  1. Stable Isotope Peptide Mass Spectrometry To Decipher Amino Acid Metabolism in Dehalococcoides Strain CBDB1

    PubMed Central

    Marco-Urrea, Ernest; Seifert, Jana; von Bergen, Martin

    2012-01-01

    Dehalococcoides species are key players in the anaerobic transformation of halogenated solvents at contaminated sites. Here, we analyze isotopologue distributions in amino acid pools from peptides of Dehalococcoides strain CBDB1 after incubation with 13C-labeled acetate or bicarbonate as a carbon source. The resulting data were interpreted with regard to genome annotations to identify amino acid biosynthesis pathways. In addition to using gas chromatography-mass spectrometry (GC-MS) for analyzing derivatized amino acids after protein hydrolysis, we introduce a second, much milder method, in which we directly analyze peptide masses after tryptic digest and peptide fragments by nano-liquid chromatography-electrospray ionization-tandem mass spectrometry (nano-LC-ESI-MS/MS). With this method, we identify isotope incorporation patterns for 17 proteinaceous amino acids, including proline, cysteine, lysine, and arginine, which escaped previous analyses in Dehalococcoides. Our results confirmed lysine biosynthesis via the α-aminoadipate pathway, precluding lysine formation from aspartate. Similarly, the isotopologue pattern obtained for arginine provided biochemical evidence of its synthesis from glutamate. Direct peptide MS/MS analysis of the labeling patterns of glutamine and asparagine, which were converted to glutamate and aspartate during protein hydrolysis, gave biochemical evidence of their precursors and confirmed glutamate biosynthesis via a Re-specific citrate synthase. By addition of unlabeled free amino acids to labeled cells, we show that in strain CBDB1 none of the 17 tested amino acids was incorporated into cell mass, indicating that they are all synthesized de novo. Our approach is widely applicable and provides a means to analyze amino acid metabolism by studying specific proteins even in mixed consortia. PMID:22661690

  2. Design of a Multicomponent Peptide-Woven Nanocomplex for Delivery of siRNA

    PubMed Central

    Jun, Eunsung; Kim, Soyoun; Kim, Jong-Ho; Cha, Kiweon; So, In-Seop; Son, Hye-Nam; Lee, Byung-Heon; Kim, Kwangmeyung; Kwon, Ick Chan; Kim, Sang Yoon; Kim, In-San

    2015-01-01

    We developed and tested a multicomponent peptide-woven siRNA nanocomplex (PwSN) comprising different peptides designed for efficient cellular targeting, endosomal escape, and release of siRNA. To enhance tumor-specific cellular uptake, we connected an interleukin-4 receptor-targeting peptide (I4R) to a nine-arginine peptide (9r), yielding I4R-9r. To facilitate endosomal escape, we blended endosomolytic peptides into the I4R-9r to form a multicomponent nanocomplex. Lastly, we modified 9r peptides by varying the number and positions of positive charges to obtain efficient release of siRNA from the nanocomplex in the cytosol. Using this step-wise approach for overcoming the biological challenges of siRNA delivery, we obtained an optimized PwSN with significant biological activity in vitro and in vivo. Interestingly, surface plasmon resonance analyses and three-dimensional peptide models demonstrated that our designed peptide adopted a unique structure that was correlated with faster complex disassembly and a better gene-silencing effect. These studies further elucidate the siRNA nanocomplex delivery pathway and demonstrate the applicability of our stepwise strategy to the design of siRNA carriers capable of overcoming multiple challenges and achieving efficient delivery. PMID:25705892

  3. Peptide design by artificial neural networks and computer-based evolutionary search

    PubMed Central

    Schneider, Gisbert; Schrödl, Wieland; Wallukat, Gerd; Müller, Johannes; Nissen, Eberhard; Rönspeck, Wolfgang; Wrede, Paul; Kunze, Rudolf

    1998-01-01

    A technique for systematic peptide variation by a combination of rational and evolutionary approaches is presented. The design scheme consists of five consecutive steps: (i) identification of a “seed peptide” with a desired activity, (ii) generation of variants selected from a physicochemical space around the seed peptide, (iii) synthesis and testing of this biased library, (iv) modeling of a quantitative sequence-activity relationship by an artificial neural network, and (v) de novo design by a computer-based evolutionary search in sequence space using the trained neural network as the fitness function. This strategy was successfully applied to the identification of novel peptides that fully prevent the positive chronotropic effect of anti-β1-adrenoreceptor autoantibodies from the serum of patients with dilated cardiomyopathy. The seed peptide, comprising 10 residues, was derived by epitope mapping from an extracellular loop of human β1-adrenoreceptor. A set of 90 peptides was synthesized and tested to provide training data for neural network development. De novo design revealed peptides with desired activities that do not match the seed peptide sequence. These results demonstrate that computer-based evolutionary searches can generate novel peptides with substantial biological activity. PMID:9770460

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

    SciTech Connect

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

    2008-10-03

    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.

  5. Design of Electrospun Hydrogel Fibers Containing Multivalent Peptide Conjugates for Cardiac Tissue Engineering

    NASA Astrophysics Data System (ADS)

    Rode, Nikhil Ajit

    A novel material was designed using biomimetic engineering principles to recreate the chemical and physical environment of the extracellular matrix for cardiac tissue engineering applications. In order to control the chemical and specific bioactive signals provided by the material, a multivalent conjugate of a RGD-containing cell-binding peptide with hyaluronic acid was synthesized. These conjugates were characterized using in-line size exclusion chromatography with static multi-angle light scattering, UV absorbance, and differential refractive index measurements (SEC-MALS-UV-RI) to determine their molecular weight and valency, as well as the distributions of each. These conjugates were electrospun with poly(ethylene glycol) and poly(ethylene glycol) diacrylate to create a nanofibrous hydrogel material embedded with bioinstructive macromolecules. This electrospinning process was explored and optimized to create well-formed nanofibers. The diameter and orientation of the fibers was controlled to closely mimic the nanostructure of the extracellular matrix of the myocardium. Further characterization of the material was performed to ensure that its mechanical properties resemble those found in the myocardium. The availability of the peptides embedded in the hydrogel material was confirmed by measuring peptides released by trypsin incubation and was found to be sufficient to cause cell adhesion. This material was capable of supporting cell culture, maintaining the viability of cultured fibroblasts and cardiomyocytes, and preserving cardiomyocyte functionality. In this way, this material shows promise of serving as a biomimetic in vitro scaffold for generation of functional myocardial tissue, with possible applications as an in vivo cardiac patch for repair of the damage myocardium post-myocardial infarction.

  6. Question 1: Peptide Nucleic Acids and the Origin and Homochirality of Life

    NASA Astrophysics Data System (ADS)

    Nielsen, Peter E.

    2007-10-01

    The possibilities of pseudo peptide DNA mimics like PNA (peptide nucleic acid) having a role for the prebiotic origin of life prior to an RNA world is discussed. In particular a scenario is proposed in which protocells with an achiral genetic material through several generations stepwise is converted into a chiral genetic material, e.g., by incorporation of RNA units. Provided that a sufficiently large sequence space is occupied, a selection process based on catalytic function in which a single cell (first common ancestor) has a definite evolutionary advantage, selection of this cell would by contingency also lock it into homochirality.

  7. Amino acids and peptides activate at least five members of the human bitter taste receptor family.

    PubMed

    Kohl, Susann; Behrens, Maik; Dunkel, Andreas; Hofmann, Thomas; Meyerhof, Wolfgang

    2013-01-01

    Amino acids and peptides represent important flavor molecules eliciting various taste sensations. Here, we present a comprehensive assessment of the interaction of various peptides and all proteinogenic amino acids with the 25 human TAS2Rs expressed in cell lines. L-Phenylalanine and L-tryptophan activate TAS2R1 and TAS2R4, respectively, whereas TAS2R4 and TAS2R39 responded to D-tryptophan. Structure-function analysis uncovered the basis for the lack of stereoselectivity of TAS2R4. The same three TAS2Rs or subsets thereof were also sensitive to various dipeptides containing L-tryptophan, L-phenylalanine, or L-leucine and to Trp-Trp-Trp, whereas Leu-Leu-Leu specifically activated TAS2R4. Trp-Trp-Trp also activated TAS2R46 and TAS2R14. Two key bitter peptides from Gouda cheese, namely, Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His-Asn-Ser and Leu-Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His-Asn, both activated TAS2R1 and TAS2R39. Thus, the data demonstrate that the bitterness of amino acids and peptides is not mediated by specifically tuned TAS2Rs but rather is brought about by an unexpectedly complex pattern of sensitive TAS2Rs. PMID:23214402

  8. Acidity-Mediated, Electrostatic Tuning of Asymmetrically Charged Peptides Interactions with Protein Nanopores.

    PubMed

    Asandei, Alina; Chinappi, Mauro; Kang, Hee-Kyoung; Seo, Chang Ho; Mereuta, Loredana; Park, Yoonkyung; Luchian, Tudor

    2015-08-01

    Despite success in probing chemical reactions and dynamics of macromolecules on submillisecond time and nanometer length scales, a major impasse faced by nanopore technology is the need to cheaply and controllably modulate macromolecule capture and trafficking across the nanopore. We demonstrate herein that tunable charge separation engineered at the both ends of a macromolecule very efficiently modulates the dynamics of macromolecules capture and traffic through a nanometer-size pore. In the proof-of-principle approach, we employed a 36 amino acids long peptide containing at the N- and C-termini uniform patches of glutamic acids and arginines, flanking a central segment of asparagines, and we studied its capture by the α-hemolysin (α-HL) and the mean residence time inside the pore in the presence of a pH gradient across the protein. We propose a solution to effectively control the dynamics of peptide interaction with the nanopore, with both association and dissociation reaction rates of peptide-α-HL interactions spanning orders of magnitude depending upon solution acidity on the peptide addition side and the transmembrane electric potential, while preserving the amplitude of the blockade current signature. PMID:26144534

  9. Design and synthesis of human ABCB1 (P-glycoprotein) inhibitors by peptide coupling of diverse chemical scaffolds on carboxyl and amino termini of (S)-valine-derived thiazole amino acid.

    PubMed

    Singh, Satyakam; Prasad, Nagarajan Rajendra; Chufan, Eduardo E; Patel, Bhargav A; Wang, Yi-Jun; Chen, Zhe-Sheng; Ambudkar, Suresh V; Talele, Tanaji T

    2014-05-22

    P-glycoprotein (P-gp) serves as a therapeutic target for the development of multidrug resistance reversal agents. In this study, we synthesized 21 novel compounds by peptide coupling at corresponding carboxyl and amino termini of (S)-valine-based bis-thiazole and monothiazole derivatives with diverse chemical scaffolds. Using calcein-AM efflux assay, we identified compound 28 (IC50 = 1.0 μM) carrying 3,4,5-trimethoxybenzoyl and 2-aminobenzophenone groups, respectively, at the amino and carboxyl termini of the monothiazole zwitter-ion. Compound 28 inhibited the photolabeling of P-gp with [(125)I]-iodoarylazidoprazosin with IC50 = 0.75 μM and stimulated the basal ATP hydrolysis of P-gp in a concentration-dependent manner (EC50 ATPase = 0.027 μM). Compound 28 at 3 μM reduced resistance in cytotoxicity assay to paclitaxel in P-gp-expressing SW620/Ad300 and HEK/ABCB1 cell lines. Biochemical and docking studies showed site-1 to be the preferable binding site for 28 within the drug-binding pocket of human P-gp. PMID:24773054

  10. Complete amino acid analysis of peptides and proteins after hydrolysis by a mixture of Sepharose-bound peptidases

    PubMed Central

    Bennett, H. P. J.; Elliott, D. F.; Evans, B. E.; Lowry, P. J.; McMartin, C.

    1972-01-01

    Incubation with a mixture of Sepharose-bound peptidases was shown to result in the quantitative release of amino acids from certain peptides and S-aminoethylated proteins. Subtraction of the low background values of amino acids generated by the enzymes enables amino acid ratios of corticotrophin-(1–24)-tetracosapeptide to be determined with a standard deviation on repeat digestions of 3–5%. Good values were obtained for amino acids that are completely or partially destroyed on acid hydrolysis, i.e. tryptophan, tyrosine, serine, asparagine and glutamine. Experiments with peptides containing d-amino acids showed that the enzyme mixture is stereospecific and could therefore be used to detect the presence of d-residues in peptides. The enzyme mixture completely hydrolyses peptide fragments obtained after Edman degradation and should therefore be useful for determining sequences of peptides containing acid-labile amino acid residues. The activities of the bound enzymes were unaltered over a period of 7 months and they provide a simple, reproducible procedure for the quantitative determination of amino acids in peptides and proteins containing l-amino acids. PMID:4349115

  11. Sustained delivery of VEGF from designer self-assembling peptides improves cardiac function after myocardial infarction

    SciTech Connect

    Guo, Hai-dong; Cui, Guo-hong; Yang, Jia-jun; Wang, Cun; Zhu, Jing; Zhang, Li-sheng; Jiang, Jun; Shao, Shui-jin

    2012-07-20

    Highlights: Black-Right-Pointing-Pointer The designer peptide LRKKLGKA could self-assemble into nanofibers. Black-Right-Pointing-Pointer Injection of LRKKLGKA peptides could promote the sustained delivery of VEGF. Black-Right-Pointing-Pointer Injection of VEGF with LRKKLGKA peptides lead to sufficient angiogenesis. Black-Right-Pointing-Pointer Injection of VEGF with LRKKLGKA peptides improves heart function. -- Abstract: Poor vascularization and insufficient oxygen supply are detrimental to the survival of residual cardiomyocytes or transplanted stem cells after myocardial infarction. To prolong and slow the release of angiogenic factors, which stimulate both angiogenesis and vasculogenesis, we constructed a novel self-assembling peptide by attaching the heparin-binding domain sequence LRKKLGKA to the self-assembling peptide RADA16. This designer self-assembling peptide self-assembled into nanofiber scaffolds under physiological conditions, as observed by atomic force microscopy. The injection of designer self-assembling peptides can efficiently provide the sustained delivery of VEGF for at least 1 month. At 4 weeks after transplantation, cardiac function was improved, and scar size and collagen deposition were markedly reduced in the group receiving VEGF with the LRKKLGKA scaffolds compared with groups receiving VEGF alone, LRKKLGKA scaffolds alone or VEGF with RADA16 scaffolds. The microvessel density in the VEGF with LRKKLGKA group was higher than that in the VEGF with RADA16 group. TUNEL and cleaved caspase-3 expression assays showed that the transplantation of VEGF with LRKKLGKA enhanced cell survival in the infarcted heart. These results present the tailor-made peptide scaffolds as a new generation of sustained-release biomimetic biomaterials and suggest that the use of angiogenic factors along with designer self-assembling peptides can lead to myocardial protection, sufficient angiogenesis, and improvement in cardiac function.

  12. Rational design of a low-affinity peptide for the detection of cystatin C in a fast homogeneous immunoassay.

    PubMed

    Dobslaff, Kristin; Zscharnack, Kristin; Kreisig, Thomas; Zuchner, Thole

    2016-02-01

    Immunoassays play an essential role in current research and diagnostics resulting in a variety of detection principles. Thereby, homogeneous assays are often used for a fast signal response as demanded for example in point-of-care diagnostics. These systems often rely on a competitive assay design where the sample analyte and the corresponding dye-labeled substance are competing for binding sites on an antibody present in limited amounts. Due to the similar affinities of the antibody towards the sample analyte and the competitor, both sensitivity and assay time are limited. As a consequence, a competitor with a slightly reduced affinity towards the antibody can potentially overcome these drawbacks. Here, we present the rational design of a low-affinity peptide (donor peptide) as a specific analyte competitor for a FRET-based homogeneous immunoassay for the analysis of the protein cystatin C. Thereby, the strategy of peptide-induced antibody generation was combined with the selective variation of the immunization sequence in order to achieve a lower affinity towards the antibody. We could show that shortened donor peptides improved the resulting quenching efficiency in the immunoassay. In addition, the substitution of small hydrophobic amino acids by those with a higher steric demand appeared to be the most promising strategy providing a fast assay response for cystatin C of only 90 s. PMID:26403846

  13. PepComposer: computational design of peptides binding to a given protein surface.

    PubMed

    Obarska-Kosinska, Agnieszka; Iacoangeli, Alfredo; Lepore, Rosalba; Tramontano, Anna

    2016-07-01

    There is a wide interest in designing peptides able to bind to a specific region of a protein with the aim of interfering with a known interaction or as starting point for the design of inhibitors. Here we describe PepComposer, a new pipeline for the computational design of peptides binding to a given protein surface. PepComposer only requires the target protein structure and an approximate definition of the binding site as input. We first retrieve a set of peptide backbone scaffolds from monomeric proteins that harbor the same backbone arrangement as the binding site of the protein of interest. Next, we design optimal sequences for the identified peptide scaffolds. The method is fully automatic and available as a web server at http://biocomputing.it/pepcomposer/webserver. PMID:27131789

  14. Inspiration from the mirror: D-amino acid containing peptides in biomedical approaches.

    PubMed

    Feng, Zhaoqianqi; Xu, Bing

    2016-06-01

    D-amino acids, the enantiomers of naturally abundant L-amino acids, bear unique stereochemistry properties that lead to the resistance towards most of the endogenous enzymes. Previous works have demonstrated applications of D-amino acids in therapeutic development with the aid of mirror-image phage display and retro-inverso peptide synthesis. In this review, we highlight the recent progress and challenges in the exploration of D-amino acids at the interface of chemistry and life science. First, we will introduce some progress made in traditional application of D-amino acids to enhance biostability of peptide therapeutics. Then, we discuss some works that explore the relatively underexplored interactions between the enzyme and D-amino acids and enzymatic reactions of D-amino acids. To highlight the enzymatic reactions of D-amino acids, we will describe several emerging works on the enzyme-instructed self-assembly (EISA) and their potential application in selective anti-inflammatory or anticancer therapies. At the end, we briefly mention the challenges and possible future directions. PMID:27159920

  15. Mass spectral study of hybrid peptides derived from (R)-aminoxy ester and [beta]-amino acids: The influence of aminoxy peptide bond (CO-NH-O) on peptide fragmentation under electrospray ionization conditions

    NASA Astrophysics Data System (ADS)

    Ramesh, V.; Ramesh, M.; Srinivas, R.; Sharma, G. V. M.; Manohar, V.

    2009-04-01

    A new class of Boc-protected aminoxy hybrid peptides containing repeats of [beta]-hAla-(R)-Ama-, and [beta]-Caa-(R)-Ama- ([beta]-hAla = [beta]3-(S)-hAlanine, (R)-Ama = (R)-aminoxy ester, and [beta]-Caa = (R)-C-linked carbo-[beta]3-amino acid) have been studied by electrospray ionization (ESI) ion-trap and quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS) of their protonated, cationized, and negative ions. MS3 CID of protonated aminoxy peptides of [beta]-hAla-(R)-Ama- yield intense [beta]-amino acid characteristic retro-Mannich fragmentation. The bn+ and [bn-methyl imine]+ (n = 3, 5) ions formed by cleavage of aminoxy peptide bond (CO-NH-O) are more intense than bn+ (n = 2, 4) formed by that of peptide bond (CO-NH-C) cleavage. Another characteristic ion observed is due to loss of H3NO from yn+ ions. The cationized (Li+, and Na+) peptides dissociate differently compared to protonated peptides. Intense cationized cn and zn ions are formed due to the cleavage of N-O bond. The deprotonated peptides also show abundant cn- and zn- ions (n = 1, 3, 5) and do not form any yn- ions. All these results clearly indicate the influence of aminoxy peptide bond on fragmentation of these hybrid peptides.

  16. Understanding Binding Peptide Design Using a Synthesis of Residue Physicality and Energetic Frustration

    NASA Astrophysics Data System (ADS)

    Flowers, Lenaya; Tripathi, Swarnendu; Cheung, Margaret

    The ubiquitous nature of Calmodulin (CaM) allows it to bind to numerous peptides, thus altering the function of a protein complex. Variations in CaM's function are a product of the numerous binding targets (BT) and their significant biological pathways. Given that CaM is a well-studied protein, we have found that certain amino acids in CaM's sequence play an important role in the event of protein binding. 36 CaM binding targets were analyzed to find sequential, physical commonalities. Using the Frustrometer (frustrometer.tk), we obtained z-scores (a numerical value for level of frustration) for each amino acids in a given binding target sequence. From those results, we were able to identify which residues show a highly favorable energetic change after binding and those that do not. We have found charged residues show the most prominent change when bound to CaM, these amino acids may provide a critical role in the overall design and function of a CaM-BT complex.

  17. Computer-aided design of peptide near infrared fluorescent probe for tumor diagnosis

    NASA Astrophysics Data System (ADS)

    Zhang, Congying; Gu, Yueqing

    2014-09-01

    Integrin αvβ3 receptors are expressed on activated endothelial cells during neovascularization to maintain tumor growth, so they become hot research tagets in cancer diagnosis. Peptides possess several attractive features when compared to protein and small molecule, such as small size and high structural compatibility with target proteins. Efficient design of high-affinity peptide ligands to Integrin αvβ3 receptors has been an important problem. Designed peptides in silico provide a valuable and high-selectivity peptide, meanwhile decrease the time of drug screening. In this study, we design peptide which can bind with integrin αvβ3 via computer, and then synthesis near infrared fluorescent probe. The characterization of this near infrared fluorescent probe was detected by UV. To investigate the tumor cell targeting of this probe, it was labeled with visible fluorescent dye Rhodamine B (RhB) for microscopy. To evaluate the targeting capability of this near infrared fluorescent probe, mice bearing integrin αvβ3 positive tumor xenografts were used. In vitro cellular experiments indicated that this probe have a clear binding affinity to αvβ3-positive tumor cells. In vivo experiments confirmed the receptor binding specificity of this probe. The peptide of computational design can bind with integrin αvβ3. Combined peptide near-infrared fluorescent probe with imaging technology use for clinical and tumor diagnosis have a greater development in future.

  18. MINIATURE ACID CONDENSATION SYSTEM: DESIGN AND OPERATION

    EPA Science Inventory

    An extractive source sampling system was designed and constructed. The sampling system measures gaseous sulfuric acid and sulfur dioxide in combustion emissions. The miniature acid condensation system (MACS) includes a high-temperature quartz probe and quartz-filter holder. Since...

  19. Antimicrobial Peptides in 2014

    PubMed Central

    Wang, Guangshun; Mishra, Biswajit; Lau, Kyle; Lushnikova, Tamara; Golla, Radha; Wang, Xiuqing

    2015-01-01

    This article highlights new members, novel mechanisms of action, new functions, and interesting applications of antimicrobial peptides reported in 2014. As of December 2014, over 100 new peptides were registered into the Antimicrobial Peptide Database, increasing the total number of entries to 2493. Unique antimicrobial peptides have been identified from marine bacteria, fungi, and plants. Environmental conditions clearly influence peptide activity or function. Human α-defensin HD-6 is only antimicrobial under reduced conditions. The pH-dependent oligomerization of human cathelicidin LL-37 is linked to double-stranded RNA delivery to endosomes, where the acidic pH triggers the dissociation of the peptide aggregate to release its cargo. Proline-rich peptides, previously known to bind to heat shock proteins, are shown to inhibit protein synthesis. A model antimicrobial peptide is demonstrated to have multiple hits on bacteria, including surface protein delocalization. While cell surface modification to decrease cationic peptide binding is a recognized resistance mechanism for pathogenic bacteria, it is also used as a survival strategy for commensal bacteria. The year 2014 also witnessed continued efforts in exploiting potential applications of antimicrobial peptides. We highlight 3D structure-based design of peptide antimicrobials and vaccines, surface coating, delivery systems, and microbial detection devices involving antimicrobial peptides. The 2014 results also support that combination therapy is preferred over monotherapy in treating biofilms. PMID:25806720

  20. Antimicrobial peptides in 2014.

    PubMed

    Wang, Guangshun; Mishra, Biswajit; Lau, Kyle; Lushnikova, Tamara; Golla, Radha; Wang, Xiuqing

    2015-01-01

    This article highlights new members, novel mechanisms of action, new functions, and interesting applications of antimicrobial peptides reported in 2014. As of December 2014, over 100 new peptides were registered into the Antimicrobial Peptide Database, increasing the total number of entries to 2493. Unique antimicrobial peptides have been identified from marine bacteria, fungi, and plants. Environmental conditions clearly influence peptide activity or function. Human α-defensin HD-6 is only antimicrobial under reduced conditions. The pH-dependent oligomerization of human cathelicidin LL-37 is linked to double-stranded RNA delivery to endosomes, where the acidic pH triggers the dissociation of the peptide aggregate to release its cargo. Proline-rich peptides, previously known to bind to heat shock proteins, are shown to inhibit protein synthesis. A model antimicrobial peptide is demonstrated to have multiple hits on bacteria, including surface protein delocalization. While cell surface modification to decrease cationic peptide binding is a recognized resistance mechanism for pathogenic bacteria, it is also used as a survival strategy for commensal bacteria. The year 2014 also witnessed continued efforts in exploiting potential applications of antimicrobial peptides. We highlight 3D structure-based design of peptide antimicrobials and vaccines, surface coating, delivery systems, and microbial detection devices involving antimicrobial peptides. The 2014 results also support that combination therapy is preferred over monotherapy in treating biofilms. PMID:25806720

  1. A novel sea anemone peptide that inhibits acid-sensing ion channels.

    PubMed

    Rodríguez, Armando Alexei; Salceda, Emilio; Garateix, Anoland Georgina; Zaharenko, André Junqueira; Peigneur, Steve; López, Omar; Pons, Tirso; Richardson, Michael; Díaz, Maylín; Hernández, Yasnay; Ständker, Ludger; Tytgat, Jan; Soto, Enrique

    2014-03-01

    Sea anemones produce ion channels peptide toxins of pharmacological and biomedical interest. However, peptides acting on ligand-gated ion channels, including acid-sensing ion channel (ASIC) toxins, remain poorly explored. PhcrTx1 is the first compound characterized from the sea anemone Phymanthus crucifer, and it constitutes a novel ASIC inhibitor. This peptide was purified by gel filtration, ion-exchange and reversed-phase chromatography followed by biological evaluation on ion channels of isolated rat dorsal root ganglia (DRG) neurons using patch clamp techniques. PhcrTx1 partially inhibited ASIC currents (IC50∼100 nM), and also voltage-gated K(+) currents but the effects on the peak and on the steady state currents were lower than 20% in DRG neurons, at concentrations in the micromolar range. No significant effect was observed on Na(+) voltage-gated currents in DRG neurons. The N-terminal sequencing yielded 32 amino acid residues, with a molecular mass of 3477 Da by mass spectrometry. No sequence identity to other sea anemone peptides was found. Interestingly, the bioinformatic analysis of Cys-pattern and secondary structure arrangement suggested that this peptide presents an Inhibitor Cystine Knot (ICK) scaffold, which has been found in other venomous organisms such as spider, scorpions and cone snails. Our results show that PhcrTx1 represents the first member of a new structural group of sea anemones toxins acting on ASIC and, with much lower potency, on Kv channels. Moreover, this is the first report of an ICK peptide in cnidarians, suggesting that the occurrence of this motif in venomous animals is more ancient than expected. PMID:23764262

  2. Nanoparticle Formation from Hybrid, Multiblock Copolymers of Poly(Acrylic Acid) and VPGVG Peptide

    PubMed Central

    Grieshaber, Sarah E.; Paik, Bradford A.; Bai, Shi; Kiick, Kristi L.; Jia, Xinqiao

    2012-01-01

    Elastin-mimetic hybrid copolymers with an alternating molecular architecture were synthesized via the step growth polymerization of azide-functionalized, telechelic poly(tert-butyl acrylate) (PtBA) and an alkyne-terminated, valine and glycine-rich peptide with a sequence of (VPGVG)2 (VG2). The resultant hybrid copolymer, [PtBA-VG2]n, contains up to six constituent building blocks and has a polydispersity index (PDI) of ~1.9. Trifluoroacetic acid (TFA) treatment of [PtBA-VG2]n gave rise to an alternating copolymer of poly(acrylic acid) (PAA) and VG2 ([PAA-VG2]n). The modular design permits facile adjustment of the copolymer composition by varying the molecular weight of PAA (22 and 63 repeat units). Characterization by dynamic light scattering indicated that the multiblock copolymers formed discrete nanoparticles at room temperature in aqueous solution at pH 3.8, with an average diameter of 250-270 nm and a particle size distribution of 0.34 for multiblock copolymers containing PAA22 and 0.17 for those containing PAA63. Upon increasing the pH to 7.4, both types of particles were able to swell without being disintegrated, reaching an average diameter of 285-300 nm for [PAA22-VG2]n and 330-350 nm for [PAA63-VG2]n, respectively. The nanoparticles were not dissociated upon the addition of urea, further confirming their unusual stability. The nanoparticles were capable of sequestering a hydrophobic fluorescent dye (pyrene), and the critical aggregation concentration (CAC) was determined to be 1.09 × 10-2 or 1.05 × 10-2 mg/mL for [PAA22-VG2]n and [PAA63-VG2]n, respectively. We suggest that the multiblock copolymers form through collective H-bonding and hydrophobic interactions between the PAA and VG2 peptide units, and that the unusual stability of the multiblock nanoparticles is conferred by the multiblock architecture. These hybrid multiblock copolymers are potentially useful as pH-responsive drug delivery vehicles, with the possibility of drug loading through

  3. Cell Penetrating Peptide Conjugated Chitosan for Enhanced Delivery of Nucleic Acid

    PubMed Central

    Layek, Buddhadev; Lipp, Lindsey; Singh, Jagdish

    2015-01-01

    Gene therapy is an emerging therapeutic strategy for the cure or treatment of a spectrum of genetic disorders. Nevertheless, advances in gene therapy are immensely reliant upon design of an efficient gene carrier that can deliver genetic cargoes into the desired cell populations. Among various nonviral gene delivery systems, chitosan-based carriers have gained increasing attention because of their high cationic charge density, excellent biocompatibility, nearly nonexistent cytotoxicity, negligible immune response, and ideal ability to undergo chemical conjugation. However, a major shortcoming of chitosan-based carriers is their poor cellular uptake, leading to inadequate transfection efficiency. The intrinsic feature of cell penetrating peptides (CPPs) for transporting diverse cargoes into multiple cell and tissue types in a safe manner suggests that they can be conjugated to chitosan for improving its transfection efficiency. In this review, we briefly discuss CPPs and their classification, and also the major mechanisms contributing to the cellular uptake of CPPs and cargo conjugates. We also discuss immense improvements for the delivery of nucleic acids using CPP-conjugated chitosan-based carriers with special emphasis on plasmid DNA and small interfering RNA. PMID:26690119

  4. Fluorescence imaging of siRNA delivery by peptide nucleic acid-based probe.

    PubMed

    Sato, Takaya; Sato, Yusuke; Iwai, Kenta; Kuge, Shusuke; Teramae, Norio; Nishizawa, Seiichi

    2015-01-01

    We report on the use of a peptide nucleic acid (PNA)-based fluorescent probe for the analysis of siRNA delivery to living cells. The probe, Py-AA-TO, possesses thiazole orange (TO) and pyrene moieties in the C- and N-termini of PNA, and can function as a light-up probe capable of selective binding to 3'-overhanging nucleotides of target siRNAs. The affinity-labeling of the siRNAs with Py-AA-TO facilitates fluorescence imaging of cellular uptake of polymer-based carriers encapsulating the siRNAs (polyplexes) through endocytosis and subsequent sequestration into lysosome. In addition, flow cytometric measurements reveal that the monitoring of Py-AA-TO fluorescence inside the cells is successfully applicable to the analysis of the polyplex disassembly. These promising functions of Py-AA-TO are presented and discussed as a basis for the design of molecular probes for fluorescent imaging and quantitative analysis of the siRNA delivery process. PMID:25864675

  5. A Sensitive Peptide Nucleic Acid Probe Assay for Detection of BRAF V600 Mutations in Melanoma.

    PubMed

    Chen, Tai-Long; Chang, John Wen-Cheng; Hsieh, Jia-Juan; Cheng, Hsin-Yi; Chiou, Chiuan-Chian

    Mutated v-Raf murine sarcoma viral oncogene homolog B (BRAF) is an important biomarker for the prediction of therapeutic efficacy of several anticancer drugs. The detection of BRAF mutation faces two challenges: Firstly, there are multiple types of mutations, and secondly, tumor samples usually contain various amounts of wild-type, normal tissues. Here, we describe a newly established method for sensitive detection of multiple types of BRAF V600 mutations in excess wild-type background. The method introduced a fluorophore-tagged peptide nucleic acid (PNA) to serve as both polymerase chain reaction (PCR) clamp and sensor probe, which inhibited the amplification of wild-type templates during PCR and revealed multiple types of mutant signals during melting analysis. We demonstrated the design and optimization process of the method, and applied it in the detection of BRAF mutations in 49 melanoma samples. This PNA probe assay method detected three types of mutations in 17 samples, and was much more sensitive than conventional PCR plus Sanger sequencing. PMID:27566656

  6. Structure-based design of small peptide inhibitors of protein kinase CK2 subunit interaction

    PubMed Central

    Laudet, Béatrice; Barette, Caroline; Dulery, Vincent; Renaudet, Olivier; Dumy, Pascal; Metz, Alexandra; Prudent, Renaud; Deshiere, Alexandre; Dideberg, Otto; Filhol, Odile; Cochet, Claude

    2007-01-01

    X-ray crystallography studies, as well as live-cell fluorescent imaging, have recently challenged the traditional view of protein kinase CK2. Unbalanced expression of catalytic and regulatory CK2 subunits has been observed in a variety of tissues and tumours. Thus the potential intersubunit flexibility suggested by these studies raises the likely prospect that the CK2 holoenzyme complex is subject to disassembly and reassembly. In the present paper, we show evidence for the reversible multimeric organization of the CK2 holoenzyme complex in vitro. We used a combination of site-directed mutagenesis, binding experiments and functional assays to show that, both in vitro and in vivo, only a small set of primary hydrophobic residues of CK2β which contacts at the centre of the CK2α/CK2β interface dominates affinity. The results indicate that a double mutation in CK2β of amino acids Tyr188 and Phe190, which are complementary and fill up a hydrophobic pocket of CK2α, is the most disruptive to CK2α binding both in vitro and in living cells. Further characterization of hotspots in a cluster of hydrophobic amino acids centred around Tyr188–Phe190 led us to the structure-based design of small-peptide inhibitors. One conformationally constrained 11-mer peptide (Pc) represents a unique CK2β-based small molecule that was particularly efficient (i) to antagonize the interaction between the CK2 subunits, (ii) to inhibit the assembly of the CK2 holoenzyme complex, and (iii) to strongly affect its substrate preference. PMID:17714077

  7. Structure-based design of small peptide inhibitors of protein kinase CK2 subunit interaction.

    PubMed

    Laudet, Béatrice; Barette, Caroline; Dulery, Vincent; Renaudet, Olivier; Dumy, Pascal; Metz, Alexandra; Prudent, Renaud; Deshiere, Alexandre; Dideberg, Otto; Filhol, Odile; Cochet, Claude

    2007-12-15

    X-ray crystallography studies, as well as live-cell fluorescent imaging, have recently challenged the traditional view of protein kinase CK2. Unbalanced expression of catalytic and regulatory CK2 subunits has been observed in a variety of tissues and tumours. Thus the potential intersubunit flexibility suggested by these studies raises the likely prospect that the CK2 holoenzyme complex is subject to disassembly and reassembly. In the present paper, we show evidence for the reversible multimeric organization of the CK2 holoenzyme complex in vitro. We used a combination of site-directed mutagenesis, binding experiments and functional assays to show that, both in vitro and in vivo, only a small set of primary hydrophobic residues of CK2beta which contacts at the centre of the CK2alpha/CK2beta interface dominates affinity. The results indicate that a double mutation in CK2beta of amino acids Tyr188 and Phe190, which are complementary and fill up a hydrophobic pocket of CK2alpha, is the most disruptive to CK2alpha binding both in vitro and in living cells. Further characterization of hotspots in a cluster of hydrophobic amino acids centred around Tyr188-Phe190 led us to the structure-based design of small-peptide inhibitors. One conformationally constrained 11-mer peptide (Pc) represents a unique CK2beta-based small molecule that was particularly efficient (i) to antagonize the interaction between the CK2 subunits, (ii) to inhibit the assembly of the CK2 holoenzyme complex, and (iii) to strongly affect its substrate preference. PMID:17714077

  8. Biological Activity of Aminophosphonic Acids and Their Short Peptides

    NASA Astrophysics Data System (ADS)

    Lejczak, Barbara; Kafarski, Pawel

    The biological activity and natural occurrence of the aminophosphonic acids were described half a century ago. Since then the chemistry and biology of this class of compounds have developed into the separate field of phosphorus chemistry. Today it is well acknowledged that these compounds possess a wide variety of promising, and in some cases commercially useful, physiological activities. Thus, they have found applications ranging from agrochemical (with the herbicides glyphosate and bialaphos being the most prominent examples) to medicinal (with the potent antihypertensive fosinopril and antiosteoporetic bisphosphonates being examples).

  9. Amino Acid Metaclusters: Implications of Growth Trends on Peptide Self-Assembly and Structure.

    PubMed

    Do, Thanh D; de Almeida, Natália E C; LaPointe, Nichole E; Chamas, Ali; Feinstein, Stuart C; Bowers, Michael T

    2016-01-01

    Ion-mobility mass spectrometry is utilized to examine the metacluster formation of serine, asparagine, isoleucine, and tryptophan. These amino acids are representative of different classes of noncharged amino acids. We show that they can form relatively large metaclusters in solution that are difficult or impossible to observe by traditional solution techniques. We further demonstrate, as an example, that the formation of Ser metaclusters is not an ESI artifact because large metaclusters can be detected in negative polarity and low concentration with similar cross sections to those measured in positive polarity and higher concentration. The growth trends of tryptophan and isoleucine metaclusters, along with serine, asparagine, and the previously studied phenylalanine, are balanced among various intrinsic properties of individual amino acids (e.g., hydrophobicity, size, and shape). The metacluster cross sections of hydrophilic residues (Ser, Asn, Trp) tend to stay on or fall below the isotropic model trend lines whereas those of hydrophobic amino acids (Ile, Phe) deviate positively from the isotropic trend lines. The growth trends correlate well to the predicted aggregation propensity of individual amino acids. From the metacluster data, we introduce a novel approach to score and predict aggregation propensity of peptides, which can offer a significant improvement over the existing methods in terms of accuracy. Using a set of hexapeptides, we show that the strong negative deviations of Ser metaclusters from the isotropic model leads a prediction of microcrystalline formation for the SFSFSF peptide, whereas the strong positive deviation of Ile leads to prediction or fibril formation for the NININI peptide. Both predictions are confirmed experimentally using ion mobility and TEM measurements. The peptide SISISI is predicted to only weakly aggregate, a prediction confirmed by TEM. PMID:26632663

  10. Peptide sequencing by using a combination of partial acid hydrolysis and fast-atom-bombardment mass spectrometry.

    PubMed Central

    De Angelis, F; Botta, M; Ceccarelli, S; Nicoletti, R

    1986-01-01

    To overcome the limit of the intensity of ions carrying sequence information in structural determinations of peptides by fast-atom-bombardment m.s., we have developed a method that consists in taking spectra of the peptide acid hydrolysates at different hydrolysis times. Peaks correspond to the oligomers arising from the peptide partial hydrolysis. The sequence can then be identified from the structurally overlapping fragments. PMID:2428356

  11. Coupling molecular dynamics simulations with experiments for the rational design of indolicidin-analogous antimicrobial peptides.

    PubMed

    Tsai, Ching-Wei; Hsu, Ning-Yi; Wang, Chang-Hsu; Lu, Chia-Yu; Chang, Yung; Tsai, Hui-Hsu Gavin; Ruaan, Rouh-Chyu

    2009-09-25

    of the erythrocyte membrane. In addition, through simulations, we identified the amino acids that are most responsible for the adsorption onto or insertion into the two model membranes. Positive charges are critical to the peptide's adsorption, whereas the presence of hydrophobic Trp8 and Trp9 leads to its deeper insertion. Combining the hypothetical relationships between the membrane disordering and the antimicrobial and hemolytical activities with the simulated results, we designed three new IL-analogous peptides: IL-K7 (Pro7-->Lys), IL-F89 (Trp8 and Trp9-->Phe), and IL-K7F89 (Pro7-->Lys; Trp8 and Trp9-->Phe). The hemolytic activity of IL-F89 is considerably lower than that of IL, whereas the antimicrobial activity of IL-K7 is greatly enhanced. In particular, the de novo peptide IL-K7F89 exhibits higher antimicrobial activity against Escherichia coli; its hemolytic activity decreased to only 10% of that of IL. Our simulated and experimental results correlated well. This approach-coupling MD simulations with experimental design-is a useful strategy toward the rational design of AMPs for potential therapeutic use. PMID:19576903

  12. Fatty acid composition modulates sensitivity of Legionella pneumophila to warnericin RK, an antimicrobial peptide.

    PubMed

    Verdon, Julien; Labanowski, Jérome; Sahr, Tobias; Ferreira, Thierry; Lacombe, Christian; Buchrieser, Carmen; Berjeaud, Jean-Marc; Héchard, Yann

    2011-04-01

    Warnericin RK is an antimicrobial peptide, produced by a Staphyloccocus warneri strain, described to be specifically active against Legionella, the pathogenic bacteria responsible for Legionnaires' disease. Warnericin RK is an amphiphilic alpha-helical peptide, which possesses a detergent-like mode of action. Two others peptides, δ-hemolysin I and II, produced by the same S. warneri strain, are highly similar to S. aureus δ-hemolysin and also display anti-Legionella activity. It has been recently reported that S. aureus δ-hemolysin activity on vesicles is likewise related to phospholipid acyl-chain structure, such as chain length and saturation. As staphylococcal δ-hemolysins were highly similar, we thus hypothesized that fatty acid composition of Legionella's membrane might influence the sensitivity of the bacteria to warnericin RK. Relationship between sensitivity to the peptide and fatty acid composition was then followed in various conditions. Cells in stationary phase, which were already described as less resistant than cells in exponential phase, displayed higher amounts of branched-chain fatty acids (BCFA) and short chain fatty acids. An adapted strain, able to grow at a concentration 33 fold higher than minimal inhibitory concentration of the wild type (i.e. 1μM), was isolated after repeated transfers of L. pneumophila in the presence of increased concentrations of warnericin RK. The amount of BCFA was significantly higher in the adapted strain than in the wild type strain. Also, a transcriptomic analysis of the wild type and adapted strains showed that two genes involved in fatty acid biosynthesis were repressed in the adapted strain. These genes encode enzymes involved in desaturation and elongation of fatty acids respectively. Their repression was in agreement with the decrease of unsaturated fatty acids and fatty acid chain length in the adapted strain. Conclusively, our results indicate that the increase of BCFA and the decrease of fatty acid

  13. Self-Assembled Antibody Multimers through Peptide Nucleic Acid Conjugation

    PubMed Central

    Kazane, Stephanie A.; Axup, Jun Y; Kim, Chan Hyuk; Ciobanu, Mihai; Wold, Erik D.; Barluenga, Sofia; Hutchins, Benjamin A.; Schultz, Peter G.; Winssinger, Nicolas; Smider, Vaughn V.

    2013-01-01

    With the recent clinical success of bispecific antibodies, a strategy to rapidly synthesize and evaluate bispecific or higher order multispecific molecules could facilitate the discovery of new therapeutic agents. Here we show that unnatural amino acids (UAAs) with orthogonal chemical reactivity can be used to generate site-specific antibody-oligonucleotide conjugates. These constructs can then be self-assembled into multimeric complexes with defined composition, valency and geometry. Using this approach, we generated potent bispecific antibodies that recruit cytotoxic T lymphocytes to Her2 and CD20 positive cancer cells, as well as multimeric antibody fragments with enhanced activity. This strategy should accelerate the synthesis and in vitro characterization of antibody constructs with unique specificities and molecular architectures. PMID:23210862

  14. Cation-halide transport through peptide pores containing aminopicolinic acid.

    PubMed

    Basak, Debajyoti; Sridhar, Sucheta; Bera, Amal K; Madhavan, Nandita

    2016-05-18

    Synthetic pores that selectively transport ions of biological significance through membranes could be potentially used in medical diagnostics or therapeutics. Herein, we report cation-selective octapeptide pores derived from alanine and aminopicolinic acid. The ion transport mechanism through the pores has been established to be a cation-chloride symport. The cation-chloride co-transport is biologically essential for the efficient functioning of the central nervous system and has been implicated in diseases such as epilepsy. The pores formed in synthetic lipid bilayers do not exhibit any closing events. The ease of synthesis as well as infinite lifetimes of these pores provides scope for modifying their transport behaviour to develop sensors. PMID:27137995

  15. Design and Characterization of a Peptide Mimotope of the HIV-1 gp120 Bridging Sheet

    PubMed Central

    Schiavone, Marco; Fiume, Giuseppe; Caivano, Antonella; de Laurentiis, Annamaria; Falcone, Cristina; Masci, Francesca Fasanella; Iaccino, Enrico; Mimmi, Selena; Palmieri, Camillo; Pisano, Antonio; Pontoriero, Marilena; Rossi, Annalisa; Scialdone, Annarita; Vecchio, Eleonora; Andreozzi, Concetta; Trovato, Maria; Rafay, Jan; Ferko, Boris; Montefiori, David; Lombardi, Angela; Morsica, Giulia; Poli, Guido; Quinto, Ileana; Pavone, Vincenzo; de Berardinis, Piergiuseppe; Scala, Giuseppe

    2012-01-01

    The Bridging Sheet domain of HIV-1 gp120 is highly conserved among the HIV-1 strains and allows HIV-1 binding to host cells via the HIV-1 coreceptors. Further, the bridging sheet domain is a major target to neutralize HIV-1 infection. We rationally designed four linear peptide epitopes that mimic the three-dimensional structure of bridging sheet by using molecular modeling. Chemically synthesized peptides BS3 and BS4 showed a fair degree of antigenicity when tested in ELISA with IgG purified from HIV+ broadly neutralizing sera while the production of synthetic peptides BS1 and BS2 failed due to their high degree of hydrophobicity. To overcome this limitation, we linked all four BS peptides to the COOH-terminus of GST protein to test both their antigenicity and immunogenicity. Only the BS1 peptide showed good antigenicity; however, no envelope specific antibodies were elicited upon mice immunization. Therefore we performed further analyses by linking BS1 peptide to the NH2-terminus of the E2 scaffold from the Geobacillus Stearothermophylus PDH complex. The E2-BS1 fusion peptide showed good antigenic results, however only one immunized rabbit elicited good antibody titers towards both the monomeric and oligomeric viral envelope glycoprotein (Env). In addition, moderate neutralizing antibodies response was elicited against two HIV-1 clade B and one clade C primary isolates. These preliminary data validate the peptide mimotope approach as a promising tool to obtain an effective HIV-1 vaccine. PMID:22754323

  16. Enzymatic generation of peptides flanked by basic amino acids to obtain MS/MS spectra with 2× sequence coverage

    PubMed Central

    Ebhardt, H Alexander; Nan, Jie; Chaulk, Steven G; Fahlman, Richard P; Aebersold, Ruedi

    2014-01-01

    RATIONALE Tandem mass (MS/MS) spectra generated by collision-induced dissociation (CID) typically lack redundant peptide sequence information in the form of e.g. b- and y-ion series due to frequent use of sequence-specific endopeptidases cleaving C- or N-terminal to Arg or Lys residues. METHODS Here we introduce arginyl-tRNA protein transferase (ATE, EC 2.3.2.8) for proteomics. ATE recognizes acidic amino acids or oxidized Cys at the N-terminus of a substrate peptide and conjugates an arginine from an aminoacylated tRNAArg onto the N-terminus of the substrate peptide. This enzymatic reaction is carried out under physiological conditions and, in combination with Lys-C/Asp-N double digest, results in arginylated peptides with basic amino acids on both termini. RESULTS We demonstrate that in vitro arginylation of peptides using yeast arginyl tRNA protein transferase 1 (yATE1) is a robust enzymatic reaction, specific to only modifying N-terminal acidic amino acids. Precursors originating from arginylated peptides generally have an increased protonation state compared with their non-arginylated forms. Furthermore, the product ion spectra of arginylated peptides show near complete 2× fragment ladders within the same MS/MS spectrum using commonly available electrospray ionization peptide fragmentation modes. Unexpectedly, arginylated peptides generate complete y- and c-ion series using electron transfer dissociation (ETD) despite having an internal proline residue. CONCLUSIONS We introduce a rapid enzymatic method to generate peptides flanked on either terminus by basic amino acids, resulting in a rich, redundant MS/MS fragment pattern. © 2014 The Authors. Rapid Communications in Mass Spectrometry published by John Wiley & Sons Ltd. PMID:25380496

  17. Designing peptidic inhibitors of serum amyloid A aggregation process.

    PubMed

    Sosnowska, Marta; Skibiszewska, Sandra; Kamińska, Emilia; Wieczerzak, Ewa; Jankowska, Elżbieta

    2016-04-01

    Amyloid A amyloidosis is a life-threatening complication of a wide range of chronic inflammatory, infectious and neoplastic diseases, and the most common form of systemic amyloidosis worldwide. It is characterized by extracellular tissue deposition of fibrils that are composed of fragments of serum amyloid A protein (SAA), a major acute-phase reactant protein, produced predominantly by hepatocytes. Currently, there are no approved therapeutic agents directed against the formation of fibrillar SAA assemblies. We attempted to develop peptidic inhibitors based on their similarity and complementarity to the regions critical for SAA self-association, which they should interact with and block their assembly into amyloid fibrils. Inh1 and inh4 which are comprised of the residues from the amyloidogenic region of SAA1.1 protein and Aβ peptide, respectively, were found by us as capable to significantly suppress aggregation of the SAA1-12 peptide. It was chosen as an aggregation model that mimicks the amyloidogenic nucleus of SAA protein. We suppose that aromatic interactions may be responsible for inhibitory activity of both compounds. We also recognized that aromatic residues are involved in self-association of SAA1-12. PMID:26759015

  18. Chiral Heteroditopic Baskets Designed from Triazolated Calixarenes and Short Peptides.

    PubMed

    Gorbunov, Alexander; Sokolova, Nadezhda; Kudryashova, Elena; Nenajdenko, Valentine; Kovalev, Vladimir; Vatsouro, Ivan

    2016-08-22

    Cone calix[4]arenes and calix[6]arenes bearing two, three, and four short peptide units each having two chiral carbon atoms were prepared. The syntheses were performed by using an efficient modular approach that includes the Ugi preparation of the azido-peptide followed by its reactions with the propargylated calixarenes under CuAAC (Cu(I) -catalyzed azide-alkyne cycloaddition) conditions. The three novel multitopic hosts were probed for their ability to bind metal ions by UV titration, and showed the highest complexation efficiency towards copper(II) and lead(II). These two cations possessed quite different complexation modes with copper(II) bound predominantly by multiple-triazole sites, in contrast to lead(II), which is stabilized mainly by multiple interactions with amide groups of the peptide units. Circular dichroism data for the free chiral hosts, their equimolar mixtures with copper(II) perchlorate and lead(II) perchlorate, and for tertiary mixtures of all three compounds showed the formation of mono- and binuclear complexes, or a switching behavior, depending on the structure of the host and the addition order of the cations. PMID:27444143

  19. Design of an epitope-based peptide vaccine against spike protein of human coronavirus: an in silico approach

    PubMed Central

    Oany, Arafat Rahman; Emran, Abdullah-Al; Jyoti, Tahmina Pervin

    2014-01-01

    Human coronavirus (HCoV), a member of Coronaviridae family, is the causative agent of upper respiratory tract infections and “atypical pneumonia”. Despite severe epidemic outbreaks on several occasions and lack of antiviral drug, not much progress has been made with regard to an epitope-based vaccine designed for HCoV. In this study, a computational approach was adopted to identify a multiepitope vaccine candidate against this virus that could be suitable to trigger a significant immune response. Sequences of the spike proteins were collected from a protein database and analyzed with an in silico tool, to identify the most immunogenic protein. Both T cell immunity and B cell immunity were checked for the peptides to ensure that they had the capacity to induce both humoral and cell-mediated immunity. The peptide sequence from 88–94 amino acids and the sequence KSSTGFVYF were found as the most potential B cell and T cell epitopes, respectively. Furthermore, conservancy analysis was also done using in silico tools and showed a conservancy of 64.29% for all epitopes. The peptide sequence could interact with as many as 16 human leukocyte antigens (HLAs) and showed high cumulative population coverage, ranging from 75.68% to 90.73%. The epitope was further tested for binding against the HLA molecules, using in silico docking techniques, to verify the binding cleft epitope interaction. The allergenicity of the epitopes was also evaluated. This computational study of design of an epitope-based peptide vaccine against HCoVs allows us to determine novel peptide antigen targets in spike proteins on intuitive grounds, albeit the preliminary results thereof require validation by in vitro and in vivo experiments. PMID:25187696

  20. Conformational flexibility in designing peptides for immunology: the molecular dynamics approach.

    PubMed

    Stavrakoudis, Athanassios

    2010-09-01

    Computational modeling techniques and computer simulations have become a routine in biological sciences and have gained great attention from researchers. Molecular dynamics simulation is a valuable tool towards an understanding of the complex structure of biological systems, especially in the study of the flexibility of the biological molecules such as peptides or proteins. Peptides play a very important role in human physiology and control many of the processes involved in the immune system response. Designing new and optimal peptide vaccines is one of the hottest challenges of the 21(st) century science and it brings together researchers from different fields. Molecular dynamics simulations have proven to be a helpful tool assisting laboratory work, saving financial sources and opening possibilities for exploring properties of the molecular systems that are hardly accessible by conventional experimental methods. Present review is dedicated to the recent contributions in applications of molecular dynamics simulations in peptide design for immunological purposes, such as B or T cell epitopes. PMID:20412039

  1. Synthesis of all nineteen appropriately protected chiral alpha-hydroxy acid equivalents of the alpha-amino acids for Boc solid-phase depsi-peptide synthesis.

    PubMed

    Deechongkit, Songpon; You, Shu-Li; Kelly, Jeffery W

    2004-02-19

    [reaction: see text] The preparation of depsi-peptides, amide-to-ester-substituted peptides used to probe the role of hydrogen bonding in protein folding energetics, is accomplished by replacing specific l-alpha-amino acid residues by their alpha-hydroxy acid counterparts in a solid-phase synthesis employing a t-Boc strategy. Herein we describe the efficient stereoselective synthesis of all 19 appropriately protected alpha-hydroxy acid equivalents of the l-alpha-amino acids, employing commercially available materials, expanding the number of available alpha-hydroxy acids from 9 to 19. PMID:14961607

  2. Combined Bioinformatic and Rational Design Approach To Develop Antimicrobial Peptides against Mycobacterium tuberculosis.

    PubMed

    Pearson, C Seth; Kloos, Zachary; Murray, Brian; Tabe, Ebot; Gupta, Monica; Kwak, Jun Ha; Karande, Pankaj; McDonough, Kathleen A; Belfort, Georges

    2016-05-01

    Drug-resistant pathogens are a growing problem, and novel strategies are needed to combat this threat. Among the most significant of these resistant pathogens is Mycobacterium tuberculosis, which is an unusually difficult microbial target due to its complex membrane. Here, we design peptides for specific activity against M. tuberculosis using a combination of "database filtering" bioinformatics, protein engineering, and de novo design. Several variants of these peptides are structurally characterized to validate the design process. The designed peptides exhibit potent activity (MIC values as low as 4 μM) against M. tuberculosis and also exhibit broad activity against a host of other clinically relevant pathogenic bacteria such as Gram-positive bacteria (Streptococcus) and Gram-negative bacteria (Escherichia coli). They also display excellent selectivity, with low cytotoxicity against cultured macrophages and lung epithelial cells. These first-generation antimicrobial peptides serve as a platform for the design of antibiotics and for investigating structure-activity relationships in the context of the M. tuberculosis membrane. The antimicrobial peptide design strategy is expected to be generalizable for any pathogen for which an activity database can be created. PMID:26902758

  3. Design of self-assembling peptide hydrogelators amenable to bacterial expression.

    PubMed

    Sonmez, Cem; Nagy, Katelyn J; Schneider, Joel P

    2015-01-01

    Hydrogels formed from self-assembling peptides are finding use in tissue engineering and drug delivery applications. Given the notorious difficulties associated with producing self-assembling peptides by recombinant expression, most are typically prepared by chemical synthesis. Herein, we report the design of a family of self-assembling β-hairpin peptides amenable to efficient production using an optimized bacterial expression system. Expressing peptides, EX1, EX2 and EX3 contain identical eight-residue amphiphilic β-strands connected by varying turn sequences that are responsible for ensuring chain reversal and the proper intramolecular folding and consequent self-assembly of the peptide into a hydrogel network under physiological conditions. EX1 was initially used to establish and optimize the bacterial expression system by which all the peptides could be eventually individually expressed. Expression clones were designed to allow exploration of possible fusion partners and investigate both enzymatic and chemical cleavage as means to liberate the target peptide. A systematic analysis of possible expression systems followed by fermentation optimization lead to a system in which all three peptides could be expressed as fusions with BAD-BH3, the BH3 domain of the proapoptotic BAD (Bcl-2 Associated Death) Protein. CNBr cleavage followed by purification afforded 50, 31, and 15 mg/L yields of pure EX1, EX2 and EX3, respectively. CD spectroscopy, TEM, and rheological analysis indicate that these peptides fold and assembled into well-defined fibrils that constitute hydrogels having shear-thin/recovery properties. PMID:25453938

  4. Design of Self-Assembling Peptide Hydrogelators Amenable to Bacterial Expression

    PubMed Central

    Sonmez, Cem; Nagy, Katelyn J.; Schneider, Joel P.

    2014-01-01

    Hydrogels formed from self-assembling peptides are finding use in tissue engineering and drug delivery applications. Given the notorious difficulties associated with producing self-assembling peptides by recombinant expression, most are typically prepared by chemical synthesis. Herein, we report the design of a family of self-assembling β-hairpin peptides amenable to efficient production using an optimized bacterial expression system. Expressing peptides, EX1, EX2 and EX3 contain identical eight-residue amphiphilic β-strands connected by varying turn sequences that are responsible for ensuring chain reversal and the proper intramolecular folding and consequent self-assembly of the peptide into a hydrogel network under physiological conditions. EX1 was initially used to establish and optimize the bacterial expression system by which all the peptides could be eventually individually expressed. Expression clones were designed to allow exploration of possible fusion partners and investigate both enzymatic and chemical cleavage as means to liberate the target peptide. A systematic analysis of possible expression systems followed by fermentation optimization lead to a system in which all three peptides could be expressed as fusions with BAD-BH3, the BH3 domain of the proapoptotic BAD (Bcl-2 Associated Death) Protein. CNBr cleavage followed by purification afforded 50, 31, and 15 mg/L yields of pure EX1, EX2 and EX3, respectively. CD spectroscopy, TEM, and rheological analysis indicate that these peptides fold and assembled into well-defined fibrils that constitute hydrogels having shear-thin/recovery properties. PMID:25453938

  5. PLGA nanofibers blended with designer self-assembling peptides for peripheral neural regeneration.

    PubMed

    Nune, Manasa; Krishnan, Uma Maheswari; Sethuraman, Swaminathan

    2016-05-01

    Electrospun nanofibers are attractive candidates for neural regeneration due to similarity to the extracellular matrix. Several synthetic polymers have been used but they lack in providing the essential biorecognition motifs on their surfaces. Self-assembling peptide nanofiber scaffolds (SAPNFs) like RADA16 and recently, designer SAPs with functional motifs RADA16-I-BMHP1 areexamples, which showed successful spinal cord regeneration. But these peptide nanofiber scaffolds have poor mechanical properties and faster degradation rates that limit their use for larger nerve defects. Hence, we have developed a novel hybrid nanofiber scaffold of polymer poly(L-lactide-co-glycolide) (PLGA) and RADA16-I-BMHP1. The scaffolds were characterized for the presence of peptides both qualitatively and quantitatively using several techniques like SEM, EDX, FTIR, CHN analysis, Circular Dichroism analysis, Confocal and thermal analysis. Peptide self-assembly was retained post-electrospinning and formed rod-like nanostructures on PLGA nanofibers. In vitro cell compatibility was studied using rat Schwann cells and their adhesion, proliferation and gene expression levels on the designed scaffolds were evaluated. Our results have revealed the significant effects of the peptide blended scaffolds on promoting Schwann cell adhesion, extension and phenotypic expression. Neural development markers (SEM3F, NRP2 & PLX1) gene expression levels were significantly upregulated in peptide blended scaffolds compared to the PLGA scaffolds. Thus the hybrid blended novel designer scaffolds seem to be promising candidates for successful and functional regeneration of the peripheral nerve. PMID:26952431

  6. Rational Design of Peptide Vaccines Against Multiple Types of Human Papillomavirus

    PubMed Central

    Dey, Sumanta; De, Antara; Nandy, Ashesh

    2016-01-01

    Human papillomavirus (HPV) occurs in many types, some of which cause cervical, genital, and other cancers. While vaccination is available against the major cancer-causing HPV types, many others are not covered by these preventive measures. Herein, we present a bioinformatics study for the designing of multivalent peptide vaccines against multiple HPV types as an alternative strategy to the virus-like particle vaccines being used now. Our technique of rational design of peptide vaccines is expected to ensure stability of the vaccine against many cycles of mutational changes, elicit immune response, and negate autoimmune possibilities. Using the L1 capsid protein sequences, we identified several peptides for potential vaccine design for HPV 16, 18, 33, 35, 45, and 11 types. Although there are concerns about the epitope-binding affinities for the peptides identified in this process, the technique indicates possibilities of multivalent, adjuvanted, peptide vaccines against a wider range of HPV types, and tailor-made different combinations of the peptides to address frequency variations of types over different population groups as required for prophylaxis and at lower cost than are in use at the present time. PMID:27279731

  7. Rational Design of Peptide Vaccines Against Multiple Types of Human Papillomavirus.

    PubMed

    Dey, Sumanta; De, Antara; Nandy, Ashesh

    2016-01-01

    Human papillomavirus (HPV) occurs in many types, some of which cause cervical, genital, and other cancers. While vaccination is available against the major cancer-causing HPV types, many others are not covered by these preventive measures. Herein, we present a bioinformatics study for the designing of multivalent peptide vaccines against multiple HPV types as an alternative strategy to the virus-like particle vaccines being used now. Our technique of rational design of peptide vaccines is expected to ensure stability of the vaccine against many cycles of mutational changes, elicit immune response, and negate autoimmune possibilities. Using the L1 capsid protein sequences, we identified several peptides for potential vaccine design for HPV 16, 18, 33, 35, 45, and 11 types. Although there are concerns about the epitope-binding affinities for the peptides identified in this process, the technique indicates possibilities of multivalent, adjuvanted, peptide vaccines against a wider range of HPV types, and tailor-made different combinations of the peptides to address frequency variations of types over different population groups as required for prophylaxis and at lower cost than are in use at the present time. PMID:27279731

  8. Stoichiometric inhibition of amyloid beta-protein aggregation with peptides containing alternating alpha,alpha-disubstituted amino acids.

    PubMed

    Etienne, Marcus A; Aucoin, Jed P; Fu, Yanwen; McCarley, Robin L; Hammer, Robert P

    2006-03-22

    We have prepared two peptides based on the hydrophobic core (Lys-Leu-Val-Phe-Phe) of amyloid beta-protein (Abeta) that contain alpha,alpha-disubstituted amino acids at alternating positions, but differ in the positioning of the oligolysine chain (AMY-1, C-terminus; AMY-2, N-terminus). We have studied the effects of AMY-1 and AMY-2 on the aggregation of Abeta and find that, at stoichiometric concentrations, both peptides completely stop Abeta fibril growth. Equimolar mixtures of AMY-1 and Abeta form only globular aggregates as imaged by scanning force microscopy and transmission electron microscopy. These samples show no signs of protofibrillar or fibrillar material even after prolonged periods of time (4.5 months). Also, 10 mol % of AMY-1 prevents Abeta self-assembly for long periods of time; aged samples (4.5 months) show only a few protofibrillar or fibrillar aggregates. Circular dichroism spectroscopy of equimolar mixtures of AMY-1 and Abeta show that the secondary structure of the mixture changes over time and progresses to a predominantly beta-sheet structure, which is consistent with the design of these inhibitors preferring a sheet-like conformation. Changing the position of the charged tail on the peptide, AMY-2 interacts with Abeta differently in that equimolar mixtures form large ( approximately 1 mum) globular aggregates which do not progress to fibrils, but precipitate out of solution. The differences in the aggregation mediated by the two peptides is discussed in terms of a model where the inhibitors act as cosurfactants that interfere with the native ability of Abeta to self-assemble by disrupting hydrophobic interactions either at the C-terminus or N-terminus of Abeta. PMID:16536517

  9. Absorption of amino acids and peptides from a complex mixture in the isolated small intestine of the rat.

    PubMed Central

    Gardner, M L

    1975-01-01

    Amino acid and peptide absorption from a pancreatic digest of casein at low concentration by an isolated preparation of perfused rat small intestine has been measured. 2. The rate of absorption of each amino acid (free or peptide-bound) is closely proportional to its concentration in the perfusate; this implies a constant Vmax/Km ration for all amino acids in the mixture. 3. There is a high correlation between the compositions of luminal perfusate and secretion into the tissue fluid (apart from the content of glutamic and aspartic acids and alanine). 4. The concentrations of each free amino acid are, on average, 9 times as great in secretion as in lumen; the total peptide-N concentration in secretion is approximately 4 times that in the lumen. 5. The rate of absorption of each free amino acid is highly negatively dependent on the rate of absorption of that amino acid in peptide-bound form, in addition to being positively dependent on the perfusate concentration of free amino acid. 6. While peptide-bound proline appears to be well absorbed, free proline liberated by hydrolysis appears to pass back into the lumen as well as into the tissue fluid. Substantial back flux of hydrolysis products may occur for all amino acids. 7. About one-third of the amino acids appearing in the secretion on the serosal surface are peptide-bound. 8. The rate of absorption of peptides appears to determine the rate of their hydrolysis which probably occurs mainly after entry into the mucosal cells. PMID:1204629

  10. Amino acid absorption and homeostasis in mice lacking the intestinal peptide transporter PEPT1.

    PubMed

    Nässl, Anna-Maria; Rubio-Aliaga, Isabel; Fenselau, Henning; Marth, Mena Katharina; Kottra, Gabor; Daniel, Hannelore

    2011-07-01

    The intestinal peptide transporter PEPT1 mediates the uptake of di- and tripeptides derived from dietary protein breakdown into epithelial cells. Whereas the transporter appears to be essential to compensate for the reduced amino acid delivery in patients with mutations in amino acid transporter genes, such as in cystinuria or Hartnup disease, its physiological role in overall amino acid absorption is still not known. To assess the quantitative importance of PEPT1 in overall amino acid absorption and metabolism, PEPT1-deficient mice were studied by using brush border membrane vesicles, everted gut sacs, and Ussing chambers, as well as by transcriptome and proteome analysis of intestinal tissue samples. Neither gene expression nor proteome profiling nor functional analysis revealed evidence for any compensatory changes in the levels and/or function of transporters for free amino acids in the intestine. However, most plasma amino acid levels were increased in Pept1(-/-) compared with Pept1(+/+) animals, suggesting that amino acid handling is altered. Plasma appearance rates of (15)N-labeled amino acids determined after intragastric administration of a low dose of protein remained unchanged, whereas administration of a large protein load via gavage revealed marked differences in plasma appearance of selected amino acids. PEPT1 seems, therefore, important for overall amino acid absorption only after high dietary protein intake when amino acid transport processes are saturated and PEPT1 can provide additional absorption capacity. Since renal amino acid excretion remained unchanged, elevated basal concentrations of plasma amino acids in PEPT1-deficient animals seem to arise mainly from alterations in hepatic amino acid metabolism. PMID:21350187

  11. Negative electron-transfer dissociation nLC-MS/MS facilitates the analysis of thousands of acidic peptides

    PubMed Central

    McAlister, Graeme C.; Russell, Jason D.; Rumachik, Neil G.; Hebert, Alexander S.; Syka, John E. P.; Geer, Lewis Y.; Westphall, Michael S.; Pagliarini, David J.; Coon, Joshua J.

    2012-01-01

    We describe the first implementation of negative electron-transfer dissociation (NETD) on a hybrid ion trap-orbitrap mass spectrometer and its application to high-throughput sequencing of peptide anions. NETD – coupled with high pH separations, negative electrospray ionization (ESI), and an NETD compatible version of OMSSA – is part of a complete workflow that includes the formation, interrogation and sequencing of peptide anions. Together these interlocking pieces facilitated the identification of more than 2,000 unique peptides from Saccharomyces cerevisiae representing the most comprehensive analysis of peptide anions by tandem mass spectrometry to date. The same S. cerevisiae samples were interrogated using traditional, positive modes of peptide LC-MS/MS analysis (e.g., acidic LC separations, positive ESI, and collision activated dissociation), and the resulting peptide identifications of the different workflows were compared. Due to a decreased flux of peptide anions, and a tendency to produced lowly charged precursors, the NETD-based LC-MS/MS workflow was not as sensitive as the positive mode methods. However, the use of NETD readily permits access to underrepresented acidic portions of the proteome by identifying peptides that tended to have lower pI values. As such NETD improves sequence coverage, filling out the acidic portions of proteins that are often overlooked by the other methods. PMID:22335612

  12. A Peptide Mimetic of 5-Acetylneuraminic Acid-Galactose Binds with High Avidity to Siglecs and NKG2D

    PubMed Central

    Eggink, Laura L.; Spyroulias, Georgios A.; Jones, Norman G.; Hanson, Carl V.; Hoober, J. Kenneth

    2015-01-01

    We previously identified several peptide sequences that mimicked the terminal sugars of complex glycans. Using plant lectins as analogs of lectin-type cell-surface receptors, a tetravalent form of a peptide with the sequence NPSHPLSG, designated svH1C, bound with high avidity to lectins specific for glycans with terminal 5-acetylneuraminic acid (Neu5Ac)-galactose (Gal)/N-acetylgalactosamine (GalNAc) sequences. In this report, we show by circular dichroism and NMR spectra that svH1C lacks an ordered structure and thus interacts with binding sites from a flexible conformation. The peptide binds with high avidity to several recombinant human siglec receptors that bind preferentially to Neu5Ac(α2,3)Gal, Neu5Ac(α2,6)GalNAc or Neu5Ac(α2,8)Neu5Ac ligands. In addition, the peptide bound the receptor NKG2D, which contains a lectin-like domain that binds Neu5Ac(α2,3)Gal. The peptide bound to these receptors with a KD in the range of 0.6 to 1 μM. Binding to these receptors was inhibited by the glycoprotein fetuin, which contains multiple glycans that terminate in Neu5Ac(α2,3)Gal or Neu5Ac(α2,6)Gal, and by sialyllactose. Binding of svH1C was not detected with CLEC9a, CLEC10a or DC-SIGN, which are lectin-type receptors specific for other sugars. Incubation of neuraminidase-treated human peripheral blood mononuclear cells with svH1C resulted in binding of the peptide to a subset of the CD14+ monocyte population. Tyrosine phosphorylation of siglecs decreased dramatically when peripheral blood mononuclear cells were treated with 100 nM svH1C. Subcutaneous, alternate-day injections of svH1C into mice induced several-fold increases in populations of several types of immune cells in the peritoneal cavity. These results support the conclusion that svH1C mimics Neu5Ac-containing sequences and interacts with cell-surface receptors with avidities sufficient to induce biological responses at low concentrations. The attenuation of inhibitory receptors suggests that svH1C has

  13. Peptides in Receptor-Mediated Radiotherapy: From Design to the Clinical Application in Cancers

    PubMed Central

    Lozza, Catherine; Navarro-Teulon, Isabelle; Pèlegrin, André; Pouget, Jean-Pierre; Vivès, Eric

    2013-01-01

    Short peptides can show high affinity for specific receptors overexpressed on tumor cells. Some of these are already used in cancerology as diagnostic tools and others are in clinical trials for therapeutic applications. Therefore, peptides exhibit great potential as a diagnostic tool but also as an alternative or an additional antitumoral approach upon the covalent attachment of a therapeutic moiety such as a radionuclide or a cytotoxic drug. The chemistry offers flexibility to graft onto the targeting-peptide either fluorine or iodine directly, or metallic radionuclides through appropriate chelating agent. Since short peptides are straightforward to synthesize, there is an opportunity to further improve existing peptides or to design new ones for clinical applications. However, several considerations have to be taken into account to optimize the recognition properties of the targeting-peptide to its receptor, to improve its stability in the biological fluids and its residence in the body, or to increase its overall therapeutic effect. In this review, we highlight the different aspects which need to be considered for the development of an efficient peptide receptor-mediated radionuclide therapy in different neoplasms. PMID:24093086

  14. Structure-Based Design of Dendritic Peptide Bolaamphiphiles for siRNA Delivery

    PubMed Central

    2015-01-01

    Development of safe and effective delivery vectors is a critical challenge for the application of RNA interference (RNAi)-based biotechnologies. In this study we show the rational design of a series of novel dendritic peptide bolaamphiphile vectors that demonstrate high efficiency for the delivery of small interfering RNA (siRNA) while exhibiting low cytotoxicity and hemolytic activity. Systematic investigation into structure–property relationships revealed an important correlation between molecular design, self-assembled nanostructure, and biological activity. The unique bolaamphiphile architecture proved a key factor for improved complex stability and transfection efficiency. The optimal vector contains a fluorocarbon core and exhibited enhanced delivery efficiency to a variety of cell lines and improved serum resistance when compared to hydrocarbon analogues and lipofectamine RNAiMAX. In addition to introducing a promising new vector system for siRNA delivery, the structure–property relationships and “fluorocarbon effect” revealed herein offer critical insight for further development of novel materials for nucleic acid delivery and other biomaterial applications. PMID:26436138

  15. Design, synthesis and biological evaluation of novel peptide MC2 analogues from Momordica charantia as potential anti-diabetic agents.

    PubMed

    Yang, Baowei; Li, Xue; Zhang, Chenyu; Yan, Sijia; Wei, Wei; Wang, Xuekun; Deng, Xin; Qian, Hai; Lin, Haiyan; Huang, Wenlong

    2015-04-21

    Three series of Momordica charantia (MC)2 analogues were designed, synthesized and evaluated for their anti-hyperglycaemic effects. Alanine scanning focusing on the peptide MC2 indicated the importance of the residues proline (Pro)(3), serine (Ser)(6), isoleucine (Ile)(7) and Ser(10) for anti-hyperglycaemic effects. Among the first series of MC2 analogues, peptide I-4 exhibited a better anti-hyperglycaemic effect and was chosen for further modification. A further two series of conformationally constrained analogues were designed by scanning the residues Pro(3), Ser(6), Ile(7), and Ser(10) with an i - (i + 2) lactam bridge consisting of a glutamic acid-Xaa-lysine (Glu-Xaa-Lys) scaffold and a diproline fragment. By screening in normal mice and mice with diabetes mellitus, peptides II-1, II-2 and III-3 showed a significant improvement in anti-hyperglycaemic and anti-oxidative activities compared with I-4. These data suggest that II-1, II-2 and III-3 could be candidates for future treatment of diabetes mellitus. PMID:25778708

  16. Receptor Binding by Cholera Toxin B-Subunit and Amino Acid Modification Improves Minimal Peptide Immunogenicity

    PubMed Central

    Boberg, Andreas; Stålnacke, Alexandra; Bråve, Andreas; Hinkula, Jorma; Wahren, Britta; Carlin, Nils

    2012-01-01

    We increase our understanding of augmenting a cellular immune response, by using an HIV-1 protease-derived epitope (PR75–84), and variants thereof, coupled to the C-terminal, of the B subunit of cholera toxin (CTB). Fusion proteins were used for immunizations of HLA-A0201 transgenic C57BL/6 mice. We observed different capacities to elicit a cellular immune response by peptides with additions of five to ten amino acids to the PR epitope. There was a positive correlation between the magnitude of the elicited cellular immune response and the capacity of the fusion protein to bind GM-1. This binding capacity is affected by its ability to form natural pentamers of CTB. Our results suggest that functional CTB pentamers containing a foreign amino acid-modified epitope is a novel way to overcome the limited cellular immunogenicity of minimal peptide antigens. This way of using a functional assay as readout for improved cellular immunogenicity might become highly valuable for difficult immunogens such as short peptides (epitopes).

  17. Manipulating the pH response of 2,3-diaminopropionic acid rich peptides to mediate highly effective gene silencing with low-toxicity☆

    PubMed Central

    Abbate, Vincenzo; Liang, Wanling; Patel, Jayneil; Lan, Yun; Capriotti, Luigi; Iacobucci, Valentina; Bui, Tam T.; Chaudhuri, Poulami; Kudsiova, Laila; Vermeer, Louic S.; Chan, Patrick F.L.; Kong, Xiaole; Drake, Alex F.; Lam, Jenny K.W.; Bansal, Sukhvinder S.; Mason, A. James

    2013-01-01

    Cationic amphipathic pH responsive peptides possess high in vitro and in vivo nucleic acid delivery capabilities and function by forming a non-covalent complex with cargo, protecting it from nucleases, facilitating uptake via endocytosis and responding to endosomal acidification by being released from the complex and inserting into and disordering endosomal membranes. We have designed and synthesised peptides to show how Coulombic interactions between ionizable 2,3-diaminopropionic acid (Dap) side chains can be manipulated to tune the functional pH response of the peptides to afford optimal nucleic acid transfer and have modified the hydrogen bonding capabilities of the Dap side chains in order to reduce cytotoxicity. When compared with benchmark delivery compounds, the peptides are shown to have low toxicity and are highly effective at mediating gene silencing in adherent MCF-7 and A549 cell lines, primary human umbilical vein endothelial cells and both differentiated macrophage-like and suspension monocyte-like THP-1 cells. PMID:24144917

  18. Designing High-Affinity Peptides for Organic Molecules by Explicit Solvent Molecular Dynamics.

    PubMed

    Gladich, Ivan; Rodriguez, Alex; Hong Enriquez, Rolando P; Guida, Filomena; Berti, Federico; Laio, Alessandro

    2015-10-15

    Short peptides offer a cheap alternative to antibodies for developing sensing units in devices for concentration measurement. We here describe a computational procedure that allows designing peptides capable of binding with high affinity a target organic molecule in aqueous or nonstandard solvent environments. The algorithm is based on a stochastic search in the space of the possible sequences of the peptide, and exploits finite temperature molecular dynamics simulations in explicit solvent to check if a proposed mutation improves the binding affinity or not. The procedure automatically produces peptides which form thermally stable complexes with the target. The estimated binding free energy reaches the 13 kcal/mol for Irinotecan anticancer drug, the target considered in this work. These peptides are by construction solvent specific; namely, they recognize the target only in the solvent in which they have been designed. This feature of the algorithm calls for applications in devices in which the peptide-based sensor is required to work in denaturants or under extreme conditions of pressure and temperature. PMID:26398715

  19. Antibacterial and anticancer activity of a series of novel peptides incorporating cyclic tetra-substituted C(α) amino acids.

    PubMed

    Hicks, Rickey P

    2016-09-15

    Eleven antimicrobial peptides (AMP) based on the incorporation of cyclic tetra substituted C(α) amino acids, as well as other unnatural amino acids were designed, synthesized and screened for in vitro activity against 18 strains of bacteria as well as 12 cancer cell lines. The AMPs discussed herein are derived from the following peptide sequence: Ac-GF(X)G(X)B(X)G(X)F(X)G(X)GB(X)BBBB-amide, X=any one of the following residues, A5c, A6c, Tic or Oic and B=any one of the following residues, Arg, Lys, Orn, Dpr or Dab. A diversity of in vitro inhibitory activity was observed for these AMPs. Several analogs exhibited single digit μM activity against drug resistant bacteria including; multiple drug resistant Mycobacterium tuberculosis, extremely drug resistant Mycobacterium tuberculosis and MRSA. The physicochemical properties of the basic amino acid residues incorporated into these AMPs seem to play a major role in defining antibacterial activity. Overall hydrophobicity seems to play a limited role in defining antibacterial activity. The ESKAPE pathogens were used to compare the activity of these AMPs to another family of synthetic AMPs incorporating the unnatural amino acids Tic and Oic. In most cases similarly substituted members of both families exhibited similar inhibitory activity against the ESKAPE pathogens. In specific cases differences in activity as high as 15 fold were observed between analogs. In addition four of these AMPs exhibited promising IC50 (<7.5μM) values against 12 different and diverse cancer cell lines. Five other AMPs exhibited promising IC50 (<7.5μM) values against selected cancer cell lines. PMID:27387357

  20. Slow peptide bond formation by proline and other N-alkylamino acids in translation.

    PubMed

    Pavlov, Michael Y; Watts, Richard E; Tan, Zhongping; Cornish, Virginia W; Ehrenberg, Måns; Forster, Anthony C

    2009-01-01

    Proteins are made from 19 aa and, curiously, one N-alkylamino acid ("imino acid"), proline (Pro). Pro is thought to be incorporated by the translation apparatus at the same rate as the 19 aa, even though the alkyl group in Pro resides directly on the nitrogen nucleophile involved in peptide bond formation. Here, by combining quench-flow kinetics and charging of tRNAs with cognate and noncognate amino acids, we find that Pro incorporates in translation significantly more slowly than Phe or Ala and that other N-alkylamino acids incorporate much more slowly. Our results show that the slowest step in incorporation of N-alkylamino acids is accommodation/peptidyl transfer after GTP hydrolysis on EF-Tu. The relative incorporation rates correlate with expectations from organic chemistry, suggesting that amino acid sterics and basicities affect translation rates at the peptidyl transfer step. Cognate isoacceptor tRNAs speed Pro incorporation to rates compatible with in vivo, although still 3-6 times slower than Phe incorporation from Phe-tRNA(Phe) depending on the Pro codon. Results suggest that Pro is the only N-alkylamino acid in the genetic code because it has a privileged cyclic structure that is more reactive than other N-alkylamino acids. Our data on the variation of the rate of incorporation of Pro from native Pro-tRNA(Pro) isoacceptors at 4 different Pro codons help explain codon bias not accounted for by the "tRNA abundance" hypothesis. PMID:19104062

  1. Structural Stability and Binding Strength of a Designed Peptide-Carbon Nanotube Hybrid

    PubMed Central

    Roxbury, Daniel; Zhang, Shao-Qing; Mittal, Jeetain; DeGrado, William F.; Jagota, Anand

    2014-01-01

    Biological polymers hybridized with single-walled carbon nanotubes (SWCNTs) have elicited much interest recently for applications in SWCNT-based sorting as well as biomedical imaging, sensing, and drug delivery. Recently, de novo designed peptides forming a coiled-coil structure have been engineered to selectively disperse SWCNT of a certain diameter. Here we report on a study of the binding strength and structural stability of the hybrid between such a “HexCoil-Ala” peptide and the (6,5)-SWCNT. Using the competitive binding of a surfactant, we find that affinity strength of the peptide ranks in comparison to that of two single-stranded DNA sequences as (GT)30-DNA > HexCoil-Ala > (TAT)4T-DNA. Further, using replica exchange molecular dynamics (REMD), we show that the hexamer peptide complex has both similarities with and differences from the original design. While one of two distinct helix-helix interfaces of the original model was largely retained, a second interface showed much greater variability. These conformational differences allowed an aromatic tyrosine residue designed to lie along the solvent-exposed surface of the protein instead to penetrate between the two helices and directly contact the SWCNT. These insights will inform future designs of SWCNT-interacting peptides. PMID:24466357

  2. Altering behavioral responses and dopamine transporter protein with antisense peptide nucleic acids.

    PubMed

    Tyler-McMahon, B M; Stewart, J A; Jackson, J; Bitner, M D; Fauq, A; McCormick, D J; Richelson, E

    2001-10-01

    The dopamine transporter (DAT) plays a role in locomotion and is an obligatory target for amphetamines. We designed and synthesized an antisense peptide nucleic acid (PNA) to rat DAT to examine the effect of this antisense molecule on locomotion and on responsiveness to amphetamines. Rats were injected intraperitoneally daily for 9 days with either saline, an antisense DAT PNA, a scrambled DAT PNA, or a mismatch DAT PNA. On days 7 and 9 after initial motility measurements were taken, the animals were challenged with 10 mg/kg of amphetamine and scored for motility. On day 7, there was no significant difference between the baseline levels of activity of any of the groups or their responses to amphetamine. On day 9, the antisense PNA-treated rats showed a statistically significant increase in their resting motility (P < 0.01). When these rats were challenged with amphetamine, motility of the saline-, scrambled PNA-, and mismatch PNA-treated animals showed increases of 31-, 36-, and 20-fold, respectively, while the antisense PNA-treated animals showed increases of only 3.4-fold (P < 0.01). ELISA results revealed a 32% decrease in striatal DAT in antisense PNA-treated rats compared with the saline, scrambled PNA, and mismatch PNA controls (P < 0.001). These results extend our previous findings that brain proteins can be knocked down in a specific manner by antisense molecules administered extracranially. Additionally, these results suggest some novel approaches for the treatment of diseases dependent upon the function of the dopamine transporter. PMID:11543728

  3. Construction of a novel peptide nucleic acid piezoelectric gene sensor microarray detection system.

    PubMed

    Chen, Ming; Liu, Minghua; Yu, Lili; Cai, Guoru; Chen, Qinghai; Wu, Rong; Wang, Feng; Zhang, Bo; Jiang, Tianlun; Fu, Welling

    2005-08-01

    A novel 2 x 5 clamped style piezoelectric gene sensor microarray has been successfully constructed. Every crystal unit of the fabricated gene sensor can oscillate independently without interfering with each other. The bis-peptide nucleic acid (bis-PNA) probe, which can combine with target DNA or RNA sequences more effectively and specifically than a DNA probe, was designed and immobilized on the surface of the gene sensor microarray to substitute the conventional DNA probe for direct detection of the hepatitis B virus (HBV) genomic DNA. Detection conditions were then explored and optimized. Results showed that PBS buffer of pH 6.8, an ion concentration of 20 mmol/liter, and a probe concentration of 1.5 micromol/liter were optimal for the detection system. Under such optimized experimental conditions, the specificity of bis-PNA was proved much higher than that of DNA probe. The relationship between quantity of target and decrease of frequency showed a typical saturation curve when concentrations of target HBV DNA varied from 10 pg/liter to 100 microg/liter, and 10 microg/liter was the watershed, with a statistic linear regression equation of I gC = -2.7455 + 0.0691 deltaF and the correlating coefficient of 0.9923. Fortunately, this is exactly the most ordinary variant range of the HBV virus concentration in clinical hepatitis samples. So, a good technical platform is successfully constructed and it will be applied to detect HBV quantitatively in clinical samples. PMID:16193990

  4. Programmable Multivalent Display of Receptor Ligands using Peptide Nucleic Acid Nanoscaffolds

    PubMed Central

    Englund, Ethan A.; Wang, Deyun; Fujigaki, Hidetsugu; Sakai, Hiroyasu; Micklitsch, Christopher M.; Ghirlando, Rodolfo; Martin-Manso, Gema; Pendrak, Michael L.; Roberts, David D.; Durell, Stewart R.; Appella, Daniel H.

    2012-01-01

    Multivalent effects dictate the binding affinity of multiple ligands on one molecular entity to receptors. Integrins are receptors that mediate cell attachment through multivalent binding to peptide sequences within the extracellular matrix, and overexpression promotes the metastasis of some cancers. Multivalent display of integrin antagonists enhances their efficacy, but current scaffolds have limited ranges and precision for the display of ligands. Here we present an approach to study multivalent effects across wide ranges of ligand number, density, and three-dimensional arrangement. Using L-lysine γ-substituted peptide nucleic acids, the multivalent effects of an integrin antagonist were examined over a range of 1 to 45 ligands. The optimal construct improves the inhibitory activity of the antagonist by two orders of magnitude against the binding of melanoma cells to the extracellular matrix in both in vitro and in vivo models. PMID:22233624

  5. Formation of peptides from amino acids by single or multiple additions of ATP to suspensions of nucleoproteinoid microparticles

    NASA Technical Reports Server (NTRS)

    Nakashima, T.; Fox, S. W.

    1981-01-01

    The synthesis of peptides from individual amino acids or pairs of amino acids and ATP in the presence of catalysis by nucleoproteinoid microparticles is investigated. Experiments were performed with suspensions formed from the condensation of lysine-rich and acidic proteinoids with polyadenylic acid, to which were added glycine, phenylalanine, proline, lysine or glycine-phenylalanine mixtures, and ATP either at once or serially. Peptide yields are found to be greatest for equal amounts of acidic and basic proteinoids. The addition of imidazole is found to alter the preference of glycine-phenylalanine mixtures to form mixed heteropeptides rather than homopeptides. A rapid ATP decay in the peptide synthesis reaction is observed, and a greater yield is obtained for repeated small additions than for a single addition of ATP. The experimental system has properties similar to modern cells, and represents an organizational unit ready for the evolution of associated biochemical pathways.

  6. Hybrid peptides endomorphin-2/DAMGO: design, synthesis and biological evaluation.

    PubMed

    Mollica, Adriano; Costante, Roberto; Stefanucci, Azzurra; Pinnen, Francesco; Luisi, Grazia; Pieretti, Stefano; Borsodi, Anna; Bojnik, Engin; Benyhe, Sándor

    2013-10-01

    Endomorphin-2 [Tyr-Pro-Phe-Phe-NH2] and DAMGO [Tyr-D-Ala-Gly-(N-Me)Phe-Gly-ol] are natural (EM2) and synthetic (DAMGO) opioid peptides both selective for μ opioid receptor with high analgesic activity. In this work we report synthesis, in vitro and in vivo biological evaluation of five new hybrid EM2/DAMGO analogues, with the aim to obtain compounds with high affinity at μ-opioid receptor, high activity in animal nociception tests (hot plate and tail flick) and improved enzymatic stability. Double N-methylation on both Phe residues and C-terminal ethanolamide led to analogue 6e, which possesses a good in vitro μ affinity (Kiμ=34 nM), combined with a remarkable in vivo antinociceptive activity. PMID:23974016

  7. Solvation thermodynamics of amino acid side chains on a short peptide backbone

    SciTech Connect

    Hajari, Timir; Vegt, Nico F. A. van der

    2015-04-14

    The hydration process of side chain analogue molecules differs from that of the actual amino acid side chains in peptides and proteins owing to the effects of the peptide backbone on the aqueous solvent environment. A recent molecular simulation study has provided evidence that all nonpolar side chains, attached to a short peptide backbone, are considerably less hydrophobic than the free side chain analogue molecules. In contrast to this, the hydrophilicity of the polar side chains is hardly affected by the backbone. To analyze the origin of these observations, we here present a molecular simulation study on temperature dependent solvation free energies of nonpolar and polar side chains attached to a short peptide backbone. The estimated solvation entropies and enthalpies of the various amino acid side chains are compared with existing side chain analogue data. The solvation entropies and enthalpies of the polar side chains are negative, but in absolute magnitude smaller compared with the corresponding analogue data. The observed differences are large; however, owing to a nearly perfect enthalpy-entropy compensation, the solvation free energies of polar side chains remain largely unaffected by the peptide backbone. We find that a similar compensation does not apply to the nonpolar side chains; while the backbone greatly reduces the unfavorable solvation entropies, the solvation enthalpies are either more favorable or only marginally affected. This results in a very small unfavorable free energy cost, or even free energy gain, of solvating the nonpolar side chains in strong contrast to solvation of small hydrophobic or nonpolar molecules in bulk water. The solvation free energies of nonpolar side chains have been furthermore decomposed into a repulsive cavity formation contribution and an attractive dispersion free energy contribution. We find that cavity formation next to the peptide backbone is entropically favored over formation of similar sized nonpolar side

  8. Spectroscopic and structural elucidation of amino acid derivatives and small peptides: experimental and theoretical tools.

    PubMed

    Kolev, Tsonko; Spiteller, Michael; Koleva, Bojidarka

    2010-01-01

    This mini review deals with the modern aspects of the spectroscopy and structural elucidation of amino acid derivatives and small biologically active compounds. Free peptide bond rotation in these systems yields various conformers, which possess differing biological activities. Another phenomenon is the intermolecular or intramolecular stacking observed in aromatic small peptides. Specifically, the main aim is to illustrate the successful application of the "complex tool", consisting of a combination of the theoretical approximation methods with experimental linear polarized infrared (IR-LD) and/or Raman spectroscopy of oriented colloid suspensions in a nematic host. The possibilities and limitations of the approach for detailed vibrational assignment and structural elucidation of small peptides are discussed. Having in mind that physical and chemical properties of these systems can be precisely calculated by means of ab initio and DFT methods at Hartee-Fock, MP2 and B3LYP level of theory, varying basis sets, the results obtained allow a precise assignment of many vibrational bands to the corresponding normal modes, electronic structures and conformational state. The validity of the conclusions about the structure or vibrational properties of these systems have been supported, compared and/or additionally proved by the results from independent physical methods. In this respect (1)H and (13)C-NMR, single crystal X-ray diffraction, HPLC tandem mass spectrometry as well as thermal methods are all employed. A well ordered crystal must first be grown in order to determine the molecular structure by the absolute method of single crystal X-ray diffraction. Although the 3D structures of peptides have been determined over the past decades, peptide crystallization is still a major obstacle to X-ray diffraction work, the presence of chiral centre/s makes for this difficulty. For this reason the "complex tool" presented can be regarded as an alternative method for obtaining of

  9. Expression pattern of peptide and amino acid genes in digestive tract of transporter juvenile turbot ( Scophthalmus maximus L.)

    NASA Astrophysics Data System (ADS)

    Xu, Dandan; He, Gen; Mai, Kangsen; Zhou, Huihui; Xu, Wei; Song, Fei

    2016-04-01

    Turbot ( Scophthalmus maximus L.), a carnivorous fish species with high dietary protein requirement, was chosen to examine the expression pattern of peptide and amino acid transporter genes along its digestive tract which was divided into six segments including stomach, pyloric caeca, rectum, and three equal parts of the remainder of the intestine. The results showed that the expression of two peptide and eleven amino acid transporters genes exhibited distinct patterns. Peptide transporter 1 (PepT1) was rich in proximal intestine while peptide transporter 2 (PepT2) was abundant in distal intestine. A number of neutral and cationic amino acid transporters expressed richly in whole intestine including B0-type amino acid transporter 1 (B0AT1), L-type amino acid transporter 2 (LAT2), T-type amino acid transporter 1 (TAT1), proton-coupled amino acid transporter 1 (PAT1), y+L-type amino acid transporter 1 (y+LAT1), and cationic amino acid transporter 2 (CAT2) while ASC amino acid transporter 2 (ASCT2), sodium-coupled neutral amino acid transporter 2 (SNAT2), and y+L-type amino acid transporter 2 (y+LAT2) abundantly expressed in stomach. In addition, system b0,+ transporters (rBAT and b0,+AT) existed richly in distal intestine. These findings comprehensively characterized the distribution of solute carrier family proteins, which revealed the relative importance of peptide and amino acid absorption through luminal membrane. Our findings are helpful to understand the mechanism of the utilization of dietary protein in fish with a short digestive tract.

  10. Protective immunogenicity of two synthetic peptides selected from the amino acid sequence of Bordetella pertussis toxin subunit S1.

    PubMed Central

    Askelöf, P; Rodmalm, K; Wrangsell, G; Larsson, U; Svenson, S B; Cowell, J L; Undén, A; Bartfai, T

    1990-01-01

    Two peptides, corresponding to amino acids 1-17 and 169-186 of the amino acid sequence of pertussis toxin (PT) subunit S1, were synthesized and coupled to the diphtheria toxin cross-reactive mutant protein CRM 197 and evaluated for immunogenicity and protective capacity against PT challenge in vivo. The peptide-CRM conjugates induced high antibody titers against native toxin in mice (BALB/c, C57/Black, and outbred NMRI) as measured by ELISA. Upon PT challenge (0.5 microgram of toxin) of the NMRI mice, the CRM conjugates of peptides 1-17 and 169-186 fully protected the mice from PT-induced leukocytosis. Immunization with the corresponding bovine serum albumin conjugates of these two peptides also fully protected mice. Rabbit antiserum to the peptide 1-17-CRM conjugate was highly efficient in inhibiting the ADP-ribosylating activity of PT but did not neutralize the clustering effect of PT on Chinese hamster ovary cells. In contrast, the rabbit antiserum raised against the peptide 169-186-CRM conjugate neutralized the clustering effect of PT on Chinese hamster ovary cells but did not inhibit the enzymatic activity of PT. Peptide 169-186-CRM conjugates mimic the immunoglobulin binding properties of PT and also cause clustering of Chinese hamster ovary cells. The CRM conjugates of these two peptides constitute a synthetic pertussis vaccine candidate with the ability to provide a chemically well-defined, safe, and efficient pertussis vaccine. Images PMID:2304902

  11. Quantitative analysis of single amino acid variant peptides associated with pancreatic cancer in serum by an isobaric labeling quantitative method.

    PubMed

    Nie, Song; Yin, Haidi; Tan, Zhijing; Anderson, Michelle A; Ruffin, Mack T; Simeone, Diane M; Lubman, David M

    2014-12-01

    Single amino acid variations are highly associated with many human diseases. The direct detection of peptides containing single amino acid variants (SAAVs) derived from nonsynonymous single nucleotide polymorphisms (SNPs) in serum can provide unique opportunities for SAAV associated biomarker discovery. In the present study, an isobaric labeling quantitative strategy was applied to identify and quantify variant peptides in serum samples of pancreatic cancer patients and other benign controls. The largest number of SAAV peptides to date in serum including 96 unique variant peptides were quantified in this quantitative analysis, of which five variant peptides showed a statistically significant difference between pancreatic cancer and other controls (p-value < 0.05). Significant differences in the variant peptide SDNCEDTPEAGYFAVAVVK from serotransferrin were detected between pancreatic cancer and controls, which was further validated by selected reaction monitoring (SRM) analysis. The novel biomarker panel obtained by combining α-1-antichymotrypsin (AACT), Thrombospondin-1 (THBS1) and this variant peptide showed an excellent diagnostic performance in discriminating pancreatic cancer from healthy controls (AUC = 0.98) and chronic pancreatitis (AUC = 0.90). These results suggest that large-scale analysis of SAAV peptides in serum may provide a new direction for biomarker discovery research. PMID:25393578

  12. Templated synthesis of peptide nucleic acids via sequence-selective base-filling reactions.

    PubMed

    Heemstra, Jennifer M; Liu, David R

    2009-08-19

    The templated synthesis of nucleic acids has previously been achieved through the backbone ligation of preformed nucleotide monomers or oligomers. In contrast, here we demonstrate templated nucleic acid synthesis using a base-filling approach in which individual bases are added to abasic sites of a peptide nucleic acid (PNA). Because nucleobase substrates in this approach are not self-reactive, a base-filling approach may reduce the formation of nontemplated reaction products. Using either reductive amination or amine acylation chemistries, we observed efficient and selective addition of each of the four nucleobases to an abasic site in the middle of the PNA strand. We also describe the addition of single nucleobases to the end of a PNA strand through base filling, as well as the tandem addition of two bases to the middle of the PNA strand. These findings represent an experimental foundation for nonenzymatic information transfer through base filling. PMID:19722647

  13. Templated Synthesis of Peptide Nucleic Acids via Sequence-Selective Base-Filling Reactions

    PubMed Central

    2009-01-01

    The templated synthesis of nucleic acids has previously been achieved through the backbone ligation of preformed nucleotide monomers or oligomers. In contrast, here we demonstrate templated nucleic acid synthesis using a base-filling approach in which individual bases are added to abasic sites of a peptide nucleic acid (PNA). Because nucleobase substrates in this approach are not self-reactive, a base-filling approach may reduce the formation of nontemplated reaction products. Using either reductive amination or amine acylation chemistries, we observed efficient and selective addition of each of the four nucleobases to an abasic site in the middle of the PNA strand. We also describe the addition of single nucleobases to the end of a PNA strand through base filling, as well as the tandem addition of two bases to the middle of the PNA strand. These findings represent an experimental foundation for nonenzymatic information transfer through base filling. PMID:19722647

  14. Highly efficient peptide formation from N-acetylaminoacyl-AMP anhydride and free amino acid

    NASA Technical Reports Server (NTRS)

    Mullins, D. W., Jr.; Lacey, J. C., Jr.

    1983-01-01

    The kinetics of formation of the N-blocked dipeptide, N-acetylglycylglycine, from N-acetylglycyl adenylate anhydride and glycine in aqueous solution at 25 C, and at various PH's are reported. The reaction is of interest in that over a physiologically relevant pH range (6-8), peptide synthesis proceeds more rapidly than hydrolysis, even at those pH's at which this compound becomes increasingly susceptible to base-catalyzed hydrolysis. Under similar conditions, the corresponding unblocked aminoacyl adenylate anhydrides are considerably more unstable, and undergo appreciable hydrlysis in the presence of free amino acid. Because N-blocked aminoacyl adenylate anhydrides serve as model compounds of peptidyl adenylate anhydrides, these results suggest that primitive amino acid polymerization systems may have operated by cyclic reactivation of the peptidyl carboxyl group, rather than that of the incoming amino acid.

  15. Wide range of racemization of amino acids in peptides from human fossil bone and its implications for amino acid racemization dating

    SciTech Connect

    Kimber, R.W.L.; Hare, P.E. )

    1992-02-01

    Aspartic acid from an HCl hydrolyzed portion of 20-25th Dynasty Egyptian bone gave a D/L value of 0.28. Various peptide and molecular weight fractions separated before hydrolysis from another aliquot of the same bone portion yielded D/L aspartic acid values ranging from 0.09 to 0.68. Higher molecular weight and higher content of hydrophobic amino acids are factors leading to lower D/L aspartic acid values. Insoluble, high molecular weight polypeptide residues showed very low D/L aspartic acid values of 0.09. The authors propose that particularly stable peptides be isolated and characterized and then used for comparison with similarly isolated peptides from other fossil bone samples for purposes of age estimation.

  16. Molecular Design, Structures, and Activity of Antimicrobial Peptide-Mimetic Polymers

    PubMed Central

    Takahashi, Haruko; Palermo, Edmund F.; Yasuhara, Kazuma; Caputo, Gregory A.

    2014-01-01

    There is an urgent need for new antibiotics which are effective against drug-resistant bacteria without contributing to resistance development. We have designed and developed antimicrobial copolymers with cationic amphiphilic structures based on the mimicry of naturally occurring antimicrobial peptides. These copolymers exhibit potent antimicrobial activity against a broad spectrum of bacteria including methicillin-resistant Staphylococcus aureus with no adverse hemolytic activity. Notably, these polymers also did not result in any measurable resistance development in E. coli. The peptide-mimetic design principle offers significant flexibility and diversity in the creation of new antimicrobial materials and their potential biomedical applications. PMID:23832766

  17. Calcium carbonate crystal growth beneath Langmuir monolayers of acidic β-hairpin peptides.

    PubMed

    Gong, Haofei; Yang, Yi; Pluntke, Manuela; Marti, Othmar; Majer, Zsuzsa; Sewald, Norbert; Volkmer, Dirk

    2014-11-28

    Four amphiphilic peptides with designed hairpin structure were synthesized and their monolayers were employed as model systems to study biologically inspired calcium carbonate crystallization. Langmuir monolayers of hairpin peptides were investigated by surface pressure area isotherms, surface potential isotherms, Brewster angle microscopy (BAM), atomic force microscopy (AFM) and Fourier transform infrared (FTIR) spectroscopy. A β-hairpin conformation was found for all peptides at the air-water interface although their packing arrangements seem to be different. Crystallization of calcium carbonate under these peptide monolayers was investigated at different surface pressures and growth times both by in situ optical microscopy, BAM and ex situ investigations such as scanning electron microscopy (SEM) and transmission electron microscopy (TEM). An amorphous calcium carbonate precursor was found at the initial crystallization stage. The crystallization process occurred in three stages. It starts from the nucleation of amorphous particles being a kinetically controlled process. Crystal nuclei subsequently aggregate to large particles and vaterite crystals start to form inside the amorphous layer, with the monolayer fluidity exerting an important role. The third process includes the re-crystallization of vaterite to calcite, which is thermodynamically controlled by monolayer structural factors including the monolayer flexibility and packing arrangement of the polar headgroups. Thus, the kinetic factors, monolayer fluidity and flexibility as well as structure factors govern the crystal morphology and polymorph distribution simultaneously and synergistically. PMID:25292256

  18. Design of cyclic RGD-conjugated Aib-containing amphipathic helical peptides for targeted delivery of small interfering RNA.

    PubMed

    Wada, Shun-Ichi; Iwata, Masashi; Ozaki, Yuka; Ozaki, Takashi; Hayashi, Junsuke; Urata, Hidehito

    2016-09-15

    To achieve the targeted delivery of siRNA, five conjugates of Aib-containing amphipathic helical peptides with mono-, di-, and trivalent cRGDfC [cyclo(-Arg-Gly-Asp-d-Phe-Cys-)], which is known to bind to αVβ3 integrin, at several positions of the amphipathic helical peptide were designed and synthesized. Among the five conjugates, the monovalent cRGDfC conjugating at position 20 of the amino acid sequence of the helical peptide through the formation of a disulfide bond (PI) and the divalent cRGDfC conjugating at positions 2 and 14 of the amino acid sequence of the helical peptide through the formation of disulfide bonds (PIII) significantly enhanced the delivery of fluorescence-labeled siRNA into A549 cells as the peptide/siRNA complex formed by electrostatic interaction. The cellular uptake of the PI/siRNA complex was mediated by both endocytic and non-endocytic pathways, whereas that of the PIII/siRNA complex was enabled by endocytosis. Furthermore, the cellular uptake of the PI/siRNA complex might involve specific interactions of the RGD group with the αVβ3 integrin receptor. Next, the RNAi effect of the peptide/siRNA complex on luciferase expression in A549-Luc cells was examined. Luciferase expression was significantly decreased in the presence of the complex at the concentration of 1.0μM PI/10nM siRNA. In contrast, the PIII/siRNA complex did not show the RNAi effect under the same conditions. However, extending the incubation time led to the suppression of the luciferase expression in the presence of the PIII/siRNA complex. Considering that the cellular uptake of the PIII/siRNA complex is mediated by the endocytic pathway, the release of siRNA from the endosome into the cytosol might require a long time. We present herein a useful and unique tool for the delivery of siRNA. PMID:27480031

  19. Effects of 8-mer acidic peptide concentration on the morphology and photoluminescence of synthesized ZnO nanomaterials

    NASA Astrophysics Data System (ADS)

    Moon, Chung Hee; Tousi, Marzieh; Cheeney, Joseph; Ngo-Duc, Tam-Triet; Zuo, Zheng; Liu, Jianlin; Haberer, Elaine D.

    2015-11-01

    An 8-mer ZnO-binding peptide, VPGAAEHT, was identified using a M13 pVIII phage display library and employed as an additive during aqueous-based ZnO synthesis at 65 °C. Unlike most other well-studied ZnO-binding sequences which are strongly basic (pI > pH 7), the 8-mer peptide was overall acidic (pI < pH 7) in character, including only a single basic residue. The selected peptide strongly influenced ZnO nanostructure formation. Morphology and optical emission properties were found to be dependent on the concentration of peptide additive. Using lower peptide concentrations (<0.1 mM), single crystal hexagonal rods and platelets were produced, and using higher peptide concentrations (≥0.1 mM), polycrystalline layered platelets, yarn-like structures, and microspheres were assembled. Photoluminescence analysis revealed a characteristic ZnO band-edge peak, as well as sub-bandgap emission peaks. Defect-related green emission, typically associated with surface-related oxygen and zinc vacancies, was significantly reduced by the peptide additive, while blue emission, attributable to oxygen and zinc interstitials, emerged with increased peptide concentrations. Peptide-directed synthesis of ZnO materials may be useful for gas sensing and photocatalytic applications in which properly engineered morphology and defect levels have demonstrated enhanced performance.

  20. The chain length of biologically produced (R)-3-hydroxyalkanoic acid affects biological activity and structure of anti-cancer peptides.

    PubMed

    Szwej, Emilia; Devocelle, Marc; Kenny, Shane; Guzik, Maciej; O'Connor, Stephen; Nikodinovic-Runic, Jasmina; Radivojevic, Jelena; Maslak, Veselin; Byrne, Annete T; Gallagher, William M; Zulian, Qun Ren; Zinn, Manfred; O'Connor, Kevin E

    2015-06-20

    Conjugation of DP18L peptide with (R)-3-hydroxydecanoic acid, derived from the biopolymer polyhydroxyalkanoate, enhances its anti-cancer activity (O'Connor et al., 2013. Biomaterials 34, 2710-2718). However, it is unknown if other (R)-3-hydroxyalkanoic acids (R3HAs) can enhance peptide activity, if chain length affects enhancement, and what effect R3HAs have on peptide structure. Here we show that the degree of enhancement of peptide (DP18L) anti-cancer activity by R3HAs is carbon chain length dependent. In all but one example the R3HA conjugated peptides were more active against cancer cells than the unconjugated peptides. However, R3HAs with 9 and 10 carbons were most effective at improving DP18L activity. DP18L peptide variant DP17L, missing a hydrophobic amino acid (leucine residue 4) exhibited lower efficacy against MiaPaCa cells. Circular dichroism analysis showed DP17L had a lower alpha helix content and the conjugation of any R3HA ((R)-3-hydroxyhexanoic acid to (R)-3-hydroxydodecanoic acid) to DP17L returned the helix content back to levels of DP18L. However (R)-3-hydroxyhexanoic did not enhance the anti-cancer activity of DP17L and at least 7 carbons were needed in the R3HA to enhance activity of D17L. DP17L needs a longer chain R3HA to achieve the same activity as DP18L conjugated to an R3HA. As a first step to assess the synthetic potential of polyhydroxyalkanoate derived R3HAs, (R)-3-hydroxydecanoic acid was synthetically converted to (±)3-chlorodecanoic acid, which when conjugated to DP18L improved its antiproliferative activity against MiaPaCa cells. PMID:25820126

  1. Functional and Mechanistic Analyses of Biomimetic Aminoacyl Transfer Reactions in de novo Designed Coiled Coil Peptides via Rational Active Site Engineering

    PubMed Central

    Leman, Luke J.; Weinberger, Dana A.; Huang, Zheng-Zheng; Wilcoxen, Keith M.; Ghadiri, M. Reza

    2008-01-01

    Ribosomes and nonribosomal peptide synthetases (NRPSs) carry out instructed peptide synthesis through a series of directed intermodular aminoacyl transfer reactions. We recently reported the design of coiled-coil assemblies that could functionally mimic the elementary aminoacyl loading and intermodular aminoacyl transfer steps of NRPSs. These peptides were designed initially to accelerate aminoacyl transfer mainly through catalysis by approximation by closely juxtaposing four active site moieties, two each from adjacent noncovalently-associated helical modules. In our designs peptide self-assembly positions a cysteine residue that is used to covalently capture substrates from solution via transthiolesterification (substrate loading step to generate the aminoacyl donor site) adjacent to an aminoacyl acceptor site provided by a covalently tethered amino acid or modeled by the ε-amine of an active site lysine. However, through systematic functional analyses of 48 rationally designed peptide sequences, we have now determined that the substrate loading and intermodular aminoacyl transfer steps can be significantly influenced (up to ~103-fold) by engineering changes in the active site microenvironment through amino acid substitutions and variations in the inter-residue distances and geometry. Mechanistic studies based on 15N-NMR and kinetic analysis further indicate that certain active site constellations furnish an unexpectedly large pKa depression (1.5 pH units) of the aminoacyl-acceptor moiety, helping to explain the observed high rates of aminoacyl transfer in those constructs. Taken together, our studies demonstrate the feasibility of engineering efficient de novo peptide sequences possessing active sites and functions reminiscent of those in natural enzymes. PMID:17302417

  2. Computational Design of Selective Peptides to Discriminate Between Similar PDZ Domains in an Oncogenic Pathway

    PubMed Central

    Zheng, Fan; Jewell, Heather; Fitzpatrick, Jeremy; Zhang, Jian; Mierke, Dale F.; Grigoryan, Gevorg

    2016-01-01

    Reagents that target protein-protein interactions to rewire signaling are of great relevance in biological research. Computational protein design may offer a means of creating such reagents on demand, but methods for encoding targeting selectivity are sorely needed. This is especially challenging when targeting interactions with ubiquitous recognition modules—e.g., PDZ domains, which bind C-terminal sequences of partner proteins. Here we consider the problem of designing selective PDZ inhibitor peptides in the context of an oncogenic signaling pathway, in which two PDZ domains (NHERF-2 PDZ2—N2P2 and MAGI-3 PDZ6—M3P6) compete for a receptor C-terminus to differentially modulate oncogenic activities. Because N2P2 increases tumorigenicity and M3P6 decreases it, we sought to design peptides that inhibit N2P2 without affecting M3P6. We developed a structure-based computational design framework that models peptide flexibility in binding, yet is efficient enough to rapidly analyze tradeoffs between affinity and selectivity. Designed peptides showed low-micromolar inhibition constants for N2P2 and no detectable M3P6 binding. Peptides designed for reverse discrimination bound M3P6 tighter than N2P2, further testing our technology. Experimental and computational analysis of selectivity determinants revealed significant indirect energetic coupling in the binding site. Successful discrimination between N2P2 and M3P6, despite their overlapping binding preferences, is highly encouraging for computational approaches to selective PDZ targeting, especially because design relied on a homology model of M3P6. Still, we demonstrate specific deficiencies of structural modeling that must be addressed to enable truly robust design. The presented framework is general and can be applied in many scenarios to engineer selective targeting. PMID:25451599

  3. Design of an electroactive peptide probe for sensing of a protein.

    PubMed

    Shinohara, Hiroki; Kuramitz, Hideki; Sugawara, Kazuharu

    2015-08-26

    We designed a new electroactive peptide probe that has a molecular recognition function for the sensing of a protein. Ovalbumin (OVA) was the model protein, and when RNRCKGTDVQAW interacted with OVA, it conjugated with a tyrosine-rich peptide (Y4C). This peptide is electroactive, has a high degree of biocompatibility, and offers the possibility of gene expression. To measure the effect of a number of the tyrosine residues, voltammetric measurements were conducted using a series of tyrosine-rich peptides (YnC, n = 3-7) with sensitivities that ranged from 10(-9) to 10(-8) M. The electrode response of Y5C was the maximum value in the series. However, the peak current did not increase when the number of tyrosine residues was increased in a linear fashion. This may have been due to the micelles that are formed by a tyrosine-rich surfactant peptide. Thus, Y4C was suitable as an electroactive label for the construction of the peptide probe. The electrode response of Y4CRNRCKGTDVQAW obtained by a glassy carbon electrode was 100-fold that of tyrosine alone. The measurement of OVA via the peptide probe resulted in a detection on the order of 10(-12) M. In contrast, the sensitivity of OVA using RCKGTDVQAWY4C probe was at the 10(-11) M level, because the hydrophobic moiety gave it a molecular recognition function. The recoveries of the OVA using Y4CRNRCKGTDVQAW in a solution containing fetal bovine serum ranged between 98 and 101%. Consequently, the combination of a specific peptide and an electroactive element could be a powerful probe for the sensing of proteins. PMID:26347176

  4. Machine Learning Assisted Design of Highly Active Peptides for Drug Discovery

    PubMed Central

    Giguère, Sébastien; Laviolette, François; Marchand, Mario; Tremblay, Denise; Moineau, Sylvain; Liang, Xinxia; Biron, Éric; Corbeil, Jacques

    2015-01-01

    The discovery of peptides possessing high biological activity is very challenging due to the enormous diversity for which only a minority have the desired properties. To lower cost and reduce the time to obtain promising peptides, machine learning approaches can greatly assist in the process and even partly replace expensive laboratory experiments by learning a predictor with existing data or with a smaller amount of data generation. Unfortunately, once the model is learned, selecting peptides having the greatest predicted bioactivity often requires a prohibitive amount of computational time. For this combinatorial problem, heuristics and stochastic optimization methods are not guaranteed to find adequate solutions. We focused on recent advances in kernel methods and machine learning to learn a predictive model with proven success. For this type of model, we propose an efficient algorithm based on graph theory, that is guaranteed to find the peptides for which the model predicts maximal bioactivity. We also present a second algorithm capable of sorting the peptides of maximal bioactivity. Extensive analyses demonstrate how these algorithms can be part of an iterative combinatorial chemistry procedure to speed up the discovery and the validation of peptide leads. Moreover, the proposed approach does not require the use of known ligands for the target protein since it can leverage recent multi-target machine learning predictors where ligands for similar targets can serve as initial training data. Finally, we validated the proposed approach in vitro with the discovery of new cationic antimicrobial peptides. Source code freely available at http://graal.ift.ulaval.ca/peptide-design/. PMID:25849257

  5. Designed Coiled-Coil Peptides Inhibit the Type Three Secretion System of Enteropathogenic Escherichia coli

    PubMed Central

    Larzábal, Mariano; Mercado, Elsa C.; Vilte, Daniel A.; Salazar-González, Hector; Cataldi, Angel; Navarro-Garcia, Fernando

    2010-01-01

    Background Enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) are two categories of E. coli strains associated with human disease. A major virulence factor of both pathotypes is the expression of a type three secretion system (TTSS), responsible for their ability to adhere to gut mucosa causing a characteristic attaching and effacing lesion (A/E). The TTSS translocates effector proteins directly into the host cell that subvert mammalian cell biochemistry. Methods/Principal Findings We examined synthetic peptides designed to inhibit the TTSS. CoilA and CoilB peptides, both representing coiled-coil regions of the translocator protein EspA, and CoilD peptide, corresponding to a coiled–coil region of the needle protein EscF, were effective in inhibiting the TTSS dependent hemolysis of red blood cells by the EPEC E2348/69 strain. CoilA and CoilB peptides also reduced the formation of actin pedestals by the same strain in HEp-2 cells and impaired the TTSS-mediated protein translocation into the epithelial cell. Interestingly, CoilA and CoilB were able to block EspA assembly, destabilizing the TTSS and thereby Tir translocation. This blockage of EspA polymerization by CoilA or CoilB peptides, also inhibited the correct delivery of EspB and EspD as detected by immunoblotting. Interestingly, electron microscopy of bacteria incubated with the CoilA peptide showed a reduction of the length of EspA filaments. Conclusions Our data indicate that coiled-coil peptides can prevent the assembly and thus the functionality of the TTSS apparatus and suggest that these peptides could provide an attractive tool to block EPEC and EHEC pathogenesis. PMID:20140230

  6. Oxidative decarboxylation of free and peptide-linked amino acids in phagocytizing guinea pig granulocytes.

    PubMed Central

    Adeniyi-Jones, S K; Karnovsky, M L

    1981-01-01

    The oxidative decarboxylation of amino acids by a system consisting of myeloperoxidase-hydrogen peroxide-chloride has been demonstrated previously by others and the process has been considered to be part of the microbicidal armamentarium of some phagocytic leukocytes. We were able to translate these earlier observations, made on model systems, to intact guinea pig granulocytes. We could demonstrate differences in the cellular handling of peptide-linked amino acids as particles, compared with free amino acids. Specific inhibitors were used to explore two routes of oxidative decarboxylation: (a) the myeloperoxidase-catalyzed direct decarboxylation-deamination reaction, and (b) oxidation of alpha-keto acids after transamination of amino acids. These inhibitors were cyanide, azide, and tapazole for the former pathway, and amino-oxyacetate for the latter. Amino-oxyacetate profoundly inhibited the decarboxylation of free 14C-amino acids (alanine and aspartate) in both resting and stimulated cells, but had only a minimal effect on 14CO2 production from ingested insoluble 14C-protein. On the other hand, the peroxidase inhibitors cyanide, azide, and tapazole dramatically inhibited the production of 14CO2 from ingested particulate 14C-protein, but had only small effects on the decarboxylation of free amino acid. Soluble, uniformly labeled 14C-protein was not significantly converted to 14CO2 even in the presence of phagocytizable polystyrene beads. These observation suggest that the amino acids taken up by phagocytosis (e.g., as denatured protein particles) are oxidatively decarboxylated and deaminated in the phagosomes by the myeloperoxidase-hydrogen peroxide-chloride system; soluble free amino acids that enter the cytoplasm by diffusion or transport are oxidatively decarboxylated after transamination by the normal cellular amino acid oxidative pathway. PMID:6267101

  7. Design of cyclic peptides featuring proline predominantly in the cis conformation under physiological conditions.

    PubMed

    Malešević, Miroslav; Schumann, Michael; Jahreis, Günther; Fischer, Gunter; Lücke, Christian

    2012-09-24

    Turns are secondary-structure elements that are omnipresent in natively folded polypeptide chains. A large variety of four-residue β-turns exist, which differ mainly in the backbone dihedral angle values of the two central residues i+1 and i+2. The βVI-type turns are of particular biological interest because the i+2 residue is always a proline in the cis conformation and might thus serve as target of peptidyl prolyl cis/trans isomerases (PPIases). We have designed cyclic hexapeptides containing two proline residues that predominantly adopt the cis conformation in aqueous solution. NMR data and MD calculations indicated that the cyclic peptide sequences c-(-DXaa-Ser-Pro-DXaa-Lys-Pro-) result in highly symmetric backbone structures when both prolines are in the cis conformation and the D-amino acids are either alanine or phenylalanine residues. Replacement of the serine residue either by phosphoserine or by tyrosine compromises this symmetry, but further increases the cis conformation content of both prolines. As a result, we obtained a cyclic hexapeptide that exists almost exclusively as the cis-Pro/cis-Pro conformer but shows no cis/trans interconversion even in the presence of the PPIase Pin1, apparently due to an energetically quite favorable but highly restricted conformational space. PMID:22969011

  8. Self-Assembling Peptide Coatings Designed for Highly Luminescent Suspension of Single-Walled Carbon Nanotubes

    PubMed Central

    Tsyboulski, Dmitri A.; Bakota, Erica L.; Witus, Leah S.; Rocha, John-David R.; Hartgerink, Jeffrey D.; Weisman, R. Bruce

    2009-01-01

    A series of self-assembling multidomain peptides have been designed, synthesized, and tested for their ability to individually suspend single-walled carbon nanotubes (SWCNTs) in water while preserving strong near-IR nanotube luminescence. Photometric and spectral measurements on individual SWCNTs revealed that emission in the common biocompatible coating agents Pluronic F127, ss-DNA, and BSA is approximately an order of magnitude weaker than in the bio-incompatible ionic surfactant SDBS. By contrast, one of the engineered peptides gave SWCNT emission ~40% as intense as in SDBS. A strong inverse correlation was also found between the spectral line widths of coated SWCNTs and the efficiency of their emission. Peptides with rationally designed self-assembly properties appear to be promising coatings that may enable SWCNT optical sensing applications in biological environments. PMID:19053447

  9. Design of a G[center dot]C-specific DNA minor groove-binding peptide

    SciTech Connect

    Geierstanger, B.H.; Wemmer, D.E. ); Mrksich, M.; Dervan, P.B. )

    1994-10-28

    A four-ring tripeptide containing alternating imidazole and pyrrole carboxamides specifically binds six-base pair 5[prime]-(A,T)GCGC(A,T)-3[prime] sites in the minor groove of DNA. The designed peptide has a specificity completely reversed from that of the tripyrrole distamycin, which binds A,T sequences. Structural studies with nuclear magnetic resonance revealed that two peptides bound side-by-side and in an antiparallel orientation in the minor groove. Each of the four imidazoles in the 2:1 ligand-DNA complex recognized a specific guanine amino group in the GCGC core through a hydrogen bond. Targeting a designated four-base pair G[center dot]C tract by this synthetic ligand supports the generality of the 2:1 peptide-DNA motif for sequence-specific minor groove recognition of DNA. 24 refs., 4 figs., 1 tab.

  10. Structure-based identification of CaMKIIα-interacting MUPP1 PDZ domains and rational design of peptide ligands to target such interaction in human fertilization.

    PubMed

    Zhang, Yi-Le; Han, Zhao-Feng; Sun, Ying-Pu

    2016-06-01

    The recognition and association between Ca(2+)/calmodulin-activated protein kinase II-α (CaMKIIα) and multi-PDZ domain protein 1 (MUPP1) plays an important role in sperm acrosome reaction and human fertilization, which is mediated by the binding of CaMKIIα's C-terminal tail to one or more PDZ domains of the scaffolding protein MUPP1. In this study, we attempt to identify the CaMKIIα-interacting MUPP1 PDZ domains and to design peptide ligands that can potently target and then competitively disrupt such interaction. Here, a synthetic biology approach was proposed to systematically characterize the structural basis, energetic property, dynamic behavior and biological implication underlying the intermolecular interactions between the C-terminal peptide of CaMKIIα and all the 13 PDZ domains of MUPP1. These domains can be grouped into four clusters in terms of their sequence, structure and physiochemical profile; different clusters appear to recognize different classes of PDZ-binding motifs. The cluster 3 includes two members, i.e. MUPP1 PDZ 5 and 11 domains, which were suggested to bind class II motif Φ-X-Φ(-COOH) of the C-terminal peptide SGAPSV(-COOH) of CaMKIIα. Subsequently, the two domains were experimentally measured as the moderate- and high-affinity binders of the peptide by using fluorescence titration (dissociation constants K d = 25.2 ± 4.6 and 0.47 ± 0.08 µM for peptide binding to PDZ 5 and 11, respectively), which was in line with theoretical prediction (binding free energies ΔG total = -7.6 and -9.2 kcal/mol for peptide binding to PDZ 5 and 11, respectively). A systematic mutation of SGAPSV(-COOH) residues suggested few favorable amino acids at different residue positions of the peptide, which were then combined to generate a number of potent peptide mutants for PDZ 11 domain. Consequently, two peptides (SIAPNV(-COOH) and SIVMNV(-COOH)) were identified to have considerably improved affinity with K d increase by ~tenfold relative to

  11. Aliphatic acid-conjugated antimicrobial peptides--potential agents with anti-tumor, multidrug resistance-reversing activity and enhanced stability.

    PubMed

    Deng, Xin; Qiu, Qianqian; Ma, Ke; Wang, Xuekun; Huang, Wenlong; Qian, Hai

    2015-07-28

    Compared with traditional therapeutics, antimicrobial peptides as novel anti-tumor agents have prominent advantages of higher specificity and circumvention of multi-drug resistance. In a previous study, we found that B1, an antimicrobial peptide derived from Cathelicidin-BF15, presented specific anti-tumor activity against several tumor cells. Since aliphatic chain-conjugated peptides have shown ameliorative activity and stability, we conjugated aliphatic acids with different lengths to the amino terminal of B1. All the conjugated peptides exhibited improved anti-tumor activity over B1. Further investigations revealed that the peptides were capable of disrupting the cell membrane, stimulating cytochrome c release into the cytosol, which results in apoptosis. The peptides also acted against multidrug resistant cells and had multidrug resistance-reversing effects. Additionally, conjugation of aliphatic acid enhanced the peptide stability in plasma. In summary, aliphatic acid-modified peptides might be promising anti-tumor agents in the future. PMID:26083110

  12. Peptide nucleic acids targeting β-globin mRNAs selectively inhibit hemoglobin production in murine erythroleukemia cells.

    PubMed

    Montagner, Giulia; Gemmo, Chiara; Fabbri, Enrica; Manicardi, Alex; Accardo, Igea; Bianchi, Nicoletta; Finotti, Alessia; Breveglieri, Giulia; Salvatori, Francesca; Borgatti, Monica; Lampronti, Ilaria; Bresciani, Alberto; Altamura, Sergio; Corradini, Roberto; Gambari, Roberto

    2015-01-01

    In the treatment of hemoglobinopathies, amending altered hemoglobins and/or globins produced in excess is an important part of therapeutic strategies and the selective inhibition of globin production may be clinically beneficial. Therefore the development of drug-based methods for the selective inhibition of globin accumulation is required. In this study, we employed peptide nucleic acids (PNAs) to alter globin gene expression. The main conclusion of the present study was that PNAs designed to target adult murine β-globin mRNA inhibit hemoglobin accumulation and erythroid differentiation of murine erythroleukemia (MEL) cells with high efficiency and fair selectivity. No major effects were observed on cell proliferation. Our study supports the concept that PNAs may be used to target mRNAs that, similar to globin mRNAs, are expressed at very high levels in differentiating erythroid cells. Our data suggest that PNAs inhibit the excess production of globins involved in the pathophysiology of hemoglobinopathies. PMID:25405921

  13. Convenient and Scalable Synthesis of Fmoc-Protected Peptide Nucleic Acid Backbone

    PubMed Central

    Feagin, Trevor A.; Shah, Nirmal I.; Heemstra, Jennifer M.

    2012-01-01

    The peptide nucleic acid backbone Fmoc-AEG-OBn has been synthesized via a scalable and cost-effective route. Ethylenediamine is mono-Boc protected, then alkylated with benzyl bromoacetate. The Boc group is removed and replaced with an Fmoc group. The synthesis was performed starting with 50 g of Boc anhydride to give 31 g of product in 32% overall yield. The Fmoc-protected PNA backbone is a key intermediate in the synthesis of nucleobase-modified PNA monomers. Thus, improved access to this molecule is anticipated to facilitate future investigations into the chemical properties and applications of nucleobase-modified PNA. PMID:22848796

  14. Convenient and scalable synthesis of fmoc-protected Peptide nucleic Acid backbone.

    PubMed

    Feagin, Trevor A; Shah, Nirmal I; Heemstra, Jennifer M

    2012-01-01

    The peptide nucleic acid backbone Fmoc-AEG-OBn has been synthesized via a scalable and cost-effective route. Ethylenediamine is mono-Boc protected, then alkylated with benzyl bromoacetate. The Boc group is removed and replaced with an Fmoc group. The synthesis was performed starting with 50 g of Boc anhydride to give 31 g of product in 32% overall yield. The Fmoc-protected PNA backbone is a key intermediate in the synthesis of nucleobase-modified PNA monomers. Thus, improved access to this molecule is anticipated to facilitate future investigations into the chemical properties and applications of nucleobase-modified PNA. PMID:22848796

  15. Incorporation of Naked Peptide Nucleic Acids into Liposomes Leads to Fast and Efficient Delivery.

    PubMed

    Avitabile, Concetta; Accardo, Antonella; Ringhieri, Paola; Morelli, Giancarlo; Saviano, Michele; Montagner, Giulia; Fabbri, Enrica; Gallerani, Eleonora; Gambari, Roberto; Romanelli, Alessandra

    2015-08-19

    The delivery of peptide nucleic acids (PNAs) to cells is a very challenging task. We report here that a liposomal formulation composed of egg PC/cholesterol/DSPE-PEG2000 can be loaded, according to different encapsulation techniques, with PNA or fluorescent PNA oligomers. PNA loaded liposomes efficiently and quickly promote the uptake of a PNA targeting the microRNA miR-210 in human erythroleukemic K562 cells. By using this innovative delivery system for PNA, down-regulation of miR-210 is achieved at a low PNA concentration. PMID:26176882

  16. In Silico Design of Novel Anticoagulant Peptides targeting Blood Coagulation Factor VIIa

    PubMed Central

    Al-Amri, Manal S Q; Alrasadi, Khalid; Bayoumi, Riad; Banerjee, Yajnavalka

    2011-01-01

    Objectives: The coagulation cascade initiated during vascular injury prevents bleeding. Unwanted clot formation is however detrimental and requires the use of anticoagulants for prophylaxis and treatment. Anticoagulants targeting a specific step or an enzyme in the clotting process are most preferred as they minimise disadvantageous side-effects. A principal step in the discovery of novel anticoagulants encompasses the in silico design of potential leads. This study depicts the in silico design of peptide anticoagulants targeting coagulation factor VIIa. Methods: Applying the proline bracket rule and using various bioinformatics tools: the basic alignment search tool (BLAST) of National Center for Biotechnology Information; the T-coffee module provided by European Molecular Biology Laboratory-European Bioinformatics Institute, and several modules available on the ExPASy server, we designed five bivalent chimeric anticoagulants targeting factor VIIa, using factor VIIa inhibitors – hemextin A from Hemachatus haemachatus (African Ringhals cobra) venom and factor VIIa exosite-inhibitor peptide as templates. Six peptides were derived from hemextin A, which were concomitantly fused with factor VIIa exosite-inhibitor peptide intermediated by a polyalanine spacer, and analysed for structural stability using the SWISS-MODEL software developed at the Swiss Institute of Bioinformatics and WebLab ViewerPro (Version 4.2). Results: Twelve chimeric peptides were obtained; only five exhibited stable structures in silico. Conclusion: The five peptides obtained are probable anticoagulant leads that should be further evaluated using suitable in vitro and in vivo assays. Further, this study shows how simple web-based modules can be used for the rational design of probable leads targeting specific physiological molecular targets. PMID:21509213

  17. Topical Delivery of Hyaluronic Acid into Skin using SPACE-peptide Carriers

    PubMed Central

    Chen, Ming; Gupta, Vivek; Anselmo, Aaron C.; Muraski, John A.; Mitragotri, Samir

    2014-01-01

    Topical penetration of macromolecules into skin is limited by their low permeability. Here, we report the use of a skin penetrating peptide, SPACE peptide, to enhance topical delivery of a macromolecule, hyaluronic acid (HA, MW: 200–325 kDa). The peptide was conjugated to phospholipids and used to prepare an ethosomal carrier system (~110 nm diameter), encapsulating HA. The SPACE-ethosomal system (SES) enhanced HA penetration into porcine skin in vitro by 7.8+/−1.1-fold compared to PBS. The system also enhanced penetration of HA in human skin in vitro, penetrating deep into the epidermis and dermis in skin of both species. In vivo experiments performed using SKH1 hairless mice also confirmed increased dermal penetration of HA using the delivery system; a 5-fold enhancement in penetration was found compared to PBS control. Concentrations of HA in skin were about 1000-fold higher than those in blood; confirming the localized nature of HA delivery into skin. The SPACE-ethosomal delivery system provides a formulation for topical delivery of macromolecules that are otherwise difficult to deliver into skin. PMID:24129342

  18. Enhanced Epimerization of Glycosylated Amino Acids During Solid Phase Peptide Synthesis

    PubMed Central

    Zhang, Yalong; Muthana, Saddam M.; Farnsworth, David; Ludek, Olaf; Adams, Kristie; Barchi, Joseph J.; Gildersleeve, Jeffrey C.

    2012-01-01

    Glycopeptides are extremely useful for basic research and clinical applications, but access to structurally-defined glycopeptides is limited by the difficulties in synthesizing this class of compounds. In this study, we demonstrate that many common peptide coupling conditions used to prepare O-linked glycopeptides result in substantial amounts of epimerization at the alpha position. In fact, epimerization resulted in up to 80% of the non-natural epimer, indicating that it can be the major product in some reactions. Through a series of mechanistic studies, we demonstrate that the enhanced epimerization relative to non-glycosylated amino acids is due to a combination of factors, including a faster rate of epimerization, an energetic preference for the unnatural epimer over the natural epimer, and a slower overall rate of peptide coupling. In addition, we demonstrate that use of 2,4,6-trimethylpyridine (TMP) as the base in peptide couplings produces glycopeptides with high efficiency and low epimerization. The information and improved reaction conditions will facilitate the preparation of glycopeptides as therapeutic compounds and vaccine antigens. PMID:22390544

  19. Black mamba venom peptides target acid-sensing ion channels to abolish pain.

    PubMed

    Diochot, Sylvie; Baron, Anne; Salinas, Miguel; Douguet, Dominique; Scarzello, Sabine; Dabert-Gay, Anne-Sophie; Debayle, Delphine; Friend, Valérie; Alloui, Abdelkrim; Lazdunski, Michel; Lingueglia, Eric

    2012-10-25

    Polypeptide toxins have played a central part in understanding physiological and physiopathological functions of ion channels. In the field of pain, they led to important advances in basic research and even to clinical applications. Acid-sensing ion channels (ASICs) are generally considered principal players in the pain pathway, including in humans. A snake toxin activating peripheral ASICs in nociceptive neurons has been recently shown to evoke pain. Here we show that a new class of three-finger peptides from another snake, the black mamba, is able to abolish pain through inhibition of ASICs expressed either in central or peripheral neurons. These peptides, which we call mambalgins, are not toxic in mice but show a potent analgesic effect upon central and peripheral injection that can be as strong as morphine. This effect is, however, resistant to naloxone, and mambalgins cause much less tolerance than morphine and no respiratory distress. Pharmacological inhibition by mambalgins combined with the use of knockdown and knockout animals indicates that blockade of heteromeric channels made of ASIC1a and ASIC2a subunits in central neurons and of ASIC1b-containing channels in nociceptors is involved in the analgesic effect of mambalgins. These findings identify new potential therapeutic targets for pain and introduce natural peptides that block them to produce a potent analgesia. PMID:23034652

  20. Self-assembling peptide of D-amino acids boosts selectivity and antitumor efficacy of 10-hydroxycamptothecin.

    PubMed

    Liu, Jianfeng; Liu, Jinjian; Chu, Liping; Zhang, Yumin; Xu, Hongyan; Kong, Deling; Yang, Zhimou; Yang, Cuihong; Ding, Dan

    2014-04-23

    D-peptides, which consist of D-amino acids and can resist the hydrolysis catalyzed by endogenous peptidases, are one of the promising candidates for construction of peptide materials with enhanced biostability in vivo. In this paper, we report on a self-assembling supramolecular nanostructure of D-amino acid-based peptide Nap-G(D)F(D)F(D)YGRGD (D-fiber, (D)F meant D-phenylalanine, (D)Y meant D-tyrosine), which were used as carriers for 10-hydroxycamptothecin (HCPT). Transmission electron microscopy observations demonstrated the filamentous morphology of the HCPT-loaded peptides (d-fiber-HCPT). The better selectivity and antitumor activity of D-fiber-HCPT than L-fiber-HCPT were found in the in vitro and in vivo antitumor studies. These results highlight that this model D-fiber system holds great promise as vehicles of hydrophobic drugs for cancer therapy. PMID:24660962

  1. Design of a novel metal binding peptide by molecular dynamics simulation to sequester Cu and Zn ions

    PubMed Central

    Mahnam, K.; Saffar, B.; Mobini-Dehkordi, M.; Fassihi, A.; Mohammadi, A.

    2014-01-01

    Heavy metal toxicity has serious adverse effects on the environment. The metal sequestering characteristics of a novel metal binding peptide (Glu-Cys)11 Gly+linker+hexahistidine (EC11:His6) was investigated to determine if it can absorb Cu2+ or Zn2+ cations. Molecular dynamics simulations were carried out using a model of 6 Cu2+ or Zn2+ and other ions enclosed in a fully hydrated simulation box with the designed peptide. Totally, 240 nano second (ns) simulations were done in three phases. Results showed that the selected linker is able to separate two domains of this peptide and that the carboxyl oxygens of Glu residues of EC11 in the designed peptide can absorb these ions. Sequestration of Cu2+ or Zn2+ ions by the designed peptide does not change overall tertiary and secondary structures of peptide. PMID:25598801

  2. Development of a method for environmentally friendly chemical peptide synthesis in water using water-dispersible amino acid nanoparticles

    PubMed Central

    2011-01-01

    Due to the vast importance of peptides in biological processes, there is an escalating need for synthetic peptides to be used in a wide variety of applications. However, the consumption of organic solvent is extremely large in chemical peptide syntheses because of the multiple condensation steps in organic solvents. That is, the current synthesis method is not environmentally friendly. From the viewpoint of green sustainable chemistry, we focused on developing an organic solvent-free synthetic method using water, an environmentally friendly solvent. Here we described in-water synthesis technology using water-dispersible protected amino acids. PMID:21867548

  3. Characterization and in vitro activity of a branched peptide boronic acid that interacts with HIV-1 RRE RNA.

    PubMed

    Wynn, Jessica E; Zhang, Wenyu; Tebit, Denis M; Gray, Laurie R; Hammarskjold, Marie-Louise; Rekosh, David; Santos, Webster L

    2016-09-01

    A branched peptide containing multiple boronic acids was found to bind RRE IIB selectively and inhibit HIV-1 p24 capsid production in a dose-dependent manner. Structure-activity relationship studies revealed that branching in the peptide is crucial for the low micromolar binding towards RRE IIB, and the peptide demonstrates selectivity towards RRE IIB in the presence of tRNA. Footprinting studies suggest a binding site on the upper stem and internal loop regions of the RNA, which induces enzymatic cleavage of the internal loops of RRE IIB upon binding. PMID:27091070

  4. Design of Compact Biomimetic Cellulose Binding Peptides as Carriers for Cellulose Catalytic Degradation.

    PubMed

    Khazanov, Netaly; Iline-Vul, Taly; Noy, Efrat; Goobes, Gil; Senderowitz, Hanoch

    2016-01-21

    The conversion of biomass into biofuels can reduce the strategic vulnerability of petroleum-based systems and at the same time have a positive effect on global climate issues. Lignocellulose is the cheapest and most abundant source of biomass and consequently has been widely considered as a source for liquid fuel. However, despite ongoing efforts, cellulosic biofuels are still far from commercial realization, one of the major bottlenecks being the hydrolysis of cellulose into simpler sugars. Inspired by the structural and functional modularity of cellulases used by many organisms for the breakdown of cellulose, we propose to mimic the cellulose binding domain (CBD) and the catalytic domain of these proteins by small molecular entities. Multiple copies of these mimics could subsequently be tethered together to enhance hydrolytic activity. In this work, we take the first step toward achieving this goal by applying computational approaches to the design of efficient, cost-effective mimetics of the CBD. The design is based on low molecular weight peptides that are amenable to large-scale production. We provide an optimized design of four short (i.e., ∼18 residues) peptide mimetics based on the three-dimensional structure of a known CBD and demonstrate that some of these peptides bind cellulose as well as or better than the full CBD. The structures of these peptides were studied by circular dichroism and their interactions with cellulose by solid phase NMR. Finally, we present a computational strategy for predicting CBD/peptide-cellulose binding free energies and demonstrate its ability to provide values in good agreement with experimental data. Using this computational model, we have also studied the dissociation pathway of the CBDs/peptides from the surface of cellulose. PMID:26691055

  5. Designer Self-Assembling Peptide Nanofiber Scaffolds for Adult Mouse Neural Stem Cell 3-Dimensional Cultures

    PubMed Central

    Gelain, Fabrizio; Bottai, Daniele; Vescovi, Angleo; Zhang, Shuguang

    2006-01-01

    Biomedical researchers have become increasingly aware of the limitations of conventional 2-dimensional tissue cell culture systems, including coated Petri dishes, multi-well plates and slides, to fully address many critical issues in cell biology, cancer biology and neurobiology, such as the 3-D microenvironment, 3-D gradient diffusion, 3-D cell migration and 3-D cell-cell contact interactions. In order to fully understand how cells behave in the 3-D body, it is important to develop a well-controlled 3-D cell culture system where every single ingredient is known. Here we report the development of a 3-D cell culture system using a designer peptide nanofiber scaffold with mouse adult neural stem cells. We attached several functional motifs, including cell adhesion, differentiation and bone marrow homing motifs, to a self-assembling peptide RADA16 (Ac-RADARADARADARADA-COHN2). These functionalized peptides undergo self-assembly into a nanofiber structure similar to Matrigel. During cell culture, the cells were fully embedded in the 3-D environment of the scaffold. Two of the peptide scaffolds containing bone marrow homing motifs significantly enhanced the neural cell survival without extra soluble growth and neurotrophic factors to the routine cell culture media. In these designer scaffolds, the cell populations with β-Tubulin+, GFAP+ and Nestin+ markers are similar to those found in cell populations cultured on Matrigel. The gene expression profiling array experiments showed selective gene expression, possibly involved in neural stem cell adhesion and differentiation. Because the synthetic peptides are intrinsically pure and a number of desired function cellular motifs are easy to incorporate, these designer peptide nanofiber scaffolds provide a promising controlled 3-D culture system for diverse tissue cells, and are useful as well for general molecular and cell biology. PMID:17205123

  6. Comparison of amino acid v peptide based enteral diets in active Crohn's disease: clinical and nutritional outcome.

    PubMed Central

    Royall, D; Jeejeebhoy, K N; Baker, J P; Allard, J P; Habal, F M; Cunnane, S C; Greenberg, G R

    1994-01-01

    Elemental diets are considered an effective primary treatment for active Crohn's disease. This study examined the hypothesis that improvement occurs because of the presence of amino acids or the low fat content, or both. A randomised, controlled trial was undertaken in 40 patients with active Crohn's disease to evaluate clinical and nutritional outcomes after an amino acid based diet containing 3% fat was given by a feeding tube compared with a peptide based diet containing 33% fat. After three weeks' treatment, clinical remission occurred in 84% of patients who were given the amino acid diet and 75% of patients who received the peptide diet (p = 0.38). Plasma linoleic acid concentration was reduced after the amino acid but not the peptide diet. An increase in total body nitrogen was associated with the magnitude of nutritional depletion before treatment and at six months' follow up, only patients who showed gains in total body nitrogen after enteral nutrition had a sustained clinical remission. This study shows that peptide based high fat diets are as effective as amino acid low fat diets for achieving clinical remission in active Crohn's disease. Improved total body protein stores but not essential fatty acid depletion may be an important indicator of a sustained remission. PMID:8020806

  7. Adapting and testing a portable Raman spectrometer for SERS analysis of amino acids and small peptides

    NASA Astrophysics Data System (ADS)

    Brambilla, A.; Philippidis, A.; Nevin, A.; Comelli, D.; Valentini, G.; Anglos, D.

    2013-07-01

    Surface-Enhanced Raman Spectroscopy (SERS), a powerful spectrochemical technique enabling highly sensitive analysis of organic and biological materials, is investigated for applications in the analysis of archaeological materials including in situ screening. In this work, a compact mobile Raman spectrometer is employed for acquiring Surface-Enhanced Raman spectra from natural amino acids (L-Arg, L-Phe, L-Met) and a tripeptide (Glutathione), adsorbed on silver colloids. The detection limits of the portable Raman spectrometer, together with an optimization of sample preparation and experimental parameters, are reported. The collection and interpretation of SER spectra of amino acids and peptides is a starting point for the optimization of the instrumentation and its application in the study of more complex biological molecules in the context of detection and analysis of archaeological materials and residues.

  8. Information transfer from peptide nucleic acids to RNA by template-directed syntheses

    NASA Technical Reports Server (NTRS)

    Schmidt, J. G.; Nielsen, P. E.; Orgel, L. E.; Bada, J. L. (Principal Investigator)

    1997-01-01

    Peptide nucleic acids (PNAs) are uncharged analogs of DNA and RNA in which the ribose-phosphate backbone is substituted by a backbone held together by amide bonds. PNAs are interesting as models of alternative genetic systems because they form potentially informational base paired helical structures. A PNA C10 oligomer has been shown to act as template for efficient formation of oligoguanylates from activated guanosine ribonucleotides. In a previous paper we used heterosequences of DNA as templates in sequence-dependent polymerization of PNA dimers. In this paper we show that information can be transferred from PNA to RNA. We describe the reactions of activated mononucleotides on heterosequences of PNA. Adenylic, cytidylic and guanylic acids were incorporated into the products opposite their complement on PNA, although less efficiently than on DNA templates.

  9. Bioactive Molecules Released in Food by Lactic Acid Bacteria: Encrypted Peptides and Biogenic Amines

    PubMed Central

    Pessione, Enrica; Cirrincione, Simona

    2016-01-01

    Lactic acid bacteria (LAB) can produce a huge amount of bioactive compounds. Since their elective habitat is food, especially dairy but also vegetal food, it is frequent to find bioactive molecules in fermented products. Sometimes these compounds can have adverse effects on human health such as biogenic amines (tyramine and histamine), causing allergies, hypertensive crises, and headache. However, some LAB products also display benefits for the consumers. In the present review article, the main nitrogen compounds produced by LAB are considered. Besides biogenic amines derived from the amino acids tyrosine, histidine, phenylalanine, lysine, ornithine, and glutamate by decarboxylation, interesting peptides can be decrypted by the proteolytic activity of LAB. LAB proteolytic system is very efficient in releasing encrypted molecules from several proteins present in different food matrices. Alpha and beta-caseins, albumin and globulin from milk and dairy products, rubisco from spinach, beta-conglycinin from soy and gluten from cereals constitute a good source of important bioactive compounds. These encrypted peptides are able to control nutrition (mineral absorption and oxidative stress protection), metabolism (blood glucose and cholesterol lowering) cardiovascular function (antithrombotic and hypotensive action), infection (microbial inhibition and immunomodulation) and gut-brain axis (opioids and anti-opioids controlling mood and food intake). Very recent results underline the role of food-encrypted peptides in protein folding (chaperone-like molecules) as well as in cell cycle and apoptosis control, suggesting new and positive aspects of fermented food, still unexplored. In this context, the detailed (transcriptomic, proteomic, and metabolomic) characterization of LAB of food interest (as starters, biocontrol agents, nutraceuticals, and probiotics) can supply a solid evidence-based science to support beneficial effects and it is a promising approach as well to obtain

  10. Bioactive Molecules Released in Food by Lactic Acid Bacteria: Encrypted Peptides and Biogenic Amines.

    PubMed

    Pessione, Enrica; Cirrincione, Simona

    2016-01-01

    Lactic acid bacteria (LAB) can produce a huge amount of bioactive compounds. Since their elective habitat is food, especially dairy but also vegetal food, it is frequent to find bioactive molecules in fermented products. Sometimes these compounds can have adverse effects on human health such as biogenic amines (tyramine and histamine), causing allergies, hypertensive crises, and headache. However, some LAB products also display benefits for the consumers. In the present review article, the main nitrogen compounds produced by LAB are considered. Besides biogenic amines derived from the amino acids tyrosine, histidine, phenylalanine, lysine, ornithine, and glutamate by decarboxylation, interesting peptides can be decrypted by the proteolytic activity of LAB. LAB proteolytic system is very efficient in releasing encrypted molecules from several proteins present in different food matrices. Alpha and beta-caseins, albumin and globulin from milk and dairy products, rubisco from spinach, beta-conglycinin from soy and gluten from cereals constitute a good source of important bioactive compounds. These encrypted peptides are able to control nutrition (mineral absorption and oxidative stress protection), metabolism (blood glucose and cholesterol lowering) cardiovascular function (antithrombotic and hypotensive action), infection (microbial inhibition and immunomodulation) and gut-brain axis (opioids and anti-opioids controlling mood and food intake). Very recent results underline the role of food-encrypted peptides in protein folding (chaperone-like molecules) as well as in cell cycle and apoptosis control, suggesting new and positive aspects of fermented food, still unexplored. In this context, the detailed (transcriptomic, proteomic, and metabolomic) characterization of LAB of food interest (as starters, biocontrol agents, nutraceuticals, and probiotics) can supply a solid evidence-based science to support beneficial effects and it is a promising approach as well to obtain

  11. Computational Design of the Sequence and Structure of a Protein-Binding Peptide

    PubMed Central

    Sammond, Deanne W.; Bosch, Dustin E.; Butterfoss, Glenn L.; Purbeck, Carrie; Machius, Mischa; Siderovski, David P.; Kuhlman, Brian

    2011-01-01

    The de novo design of protein-binding peptides is challenging, because it requires identifying both a sequence and a backbone conformation favorable for binding. We used a computational strategy that iterates between structure and sequence optimization to redesign the C-terminal portion of the RGS14 GoLoco motif peptide so that it adopts a new conformation when bound to Gαi1. An X-ray crystal structure of the redesigned complex closely matches the computational model, with a backbone RMSD of 1.1 Å. PMID:21388199

  12. Design and Characterization of a Multifunctional pH-Triggered Peptide C8 for Selective Anticancer Activity.

    PubMed

    Lu, Sheng; Bennett, W F Drew; Ding, Yong; Zhang, Lei; Fan, Helen Y; Zhao, Danyang; Zheng, Tao; Ouyang, Ping-Kai; Li, Jason; Wu, Yan; Xu, Wen; Chu, Dafeng; Yuan, Yongfang; Heerklotz, Heiko; Karttunen, Mikko; Chen, P

    2015-12-01

    Most drug delivery systems have been developed for efficient delivery to tumor sites via targeting and on-demand strategies, but the carriers rarely execute synergistic therapeutic actions. In this work, C8, a cationic, pH-triggered anticancer peptide, is developed by incorporating histidine-mediated pH-sensitivity, amphipathic helix, and amino acid pairing self-assembly design. We designed C8 to function as a pH-responsive nanostructure whose cytotoxicity can be switched on and off by its self-assembly: Noncytotoxic β-sheet fibers at high pH with neutral histidines, and positively charged monomers with membrane lytic activity at low pH. The selective activity of C8, tested for three different cancer cell lines and two noncancerous cell lines, is shown. Based on liposome leakage assays and multiscale computer simulations, its physical mechanisms of pore-forming action and selectivity are proposed, which originate from differences in the lipid composition of the cellular membrane and changes in hydrogen bonding. C8 is then investigated for its potential as a drug carrier. C8 forms a nanocomplex with ellipticine, a nonselective model anticancer drug. It selectively targets cancer cells in a pH-responsive manner, demonstrating enhanced efficacy and selectivity. This study provides a novel powerful strategy for the design and development of multifunctional self-assembling peptides for therapeutic and drug delivery applications. PMID:26474414

  13. Peptide nucleic acids rather than RNA may have been the first genetic molecule

    NASA Technical Reports Server (NTRS)

    Nelson, K. E.; Levy, M.; Miller, S. L.

    2000-01-01

    Numerous problems exist with the current thinking of RNA as the first genetic material. No plausible prebiotic processes have yet been demonstrated to produce the nucleosides or nucleotides or for efficient two-way nonenzymatic replication. Peptide nucleic acid (PNA) is a promising precursor to RNA, consisting of N-(2-aminoethyl)glycine (AEG) and the adenine, uracil, guanine, and cytosine-N-acetic acids. However, PNA has not yet been demonstrated to be prebiotic. We show here that AEG is produced directly in electric discharge reactions from CH(4), N(2), NH(3), and H(2)O. Electric discharges also produce ethylenediamine, as do NH(4)CN polymerizations. AEG is produced from the robust Strecker synthesis with ethylenediamine. The NH(4)CN polymerization in the presence of glycine leads to the adenine and guanine-N(9)-acetic acids, and the cytosine and uracil-N(1)-acetic acids are produced in high yield from the reaction of cyanoacetaldehyde with hydantoic acid, rather than urea. Preliminary experiments suggest that AEG may polymerize rapidly at 100 degrees C to give the polypeptide backbone of PNA. The ease of synthesis of the components of PNA and possibility of polymerization of AEG reinforce the possibility that PNA may have been the first genetic material.

  14. Amino acid and peptide absorption in patients with coeliac disease and dermatitis herpetiformis

    PubMed Central

    Silk, D. B. A.; Kumar, Parveen J.; Perrett, D.; Clark, M. L.; Dawson, A. M.

    1974-01-01

    A double-lumen perfusion technique has been used to study amino acid and peptide absorption in eight normal control subjects, 13 patients with untreated adult coeliac disease, and 16 patients with dermatitis herpetiformis who had varying morphological abnormalities of the small bowel. All subjects were perfused with isotonic solutions containing 10 mM glycyl-L-alanine and 10 mM glycine + 10 mM L-alanine. Patients with adult coeliac disease had impaired absorption of glycine (p < 0·01) and L-alanine (p < 0·05) from the amino acid solution compared with the control subjects. Amino acid uptake from the dipeptide solution was not significantly impaired, although four individual patients had impaired uptake of both amino acids. In contrast to these findings, very few patients with dermatitis herpetiformis had impaired amino acid absorption from either solution. Sodium absorption was impaired from both solutions when the groups of patients with adult coeliac disease and dermatitis herpetiformis with subtotal villous atrophy and partial villous atrophy were studied, and there were patients in each group who secreted sodium and water. The results suggest that malabsorption of dietary protein is unlikely to occur in dermatitis herpetiformis but may occur and contribute to protein deficiency seen in some severe cases of adult coeliac disease. The impairment of sodium and water absorption provides evidence that there may be functional impairment of the jejunal mucosa in dermatitis herpetiformis as well as in adult coeliac disease. PMID:4820629

  15. Template tailoring: Accurate determination of heterozygous alleles using peptide nucleic acid and dideoxyNTP

    PubMed Central

    Tariq, Muhammad Akram; Pourmand, Nader

    2010-01-01

    Measurement of the length of DNA fragments plays a pivotal role in genetic mapping, disease diagnostics, human identification and forensic applications. PCR followed by electrophoresis is used for DNA length measurement of STRs, a process that requires labeled primers and allelic ladders as standards to avoid machine error. Sequencing-based approaches can be used for STR analysis to eliminate the requirement of labeled primers and allelic ladder. However, the limiting factor with this approach is unsynchronized polymerization in heterozygous sample analysis, in which alleles with different lengths can lead to imbalanced heterozygote peak height ratios. We have developed a rapid DNA length measurement method using peptide nucleic acid and dideoxy dNTPs to “tailor” DNA templates for accurate sequencing to overcome this hurdle. We also devised an accelerated “dyad” pyrosequencing strategy, such that the combined approach can be used as a faster, more accurate alternative to de novo sequencing. Dyad sequencing interrogates two bases at a time by allowing the polymerase to incorporate two nucleotides to DNA template, cutting the analysis time in half. In addition, for the first time, we show the effect of peptide nucleic acid as a blocking probe to stop polymerization, which is essential to analyze the heterozygous samples by sequencing. This approach provides a new platform for rapid and cost-effective DNA length measurement for STRs and resequencing of small DNA fragments. PMID:20408144

  16. Fabrication of Odor Sensor Using Peptide

    NASA Astrophysics Data System (ADS)

    Hotokebuchi, Yuta; Hayashi, Kenshi; Toko, Kiyoshi; Chen, Ronggang; Ikezaki, Hidekazu

    We report fabrication of an odor sensor using peptides. Peptides were designed to acquire the specific reception for a target odor molecule. Au surface of the sensor electrode was coated by the designed peptide using the method of self assembled monolayers (SAMs). Functionalized Au surfaces by the peptides were confirmed by ellipsometry and cyclic voltammetry. The odorants of vanillin, phenethyl alcohol and hexanol were discriminated by QCM sensor with the peptide surface. Moreover, we verified specific interaction between amino acid (Trp) and vanillin by fluorescence assay.

  17. Conformational characterization of the 1-aminocyclobutane-1-carboxylic acid residue in model peptides.

    PubMed

    Gatos, M; Formaggio, F; Crisma, M; Toniolo, C; Bonora, G M; Benedetti, Z; Di Blasio, B; Iacovino, R; Santini, A; Saviano, M; Kamphuis, J

    1997-01-01

    A series of N- and C-protected, monodispersed homo-oligopeptides (to the dodecamer level) from the small-ring alicyclic C alpha, alpha-dialkylated glycine 1-aminocyclobutane-1-carboxylic acid (Ac4c) and two Ala/Ac4c tripeptides were synthesized by solution methods and fully characterized. The conformational preferences of all the model peptides were determined in deuterochloroform solution by FT-IR absorption and 1H-NMR. The molecular structures of the amino acid derivatives Z-Ac4c-OH and Z2-Ac4c-OH, the tripeptides Z-(Ac4c)3-OtBu, Z-Ac4c-(L-Ala)2-OMe and Z-L-Ala-Ac4c-L-Ala-OMe, and the tetrapeptide Z-(Ac4c)4-OtBu were determined in the crystal state by X-ray diffraction. The average geometry of the cyclobutyl moiety of the Ac4c residue was assessed and the tau(N-C alpha-C') bond angle was found to be significantly expanded from the regular tetrahedral value. The conformational data are strongly in favour of the conclusion that the Ac4c residue is an effective beta-turn and helix former. A comparison with the structural propensities of alpha-aminoisobutyric acid, the prototype of C alpha, alpha-dialkylated glycines, and the other extensively investigated members of the family of 1-aminocycloalkane-1-carboxylic acids (Acnc, with n = 3, 5-8) is made and the implications for the use of the Ac4c residue in conformationally constrained peptide analogues are briefly examined. PMID:9230476

  18. Design, recombinant expression, and antibacterial activity of the cecropins-melittin hybrid antimicrobial peptides.

    PubMed

    Cao, Yu; Yu, Rong Qing; Liu, Yi; Zhou, Huo Xiang; Song, Ling Ling; Cao, Yi; Qiao, Dai Rong

    2010-09-01

    In order to evaluate their antibacterial activities and toxicities, the cecropins-melittin hybrid antimicrobial peptide, CA(1-7)-M(4-11) (CAM) and CB(1-7)-M(4-11) (CBM), were designed by APD2 database. The recombinant hybrid antimicrobial peptides were successfully expressed and purified in Pichia pastoris. Antimicrobial activity assay showed that both of the two hybrid antimicrobial peptides had strong antibacterial abilities against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Bacillus subtilis, Bacillus thuringiensis, and Salmonella derby. The potency of CAM and CBM to E. coli 25922 were 0.862 and 0.849, respectively, slightly lower than Amp's 0.957. The hemolytic assays indicated CAM and CBM had no hemolytic in vivo and in vitro, and so they had a good application prospect. PMID:20111863

  19. Copper-binding peptides from human prion protein and newly designed peroxidative biocatalysts.

    PubMed

    Kagenishi, Tomoko; Yokawa, Ken; Kadono, Takashi; Uezu, Kazuya; Kawano, Tomonori

    2011-01-01

    A previous work suggested that peptides from the histidine-containing copper-binding motifs in human prion protein (PrP) function as peroxidase-like biocatalysts catalyzing the generation of superoxide anion radicals in the presence of neurotransmitters (aromatic monoamines) and phenolics such as tyrosine and tyrosyl residues on proteins. In this study, using various phenolic substrates, the phenol-dependent superoxide-generating activities of PrP-derived peptide sequences were compared. Among the peptides tested, the GGGTH pentapeptide was shown to be the most active catalyst for phenol-dependent reactions. Based on these results, we designed a series of oligoglycyl-histidines as novel peroxidative biocatalysts, and their catalytic performances including kinetics, heat tolerance, and freezing tolerance were analysed. PMID:21630593

  20. A novel antimicrobial peptide derived from modified N-terminal domain of bovine lactoferrin: design, synthesis, activity against multidrug-resistant bacteria and Candida.

    PubMed

    Mishra, Biswajit; Leishangthem, Geeta Devi; Gill, Kamaldeep; Singh, Abhay K; Das, Swagata; Singh, Kusum; Xess, Immaculata; Dinda, Amit; Kapil, Arti; Patro, Ishan K; Dey, Sharmistha

    2013-02-01

    Lactoferrin (LF) is believed to contribute to the host's defense against microbial infections. This work focuses on the antibacterial and antifungal activities of a designed peptide, L10 (WFRKQLKW) by modifying the first eight N-terminal residues of bovine LF by selective homologous substitution of amino acids on the basis of hydrophobicity, L10 has shown potent antibacterial and antifungal properties against clinically isolated extended spectrum beta lactamases (ESBL), producing gram-negative bacteria as well as Candida strains with minimal inhibitory concentrations (MIC) ranging from 1 to 8 μg/mL and 6.5 μg/mL, respectively. The peptide was found to be least hemolytic at a concentration of 800 μg/mL. Interaction with lipopolysaccharide (LPS) and lipid A (LA) suggests that the peptide targets the membrane of gram-negative bacteria. The membrane interactive nature of the peptide, both antibacterial and antifungal, was further confirmed by visual observations employing electron microscopy. Further analyses, by means of propidium iodide based flow cytometry, also supported the membrane permeabilization of Candida cells. The peptide was also found to possess anti-inflammatory properties, by virtue of its ability to inhibit cyclooxygenase-2 (COX-2). L10 therefore emerges as a potential therapeutic remedial solution for infections caused by ESBL positive, gram-negative bacteria and multidrug-resistant (MDR) fungal strains, on account of its multifunctional activities. This study may open up new approach to develop and design novel antimicrobials. PMID:23026014

  1. Role of SbmA in the uptake of peptide nucleic acid (PNA)-peptide conjugates in E. coli.

    PubMed

    Ghosal, Anubrata; Vitali, Ally; Stach, James E M; Nielsen, Peter E

    2013-02-15

    Antisense PNA oligomers targeting essential genes (acpP or ftsZ) and conjugated to the delivery peptide L((KFF)(3)K) show complete growth inhibition of wild type E. coli strain (MG1655) with submicromolar MIC. In this study we show that resistant mutants generated against such PNA-peptide conjugates had disruptions in the region of sbmA, a gene encoding an inner membrane peptide transporter. The wild type sensitivity to the PNA conjugates was re-established in the resistance mutants by complementation with sbmA. Furthermore, deletion of sbmA in E. coli AS19, a strain that is sensitive to unmodified PNA, resulted in resistance to PNA. Finally, PNA conjugated with the corresponding non-biological H-D((KFF)(3)K) peptide retained antibacterial activity in sbmA deletion strains, whereas the same conjugate with a protease-sensitive linker did not. These results clearly identify SbmA as a carrier of naked PNA over the inner bacterial membrane and thereby infer that the peptide is transporting the PNA conjugates over the outer membrane. Strains lacking SbmA were used to screen novel peptide-PNA carriers that were SbmA-independent. Four such PNA-peptide conjugates, H-D((KFF)(3)K), H-(RFR)(4)-Ahx-βAla, H-(R-Ahx-R)(4)-Ahx-βAla, and H-(R-Ahx)(6)-βAla, were identified that utilize an alternative uptake mechanism but retain their antimicrobial potency. In addition SbmA is the first protein identified to recognize PNA. PMID:23138594

  2. Oxytocic plant cyclotides as templates for peptide G protein-coupled receptor ligand design

    PubMed Central

    Koehbach, Johannes; O’Brien, Margaret; Muttenthaler, Markus; Miazzo, Marion; Akcan, Muharrem; Elliott, Alysha G.; Daly, Norelle L.; Harvey, Peta J.; Arrowsmith, Sarah; Gunasekera, Sunithi; Smith, Terry J.; Wray, Susan; Göransson, Ulf; Dawson, Philip E.; Craik, David J.; Freissmuth, Michael; Gruber, Christian W.

    2013-01-01

    Cyclotides are plant peptides comprising a circular backbone and three conserved disulfide bonds that confer them with exceptional stability. They were originally discovered in Oldenlandia affinis based on their use in traditional African medicine to accelerate labor. Recently, cyclotides have been identified in numerous plant species of the coffee, violet, cucurbit, pea, potato, and grass families. Their unique structural topology, high stability, and tolerance to sequence variation make them promising templates for the development of peptide-based pharmaceuticals. However, the mechanisms underlying their biological activities remain largely unknown; specifically, a receptor for a native cyclotide has not been reported hitherto. Using bioactivity-guided fractionation of an herbal peptide extract known to indigenous healers as “kalata-kalata,” the cyclotide kalata B7 was found to induce strong contractility on human uterine smooth muscle cells. Radioligand displacement and second messenger-based reporter assays confirmed the oxytocin and vasopressin V1a receptors, members of the G protein-coupled receptor family, as molecular targets for this cyclotide. Furthermore, we show that cyclotides can serve as templates for the design of selective G protein-coupled receptor ligands by generating an oxytocin-like peptide with nanomolar affinity. This nonapeptide elicited dose-dependent contractions on human myometrium. These observations provide a proof of concept for the development of cyclotide-based peptide ligands. PMID:24248349

  3. Design of Potent Inhibitors of Human RAD51 Recombinase Based on BRC Motifs of BRCA2 Protein: Modeling and Experimental Validation of a Chimera Peptide

    PubMed Central

    2010-01-01

    We have previously shown that a 28-amino acid peptide derived from the BRC4 motif of BRCA2 tumor suppressor inhibits selectively human RAD51 recombinase (HsRad51). With the aim of designing better inhibitors for cancer treatment, we combined an in silico docking approach with in vitro biochemical testing to construct a highly efficient chimera peptide from eight existing human BRC motifs. We built a molecular model of all BRC motifs complexed with HsRad51 based on the crystal structure of the BRC4 motif-HsRad51 complex, computed the interaction energy of each residue in each BRC motif, and selected the best amino acid residue at each binding position. This analysis enabled us to propose four amino acid substitutions in the BRC4 motif. Three of these increased the inhibitory effect in vitro, and this effect was found to be additive. We thus obtained a peptide that is about 10 times more efficient in inhibiting HsRad51−ssDNA complex formation than the original peptide. PMID:20684611

  4. Chemiluminescence detection of amino acids, peptides, and proteins using tris-2,2 prime -bipyridine ruthenium(III)

    SciTech Connect

    Li He; Cox, K.A.; Danielson, N.D. )

    1990-01-01

    The feasibility of using the tris-2-2{prime}-bipyridine ruthenium(III) chemiluminescent (CL) reaction for the detection of amino acids, peptides, and proteins has been studied. Detection limits of the amino acids as determined by flow injection analysis (FIA) ranged from 20 pmol of proline to 50 nmol of asparagine. In general, amino acids containing secondary amine groups yielded the strongest responses. A reaction mechanism for Ru(bpy){sub 3}{sup 3+} chemiluminescence of aliphatic amines has been proposed. Studies of peptide molecules and poly-prolines showed that the peptide bond barely contributes to the detection signals. The separation of hydroxyproline and proline in synthetic collagen by HPLC with Ru(bpy){sub 3}{sup 3+} chemiluminescence detection has been shown to be possible.

  5. Design and validation of an ELISA for equine infectious anemia (EIA) diagnosis using synthetic peptides.

    PubMed

    Soutullo, A; Verwimp, V; Riveros, M; Pauli, R; Tonarelli, G

    2001-03-20

    Three peptides derived from the equine infectious anemia virus (EIAV) surface proteins were synthesized to design and validate an ELISA for EIA diagnosis. Peptides identified as gp90-I and gp90-II correspond to the N- and C-terminal part of the surface glycoprotein gp90. Peptide gp45-1 overlaps the immunodominant epitope CIERTHVFC of the transmembrane glycoprotein gp45, and includes a hydrophilic chain close to the N-terminal end of this nonapeptide loop. Serum samples from 140 naturally infected horses with EIAV and a panel of 167 non-immune equine sera obtained from non-infected animals were used. Differences in reactivity between positive and negative serum samples were clearly distinguished. Samples considered weak positive to the agar gel immunodiffusion (AGID) test were "true" positive in the ELISA. These results are consistent with the improved sensitivity of the ELISA in comparison with the AGID test. The cyclic peptide that mimics the immunodominant sequence of gp45 showed excellent reactivity, thus suggesting that its functional activity depends significantly on its conformation, since very low reactivity was observed in the linear form of the peptide. The detectability indices of positive and negative sera reached 98% when gp90-II and gp45-I synthetic peptides were used in the same assay, illustrating the high specificity and sensitivity of the assay. Our study represents a first approach for the design of a diagnostic kit, which would allow the rapid analysis of a large numbers of serum samples from horses, and could be applied in endemic areas with different prevalence of infection. PMID:11230933

  6. Effects of Ti surface treatments with silane and arginylglycylaspartic acid peptide on bone cell progenitors.

    PubMed

    Chen, Wen-Cheng; Lo, Yang; Chen, Hong-Sen

    2015-09-01

    Achieving optimal aesthetic appearance is a major objective in dental implant design, and the interaction between the materials and the bone cell progenitors is an important factor in the attainment of this objective. In this study, a novel concept was evaluated by varying the surface modifications on titanium (Ti). Different levels of roughness can be attained by machine grinding (M), sand blasting, and acid etching (SLA) of the samples. The behavior of bone cell progenitors (D1) on the surfaces of Ti disks with different surface modifications was investigated. The surfaces of M or SLA disks were silanized (MS or SLAS group) through treatment with silane/Gly-Arg-Gly-Asp-Ser (GRGDS) peptide (MSP or SLASP group) and anchored particles of tetracalcium phosphate (TTCP) on the specimen surfaces (SLA-TTCP group). Physicochemical analysis was performed by metallographic microscopy, scanning electron microscopy, and contact angle analysis. The proliferation and the quantitative alkaline phosphatase (ALP) production of D1 cells on the surface of different sample groups were determined. The SLASP group had a significantly larger D1 cell proliferation than the other groups after 4 and 7 d of incubation (p < 0.05). ALP expression was a very early marker of differentiation, and was the first indication of the increasing number of cells at 7 d of culture. Among the groups in the M substrate series (i.e., M, MS, and MSP) and in the SLA series (i.e., SLA, SLAS, and SLASP), the MSP and SLASP specimens exhibited superior differentiation abilities on respective cultures until day 7 and day 10. A high number of hydrophilic surfaces dominated cell proliferation in the early stage of cell attachment. However, factors affecting the pore structure and the surface morphology can improve cell proliferation and differentiation. According to analyses of proliferation and ALP expression of bone cell progenitors D1, the original SLA implant surface can be improved with surface treatment

  7. A conjugate of the lytic peptide Hecate and gallic acid: structure, activity against cervical cancer, and toxicity.

    PubMed

    Sanches, Paulo R S; Carneiro, Bruno M; Batista, Mariana N; Braga, Ana Cláudia S; Lorenzón, Esteban N; Rahal, Paula; Cilli, Eduardo Maffud

    2015-07-01

    Conjugate compounds constitute a new class of molecules of important biological interest mainly for the treatment of diseases such as cancer. The N-terminus region of cationic peptides has been described as important for their biological activity. The aim of this study was to evaluate the lytic peptide Hecate (FALALKALKKALKKLKKALKKAL) and the effect of conjugating this macromolecule with gallic acid (C7H6O5) in terms of structure, anti-cancer activity, and toxicity. An N-terminus GA-Hecate peptide conjugate was synthesized to provide information regarding the relationship between the amino-terminal region and its charge and the secondary structure and biological activity of the peptide; and the effects of gallic acid on these parameters. Peptide secondary structure was confirmed using circular dichroism (CD). The CD measurements showed that the peptide has a high incidence of α-helical structures in the presence of SDS and LPC, while GA-Hecate presented lower incidence of α-helical structures in the same chemical environment. An evaluation of the anti-cancer activity in HeLa cancer cells indicated that both peptides are active, but that coupling gallic acid at the N-terminus decreased the activity of the free peptide. GA-Hecate showed lower activity in non-tumor keratinocyte cells but higher hemolytic activity. Our findings suggest that the N-terminus of Hecate plays an important role in its activity against cervical cancer by affecting it secondary structure, toxicity, and hemolytic activity. This study highlights the importance of the N-terminus in antitumor activity and could provide an important tool for developing new anti-cancer drugs. PMID:25868656

  8. Conformations of helical Aib peptides containing a pair of L-amino acid and D-amino acid.

    PubMed

    Demizu, Yosuke; Yabuki, Yu-U; Doi, Mitsunobu; Sato, Yukiko; Tanaka, Masakazu; Kurihara, Masaaki

    2012-07-01

    A pair of L-leucine (L-Leu) and D-leucine (D-Leu) was incorporated into a-aminoisobutyric acid (Aib) peptide segments. Thedominant conformations of four hexapeptides, Boc-L-Leu-Aib-Aib-Aib-Aib-L-Leu-OMe (1a), Boc-D-Leu-Aib-Aib-Aib-Aib-L-Leu-OMe(1b), Boc-Aib-Aib-L-Leu-L-Leu-Aib-Aib-OMe (2a), and Boc-Aib-Aib-D-Leu-L-Leu-Aib-Aib-OMe (2b), were investigated by IR,¹H NMR, CD spectra, and X-ray crystallographic analysis. All peptides 1a,b and 2a,b formed 3₁₀-helical structures in solution. X-ray crystallographic analysis revealed that right-handed (P) 3₁₀-helices were present in 1a and 1b and a mixture of right-handed(P) and left-handed (M) 3₁₀-helices was present in 2b in their crystalline states. PMID:22619002

  9. Radiolytic Modification of Sulfur Containing Acidic Amino Residues in Model Peptides: Fundamental Studies for Protein Footprinting

    SciTech Connect

    Xu,G.; Chance, M.

    2005-01-01

    Protein footprinting based on hydroxyl radical-mediated modification and quantitative mass spectroscopic analysis is a proven technique for examining protein structure, protein-ligand interactions, and structural allostery upon protein complex formation. The reactive and solvent-accessible amino acid side chains function as structural probes; however, correct structural analysis depends on the identification and quantification of all the relevant oxidative modifications within the protein sequence. Sulfur-containing amino acids are oxidized readily and the mechanisms of oxidation are particularly complex, although they have been extensively investigated by EPR and other spectroscopic methods. Here we have undertaken a detailed mass spectrometry study (using electrospray ionization mass spectrometry and tandem mass spectrometry) of model peptides containing cysteine (Cys-SH), cystine (disulfide bonded Cys), and methionine after oxidation using {gamma}-rays or synchrotron X-rays and have compared these results to those expected from oxidation mechanisms proposed in the literature. Radiolysis of cysteine leads to cysteine sulfonic acid (+48 Da mass shift) and cystine as the major products; other minor products including cysteine sulfinic acid (+32 Da mass shift) and serine (-16 Da mass shift) are observed. Radiolysis of cystine results in the oxidative opening of the disulfide bond and generation of cysteine sulfonic acid and sulfinic acid; however, the rate of oxidation is significantly less than that for cysteine. Radiolysis of methionine gives rise primarily to methionine sulfoxide (+16 Da mass shift); this can be further oxidized to methionine sulfone (+32 Da mass shift) or another product with a -32 Da mass shift likely due to aldehyde formation at the {gamma}-carbon. Due to the high reactivity of sulfur-containing amino acids, the extent of oxidation is easily influenced by secondary oxidation events or the presence of redox reagents used in standard proteolytic

  10. Slow peptide bond formation by proline and other N-alkylamino acids in translation

    PubMed Central

    Pavlov, Michael Y.; Watts, Richard E.; Tan, Zhongping; Cornish, Virginia W.; Ehrenberg, Måns; Forster, Anthony C.

    2009-01-01

    Proteins are made from 19 aa and, curiously, one N-alkylamino acid (“imino acid”), proline (Pro). Pro is thought to be incorporated by the translation apparatus at the same rate as the 19 aa, even though the alkyl group in Pro resides directly on the nitrogen nucleophile involved in peptide bond formation. Here, by combining quench-flow kinetics and charging of tRNAs with cognate and noncognate amino acids, we find that Pro incorporates in translation significantly more slowly than Phe or Ala and that other N-alkylamino acids incorporate much more slowly. Our results show that the slowest step in incorporation of N-alkylamino acids is accommodation/peptidyl transfer after GTP hydrolysis on EF-Tu. The relative incorporation rates correlate with expectations from organic chemistry, suggesting that amino acid sterics and basicities affect translation rates at the peptidyl transfer step. Cognate isoacceptor tRNAs speed Pro incorporation to rates compatible with in vivo, although still 3–6 times slower than Phe incorporation from Phe-tRNAPhe depending on the Pro codon. Results suggest that Pro is the only N-alkylamino acid in the genetic code because it has a privileged cyclic structure that is more reactive than other N-alkylamino acids. Our data on the variation of the rate of incorporation of Pro from native Pro-tRNAPro isoacceptors at 4 different Pro codons help explain codon bias not accounted for by the “tRNA abundance” hypothesis. PMID:19104062

  11. Design of immobile nucleic acid junctions.

    PubMed Central

    Seeman, N C; Kallenbach, N R

    1983-01-01

    Nucleic acids that interact to generate structures in which three or more double helices emanate from a single point are said to form a junction. Such structures arise naturally as intermediates in DNA replication and recombination. It has been proposed that stable junctions can be created by synthesizing sets of oligonucleotides of defined sequence that can associate by maximizing Watson-Crick complementarity (Seeman N. C., 1981, Biomolecular Stereodynamics. Adenine Press, New York. 1: 269-278; Seeman, N. C., 1982, J. Theor. Biol. 99:237-247.) To make it possible to design molecules that will form junctions of specific architecture, we present here an efficient algorithm for generating nucleic acid sequences that optimize two fundamental properties: fidelity and stability. Fidelity refers to the relative probability of forming the junction complex relative to all alternative paired structures. Calculations are described that permit approximate prediction of the melting curves for junction complexes. PMID:6197102

  12. Reversible Major Histocompatibility Complex I-Peptide Multimers Containing Ni2+-Nitrilotriacetic Acid Peptides and Histidine Tags Improve Analysis and Sorting of CD8+ T Cells*

    PubMed Central

    Schmidt, Julien; Guillaume, Philippe; Irving, Melita; Baumgaertner, Petra; Speiser, Daniel; Luescher, Immanuel F.

    2011-01-01

    MHC-peptide multimers containing biotinylated MHC-peptide complexes bound to phycoerythrin (PE) streptavidin (SA) are widely used for analyzing and sorting antigen-specific T cells. Here we describe alternative T cell-staining reagents that are superior to conventional reagents. They are built on reversible chelate complexes of Ni2+-nitrilotriacetic acid (NTA) with oligohistidines. We synthesized biotinylated linear mono-, di-, and tetra-NTA compounds using conventional solid phase peptide chemistry and studied their interaction with HLA-A*0201-peptide complexes containing a His6, His12, or 2×His6 tag by surface plasmon resonance on SA-coated sensor chips and equilibrium dialysis. The binding avidity increased in the order His6 < His12 < 2×His6 and NTA1 < NTA2 < NTA4, respectively, depending on the configuration of the NTA moieties and increased to picomolar KD for the combination of a 2×His6 tag and a 2×Ni2+-NTA2. We demonstrate that HLA-A2–2×His6-peptide multimers containing either Ni2+-NTA4-biotin and PE-SA- or PE-NTA4-stained influenza and Melan A-specific CD8+ T cells equal or better than conventional multimers. Although these complexes were highly stable, they very rapidly dissociated in the presence of imidazole, which allowed sorting of bona fide antigen-specific CD8+ T cells without inducing T cell death as well as assessment of HLA-A2-peptide monomer dissociation kinetics on CD8+ T cells. PMID:21990358

  13. Vibrational analysis of amino acids and short peptides in hydrated media. II. Role of KLLL repeats to induce helical conformations in minimalist LK-peptides.

    PubMed

    Guiffo-Soh, Guy; Hernandez, Belén; Coïc, Yves-Marie; Boukhalfa-Heniche, Fatima-Zohra; Ghomi, Mahmoud

    2007-11-01

    Aqueous solution secondary structures of minimalist LK-peptides, with the generic sequence defined as KLL(KLLL)nKLLK, have been analyzed by means of circular dichroism (CD) and Raman scattering techniques. Our discussion in the present paper is mainly focused on four synthetic peptides (from 5 to 19 amino acids), KLLLK, KLLKLLLKLLK, KLLKLLLKLLLKLLK, and KLLKLLLKLLLKLLLKLLK, corresponding to the repeat unit, and to the peptide chains with the values of n = 1-3, respectively. CD and Raman spectra were analyzed in order to study both structural features of the peptide chains and their capability to form aggregates. On the basis of the obtained results it was concluded that the conformational flexibility of the shortest peptides (5-mer and 11-mer) is high enough to adopt random, beta-type, and helical chains in aqueous solution. However, the 11-mer shows a clear tendency to form beta-strands in phosphate buffer. The conformational equilibrium can be completely shifted to beta-type structures upon increasing ionic strength, i.e., in PBS and tris buffers. This equilibrium can also be shifted toward helical chains in the presence of methanol. Finally, the longest peptides (15-mer and 19-mer) are shown to form alpha-helical chains with an amphipathic character in aqueous solution. The possibility of bundle formation between helical chains is discussed over the temperature-dependent H-D exchange on labile hydrogens and particularly by considering the particular behavior of an intense Raman mode at 1127 cm-1 originating from the leucine residue side chain. The conformational dependence of this mode observed upon selective deuteration has never been documented up to now. PMID:17918991

  14. Hydration studies of electrospray ions from amino acids and small peptides

    NASA Astrophysics Data System (ADS)

    Nguyen, Chuong (Steve)

    This project was undertaken to gain a better understanding of the hydration behaviors of gas phase ions from solutions containing amino acids and peptides. In order to characterize their hydration behavior, the molecules of interest in solutions were first converted into gas phase ions by electrospray ionization (ESI). The completely desolvated ions were then deliberately dispersed into an inert bath gas, usually nitrogen, containing accurately known concentrations of solvent vapor. The resulting mixtures of ions and bath gas were subsequently passed into a vacuum chamber by way of an adiabatic supersonic free jet expansion. The cooling during that expansion caused solvation of the ions, the extent of which was determined by a quadrupole mass analyzer. Mass analysis of the solute ions in the absence of vapor showed peaks with the mass to charge ratios corresponding to the desolvated ions. On the other hand, mass spectrometric analyses of ions in the presence of solvent vapor showed sequences of peaks corresponding to the solvated ions with varying numbers of water molecules. The extent of the ion solvation was controlled by varying the concentration of solvent vapor in the bath gas. Two different scales were proposed for the evaluation of the relative affinities of amino acids for water molecules. One was based primarily on the assumption that the affinities of amino acids for water molecules are directly proportional to their gas phase solvation rate constants ( k). An alternative approach produced an affinity scale based on the extent of ion hydration occurred during the free jet expansion. It was found that the addition of a polar solvent vapor to the bath gas at low concentrations substantially enhanced the production of the bare solute ions from the evaporating charged droplets. This remarkable result not only provided a means to increase the ion production and thus detection sensitivity of mass spectrometric analyses, but also yielded important information

  15. Design of minimally strained nucleic Acid nanotubes.

    PubMed

    Sherman, William B; Seeman, Nadrian C

    2006-06-15

    A practical theoretical framework is presented for designing and classifying minimally strained nucleic acid nanotubes. The structures are based on the double crossover motif where each double-helical domain is connected to each of its neighbors via two or more Holliday-junction-like reciprocal exchanges, such that each domain is parallel to the main tube axis. Modeling is based on a five-parameter characterization of the segmented double-helical structure. Once the constraint equations have been derived, the primary design problem for a minimally strained N-domain structure is reduced to solving three simultaneous equations in 2N+2 variables. Symmetry analysis and tube merging then allow for the design of a wide variety of tubes, which can be tailored to satisfy requirements such as specific inner and outer radii, or multiple lobed structures. The general form of the equations allows similar techniques to be applied to various nucleic acid helices: B-DNA, A-DNA, RNA, DNA-PNA, or others. Possible applications for such tubes include nanoscale scaffolding as well as custom-shaped enclosures for other nano-objects. PMID:16581842

  16. Self-assembly of short peptides composed of only aliphatic amino acids and a combination of aromatic and aliphatic amino acids.

    PubMed

    Subbalakshmi, Chilukuri; Manorama, Sunkara V; Nagaraj, Ramakrishnan

    2012-05-01

    The morphology of structures formed by the self-assembly of short N-terminal t-butyloxycarbonyl (Boc) and C-terminal methyl ester (OMe) protected and Boc-deprotected hydrophobic peptide esters was investigated. We have observed that Boc-protected peptide esters composed of either only aliphatic hydrophobic amino acids or aliphatic hydrophobic amino acids in combination with aromatic amino acids, formed highly organized structures, when dried from methanol solutions. Transmission and scanning electron microscopic images of the peptides Boc-Ile-Ile-OMe, Boc-Phe-Phe-Phe-Ile-Ile-OMe and Boc-Trp-Ile-Ile-OMe showed nanotubular structures. Removal of the Boc group resulted in disruption of the ability to form tubular structures though spherical aggregates were formed. Both Boc-Leu-Ile-Ile-OMe and H-Leu-Ile-Ile-OMe formed only spherical nanostructures. Dynamic light scattering studies showed that aggregates of varying dimensions were present in solution suggesting that self-assembly into ordered structures is facilitated by aggregation in solution. Fourier transform infrared spectroscopy and circular dichroism spectroscopy data show that although all four of the protected peptides adopt well-defined tertiary structures, upon removal of the Boc group, only H-Phe-Phe-Phe-Ile-Ile-OMe had the ability to adopt β-structure. Our results indicate that hydrophobic interaction is a very important determinant for self-assembly and presence of charged and aromatic amino acids in a peptide is not necessary for self-assembly. PMID:22431418

  17. Acidity and metal (Mg2+, Ca2+, Zn2+) affinity of L-γ-carboxyglutamic acid and its peptide analog

    NASA Astrophysics Data System (ADS)

    Remko, Milan; Broer, Ria; Remková, Anna; Van Duijnen, Piet Th.

    2014-10-01

    Density functional theory methods with the B3LYP and B97D functionals with triple-zeta 6-311++G(d,p) basis set have been used to study the acidity, basicity and metal affinity of L-γ-carboxyglutamic acid (GLA) and its peptide derivative [2-acetylamino-3-(methylamino)-3-oxopropyl]malonic acid (AMD-GLA). The Gibbs interaction energies of the GLA2-…M2+ and AMD-GLA2-…M2+ (M = Mg, Ca, Zn) complexes show an increasing binding affinity in the order Ca2+ < Mg2+ < Zn2+ The transition metal Zn2+ is most effectively recognized by the dianions of GLA and AMD-GLA. Of the dianions studied the AMD-GLA dianion is the strongest Lewis base. Computations that include the effect of solvation showed that in water the relative stability of GLA2-…M2+ and AMD-GLA2-…M2+ ionic bonds is rapidly diminished. The computed interaction Gibbs energy in water is small and negative.

  18. Theoretical study on application of peptide nanoring to chiral recognition of amino acid

    NASA Astrophysics Data System (ADS)

    Takeuchi, Jo; Takeda, Kyozaburo

    2016-03-01

    Chiral recognition of a guest amino acid by a host peptide nanoring (PNR) is studied by ab initio calculations. The intermolecular hydrogen bonds (HBs) between the present host and the guest force the guest amino acid to land into the host PNR, and also the side chain of the host PNR to hold the guest amino acid. Thus, the host PNR captures the guest amino acid and gives a distinct energy difference in accordance with the chirality of the captured guest d/l-SerH+. The calculated energy difference of 18 kJ/mol in Gibbs free energy is comparable to that by the host crown ether, by which Moreno et al. have succeeded in experimentally recognizing the chirality of the guest d/l-SerH+ [J. R. A. Moreno, M. M. Q. Moreno, J. J. L. González, and B. M. Haya, J. Phys. Chem. B 117, 9362 (2013)]. The PNRs could be unique biomolecular materials from the perspective that the chirality of an “amino acid” is recognized by the PNR consisting of “amino acid” residues.

  19. Absorption of Amino Acids and Peptides in a Child with a Variant of Hartnup Disease and Coexistent Coeliac Disease

    PubMed Central

    Tarlow, M. J.; Seakins, J. W. T.; Lloyd, June K.; Matthews, D. M.; Cheng, B.; Thomas, A. J.

    1972-01-01

    A child with a variant of Hartnup disease and co-existent coeliac disease is described. Oral tolerance tests with L-histidine, L-tyrosine, and glycyl-L-tyrosine, and in vitro uptake studies on a small intestinal biopsy with L-histidine and glycyl-L-histidine, showed impaired absorption of the free amino acids, and showed that absorption of tyrosine and mucosal uptake of histidine was better from the dipeptides than from the free amino acids. This supports the hypothesis that the intestinal mucosa can take up small peptides intact, and that the peptide uptake mechanism is not involved in the intestinal defect of Hartnup disease. PMID:5086513

  20. Structure-based design of non-natural amino-acid inhibitors of amyloid fibril formation

    SciTech Connect

    Sievers, Stuart A.; Karanicolas, John; Chang, Howard W.; Zhao, Anni; Jiang, Lin; Zirafi, Onofrio; Stevens, Jason T.; Münch, Jan; Baker, David; Eisenberg, David

    2011-09-20

    Many globular and natively disordered proteins can convert into amyloid fibrils. These fibrils are associated with numerous pathologies as well as with normal cellular functions, and frequently form during protein denaturation. Inhibitors of pathological amyloid fibril formation could be useful in the development of therapeutics, provided that the inhibitors were specific enough to avoid interfering with normal processes. Here we show that computer-aided, structure-based design can yield highly specific peptide inhibitors of amyloid formation. Using known atomic structures of segments of amyloid fibrils as templates, we have designed and characterized an all-D-amino-acid inhibitor of the fibril formation of the tau protein associated with Alzheimer's disease, and a non-natural L-amino-acid inhibitor of an amyloid fibril that enhances sexual transmission of human immunodeficiency virus. Our results indicate that peptides from structure-based designs can disrupt the fibril formation of full-length proteins, including those, such as tau protein, that lack fully ordered native structures. Because the inhibiting peptides have been designed on structures of dual-{beta}-sheet 'steric zippers', the successful inhibition of amyloid fibril formation strengthens the hypothesis that amyloid spines contain steric zippers.

  1. Rational design of tryptophan-rich antimicrobial peptides with enhanced antimicrobial activities and specificities.

    PubMed

    Yu, Hui-Yuan; Huang, Kuo-Chun; Yip, Bak-Sau; Tu, Chih-Hsiang; Chen, Heng-Li; Cheng, Hsi-Tsung; Cheng, Jya-Wei

    2010-11-01

    Trp-rich antimicrobial peptides play important roles in the host innate defense mechanism of many plants and animals. A series of short Trp-rich peptides derived from the C-terminal region of Bothrops asper myothoxin II, a Lys49 phospholipase A(2) (PLA(2)), were found to reproduce the antimicrobial activities of their parent molecule. Of these peptides, KKWRWWLKALAKK-designated PEM-2-was found to display improved activity against both Gram-positive and Gram-negative bacteria. To improve the antimicrobial activity of PEM-2 for potential clinical applications further, we determined the solution structure of PEM-2 bound to membrane-mimetic dodecylphosphocholine (DPC) micelles by two-dimensional NMR methods. The DPC micelle-bound structure of PEM-2 adopts an α-helical conformation and the positively charged residues are clustered together to form a hydrophilic patch. The surface electrostatic potential map indicates that two of the three tryptophan residues are packed against the peptide backbone and form a hydrophobic face with Leu7, Ala9, and Leu10. A variety of biophysical and biochemical experiments, including circular dichroism, fluorescence spectroscopy, and microcalorimetry, were used to show that PEM-2 interacted with negatively charged phospholipid vesicles and efficiently induced dye release from these vesicles, suggesting that the antimicrobial activity of PEM-2 could be due to interactions with bacterial membranes. Potent analogues of PEM-2 with enhanced antimicrobial and less pronounced hemolytic activities were designed with the aid of these structural studies. PMID:20865718

  2. Rational design and application of responsive α-helical peptide hydrogels

    PubMed Central

    Banwell, Eleanor F.; Abelardo, Edgardo S.; Adams, Dave J.; Birchall, Martin A.; Corrigan, Adam; Donald, Athene M.; Kirkland, Mark; Serpell, Louise C.; Butler, Michael F.; Woolfson, Derek N.

    2009-01-01

    Biocompatible hydrogels have a wide variety of potential applications in biotechnology and medicine, such as the controlled delivery and release of cells, cosmetics and drugs; and as supports for cell growth and tissue engineering1. Rational peptide design and engineering are emerging as promising new routes to such functional biomaterials2-4. Here we present the first examples of rationally designed and fully characterized self-assembling hydrogels based on standard linear peptides with purely α-helical structures, which we call hydrogelating self-assembling fibres (hSAFs). These form spanning networks of α-helical fibrils that interact to give self-supporting physical hydrogels of >99% water content. The peptide sequences can be engineered to alter the underlying mechanism of gelation and, consequently, the hydrogel properties. Interestingly, for example, those with hydrogen-bonded networks melt upon heating, whereas those formed via hydrophobic interactions strengthen when warmed. The hSAFs are dual-peptide systems that only gel on mixing, which gives tight control over assembly5. These properties raise possibilities for using the hSAFs as substrates in cell culture. We have tested this in comparison with the widely used Matrigel substrate, and demonstrate that, like Matrigel, hSAFs support both growth and differentiation of rat adrenal pheochromocytoma cells for sustained periods in culture. PMID:19543314

  3. Rational design and application of responsive α-helical peptide hydrogels

    NASA Astrophysics Data System (ADS)

    Banwell, Eleanor F.; Abelardo, Edgardo S.; Adams, Dave J.; Birchall, Martin A.; Corrigan, Adam; Donald, Athene M.; Kirkland, Mark; Serpell, Louise C.; Butler, Michael F.; Woolfson, Derek N.

    2009-07-01

    Biocompatible hydrogels have a wide variety of potential applications in biotechnology and medicine, such as the controlled delivery and release of cells, cosmetics and drugs, and as supports for cell growth and tissue engineering. Rational peptide design and engineering are emerging as promising new routes to such functional biomaterials. Here, we present the first examples of rationally designed and fully characterized self-assembling hydrogels based on standard linear peptides with purely α-helical structures, which we call hydrogelating self-assembling fibres (hSAFs). These form spanning networks of α-helical fibrils that interact to give self-supporting physical hydrogels of >99% water content. The peptide sequences can be engineered to alter the underlying mechanism of gelation and, consequently, the hydrogel properties. Interestingly, for example, those with hydrogen-bonded networks of fibrils melt on heating, whereas those formed through hydrophobic fibril-fibril interactions strengthen when warmed. The hSAFs are dual-peptide systems that gel only on mixing, which gives tight control over assembly. These properties raise possibilities for using the hSAFs as substrates in cell culture. We have tested this in comparison with the widely used Matrigel substrate, and demonstrate that, like Matrigel, hSAFs support both growth and differentiation of rat adrenal pheochromocytoma cells for sustained periods in culture.

  4. Engineering phage materials with desired peptide display: rational design sustained through natural selection.

    PubMed

    Merzlyak, Anna; Lee, Seung-Wuk

    2009-12-01

    Genetic engineering of phage provides novel opportunities to build various nanomaterials by displaying functional peptide motifs on its surface coat protein. However, any genetic modifications of phage coat proteins must be able to accommodate their many biological roles in the phage replication process. To express functional but inherently unfavorable peptide motifs on major coat protein pVIII, we devised a novel genetic conjugation method to circumvent bacterial biological censorship. Constraining the designed peptides among the degenerate flanking residues, we obtained a pVIII library of phage that retained the desired sequences yet could navigate through the phage replication process due to the naturally selected flanking residues. Further, we systematically analyzed the biochemical and size-related compensation mechanisms of the pVIII expressed peptides by constructing four chemically diverse (His, Trp, Glu, Lys) partial library series. Described genetic conjugation methodology can serve to improve the design of engineered phage and allow further exploitation of these particles as functional nanobiomaterials for various applications. PMID:19842621

  5. Penetration depth of surfactant peptide KL4 into membranes is determined by fatty acid saturation.

    PubMed

    Antharam, Vijay C; Elliott, Douglas W; Mills, Frank D; Farver, R Suzanne; Sternin, Edward; Long, Joanna R

    2009-05-20

    KL(4) is a 21-residue functional peptide mimic of lung surfactant protein B, an essential protein for lowering surface tension in the alveoli. Its ability to modify lipid properties and restore lung compliance was investigated with circular dichroism, differential scanning calorimetry, and solid-state NMR spectroscopy. KL(4) binds fluid lamellar phase PC/PG lipid membranes and forms an amphipathic helix that alters lipid organization and acyl chain dynamics. The binding and helicity of KL(4) is dependent on the level of monounsaturation in the fatty acid chains. At physiologic temperatures, KL(4) is more peripheral and dynamic in fluid phase POPC/POPG MLVs but is deeply inserted into fluid phase DPPC/POPG vesicles, resulting in immobilization of the peptide. Substantial increases in the acyl chain order are observed in DPPC/POPG lipid vesicles with increasing levels of KL(4), and POPC/POPG lipid vesicles show small decreases in the acyl chain order parameters on addition of KL(4). Additionally, a clear effect of KL(4) on the orientation of the fluid phase PG headgroups is observed, with similar changes in both lipid environments. Near the phase transition temperature of the DPPC/POPG lipid mixtures, which is just below the physiologic temperature of lung surfactant, KL(4) causes phase separation with the DPPC remaining in a gel phase and the POPG partitioned between gel and fluid phases. The ability of KL(4) to differentially partition into lipid lamellae containing varying levels of monounsaturation and subsequent changes in curvature strain suggest a mechanism for peptide-mediated lipid organization and trafficking within the dynamic lung environment. PMID:19450480

  6. Penetration Depth of Surfactant Peptide KL4 into Membranes Is Determined by Fatty Acid Saturation

    PubMed Central

    Antharam, Vijay C.; Elliott, Douglas W.; Mills, Frank D.; Farver, R. Suzanne; Sternin, Edward; Long, Joanna R.

    2009-01-01

    KL4 is a 21-residue functional peptide mimic of lung surfactant protein B, an essential protein for lowering surface tension in the alveoli. Its ability to modify lipid properties and restore lung compliance was investigated with circular dichroism, differential scanning calorimetry, and solid-state NMR spectroscopy. KL4 binds fluid lamellar phase PC/PG lipid membranes and forms an amphipathic helix that alters lipid organization and acyl chain dynamics. The binding and helicity of KL4 is dependent on the level of monounsaturation in the fatty acid chains. At physiologic temperatures, KL4 is more peripheral and dynamic in fluid phase POPC/POPG MLVs but is deeply inserted into fluid phase DPPC/POPG vesicles, resulting in immobilization of the peptide. Substantial increases in the acyl chain order are observed in DPPC/POPG lipid vesicles with increasing levels of KL4, and POPC/POPG lipid vesicles show small decreases in the acyl chain order parameters on addition of KL4. Additionally, a clear effect of KL4 on the orientation of the fluid phase PG headgroups is observed, with similar changes in both lipid environments. Near the phase transition temperature of the DPPC/POPG lipid mixtures, which is just below the physiologic temperature of lung surfactant, KL4 causes phase separation with the DPPC remaining in a gel phase and the POPG partitioned between gel and fluid phases. The ability of KL4 to differentially partition into lipid lamellae containing varying levels of monounsaturation and subsequent changes in curvature strain suggest a mechanism for peptide-mediated lipid organization and trafficking within the dynamic lung environment. PMID:19450480

  7. Composition of free and peptide-bound amino acids in beef chuck, loin, and round cuts.

    PubMed

    Wu, G; Cross, H R; Gehring, K B; Savell, J W; Arnold, A N; McNeill, S H

    2016-06-01

    Meat is a food for humans. However, beef consumption in the United States has steadily declined by >14% over the past decade due to a variety of factors, including insufficient knowledge of animal protein. This study quantified all proteinogenic AA as well as nutritionally and physiologically significant nonproteinogenic AA and small peptides in beef cuts from 3 subprimals (chuck, round, and loin). Beef carcasses ( = 10) were selected at 3 commercial packing plants in the United States. Retail-cut samples were analyzed for the nitrogenous substances after acid, alkaline, or enzymatic hydrolysis and after deproteinization. In these chuck, round, and loin cuts, total amounts of glutamate (free plus peptide bound) were the highest (69-75 mg/g dry weight) followed by lysine, leucine, arginine, and glutamine in descending order. This is the first study to determine aspartate, asparagine, glutamate, and glutamine in meat proteins of any animal species. In all the beef samples evaluated, glutamine was the most abundant free AA (4.0-5.7 mg/g dry weight) followed by taurine, alanine, glutamate, and β-alanine. Additionally, samples from all beef cuts had high concentrations of anserine, carnosine, and glutathione, which were 2.8 to 3.7, 15.2 to 24.2, and 0.68 to 0.79 mg/g dry weight, respectively. Beef top loin steaks appear to provide higher protein nutrition values than top round steaks and under blade roasts, but all are excellent sources of proteinogenic AA as well as antioxidant AA and peptides to improve human growth, development, and health. Our findings may help guide future decisions regarding human and animal nutrition. PMID:27285936

  8. Kinetic properties of the binding of alpha-lytic protease to peptide boronic acids.

    PubMed

    Kettner, C A; Bone, R; Agard, D A; Bachovchin, W W

    1988-10-01

    The kinetic parameters for peptide boronic acids in their interaction with alpha-lytic protease were determined and found to be similar to those of other serine proteases [Kettner, C., & Shenvi, A. B. (1984) J. Biol. Chem. 259, 15106-15114]. alpha-Lytic protease hydrolyzes substrates with either alanine or valine in the P1 site and has a preference for substrate with a P1 alanine. The most effective inhibitors are tri- and tetrapeptide analogues that have a -boroVal-OH residue in the P1 site. At pH 7.5, MeOSuc-Ala-Ala-Pro-boroVal-OH has a Ki of 6.4 nM and Boc-Ala-Pro-boroVal-OH has a Ki of 0.35 nM. Ac-boroVal-OH and Ac-Pro-boroVal-OH are 220,000- and 500-fold less effective, respectively, than the tetrapeptide analogue. The kinetic properties of the tri- and tetrapeptide analogues are consistent with the mechanism for slow-binding inhibition, E + I in equilibrium EI in equilibrium EI*, while the less effective inhibitors are simple competitive inhibitors. MeO-Suc-Ala-Ala-Pro-boroAla-OH is a simple competitive inhibitor with a Ki of 67 nM at pH 7.5. Other peptide boronic acids, which are analogues of nonsubstrates, are less effective than substrate analogues but still are effective competitive inhibitors. For example, MeOSuc-Ala-Ala-Pro-boroPhe-OH has a Ki of 0.54 microM although substrates with a phenylalanine in the P1 position are not hydrolyzed. Binding for boronic acid analogues of both substrate and nonsubstrate analogues is pH dependent with higher affinity near pH 7.5. Similar binding properties have been observed for pancreatic elastase. Both enzymes have almost identical requirements for an extended peptide inhibitor sequence in order to exhibit highly effective binding and slow-binding characteristics.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3207699

  9. Effect of the amino acid composition of cyclic peptides on their self-assembly in lipid bilayers.

    PubMed

    Danial, Maarten; Perrier, Sébastien; Jolliffe, Katrina A

    2015-02-28

    The effect of amino acid composition on the formation of transmembrane channels in lipid bilayers upon self-assembly of alt-(L,D)-α-cyclic octapeptides has been investigated. Cyclic peptides comprising D-leucine, alternating with different combinations of L-azidolysine, L-lysine(Alloc), L-lysine and L-tryptophan were synthesized and the size of pores formed via self-assembly of these molecules in lipid bilayers was elucidated using large unilamellar vesicle fluorescence assays and dynamic light scattering. Pore formation was examined in large unilamellar vesicles made up of egg yolk phosphatidylcholine or Escherichia coli total lipid extract. From these analyses, we have established that cyclic peptides with charged side chains form large pores while those with neutral side chains form unimeric pores. Furthermore, the cyclic peptides that consist of non-symmetric amino acid configurations possess a higher membrane activity than the cyclic peptides with a symmetric amino acid configuration. In addition, we have found that peptide amphiphilicity plays a vital role in selective partitioning between bilayers that consist of egg yolk phosphatidylcholine and those comprised of E. coli total lipid extract. These results suggest that selective transbilayer channel formation via self-assembly may be a viable alternative for many applications that currently use more expensive, multistep synthesis methods. PMID:25566760

  10. Increased yield of high purity recombinant human brain natriuretic peptide by acid hydrolysis of short fusion partner in Escherichia coli.

    PubMed

    Kanumuri, Radha Madhavi; Bajji, Chitra; Tummuru, Rajesh R; Tatireddigari, Venkat R R Arva; Mangamoori, Lakshmi Narasu; Panati, Kalpana; Narala, Venkata Ramireddy

    2015-07-01

    Recombinant human B-type natriuretic peptide (rhBNP) is a 32-amino acid peptide used to treat congestive heart failure. In this paper, we report a method for the increased production of rhBNP in Escherichia coli with high purity. hBNP was cloned with a short growth hormone fusion partner coupled with a unique acid-labile dipeptide linker to cleave the fusion protein to release the rhBNP. The recombinant fusion protein was expressed as an inclusion body (IB) and the fermentation process was optimized to produce on large scale. The IBs were recovered by cell lysis, and the pure IBs were directly treated with diluted acid to get the target peptide from the fusion protein and the resultant peptide was purified by reversed phase chromatography. The final purity of the rhBNP was more than 99% with yield of 50mg per liter of culture, which is ten times higher than the previous reports. The purified rhBNP exhibited specific biological activity similar to the standard peptide in producing cyclic-guanosine monophosphate. PMID:25823948

  11. Anti-biofilm activity of ultrashort cinnamic acid peptide derivatives against medical device-related pathogens.

    PubMed

    Laverty, Garry; McCloskey, Alice P; Gorman, Sean P; Gilmore, Brendan F

    2015-10-01

    The threat of antimicrobial resistance has placed increasing emphasis on the development of innovative approaches to eradicate multidrug-resistant pathogens. Biofilm-forming microorganisms, for example, Staphylococcus epidermidis and Staphylococcus aureus, are responsible for increased incidence of biomaterial infection, extended hospital stays and patient morbidity and mortality. This paper highlights the potential of ultrashort tetra-peptide conjugated to hydrophobic cinnamic acid derivatives. These peptidomimetic molecules demonstrate selective and highly potent activity against resistant biofilm forms of Gram-positive medical device-related pathogens. 3-(4-Hydroxyphenyl)propionic)-Orn-Orn-Trp-Trp-NH2 displays particular promise with minimum biofilm eradication concentration (MBEC) values of 125 µg/ml against methicillin sensitive (ATCC 29213) and resistant (ATCC 43300) S. aureus and activity shown against biofilm forms of Escherichia coli (MBEC: 1000 µg/ml). Kill kinetics confirms complete eradication of established 24-h biofilms at MBEC with 6-h exposure. Reduced cell cytotoxicity, relative to Gram-positive pathogens, was proven via tissue culture (HaCaT) and haemolysis assays (equine erythrocytes). Existing in nature as part of the immune response, antimicrobial peptides display great promise for exploitation by the pharmaceutical industry in order to increase the library of available therapeutic molecules. Ultrashort variants are particularly promising for translation as clinical therapeutics as they are more cost-effective, easier to synthesise and can be tailored to specific functional requirements based on the primary sequence allowing factors such as spectrum of activity to be varied. PMID:26310860

  12. Designed α-sheet peptides inhibit amyloid formation by targeting toxic oligomers

    PubMed Central

    Hopping, Gene; Kellock, Jackson; Barnwal, Ravi Pratap; Law, Peter; Bryers, James; Varani, Gabriele; Caughey, Byron; Daggett, Valerie

    2014-01-01

    Previous studies suggest that the toxic soluble-oligomeric form of different amyloid proteins share a common backbone conformation, but the amorphous nature of this oligomer prevents its structural characterization by experiment. Based on molecular dynamics simulations we proposed that toxic intermediates of different amyloid proteins adopt a common, nonstandard secondary structure, called α-sheet. Here we report the experimental characterization of peptides designed to be complementary to the α-sheet conformation observed in the simulations. We demonstrate inhibition of aggregation in two different amyloid systems, β-amyloid peptide (Aβ) and transthyretin, by these designed α-sheet peptides. When immobilized the α-sheet designs preferentially bind species from solutions enriched in the toxic conformer compared with non-aggregated, nontoxic species or mature fibrils. The designs display characteristic spectroscopic signatures distinguishing them from conventional secondary structures, supporting α-sheet as a structure involved in the toxic oligomer stage of amyloid formation and paving the way for novel therapeutics and diagnostics. DOI: http://dx.doi.org/10.7554/eLife.01681.001 PMID:25027691

  13. Microbial degradation rates of small peptides and amino acids in the oxygen minimum zone of Chilean coastal waters

    NASA Astrophysics Data System (ADS)

    Pantoja, Silvio; Rossel, Pamela; Castro, Rodrigo; Cuevas, L. Antonio; Daneri, Giovanni; Córdova, Candy

    2009-07-01

    We found similar microbial degradation rates of labile dissolved organic matter in oxic and suboxic waters off northern Chile. Rates of peptide hydrolysis and amino acid uptake in unconcentrated water samples were not low in the water column where oxygen concentration was depleted. Hydrolysis rates ranged from 65 to 160 nmol peptide L -1 h -1 in the top 20 m, 8-28 nmol peptide L -1 h -1 between 100 and 300 m (O 2-depleted zone), and 14-19 nmol peptide L -1 h -1 between 600 and 800 m. Dissolved free amino acid uptake rates were 9-26, 3-17, and 6 nmol L -1 h -1 at similar depth intervals. Since these findings are consistent with a model of comparable potential activity of microbes in degrading labile substrates of planktonic origin, we suggest, as do other authors, that differences in decomposition rates with high and low oxygen concentrations may be a matter of substrate lability. The comparison between hydrolysis and uptake rates indicates that microbial peptide hydrolysis occurs at similar or faster rates than amino acid uptake in the water column, and that the hydrolysis of peptides is not a rate-limiting step for the complete remineralization of labile macromolecules. Low O 2 waters process about 10 tons of peptide carbon per h, double the amount processed in surface-oxygenated water. In the oxygen minimum zone, we suggest that the C balance may be affected by the low lability of the dissolved organic matter when this is upwelled to the surface. An important fraction of dissolved organic matter is processed in the oxygen minimum layer, a prominent feature of the coastal ocean in the highly productive Humboldt Current System.

  14. D-amino acid residue in a defensin-like peptide from platypus venom: effect on structure and chromatographic properties.

    PubMed

    Torres, Allan M; Tsampazi, Chryssanthi; Geraghty, Dominic P; Bansal, Paramjit S; Alewood, Paul F; Kuchel, Philip W

    2005-10-15

    The recent discovery that the natriuretic peptide OvCNPb (Ornithorhynchus venom C-type natriuretic peptide B) from platypus (Ornithorynchus anatinus) venom contains a D-amino acid residue suggested that other D-amino-acid-containing peptides might be present in the venom. In the present study, we show that DLP-2 (defensin-like peptide-2), a 42-amino-acid residue polypeptide in the platypus venom, also contains a D-amino acid residue, D-methionine, at position 2, while DLP-4, which has an identical amino acid sequence, has all amino acids in the L-form. These findings were supported further by the detection of isomerase activity in the platypus gland venom extract that converts DLP-4 into DLP-2. In the light of this new information, the tertiary structure of DLP-2 was recalculated using a new structural template with D-Met2. The structure of DLP-4 was also determined in order to evaluate the effect of a D-amino acid at position 2 on the structure and possibly to explain the large retention time difference observed for the two molecules in reverse-phase HPLC. The solution structures of the DLP-2 and DLP-4 are very similar to each other and to the earlier reported structure of DLP-2, which assumed that all amino acids were in the L-form. Our results suggest that the incorporation of the D-amino acid at position 2 has minimal effect on the overall fold in solution. PMID:16033333

  15. Design and characterization of novel antimicrobial peptides, R-BP100 and RW-BP100, with activity against Gram-negative and Gram-positive bacteria.

    PubMed

    Torcato, Inês M; Huang, Yen-Hua; Franquelim, Henri G; Gaspar, Diana; Craik, David J; Castanho, Miguel A R B; Troeira Henriques, Sónia

    2013-03-01

    BP100 is a short cationic antimicrobial peptide with a mechanism of action dependent on peptide-lipid interactions and microbial surface charge neutralization. Although active against Gram-negative bacteria, BP100 is inactive against Gram-positive bacteria. In this study we report two newly designed BP100 analogues, RW-BP100 and R-BP100 that have the Tyr residue replaced with a Trp and/or the Lys residues replaced with an Arg. The new analogues in addition to being active against Gram-negative bacteria, possess activity against all tested Gram-positive bacteria. Mechanistic studies using atomic force microscopy, surface plasmon resonance and fluorescence methodologies reveal that the antibacterial efficiency follows the affinity for bacterial membrane. The studies suggest that the activity of BP100 and its analogues against Gram-negative bacteria is mainly driven by electrostatic interactions with the lipopolysaccharide layer and is followed by binding to and disruption of the inner membrane, whereas activity against Gram-positive bacteria, in addition to electrostatic attraction to the exposed lipoteichoic acids, requires an ability to more deeply insert in the membrane environment, which is favoured with Arg residues and is facilitated in the presence of a Trp residue. Knowledge on the mechanism of action of these antimicrobial peptides provides information that assists in the design of antimicrobials with higher efficacy and broader spectra of action, but also on the design of peptides with higher specificity if required. PMID:23246973

  16. Selective labeling of a membrane peptide with 15N-amino acids using cells grown in rich medium.

    PubMed

    Englander, Jacqueline; Cohen, Leah; Arshava, Boris; Estephan, Racha; Becker, Jeffrey M; Naider, Fred

    2006-01-01

    Nuclear magnetic resonance spectra of membrane proteins containing multiple transmembrane helices have proven difficult to resolve due to the redundancy of aliphatic and Ser/Thr residues in transmembrane domains and the low chemical shift dispersity exhibited by residues in alpha-helical structures. Although (13)C- and (15)N-labeling are useful tools in the biophysical analysis of proteins, selective labeling of individual amino acids has been used to help elucidate more complete structures and to probe ligand-protein interactions. In general, selective labeling has been performed in Escherichia coli expression systems using minimal media supplemented with a single labeled amino acid and nineteen other unlabeled amino acids and/or by using auxotrophs for specific amino acids. Growth in minimal media often results in low yields of cells or expression products. We demonstrate a method in which one labeled amino acid is added to a rich medium. These conditions resulted in high expression (> or =100 mg/L) of a test fusion protein and milligram quantities of the selectively labeled membrane peptide after cyanogen bromide cleavage to release the peptide from the fusion protein. High levels of (15)N incorporation and acceptable levels of cross-labeling into other amino acid residues of the peptide were achieved. Growth in rich media is a simple and convenient alternative to growth in supplemented minimal media and is readily applicable to the expression of proteins selectively labeled with specific amino acids. PMID:16741986

  17. Chemical structure requirements and cellular targeting of microRNA-122 by peptide nucleic acids anti-miRs

    PubMed Central

    Torres, Adrian G.; Fabani, Martin M.; Vigorito, Elena; Williams, Donna; Al-Obaidi, Naowras; Wojciechowski, Filip; Hudson, Robert H. E.; Seitz, Oliver; Gait, Michael J.

    2012-01-01

    Anti-miRs are oligonucleotide inhibitors complementary to miRNAs that have been used extensively as tools to gain understanding of specific miRNA functions and as potential therapeutics. We showed previously that peptide nucleic acid (PNA) anti-miRs containing a few attached Lys residues were potent miRNA inhibitors. Using miR-122 as an example, we report here the PNA sequence and attached amino acid requirements for efficient miRNA targeting and show that anti-miR activity is enhanced substantially by the presence of a terminal-free thiol group, such as a Cys residue, primarily due to better cellular uptake. We show that anti-miR activity of a Cys-containing PNA is achieved by cell uptake through both clathrin-dependent and independent routes. With the aid of two PNA analogues having intrinsic fluorescence, thiazole orange (TO)-PNA and [bis-o-(aminoethoxy)phenyl]pyrrolocytosine (BoPhpC)-PNA, we explored the subcellular localization of PNA anti-miRs and our data suggest that anti-miR targeting of miR-122 may take place in or associated with endosomal compartments. Our findings are valuable for further design of PNAs and other oligonucleotides as potent anti-miR agents. PMID:22070883

  18. Roles of d-Amino Acids on the Bioactivity of Host Defense Peptides.

    PubMed

    Li, Hao; Anuwongcharoen, Nuttapat; Malik, Aijaz Ahmad; Prachayasittikul, Virapong; Wikberg, Jarl E S; Nantasenamat, Chanin

    2016-01-01

    Host defense peptides (HDPs) are positively-charged and amphipathic components of the innate immune system that have demonstrated great potential to become the next generation of broad spectrum therapeutic agents effective against a vast array of pathogens and tumor. As such, many approaches have been taken to improve the therapeutic efficacy of HDPs. Amongst these methods, the incorporation of d-amino acids (d-AA) is an approach that has demonstrated consistent success in improving HDPs. Although, virtually all HDP review articles briefly mentioned about the role of d-AA, however it is rather surprising that no systematic review specifically dedicated to this topic exists. Given the impact that d-AA incorporation has on HDPs, this review aims to fill that void with a systematic discussion of the impact of d-AA on HDPs. PMID:27376281

  19. Roles of d-Amino Acids on the Bioactivity of Host Defense Peptides

    PubMed Central

    Li, Hao; Anuwongcharoen, Nuttapat; Malik, Aijaz Ahmad; Prachayasittikul, Virapong; Wikberg, Jarl E. S.; Nantasenamat, Chanin

    2016-01-01

    Host defense peptides (HDPs) are positively-charged and amphipathic components of the innate immune system that have demonstrated great potential to become the next generation of broad spectrum therapeutic agents effective against a vast array of pathogens and tumor. As such, many approaches have been taken to improve the therapeutic efficacy of HDPs. Amongst these methods, the incorporation of d-amino acids (d-AA) is an approach that has demonstrated consistent success in improving HDPs. Although, virtually all HDP review articles briefly mentioned about the role of d-AA, however it is rather surprising that no systematic review specifically dedicated to this topic exists. Given the impact that d-AA incorporation has on HDPs, this review aims to fill that void with a systematic discussion of the impact of d-AA on HDPs. PMID:27376281

  20. Enhanced lubrication on tissue and biomaterial surfaces through peptide-mediated binding of hyaluronic acid

    NASA Astrophysics Data System (ADS)

    Singh, Anirudha; Corvelli, Michael; Unterman, Shimon A.; Wepasnick, Kevin A.; McDonnell, Peter; Elisseeff, Jennifer H.

    2014-10-01

    Lubrication is key for the efficient function of devices and tissues with moving surfaces, such as articulating joints, ocular surfaces and the lungs. Indeed, lubrication dysfunction leads to increased friction and degeneration of these systems. Here, we present a polymer-peptide surface coating platform to non-covalently bind hyaluronic acid (HA), a natural lubricant in the body. Tissue surfaces treated with the HA-binding system exhibited higher lubricity values, and in vivo were able to retain HA in the articular joint and to bind ocular tissue surfaces. Biomaterials-mediated strategies that locally bind and concentrate HA could provide physical and biological benefits when used to treat tissue-lubricating dysfunction and to coat medical devices.

  1. Metallamacrocycle formation through dimerization of metal bioconjugates derived from amino acids and peptides.

    PubMed

    Álvarez, Celedonio M; García-Rodríguez, Raúl; Miguel, Daniel

    2016-01-21

    Metallamacrocycles of 12, 16, and 22 members are obtained by deprotonation of the carboxylic group of the side chain of iminopyridine complexes derived from the amino acid β-alanine, and the peptides Gly-Gly and Gly-Gly-Gly. Instead of the expected intramolecular attack to give tridentate (N,N,O) ligands, the deprotonated carboxylate attacks in an inter-molecular manner to give dimers in which the ligand acts as a bridge bonded in a κ(2)(N,N') chelating fashion to one metal and as κ(O) to the other metal. The formation of the dimers is supported by NMR spectroscopy, mass spectrometry and X-ray crystallography. PMID:26645303

  2. Adsorption of peptide nucleic acid and DNA decamers at electrically charged surfaces.

    PubMed Central

    Fojta, M; Vetterl, V; Tomschik, M; Jelen, F; Nielsen, P; Wang, J; Palecek, E

    1997-01-01

    Adsorption behavior of peptide nucleic acid (PNA) and DNA decamers (GTAGATCACT and the complementary sequence) on a mercury surface was studied by means of AC impedance measurements at a hanging mercury drop electrode. The nucleic acid was first attached to the electrode by adsorption from a 5-microliter drop of PNA (or DNA) solution, and the electrode with the adsorbed nucleic acid layer was then washed and immersed in the blank background electrolyte where the differential capacity C of the electrode double layer was measured as a function of the applied potential E. It was found that the adsorption behavior of the PNA with an electrically neutral backbone differs greatly from that of the DNA (with a negatively charged backbone), whereas the DNA-PNA hybrid shows intermediate behavior. At higher surface coverage PNA molecules associate at the surface, and the minimum value of C is shifted to negative potentials because of intermolecular interactions of PNA at the surface. Prolonged exposure of PNA to highly negative potentials does not result in PNA desorption, whereas almost all of the DNA is removed from the surface at these potentials. Adsorption of PNA decreases with increasing NaCl concentration in the range from 0 to 50 mM NaCl, in contrast to DNA, the adsorption of which increases under the same conditions. PMID:9129832

  3. Determination of the gas-phase acidities of cysteine-polyalanine peptides using the extended kinetic method.

    PubMed

    Tan, John P; Ren, Jianhua

    2007-02-01

    We determined the gas-phase acidities of two cysteine-polyalanine peptides, HSCA3 and HSCA4, using a triple-quadrupole mass spectrometer through application of the extended kinetic method with full entropy analysis. Five halogenated carboxylic acids were used as the reference acids. The negatively charged proton-bound dimers of the deprotonated peptides with the conjugate bases of the reference acids were generated by electrospray ionization. Collision-induced dissociation (CID) experiments were carried out at three collision energies. The enthalpies of deprotonation (Delta(acid)H) of the peptides were derived according to the linear relationship between the logarithms of the CID product ion branching ratios and the differences of the gas-phase acidities. The values were determined to be Delta(acid)H(HSCA3) = 317.3 +/- 2.4 kcal/mol and Delta(acid)H (HSCA4) = 316.2 +/- 3.9 kcal/mol. Large entropy effects (Delta(DeltaS) = 13-16 cal/mol K) were observed for these systems. Combining the enthalpies of deprotonation with the entropy term yielded the apparent gas-phase acidities (Delta(acid)G(app)) of 322.1 +/- 2.4 kcal/mol (HSCA3) and 320.1 +/- 3.9 kcal/mol (HSCA4), in agreement with the results obtained from the CID-bracketing experiments. Compared with that in the isolated cysteine residue, the thiol group in HSCA3,4 has a stronger gas-phase acidity by about 20 kcal/mol. This increased acidity is likely due to the stabilization of the negatively charged thiolate group through internal solvation. PMID:17067812

  4. Antibody Production with Synthetic Peptides.

    PubMed

    Lee, Bao-Shiang; Huang, Jin-Sheng; Jayathilaka, Lasanthi P; Lee, Jenny; Gupta, Shalini

    2016-01-01

    Peptides (usually 10-20 amino acid residues in length) can be used as effectively as proteins in raising antibodies producing both polyclonal and monoclonal antibodies routinely with titers higher than 20,000. Peptide antigens do not function as immunogens unless they are conjugated to proteins. Production of high quality antipeptide antibodies is dependent upon peptide sequence selection, the success of peptide synthesis, peptide-carrier protein conjugation, the humoral immune response in the host animal, the adjuvant used, the peptide dose administered, the injection method, and the purification of the antibody. Peptide sequence selection is probably the most critical step in the production of antipeptide antibodies. Although the process for designing peptide antigens is not exact, several guidelines and computational B-cell epitope prediction methods can help maximize the likelihood of producing antipeptide antibodies that recognize the protein. Antibodies raised by peptides have become essential tools in life science research. Virtually all phospho-specific antibodies are now produced using phosphopeptides as antigens. Typically, 5-20 mg of peptide is enough for antipeptide antibody production. It takes 3 months to produce a polyclonal antipeptide antibody in rabbits that yields ~100 mL of serum which corresponds to ~8-10 mg of the specific antibody after affinity purification using a peptide column. PMID:27515072

  5. Specificity and formation of unusual amino acids of an amide ligation strategy for unprotected peptides.

    PubMed

    Tam, J P; Rao, C; Liu, C F; Shao, J

    1995-03-01

    An important step in the recently developed ligation strategy known as domain ligation strategy to link unprotected peptide segments without activation is the ring formation between the C-terminal ester aldehyde and the N-terminal amino acid bearing a beta-thiol or beta-hydroxide. A new method was developed to define the specificity of this reaction using a dye-labeled alanyl ester aldehyde to react with libraries of 400 dipeptides which contained all dipeptide combinations of the 20 genetically coded amino acids. Three different ester aldehydes of the dye-labeled alanine: alpha-formylmethyl (FM), beta-formylethyl (FE), and beta,beta,beta-dimethyl and formylethyl esters (DFE), were examined. The DFE ester was overly hindered and reacted with N-terminal Cys dipeptides (Cys-X). Interestingly, it also reacted slowly with the sequences of X-Gly where Gly was the second amino acid and the X-Gly amide bond participated in the ring formation. Although the FE ester reacted similarly as the FM ester in the ring formation, the subsequent O,N-acyl transfer was at least 30-fold slower than those of the FM-ester. The FM alpha-formyl methyl ester was the most suitable ester and was reactive with dipeptides of six N-terminal amino acids: Cys, Thr, Trp, Ser, His and Asn. The order and extent of their reactivity were highly dependent on pH, solvent and neighboring participation by the adjacent amino acid. In general, they could be divided into three categories. (1) N-Terminal Cys and Thr were the most reactive.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7775013

  6. A simple three-step method for design and affinity testing of new antisense peptides: an example of erythropoietin.

    PubMed

    Štambuk, Nikola; Manojlović, Zoran; Turčić, Petra; Martinić, Roko; Konjevoda, Paško; Weitner, Tin; Wardega, Piotr; Gabričević, Mario

    2014-01-01

    Antisense peptide technology is a valuable tool for deriving new biologically active molecules and performing peptide-receptor modulation. It is based on the fact that peptides specified by the complementary (antisense) nucleotide sequences often bind to each other with a higher specificity and efficacy. We tested the validity of this concept on the example of human erythropoietin, a well-characterized and pharmacologically relevant hematopoietic growth factor. The purpose of the work was to present and test simple and efficient three-step procedure for the design of an antisense peptide targeting receptor-binding site of human erythropoietin. Firstly, we selected the carboxyl-terminal receptor binding region of the molecule (epitope) as a template for the antisense peptide modeling; Secondly, we designed an antisense peptide using mRNA transcription of the epitope sequence in the 3'→5' direction and computational screening of potential paratope structures with BLAST; Thirdly, we evaluated sense-antisense (epitope-paratope) peptide binding and affinity by means of fluorescence spectroscopy and microscale thermophoresis. Both methods showed similar Kd values of 850 and 816 µM, respectively. The advantages of the methods were: fast screening with a small quantity of the sample needed, and measurements done within the range of physicochemical parameters resembling physiological conditions. Antisense peptides targeting specific erythropoietin region(s) could be used for the development of new immunochemical methods. Selected antisense peptides with optimal affinity are potential lead compounds for the development of novel diagnostic substances, biopharmaceuticals and vaccines. PMID:24865486

  7. Introducing folding stability into the score function for computational design of RNA-binding peptides boosts the probability of success.

    PubMed

    Xiao, Xingqing; Agris, Paul F; Hall, Carol K

    2016-05-01

    A computational strategy that integrates our peptide search algorithm with atomistic molecular dynamics simulation was used to design rational peptide drugs that recognize and bind to the anticodon stem and loop domain (ASL(Lys3) ) of human tRNAUUULys3 for the purpose of interrupting HIV replication. The score function of the search algorithm was improved by adding a peptide stability term weighted by an adjustable factor λ to the peptide binding free energy. The five best peptide sequences associated with five different values of λ were determined using the search algorithm and then input in atomistic simulations to examine the stability of the peptides' folded conformations and their ability to bind to ASL(Lys3) . Simulation results demonstrated that setting an intermediate value of λ achieves a good balance between optimizing the peptide's binding ability and stabilizing its folded conformation during the sequence evolution process, and hence leads to optimal binding to the target ASL(Lys3) . Thus, addition of a peptide stability term significantly improves the success rate for our peptide design search. Proteins 2016; 84:700-711. © 2016 Wiley Periodicals, Inc. PMID:26914059

  8. Electrostatic binding and hydrophobic collapse of peptide-nucleic acid aggregates quantified using force spectroscopy.

    PubMed

    Camunas-Soler, Joan; Frutos, Silvia; Bizarro, Cristiano V; de Lorenzo, Sara; Fuentes-Perez, Maria Eugenia; Ramsch, Roland; Vilchez, Susana; Solans, Conxita; Moreno-Herrero, Fernando; Albericio, Fernando; Eritja, Ramón; Giralt, Ernest; Dev, Sukhendu B; Ritort, Felix

    2013-06-25

    Knowledge of the mechanisms of interaction between self-aggregating peptides and nucleic acids or other polyanions is key to the understanding of many aggregation processes underlying several human diseases (e.g., Alzheimer's and Parkinson's diseases). Determining the affinity and kinetic steps of such interactions is challenging due to the competition between hydrophobic self-aggregating forces and electrostatic binding forces. Kahalalide F (KF) is an anticancer hydrophobic peptide that contains a single positive charge that confers strong aggregative properties with polyanions. This makes KF an ideal model to elucidate the mechanisms by which self-aggregation competes with binding to a strongly charged polyelectrolyte such as DNA. We use optical tweezers to apply mechanical forces to single DNA molecules and show that KF and DNA interact in a two-step kinetic process promoted by the electrostatic binding of DNA to the aggregate surface followed by the stabilization of the complex due to hydrophobic interactions. From the measured pulling curves we determine the spectrum of binding affinities, kinetic barriers, and lengths of DNA segments sequestered within the KF-DNA complex. We find there is a capture distance beyond which the complex collapses into compact aggregates stabilized by strong hydrophobic forces and discuss how the bending rigidity of the nucleic acid affects this process. We hypothesize that within an in vivo context, the enhanced electrostatic interaction of KF due to its aggregation might mediate the binding to other polyanions. The proposed methodology should be useful to quantitatively characterize other compounds or proteins in which the formation of aggregates is relevant. PMID:23706043

  9. Global analysis of myocardial peptides containing cysteines with irreversible sulfinic and sulfonic acid post-translational modifications.

    PubMed

    Paulech, Jana; Liddy, Kiersten A; Engholm-Keller, Kasper; White, Melanie Y; Cordwell, Stuart J

    2015-03-01

    Cysteine (Cys) oxidation is a crucial post-translational modification (PTM) associated with redox signaling and oxidative stress. As Cys is highly reactive to oxidants it forms a range of post-translational modifications, some that are biologically reversible (e.g. disulfides, Cys sulfenic acid) and others (Cys sulfinic [Cys-SO2H] and sulfonic [Cys-SO3H] acids) that are considered "irreversible." We developed an enrichment method to isolate Cys-SO2H/SO3H-containing peptides from complex tissue lysates that is compatible with tandem mass spectrometry (MS/MS). The acidity of these post-translational modification (pKa Cys-SO3H < 0) creates a unique charge distribution when localized on tryptic peptides at acidic pH that can be utilized for their purification. The method is based on electrostatic repulsion of Cys-SO2H/SO3H-containing peptides from cationic resins (i.e. "negative" selection) followed by "positive" selection using hydrophilic interaction liquid chromatography. Modification of strong cation exchange protocols decreased the complexity of initial flowthrough fractions by allowing for hydrophobic retention of neutral peptides. Coupling of strong cation exchange and hydrophilic interaction liquid chromatography allowed for increased enrichment of Cys-SO2H/SO3H (up to 80%) from other modified peptides. We identified 181 Cys-SO2H/SO3H sites from rat myocardial tissue subjected to physiologically relevant concentrations of H2O2 (<100 μm) or to ischemia/reperfusion (I/R) injury via Langendorff perfusion. I/R significantly increased Cys-SO2H/SO3H-modified peptides from proteins involved in energy utilization and contractility, as well as those involved in oxidative damage and repair. PMID:25561502

  10. A bottom-up approach to build the hyperpolarizability of peptides and proteins from their amino acids.

    PubMed

    Duboisset, Julien; Deniset-Besseau, Ariane; Benichou, Emmanuel; Russier-Antoine, Isabelle; Lascoux, Noelle; Jonin, Christian; Hache, François; Schanne-Klein, Marie-Claire; Brevet, Pierre-François

    2013-08-29

    We experimentally demonstrate that some peptides and proteins lend themselves to an elementary analysis where their first hyperpolarizability can be decomposed into the coherent superposition of the first hyperpolarizability of their elementary units. We then show that those elementary units can be associated with the amino acids themselves in the case of nonaromatic amino acids and nonresonant second harmonic generation. As a case study, this work investigates the experimentally determined first hyperpolarizability of rat tail Type I collagen and compares it to that of the shorter peptide [(PPG)10]3, where P and G are the one-letter code for Proline and Glycine, respectively, and that of the triamino acid peptides PPG and GGG. An absolute value of (0.16 ± 0.01) × 10(-30) esu for the first hyperpolarizability of nonaromatic amino acids is then obtained by using the newly defined 0.087 × 10(-30) esu reference value for water. By using a collagen like model, the microscopic hyperpolarizability along the peptide bond can be evaluated at (0.7 ± 0.1) × 10(-30) esu. PMID:23879840

  11. Formulation of pH responsive peptides as inhalable dry powders for pulmonary delivery of nucleic acids

    PubMed Central

    Liang, Wanling; Kwok, Philip C.L.; Chow, Michael Y.T.; Tang, Patricia; Mason, A. James; Chan, Hak-Kim; Lam, Jenny. K.W.

    2013-01-01

    Nucleic acids have the potential to be used as therapies or vaccines for many different types of disease but delivery remains the most significant challenge to their clinical adoption. pH responsive peptides containing either histidine or derivatives of 2,3-diaminopropionic acid (Dap) can mediate effective DNA transfection in lung epithelial cells with the latter remaining effective even in the presence of lung surfactant containing bronchoalveolar fluid (BALF), making this class of peptides attractive candidates for delivering nucleic acids to lung tissues. To further assess the suitability of pH responsive peptides for pulmonary delivery by inhalation, dry powder formulations of pH responsive peptides and plasmid DNA, with mannitol as carrier, were produced by either spray drying (SD) or spray freeze drying (SFD). The properties of the two types of powders were characterised and compared using scanning electron microscopy (SEM), next generation impaction (NGI), gel retardation and in vitro transfection via a twin-stage impinger (TSI) following aerosolisation by a dry powder inhaler (Osmohaler™). Although the aerodynamic performance and transfection efficacy of both powders were good, the overall performance revealed SD powders to have a number of advantages over SFD powders and are the more effective formulation with potential for efficient nucleic acid delivery through inhalation. PMID:23702276

  12. Impact of the N-terminal amino acid on the formation of pyrazines from peptides in Maillard model systems.

    PubMed

    Van Lancker, Fien; Adams, An; De Kimpe, Norbert

    2012-05-01

    Only a minor part of Maillard reaction studies in the literature focused on the reaction between carbohydrates and peptides. Therefore, in continuation of a previous study in which the influence of the peptide C-terminal amino acid was investigated, this study focused on the influence of the peptide N-terminal amino acid on the production of pyrazines in model reactions of glucose, methylglyoxal, or glyoxal. Nine different dipeptides and three tripeptides were selected. It was shown that the structure of the N-terminal amino acid is determinative for the overall pyrazine production. Especially, the production of 2,5(6)-dimethylpyrazine and trimethylpyrazine was low in the case of proline, valine, or leucine at the N-terminus, whereas it was very high for glycine, alanine, or serine. In contrast to the alkyl-substituted pyrazines, unsubstituted pyrazine was always produced more in the case of experiments with free amino acids. It is clear that different mechanisms must be responsible for this observation. This study clearly illustrates the capability of peptides to produce flavor compounds such as pyrazines. PMID:22463717

  13. Bile acids induce glucagon-like peptide 2 secretion with limited effects on intestinal adaptation in early weaned pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Early weaning is a stressful event characterized by a transient period of intestinal atrophy that may be mediated by reduced secretion of glucagon-like peptide (GLP) 2. We tested whether enterally fed bile acids or plant sterols could increase nutrient-dependent GLP-2 secretion and improve intestina...

  14. Computational Design of Hypothetical New Peptides Based on a Cyclotide Scaffold as HIV gp120 Inhibitor

    PubMed Central

    Sangphukieo, Apiwat; Nawae, Wanapinun; Laomettachit, Teeraphan; Supasitthimethee, Umaporn; Ruengjitchatchawalya, Marasri

    2015-01-01

    Cyclotides are a family of triple disulfide cyclic peptides with exceptional resistance to thermal/chemical denaturation and enzymatic degradation. Several cyclotides have been shown to possess anti-HIV activity, including kalata B1 (KB1). However, the use of cyclotides as anti-HIV therapies remains limited due to the high toxicity in normal cells. Therefore, grafting anti-HIV epitopes onto a cyclotide might be a promising approach for reducing toxicity and simultaneously improving anti-HIV activity. Viral envelope glycoprotein gp120 is required for entry of HIV into CD4+ T cells. However, due to a high degree of variability and physical shielding, the design of drugs targeting gp120 remains challenging. We created a computational protocol in which molecular modeling techniques were combined with a genetic algorithm (GA) to automate the design of new cyclotides with improved binding to HIV gp120. We found that the group of modified cyclotides has better binding scores (23.1%) compared to the KB1. By using molecular dynamic (MD) simulation as a post filter for the final candidates, we identified two novel cyclotides, GA763 and GA190, which exhibited better interaction energies (36.6% and 22.8%, respectively) when binding to gp120 compared to KB1. This computational design represents an alternative tool for modifying peptides, including cyclotides and other stable peptides, as therapeutic agents before the synthesis process. PMID:26517259

  15. Modeling of Peptide Adsorption Interactions with a Poly(lactic acid) Surface

    PubMed Central

    O'Brien, C. P.; Stuart, S. J.; Bruce, D. A.; Latour, R. A.

    2009-01-01

    The biocompatibility of implanted materials and devices is governed by the conformation, orientation, and composition of the layer of proteins that adsorb to the surface of the material immediately upon implantation, so an understanding of this adsorbed protein layer is essential to the rigorous and methodical design of implant materials. In this study, novel molecular dynamics techniques were employed in order to determine the change in free energy for the adsorption of a solvated nine-residue peptide (GGGG-K-GGGG) to a crystalline polylactide surface in an effort to elucidate the fundamental mechanisms that govern protein adsorption. This system, like many others, involves two distinct types of sampling problems: a spatial sampling problem, which arises due to entropic effects creating barriers in the free energy profile, and a conformational sampling problem, which occurs due to barriers in the potential energy landscape. In a two-step process that addresses each sampling problem in turn, the technique of biased replica exchange molecular dynamics was refined and applied in order to overcome these sampling problems and, using the information available at the atomic level of detail afforded by molecular simulation, both quantify and characterize the interactions between the peptide and a relevant biomaterial surface. The results from these simulations predict a fairly strong adsorption response with an adsorption free energy of -2.5 ± 0.6 kcal/mol (mean ±95% confidence interval), with adsorption primarily due to hydrophobic interactions between the nonpolar groups of the peptide and the PLA surface. As part of a larger and ongoing effort involving both simulation and experimental investigations, this work contributes to the goal of transforming the engineering of biomaterials from one dominated by trial-and-error to one which is guided by an atomic-level understanding of the interactions that occur at the tissue-biomaterial interface. PMID:19360943

  16. Trends in the Design and Development of Specific Aptamers Against Peptides and Proteins.

    PubMed

    Tabarzad, Maryam; Jafari, Marzieh

    2016-04-01

    Aptamers are single stranded oligonucleotides, comparable to monoclonal antibodies (mAbs) in selectivity and affinity and have significant strategic properties in design, development and applications more than mAbs. Ease of design and development, simple chemical modification and the attachment of functional groups, easily handling and more adaptability with analytical methods, small size and adaptation with nanostructures are the valuable characteristics of aptamers in comparison to large protein based ligands. Among a broad range of targets that their specific aptamers developed, proteins and peptides have significant position according to the number of related studies performed so far. Since proteins control many of important physiological and pathological incidents in the living organisms, particularly human beings and because of the benefits of aptamers in clinical and analytical applications, aptamer related technologies in the field of proteins and peptides are under progress, exclusively. Currently, there is only one FDA approved therapeutic aptamer in the pharmaceutical market, which is specific to vascular endothelial growth factor and is prescribed for age related macular degenerative disease. Additionally, there are several aptamers in the different phases of clinical trials. Almost all of these aptamers are specific to clinically important peptide or protein targets. In addition, the application of protein specific aptamers in the design and development of targeted drug delivery systems and diagnostic biosensors is another intersting field of aptamer technology. In this review, significant efforts related to development and applications of aptamer technologies in proteins and peptides sciences were considered to emphasis on the importance of aptamers in medicinal and clinical applications. PMID:26984473

  17. Design and Screening of a Glial Cell-Specific, Cell Penetrating Peptide for Therapeutic Applications in Multiple Sclerosis

    PubMed Central

    Heffernan, Corey; Sumer, Huseyin; Guillemin, Gilles J.; Manuelpillai, Ursula; Verma, Paul J.

    2012-01-01

    Multiple Sclerosis (MS) is an autoimmune, neurodegenerative disease of the central nervous system (CNS) characterized by demyelination through glial cell loss. Current and proposed therapeutic strategies to arrest demyelination and/or promote further remyelination include: (i) modulation of the host immune system; and/or (ii) transplantation of myelinating/stem or progenitor cells to the circulation or sites of injury. However, significant drawbacks are inherent with both approaches. Cell penetrating peptides (CPP) are short amino acid sequences with an intrinsic ability to translocate across plasma membranes, and theoretically represent an attractive vector for delivery of therapeutic peptides or nanoparticles to glia to promote cell survival or remyelination. The CPPs described to date are commonly non-selective in the cell types they transduce, limiting their therapeutic application in vivo. Here, we describe a theoretical framework for design of a novel CPP sequence that selectively transduces human glial cells (excluding non-glial cell types), and conduct preliminary screens of purified, recombinant CPPs with immature and matured human oligodendrocytes and astrocytes, and two non-glial cell types. A candidate peptide, termed TD2.2, consistently transduced glial cells, was significantly more effective at transducing immature oligodendrocytes than matured progeny, and was virtually incapable of transducing two non-glial cell types: (i) human neural cells and (ii) human dermal fibroblasts. Time-lapse confocal microscopy confirms trafficking of TD2.2 (fused to EGFP) to mature oligodendrocytes 3–6 hours after protein application in vitro. We propose selectivity of TD2.2 for glial cells represents a new therapeutic strategy for the treatment of glial-related disease, such as MS. PMID:23049807

  18. Development of novel radiogallium-labeled bone imaging agents using oligo-aspartic acid peptides as carriers.

    PubMed

    Ogawa, Kazuma; Ishizaki, Atsushi; Takai, Kenichiro; Kitamura, Yoji; Kiwada, Tatsuto; Shiba, Kazuhiro; Odani, Akira

    2013-01-01

    (68)Ga (T 1/2 = 68 min, a generator-produced nuclide) has great potential as a radionuclide for clinical positron emission tomography (PET). Because poly-glutamic and poly-aspartic acids have high affinity for hydroxyapatite, to develop new bone targeting (68)Ga-labeled bone imaging agents for PET, we used 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) as a chelating site and conjugated aspartic acid peptides of varying lengths. Subsequently, we compared Ga complexes, Ga-DOTA-(Asp)n (n = 2, 5, 8, 11, or 14) with easy-to-handle (67)Ga, with the previously described (67)Ga-DOTA complex conjugated bisphosphonate, (67)Ga-DOTA-Bn-SCN-HBP. After synthesizing DOTA-(Asp)n by a Fmoc-based solid-phase method, complexes were formed with (67)Ga, resulting in (67)Ga-DOTA-(Asp)n with a radiochemical purity of over 95% after HPLC purification. In hydroxyapatite binding assays, the binding rate of (67)Ga-DOTA-(Asp)n increased with the increase in the length of the conjugated aspartate peptide. Moreover, in biodistribution experiments, (67)Ga-DOTA-(Asp)8, (67)Ga-DOTA-(Asp)11, and (67)Ga-DOTA-(Asp)14 showed high accumulation in bone (10.5 ± 1.5, 15.1 ± 2.6, and 12.8 ± 1.7% ID/g, respectively) but were barely observed in other tissues at 60 min after injection. Although bone accumulation of (67)Ga-DOTA-(Asp)n was lower than that of (67)Ga-DOTA-Bn-SCN-HBP, blood clearance of (67)Ga-DOTA-(Asp)n was more rapid. Accordingly, the bone/blood ratios of (67)Ga-DOTA-(Asp)11 and (67)Ga-DOTA-(Asp)14 were comparable with those of (67)Ga-DOTA-Bn-SCN-HBP. In conclusion, these data provide useful insights into the drug design of (68)Ga-PET tracers for the diagnosis of bone disorders, such as bone metastases. PMID:24391942

  19. Enzymatic recycling of ascorbic acid from dehydroascorbic acid by glutathione-like peptides in the extracellular loops of aminergic G-protein coupled receptors.

    PubMed

    Root-Bernstein, Robert; Fewins, Jenna; Rhinesmith, Tyler; Koch, Ariana; Dillon, Patrick F

    2016-07-01

    The intracellular recycling of ascorbic acid from dehydroascorbic acid by the glutathione-glutathione reductase system has been well-characterized. We propose that extracellular recycling of ascorbic acid is performed in a similar manner by cysteine-rich, glutathione-like regions of the first and second extracellular loops of some aminergic receptors including adrenergic, histaminergic, and dopaminergic receptors. Previous research in our laboratory demonstrated that ascorbic acid binds to these receptors at a site on their first or second extracellular loops, significantly enhancing ligand activity, and apparently recycling hundreds of times their own concentration of ascorbate in an enzymatic fashion. In this study, we have synthesized 25 peptides from the first and second extracellular loops of aminergic and insulin receptors and compared them directly to glutathione for their ability to prevent the oxidation of ascorbate and to regenerate ascorbate from dehydroascorbic acid. Peptide sequences that mimic glutathione in containing a cysteine and a glutamic acid-like amino acid also mimic glutathione activity in effects and in kinetics. Some (but not all) peptide sequences that contain one or more methionines instead of cysteine can significantly retard the oxidation of ascorbic acid but do not recycle it from dehydroascorbate into ascorbate. Peptides lacking both cysteines and methionines uniformly failed to alter significantly ascorbate or dehydroascorbate oxidation or reduction. We believe that this is the first proof that receptors may carry out both ligand binding and enzymatic activity extracellularly. Our results suggest the existence of a previously unknown extracellular system for recycling ascorbate. Copyright © 2016 John Wiley & Sons, Ltd. PMID:26749062

  20. Design and synthesis of basic peptides having amphipathic beta-structure and their interaction with phospholipid membranes.

    PubMed

    Ono, S; Lee, S; Mihara, H; Aoyagi, H; Kato, T; Yamasaki, N

    1990-02-28

    Basic amphipathic beta-structural peptides, Ac-(Ser-Val-Lys-Val)n-NHCH3 (1n, n = 1-3) and Ac-(Lys-Val)n-NHCH3 (2n, n = 2-4), were synthesized and their interaction with DPPC and DPPC-DPPG (3:1) bilayers was studied by CD, dye-leakage and fluorescence experiments. The CD data indicated that oligopeptides consisting of more than eight residues with alternating hydrophobic (Val) and hydrophilic amino acids (Ser and Lys) were able to form an amphipathic beta-structure in acidic phospholipid bilayers, but not or weakly in aqueous solution and in neutral phospholipid bilayers. The dye-leakage experiment showed that the basic amphipathic beta-structural peptides interact with acidic phospholipid bilayers to perturb them, but less effectively compared with basic amphipathic alpha-helical peptides. Fluorescent spectroscopic data suggest that hydrophobic side of the amphipathic peptides may immerse into membrane without deep penetration. Based on these results, we postulate that the formation of the basic amphipathic beta-structure on acidic lipid bilayers may be due to the combined effect of electrostatic and hydrophobic interactions between basic peptides and acidic lipid bilayers. PMID:2306456

  1. Molecular mechanisms underlying bile acid-stimulated glucagon-like peptide-1 secretion

    PubMed Central

    Parker, HE; Wallis, K; le Roux, CW; Wong, KY; Reimann, F; Gribble, FM

    2012-01-01

    BACKGROUND AND PURPOSE The glucagon-like peptides GLP-1 and GLP-2 are secreted from enteroendocrine L-cells following nutrient ingestion. Drugs that increase activity of the GLP-1 axis are highly successful therapies for type 2 diabetes, and boosting L-cell secretion is a potential strategy for future diabetes treatment. The aim of the present study was to further our understanding of the bile acid receptor GPBA (TGR5), an L-cell target currently under therapeutic exploration. EXPERIMENTAL APPROACH GLUTag cells and mixed primary murine intestinal cultures were exposed to bile acids and a specific agonist, GPBAR-A. Secretion was measured using hormone assays and intracellular calcium and cAMP responses were monitored using real-time imaging techniques. KEY RESULTS Bile acid-triggered GLP-1 secretion from GLUTag cells was GPBA-dependent, as demonstrated by its abolition following tgr5 siRNA transfection. Bile acids and GPBAR-A increased GLP-1 secretion from intestinal cultures, with evidence for synergy between the effects of glucose and GPBA activation. Elevation of cAMP was observed following GPBA activation in individual GLUTag cells. Direct calcium responses to GPBAR-A were small, but in the presence of the agonist, a subpopulation of cells that was previously poorly glucose-responsive exhibited robust glucose responses. In vivo, increased delivery of bile to more distal regions of the ileum augmented L-cell stimulation. CONCLUSIONS AND IMPLICATIONS GPBA signalling in L-cells involves rapid elevation of cAMP, and enhanced calcium and secretory responses to glucose. Modulation of this receptor therapeutically may be an attractive strategy to enhance GLP-1 secretion and achieve better glycaemic control in diabetic patients. PMID:21718300

  2. Design of p53-derived peptides with cytotoxicity on breast cancer.

    PubMed

    Fang, Yi; Jin, Rongzhong; Gao, Yinqi; Gao, Jidong; Wang, Jing

    2014-08-01

    The tumor suppressor p53 plays essential role in conserving stability by preventing genome mutation, which is inactivated naturally by its negative regulator MDM2. Thus, targeting p53-MDM2 protein-protein interaction has been raised as a new cancer therapy in the medicinal community. In the current study, we report a successful application of an integrative protocol to design novel p53-derived peptides with cytotoxicity on human breast cancer cells. A quantitative structure-activity relationship-improved statistical potential was used to evaluate the binding potency of totally 24,054 single- and dual-point mutants of p53 peptide to MDM2 in a high-throughput manner, from which 46 peptide mutants with high predicted affinity and typical helical feature were involved in a rigorous modeling procedure that employed molecular dynamics simulations and post-binding energy analysis to systematically investigate the structural, energetic and dynamic aspects of peptide interactions with MDM2. Subsequently, a biological analysis was performed on a number of promising peptide candidates to determine their cytotoxic effects on human breast cancer cell line MDF-7. Six dual-point mutants were found to have moderate or high activities with their IC50 values ranging from 16.3 to 137.0 μM, which are better than that of wild-type p53 peptide (IC50 = 182.6 μM) and close to that of classical anticancer agent cis-platin (IC50 = 4.3 μM). Further, the most active peptide ETFSDWWKLLAE was selected as parent to further derive new mutants on the basis of the structural and energetic profile of its complex with MDM2. Consequently, three triple-point mutants (LTFSDWWKLLAE, ESFSDWWKLLAE and ETFADWWKLLAE) were obtained, and their biological activities (IC50 = 15.1, 27.0 and 8.7 μM, respectively) were determined to be comparable or better than the parent (IC50 = 16.3 μM). PMID:24830845

  3. Improved Synthesis Strategy for Peptide Nucleic Acids (PNA) appropriate for Cell-specific Fluorescence Imaging

    PubMed Central

    Pipkorn, Rüdiger; Wiessler, Manfred; Waldeck, Waldemar; Hennrich, Ute; Nokihara, Kiyoshi; Beining, Marcel; Braun, Klaus

    2012-01-01

    Progress in genomics and proteomics attended to the door for better understanding the recent rapid expanding complex research field of metabolomics. This trend in biomedical research increasingly focuses to the development of patient-specific therapeutic approaches with higher efficiency and sustainability. Simultaneously undesired adverse reactions are avoided. In parallel, the development of molecules for molecular imaging is required not only for the imaging of morphological structures but also for the imaging of metabolic processes like the aberrant expression of the cysteine protease cathepsin B (CtsB) gene and the activity of the resulting product associated with metastasis and invasiveness of malign tumors. Finally the objective is to merge imaging and therapy at the same level. The design of molecules which fulfil these responsibilities is pivotal and requires proper chemical methodologies. In this context our modified solid phase peptide chemistry using temperature shifts during synthesis is considered as an appropriate technology. We generated highly variable conjugates which consist of molecules useful as diagnostically and therapeutically active molecules. As an example the modular PNA products with the complementary sequence to the CtsB mRNA and additionally with a cathepsin B cleavage site had been prepared as functional modules for distinction of cell lines with different CtsB gene expression. After ligation to the modular peptide-based BioShuttle carrier, which was utilized to facilitate the delivery of the functional modules into the cells' cytoplasm, the modules were scrutinized. PMID:22211082

  4. Engineering D-Amino Acid Containing Collagen Like Peptide at the Cleavage Site of Clostridium histolyticum Collagenase for Its Inhibition

    PubMed Central

    Velmurugan, Punitha; Jonnalagadda, Raghava Rao; Unni Nair, Balachandran

    2015-01-01

    Collagenase is an important enzyme which plays an important role in degradation of collagen in wound healing, cancer metastasis and even in embryonic development. However, the mechanism of this degradation has not yet been completely understood. In the field of biomedical and protein engineering, the design and development of new peptide based materials is of main concern. In the present work an attempt has been made to study the effect of DAla in collagen like peptide (imino-poor region of type I collagen) on the structure and stability of peptide against enzyme hydrolysis. Effect of replacement of DAla in the collagen like peptide has been studied using circular dichroic spectroscopy (CD). Our findings suggest that, DAla substitution leads to conformational changes in the secondary structure and favours the formation of polyproline II conformation than its L-counterpart in the imino-poor region of collagen like peptides. Change in the chirality of alanine at the cleavage site of collagenase in the imino-poor region inhibits collagenolytic activity. This may find application in design of peptides and peptidomimics for enzyme-substrate interaction, specifically with reference to collagen and other extra cellular matrix proteins. PMID:25973613

  5. Nisin-induced expression of a recombinant antihypertensive peptide in dairy lactic acid bacteria

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Peptides with antihypertensive activity have been identified from the enzymatic hydrolysis of bovine milk proteins. A 12-residue peptide (FFVAPFPEVFGK) shown to inhibit the angiotensin I-converting enzyme is released from the enzymatic breakdown of aS1-casein. A synthetic gene encoding this peptid...

  6. Computational design of peptide-Au cluster probe for sensitive detection of αIIbβ3 integrin.

    PubMed

    Zhao, Lina; Zhai, Jiao; Zhang, Xuejie; Gao, Xueyun; Fang, Xiaohong; Li, Jingyuan

    2016-02-11

    We have designed a novel peptide-Au cluster probe to specifically bind to αIIbβ3 integrin. As indicated by molecular dynamics (MD) simulations, the binding mode of the native ligand of αIIbβ3 integrin, γC peptide, can be realized by the designed probe. More importantly, the peptide-Au probe can provide multiple coating peptides to form additional salt bridges with protein, and the binding stability of the probe is comparable to the native ligand. The designed probe was then successfully synthesized. The specific binding in a cellular environment was validated by colocalization analysis of confocal microscopy. In addition, the binding affinity was confirmed by atomic force microscopy (AFM) based single molecule force spectroscopy. Our results suggest the combination of computational design and experimental verification can be a useful strategy for the development of nanoprobes. PMID:26831577

  7. Design of a RANK-Mimetic Peptide Inhibitor of Osteoclastogenesis with Enhanced RANKL-Binding Affinity

    PubMed Central

    Hur, Jeonghwan; Ghosh, Ambarnil; Kim, Kabsun; Ta, Hai Minh; Kim, Hyunju; Kim, Nacksung; Hwang, Hye-Yeon; Kim, Kyeong Kyu

    2016-01-01

    The receptor activator of nuclear factor κB (RANK) and its ligand RANKL are key regulators of osteoclastogenesis and well-recognized targets in developing treatments for bone disorders associated with excessive bone resorption, such as osteoporosis. Our previous work on the structure of the RANK-RANKL complex revealed that Loop3 of RANK, specifically the non-canonical disulfide bond at the tip, performs a crucial role in specific recognition of RANKL. It also demonstrated that peptide mimics of Loop3 were capable of interfering with the function of RANKL in osteoclastogenesis. Here, we reported the structure-based design of a smaller peptide with enhanced inhibitory efficiency. The kinetic analysis and osteoclast differentiation assay showed that in addition to the sharp turn induced by the disulfide bond, two consecutive arginine residues were also important for binding to RANKL and inhibiting osteoclastogenesis. Docking and molecular dynamics simulations proposed the binding mode of the peptide to the RANKL trimer, showing that the arginine residues provide electrostatic interactions with RANKL and contribute to stabilizing the complex. These findings provided useful information for the rational design of therapeutics for bone diseases associated with RANK/RANKL function. PMID:26923188

  8. Single Molecule Spectroscopy of Amino Acids and Peptides by Recognition Tunneling

    PubMed Central

    Zhao, Yanan; Ashcroft, Brian; Zhang, Peiming; Liu, Hao; Sen, Suman; Song, Weisi; Im, JongOne; Gyarfas, Brett; Manna, Saikat; Biswas, Sovan; Borges, Chad; Lindsay, Stuart

    2014-01-01

    The human proteome has millions of protein variants due to alternative RNA splicing and post-translational modifications, and variants that are related to diseases are frequently present in minute concentrations. For DNA and RNA, low concentrations can be amplified using the polymerase chain reaction, but there is no such reaction for proteins. Therefore, the development of single molecule protein sequencing is a critical step in the search for protein biomarkers. Here we show that single amino acids can be identified by trapping the molecules between two electrodes that are coated with a layer of recognition molecules and measuring the electron tunneling current across the junction. A given molecule can bind in more than one way in the junction, and we therefore use a machine-learning algorithm to distinguish between the sets of electronic ‘fingerprints’ associated with each binding motif. With this recognition tunneling technique, we are able to identify D, L enantiomers, a methylated amino acid, isobaric isomers, and short peptides. The results suggest that direct electronic sequencing of single proteins could be possible by sequentially measuring the products of processive exopeptidase digestion, or by using a molecular motor to pull proteins through a tunnel junction integrated with a nanopore. PMID:24705512

  9. Single-molecule spectroscopy of amino acids and peptides by recognition tunnelling.

    PubMed

    Zhao, Yanan; Ashcroft, Brian; Zhang, Peiming; Liu, Hao; Sen, Suman; Song, Weisi; Im, JongOne; Gyarfas, Brett; Manna, Saikat; Biswas, Sovan; Borges, Chad; Lindsay, Stuart

    2014-06-01

    The human proteome has millions of protein variants due to alternative RNA splicing and post-translational modifications, and variants that are related to diseases are frequently present in minute concentrations. For DNA and RNA, low concentrations can be amplified using the polymerase chain reaction, but there is no such reaction for proteins. Therefore, the development of single-molecule protein sequencing is a critical step in the search for protein biomarkers. Here, we show that single amino acids can be identified by trapping the molecules between two electrodes that are coated with a layer of recognition molecules, then measuring the electron tunnelling current across the junction. A given molecule can bind in more than one way in the junction, and we therefore use a machine-learning algorithm to distinguish between the sets of electronic 'fingerprints' associated with each binding motif. With this recognition tunnelling technique, we are able to identify D and L enantiomers, a methylated amino acid, isobaric isomers and short peptides. The results suggest that direct electronic sequencing of single proteins could be possible by sequentially measuring the products of processive exopeptidase digestion, or by using a molecular motor to pull proteins through a tunnel junction integrated with a nanopore. PMID:24705512

  10. Single-molecule spectroscopy of amino acids and peptides by recognition tunnelling

    NASA Astrophysics Data System (ADS)

    Zhao, Yanan; Ashcroft, Brian; Zhang, Peiming; Liu, Hao; Sen, Suman; Song, Weisi; Im, Jongone; Gyarfas, Brett; Manna, Saikat; Biswas, Sovan; Borges, Chad; Lindsay, Stuart

    2014-06-01

    The human proteome has millions of protein variants due to alternative RNA splicing and post-translational modifications, and variants that are related to diseases are frequently present in minute concentrations. For DNA and RNA, low concentrations can be amplified using the polymerase chain reaction, but there is no such reaction for proteins. Therefore, the development of single-molecule protein sequencing is a critical step in the search for protein biomarkers. Here, we show that single amino acids can be identified by trapping the molecules between two electrodes that are coated with a layer of recognition molecules, then measuring the electron tunnelling current across the junction. A given molecule can bind in more than one way in the junction, and we therefore use a machine-learning algorithm to distinguish between the sets of electronic `fingerprints' associated with each binding motif. With this recognition tunnelling technique, we are able to identify D and L enantiomers, a methylated amino acid, isobaric isomers and short peptides. The results suggest that direct electronic sequencing of single proteins could be possible by sequentially measuring the products of processive exopeptidase digestion, or by using a molecular motor to pull proteins through a tunnel junction integrated with a nanopore.

  11. Information transfer from DNA to peptide nucleic acids by template-directed syntheses

    NASA Technical Reports Server (NTRS)

    Schmidt, J. G.; Christensen, L.; Nielsen, P. E.; Orgel, L. E.; Bada, J. L. (Principal Investigator)

    1997-01-01

    Peptide nucleic acids (PNAs) are analogs of nucleic acids in which the ribose-phosphate backbone is replaced by a backbone held together by amide bonds. PNAs are interesting as models of alternative genetic systems because they form potentially informational base paired helical structures. Oligocytidylates have been shown to act as templates for formation of longer oligomers of G from PNA G2 dimers. In this paper we show that information can be transferred from DNA to PNA. DNA C4T2C4 is an efficient template for synthesis of PNA G4A2G4 using G2 and A2 units as substrates. The corresponding synthesis of PNA G4C2G4 on DNA C4G2C4 is less efficient. Incorporation of PNA T2 into PNA products on DNA C4A2C4 is the least efficient of the three reactions. These results, obtained using PNA dimers as substrates, parallel those obtained using monomeric activated nucleotides.

  12. Progress in nanoparticulate systems for peptide, proteins and nucleic acid drug delivery.

    PubMed

    Slomkowski, Stanislaw; Gosecki, Mateusz

    2011-11-01

    Progress in many therapies, in particular in the therapies based on peptides, proteins and nucleic acids used as bioactive compounds, strongly depends on development of appropriate carriers which would be suitable for controlled delivery of the intact abovementioned compounds to required tissues, cells and intracellular compartments. This review presents last ten years' achievements and problems in development and application of synthetic polymer nanoparticulate carriers for oral, pulmonary and nasal delivery routes of oligopeptides and proteins. Whereas some traditional synthetic polymer carriers are only briefly recalled the main attention is concentrated on nanoparticles produced from functional copolymers mostly with hydroxyl, carboxyl and amino groups, suitable for immobilization of targeting moieties and for assuring prolonged circulation of nanoparticles in blood. Formulations of various nanoparticulate systems are described, including solid particles, polymer micelles, nanovesicles and nanogels, especially systems allowing drug release induced by external stimuli. Discussed are properties of these species, in particular stability in buffers and models of body fluids, loading with drugs and with drug models, drug release processes and results of biological studies. There are also discussed systems for gene delivery with special attention devoted to polymers suitable for compacting nucleic acids into nanoparticles as well as the relations between chemical structure of polymer carriers and ability of the latter for crossing cell membranes and for endosomal escape. PMID:21902630

  13. Structure-Based Design of Inhibitors of Protein–Protein Interactions: Mimicking Peptide Binding Epitopes

    PubMed Central

    Pelay-Gimeno, Marta; Glas, Adrian; Koch, Oliver; Grossmann, Tom N

    2015-01-01

    Protein–protein interactions (PPIs) are involved at all levels of cellular organization, thus making the development of PPI inhibitors extremely valuable. The identification of selective inhibitors is challenging because of the shallow and extended nature of PPI interfaces. Inhibitors can be obtained by mimicking peptide binding epitopes in their bioactive conformation. For this purpose, several strategies have been evolved to enable a projection of side chain functionalities in analogy to peptide secondary structures, thereby yielding molecules that are generally referred to as peptidomimetics. Herein, we introduce a new classification of peptidomimetics (classes A–D) that enables a clear assignment of available approaches. Based on this classification, the Review summarizes strategies that have been applied for the structure-based design of PPI inhibitors through stabilizing or mimicking turns, β-sheets, and helices. PMID:26119925

  14. The rational design of a peptide-based hydrogel responsive to H2S.

    PubMed

    Peltier, Raoul; Chen, Ganchao; Lei, Haipeng; Zhang, Mei; Gao, Liqian; Lee, Su Seong; Wang, Zuankai; Sun, Hongyan

    2015-12-18

    The development of hydrogels that are responsive to external stimuli in a well-controlled manner is important for numerous biomedical applications. Herein we reported the first example of a hydrogel responsive to hydrogen sulphide (H2S). H2S is an important gasotransmitter whose deregulation has been associated with a number of pathological conditions. Our hydrogel design is based on the functionalization of an ultrashort hydrogelating peptide sequence with an azidobenzyl moiety, which was reported to react with H2S selectively under physiological conditions. The resulting peptide was able to produce hydrogels at a concentration as low as 0.1 wt%. It could then be fully degraded in the presence of excess H2S. We envision that the novel hydrogel developed in this study may provide useful tools for biomedical research. PMID:26463661

  15. Computational approaches for designing potent and selective analogs of peptide toxins as novel therapeutics

    PubMed Central

    Kuyucak, Serdar; Norton, Raymond S

    2015-01-01

    Peptide toxins provide valuable therapeutic leads for many diseases. As they bind to their targets with high affinity, potency is usually ensured. However, toxins also bind to off-target receptors, causing potential side effects. Thus, a major challenge in generating drugs from peptide toxins is ensuring their specificity for their intended targets. Computational methods can play an important role in solving such design problems through construction of accurate models of receptor–toxin complexes and calculation of binding free energies. Here we review the computational methods used for this purpose and their application to toxins targeting ion channels. We describe ShK and HsTX1 toxins, high-affinity blockers of the voltage-gated potassium channel Kv1.3, which could be developed as therapeutic agents for autoimmune diseases. PMID:25406005

  16. Evolving serodiagnostics by rationally designed peptide arrays: the Burkholderia paradigm in Cystic Fibrosis.

    PubMed

    Peri, Claudio; Gori, Alessandro; Gagni, Paola; Sola, Laura; Girelli, Daniela; Sottotetti, Samantha; Cariani, Lisa; Chiari, Marcella; Cretich, Marina; Colombo, Giorgio

    2016-01-01

    Efficient diagnosis of emerging and novel bacterial infections is fundamental to guide decisions on therapeutic treatments. Here, we engineered a novel rational strategy to design peptide microarray platforms, which combines structural and genomic analyses to predict the binding interfaces between diverse protein antigens and antibodies against Burkholderia cepacia complex infections present in the sera of Cystic Fibrosis (CF) patients. The predicted binding interfaces on the antigens are synthesized in the form of isolated peptides and chemically optimized for controlled orientation on the surface. Our platform displays multiple Burkholderia-related epitopes and is shown to diagnose infected individuals even in presence of superinfections caused by other prevalent CF pathogens, with limited cost and time requirements. Moreover, our data point out that the specific patterns determined by combined probe responses might provide a characterization of Burkholderia infections even at the subtype level (genomovars). The method is general and immediately applicable to other bacteria. PMID:27615705

  17. Incorporation of Acid-Labile Masking Groups for the Traceless Synthesis of C-Terminal Peptide α-Ketoacids.

    PubMed

    Thuaud, Frédéric; Rohrbacher, Florian; Zwicky, André; Bode, Jeffrey W

    2016-08-01

    An optimized protocol for the masking of α-ketoacids with acid-labile cyclic acetal protecting groups is reported. Unlike prior approaches, these new conditions allow the synthesis of protected α-ketoacids bearing aromatic, hindered alkyl, and protected polar side chains. Attachment to a Wang-type linker and solid support provides a resin that delivers fully unprotected C-terminal peptide α-ketoacids upon resin cleavage. These peptides are the key starting materials for chemical protein synthesis using the α-ketoacid-hydroxylamine ligation. PMID:27439001

  18. Identification and binding mechanism of phage displayed peptides with specific affinity to acid-alkali treated titanium.

    PubMed

    Sun, Yuhua; Tan, Jing; Wu, Baohua; Wang, Jianxin; Qu, Shuxin; Weng, Jie; Feng, Bo

    2016-10-01

    Acid-alkali treatment is one of means widely used for preparing bioactive titanium surfaces. Peptides with specific affinity to titanium surface modified by acid-alkali two-steps treatment were obtained via phage display technology. Out of the eight new unique peptides, titanium-binding peptide 54 displayed by monoclonal M13 phage at its pIII coat protein (TBP54-M13 phage) was proved to have higher binding affinity to the substrate. The binding interaction occurred at the domain from phenylalanine at position 1 to arginine at position 6 in the sequences of TBP54 (FAETHRGFHFSF) mainly via the reaction of these residues with the Ti surface. Together the coordination and electrostatic interactions controlled the specific binding of the phage to the substrate. The binding affinity was dependent on the surface basic hydroxyl group content. In addition, the phage showed a different interaction way with the Ti surface without acid-alkali treatment along with an impaired affinity. This study could provide more understanding of the interaction mechanism between the selected peptide and its specific substrate, and develop a promising method for the biofunctionalization of titanium. PMID:27371890

  19. Antioxidant capacity of hydrolyzed animal by-products and relation to amino acid composition and peptide size distribution.

    PubMed

    Damgaard, Trine; Lametsch, René; Otte, Jeanette

    2015-10-01

    The antioxidative capacity of six different tissue hydrolysates (porcine colon, heart and neck and bovine lung, kidney and pancreas) were tested by three different assays monitoring iron chelation, ABTS radical scavenging and inhibition of lipid oxidation in emulsions, respectively. The hydrolysates were also investigated with respect to amino acid composition and peptide size distribution. The hydrolysates contained peptides ranging from 20 kDa to below 100 Da with a predominance of peptides with low molecular weight (53.8 to 89.0 % below 3 kDa). All hydrolysates exhibited antioxidant activity as assessed with all three methods; inhibition of lipid oxidation ranging from 72 to 88 % (at a final protein concentration of 7 mg/mL), iron chelation capacity from 23 to 63 % (at 1.1 mg/mL), and ABTS radical scavenging from 38 to 50 % (at 10 μg /mL). The antioxidant activity did not correlate with the proportion of low molecular weight peptides in the hydrolysed tissues, but with the content of specific amino acid residues. The ABTS radical scavenging capacity of the tissues was found to correlate with the content of Trp, Tyr, Met and Arg, whereas the ability to inhibit the oxidation of lineoleic acid correlated with the content of Glu and His. The chosen animal by-products thus represent a natural source of antioxidants with potential for food application. PMID:26396396

  20. Tuning One-Dimensional Nanostructures of Bola-Like Peptide Amphiphiles by Varying the Hydrophilic Amino Acids.

    PubMed

    Zhao, Yurong; Deng, Li; Yang, Wei; Wang, Dong; Pambou, Elias; Lu, Zhiming; Li, Zongyi; Wang, Jiqian; King, Stephen; Rogers, Sarah; Xu, Hai; Lu, Jian R

    2016-08-01

    By combining experimental measurements and computer simulations, we here show that for the bola-like peptide amphiphiles XI4 X, where X=K, R, and H, the hydrophilic amino acid substitutions have little effect on the β-sheet hydrogen-bonding between peptide backbones. Whereas all of the peptides self-assemble into one dimensional (1D) nanostructures with completely different morphologies, that is, nanotubes and helical nanoribbons for KI4 K, flat and multilayered nanoribbons for HI4 H, and twisted and bilayered nanoribbons for RI4 R. These different 1D morphologies can be explained by the distinct stacking degrees and modes of the three peptide β-sheets along the x-direction (width) and the z-direction (height), which microscopically originate from the hydrogen-bonding ability of the sheets to solvent molecules and the pairing of hydrophilic amino acid side chains between β-sheet monolayers through stacking interactions and hydrogen bonding. These different 1D nanostructures have distinct surface chemistry and functions, with great potential in various applications exploiting the respective properties of these hydrophilic amino acids. PMID:27362441

  1. A Simple Three-Step Method for Design and Affinity Testing of New Antisense Peptides: An Example of Erythropoietin

    PubMed Central

    Štambuk, Nikola; Manojlović, Zoran; Turčić, Petra; Martinić, Roko; Konjevoda, Paško; Weitner, Tin; Wardega, Piotr; Gabričević, Mario

    2014-01-01

    Antisense peptide technology is a valuable tool for deriving new biologically active molecules and performing peptide–receptor modulation. It is based on the fact that peptides specified by the complementary (antisense) nucleotide sequences often bind to each other with a higher specificity and efficacy. We tested the validity of this concept on the example of human erythropoietin, a well-characterized and pharmacologically relevant hematopoietic growth factor. The purpose of the work was to present and test simple and efficient three-step procedure for the design of an antisense peptide targeting receptor-binding site of human erythropoietin. Firstly, we selected the carboxyl-terminal receptor binding region of the molecule (epitope) as a template for the antisense peptide modeling; Secondly, we designed an antisense peptide using mRNA transcription of the epitope sequence in the 3'→5' direction and computational screening of potential paratope structures with BLAST; Thirdly, we evaluated sense–antisense (epitope–paratope) peptide binding and affinity by means of fluorescence spectroscopy and microscale thermophoresis. Both methods showed similar Kd values of 850 and 816 µM, respectively. The advantages of the methods were: fast screening with a small quantity of the sample needed, and measurements done within the range of physicochemical parameters resembling physiological conditions. Antisense peptides targeting specific erythropoietin region(s) could be used for the development of new immunochemical methods. Selected antisense peptides with optimal affinity are potential lead compounds for the development of novel diagnostic substances, biopharmaceuticals and vaccines. PMID:24865486

  2. Computational design of peptide-Au cluster probe for sensitive detection of αIIbβ3 integrin

    NASA Astrophysics Data System (ADS)

    Zhao, Lina; Zhai, Jiao; Zhang, Xuejie; Gao, Xueyun; Fang, Xiaohong; Li, Jingyuan

    2016-02-01

    We have designed a novel peptide-Au cluster probe to specifically bind to αIIbβ3 integrin. As indicated by molecular dynamics (MD) simulations, the binding mode of the native ligand of αIIbβ3 integrin, γC peptide, can be realized by the designed probe. More importantly, the peptide-Au probe can provide multiple coating peptides to form additional salt bridges with protein, and the binding stability of the probe is comparable to the native ligand. The designed probe was then successfully synthesized. The specific binding in a cellular environment was validated by colocalization analysis of confocal microscopy. In addition, the binding affinity was confirmed by atomic force microscopy (AFM) based single molecule force spectroscopy. Our results suggest the combination of computational design and experimental verification can be a useful strategy for the development of nanoprobes.We have designed a novel peptide-Au cluster probe to specifically bind to αIIbβ3 integrin. As indicated by molecular dynamics (MD) simulations, the binding mode of the native ligand of αIIbβ3 integrin, γC peptide, can be realized by the designed probe. More importantly, the peptide-Au probe can provide multiple coating peptides to form additional salt bridges with protein, and the binding stability of the probe is comparable to the native ligand. The designed probe was then successfully synthesized. The specific binding in a cellular environment was validated by colocalization analysis of confocal microscopy. In addition, the binding affinity was confirmed by atomic force microscopy (AFM) based single molecule force spectroscopy. Our results suggest the combination of computational design and experimental verification can be a useful strategy for the development of nanoprobes. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr09175f

  3. A combined NMR and computational approach to investigate peptide binding to a designed Armadillo repeat protein.

    PubMed

    Ewald, Christina; Christen, Martin T; Watson, Randall P; Mihajlovic, Maja; Zhou, Ting; Honegger, Annemarie; Plückthun, Andreas; Caflisch, Amedeo; Zerbe, Oliver

    2015-05-22

    The specific recognition of peptide sequences by proteins plays an important role both in biology and in diagnostic applications. Here we characterize the relatively weak binding of the peptide neurotensin (NT) to the previously developed Armadillo repeat protein VG_328 by a multidisciplinary approach based on solution NMR spectroscopy, mutational studies, and molecular dynamics (MD) simulations, totaling 20μs for all MD runs. We describe assignment challenges arising from the repetitive nature of the protein sequence, and we present novel approaches to address them. Partial assignments obtained for VG_328 in combination with chemical shift perturbations allowed us to identify the repeats not involved in binding. Their subsequent elimination resulted in a reduced-size binder with very similar affinity for NT, for which near-complete backbone assignments were achieved. A binding mode suggested by automatic docking and further validated by explicit solvent MD simulations is consistent with paramagnetic relaxation enhancement data collected using spin-labeled NT. Favorable intermolecular interactions are observed in the MD simulations for the residues that were previously shown to contribute to binding in an Ala scan of NT. We further characterized the role of residues within the N-cap for protein stability and peptide binding. Our multidisciplinary approach demonstrates that an initial low-resolution picture for a low-micromolar-peptide binder can be refined through the combination of NMR, protein design, docking, and MD simulations to establish its binding mode, even in the absence of crystallographic data, thereby providing valuable information for further design. PMID:25816772

  4. Effect of organic acids on calcium phosphate nucleation and osteogenic differentiation of human mesenchymal stem cells on peptide functionalized nanofibers.

    PubMed

    Barati, Danial; Walters, Joshua D; Shariati, Seyed Ramin Pajoum; Moeinzadeh, Seyedsina; Jabbari, Esmaiel

    2015-05-12

    Carboxylate-rich organic acids play an important role in controlling the growth of apatite crystals and the extent of mineralization in the natural bone. The objective of this work was to investigate the effect of organic acids on calcium phosphate (CaP) nucleation on nanofiber microsheets functionalized with a glutamic acid peptide and osteogenic differentiation of human mesenchymal stem cells (hMSCs) seeded on the CaP-nucleated microsheets. High molecular weight poly(dl-lactide) (DL-PLA) was mixed with low molecular weight L-PLA conjugated with Glu-Glu-Gly-Gly-Cys peptide, and the mixture was electrospun to generate aligned nanofiber microsheets. The nanofiber microsheets were incubated in a modified simulated body fluid (mSBF) supplemented with different organic acids for nucleation and growth of CaP crystals on the nanofibers. Organic acids included citric acid (CA), hydroxycitric acid (HCA), tartaric acid (TART), malic acid (MA), ascorbic acid (AsA), and salicylic acid (SalA). HCA microsheets had the highest CaP content at 240 ± 10% followed by TART and CA with 225 ± 8% and 225 ± 10%, respectively. The Ca/P ratio and percent crystallinity of the nucleated CaP in TART microsheets was closest to that of stoichiometric hydroxyapatite. The extent of CaP nucleation and growth on the nanofiber microsheets depended on the acidic strength and number of hydrogen-bonding hydroxyl groups of the organic acids. Compressive modulus and degradation of the CaP nucleated microsheets were related to percent crystallinity and CaP content. Osteogenic differentiation of hMSCs seeded on the microsheets and cultured in osteogenic medium increased only for those microsheets nucleated with CaP by incubation in CA or AsA-supplemented mSBF. Further, only CA microsheets stimulated bone nodule formation by the seeded hMSCs. PMID:25879768

  5. Separation of proteins and peptides by capillary electrophoresis in acid buffers containing high concentrations of surfactants.

    PubMed

    Miksík, I; Deyl, Z

    1999-08-01

    Separations of proteins at acid pH in the presence of a high concentration of surfactant [sodium laurylsulfate (SDS), 50 mmol/l] was investigated. The purpose of using high concentrations of SDS as background electrolyte modifier was threefold: First, the surfactant exerts a washing effect upon the capillary wall thus preventing binding of analytes and possible clogging of the capillary. Second, it was revealed that even under very acid conditions (below pH 3) the surfactant is capable of forming associates with protein analytes which still bear considerable negative charge and can be separated on this basis. Third, the system can be applied not only for protein mixtures sufficiently soluble in neutral to alkaline media (leukocyte lysates, standard proteins), but it can be used also with proteins, that are under such conditions virtually insoluble and their solubilization is possible in acid buffers only (eggshell proteins or collagen CNBr fragments). The result was that adsorption to the capillary wall was minimized and the analytes were separated as negatively charged associates with high efficiency. With collagen fragments partition was possible on the affinity differences of the peptides to the surfactant micelles and inner wall of the capillary. Theoretical plate counts approaching 100,000 were easily achieved even with proteins which under the more conventional operation conditions exhibit considerable sticking to the capillary wall. The other feature of this system is that the associates move very rapidly to the anode. Owing to the low pH, endoosmotic flow is negligible, and therefore the system has to be operated at reversed polarity. PMID:10480258

  6. Covalent and non-covalent binding in the ion/ion charge inversion of peptide cations with benzene-disulfonic acid anions.

    PubMed

    Stutzman, John R; Luongo, Carl A; McLuckey, Scott A

    2012-06-01

    Protonated angiotensin II and protonated leucine enkephalin-based peptides, which included YGGFL, YGGFLF, YGGFLH, YGGFLK and YGGFLR, were subjected to ion/ion reactions with the doubly deprotonated reagents 4-formyl-1,3-benzenedisulfonic acid (FBDSA) and 1,3-benzenedisulfonic acid (BDSA). The major product of the ion/ion reaction is a negatively charged complex of the peptide and reagent. Following dehydration of [M + FBDSA-H](-) via collisional-induced dissociation (CID), angiotensin II (DRVYIHPF) showed evidence for two product populations, one in which a covalent modification has taken place and one in which an electrostatic modification has occurred (i.e. no covalent bond formation). A series of studies with model systems confirmed that strong non-covalent binding of the FBDSA reagent can occur with subsequent ion trap CID resulting in dehydration unrelated to the adduct. Ion trap CID of the dehydration product can result in cleavage of amide bonds in competition with loss of the FBDSA adduct. This scenario is most likely for electrostatically bound complexes in which the peptide contains both an arginine residue and one or more carboxyl groups. Otherwise, loss of the reagent species from the complex, either as an anion or as a neutral species, is the dominant process for electrostatically bound complexes. The results reported here shed new light on the nature of non-covalent interactions in gas phase complexes of peptide ions that can be used in the rationale design of reagent ions for specific ion/ion reaction applications. PMID:22707160

  7. Increase of anti-HIV activity of C-peptide fusion inhibitors using a bivalent drug design approach.

    PubMed

    Ling, Yanbo; Xue, Huifang; Jiang, Xifeng; Cai, Lifeng; Liu, Keliang

    2013-09-01

    We reported the design of fusion inhibitors with improved activity using a multivalent inhibitor design strategy. First, we chose C29 as the template sequence, which is a 29-mer peptide derived from HIV-1 gp41 CHR domain and has anti-HIV activity of IC50 118 nM in a cell-cell fusion assay. We optimized the crosslink sites and linkers of the template peptide. We found that N-terminal crosslink caused activity improvement based on the multivalent co-operative effect. Especially, the IC50 of peptide (CAcaC29)2 was improved from 49.02 (monomeric form) to 5.71 nM. Compared with long peptides, short peptides may be more suitable to analyze the co-operative effect. So we selected a shorter peptide C22 to synthesize the bivalent inhibitors. Due its weak helicity, no co-operative effect appeared. Therefore, we chose SC22EK, which were introduced salt bridges to consolidate the helicity based on the natural sequence C22. The cross-linked (CAcaSC22EK)2 was four times more potent than the monomer SC22EK in anti-HIV activity, with an IC50 value of 4.92 nM close to the high active peptide fusion inhibitor C34. The strategy used in this study may be used to design new fusion inhibitors to interfere similar processes. PMID:23906421

  8. Advances in the design and delivery of peptide subunit vaccines with a focus on Toll-like receptor agonists

    PubMed Central

    Black, Matthew; Trent, Amanda; Tirrell, Matthew; Olive, Colleen

    2010-01-01

    Considerable success has been made with many peptide antigen formulations, and peptide-based vaccines are emerging as the next generation of prophylactic and remedial immunotherapy. However, finding an optimal platform balancing all of the requirements for an effective, specific and safe immune response remains a major challenge for many infectious and chronic diseases. This review outlines how peptide immunogenicity is influenced by the way in which peptides are presented to the immune system, underscoring the need for multifunctional delivery systems that couple antigen and adjuvant into a single construct. Particular attention is given to the ability of Toll-like receptor agonists to act as adjuvants. A survey of recent approaches to developing peptide antigen delivery systems is given, many of which incorporate Toll-like receptor agonists into the design. PMID:20109027

  9. Detection of antimicrobial (poly)peptides with acid urea polyacrylamide gel electrophoresis followed by Western immunoblot.

    PubMed

    Porter, Edith; Valore, Erika V; Anouseyan, Rabin; Salzman, Nita H

    2015-01-01

    Antimicrobial (poly)peptides (AMPs) are ancient key effector molecules of innate host defense and have been identified in mammals, insects, plants, and even fungi (Nakatsuji and Gallo, J Invest Dermatol, 132: 887-895, 2012). They exhibit a cationic net charge at physiological pH and are rich in hydrophobic amino acids (Dufourc et al., Curr Protein Pept Sci, 13: 620-631, 2012). Their mode of action has been best investigated in bacteria. When assuming secondary structure the cationic and hydrophobic amino acids are sequestered creating a bipartitioned molecule in which the cationic amino acids mediate initial electrostatic interaction with the negatively charged bacterial surface and the hydrophobic amino acids mediate embedding into the bacterial membranes followed by a multitude of effects interfering with bacterial viability (Nicolas, FEBS J, 276: 6483-6496, 2009; Padovan et al., Curr Protein Pept Sci, 11: 210-219, 2010). However, immunomodulatory, antitumor, and other effects have been added to the ever increasing list of AMP functions (Pushpanathan et al., Int J Pept, 2013: 675391, 2013). Several classes of AMPs have been distinguished based on structure, namely anti-parallel beta-sheet, alpha-helical, circular, as well as disulfide bridge connectivity (Bond and Khalid, Protein Pept Lett, 17: 1313-1327, 2010). Many of the AMPs undergo posttranslational modification including further proteolysis. Biochemical analysis at the protein level is of great interest for a wide range of scientists and important when studying host-pathogen interaction, for example Salmonella invasion of the small intestine. Acid-urea polyacrylamide gel electrophoresis (AU-PAGE) followed by Western immunoblotting is an important tool for the identification and quantification of cationic AMPs. The protocol for these procedures outlined here describes, in detail, the necessary steps; including pouring the AU-gels, preparing the test samples, performing the electrophoretic separation and

  10. Protein-Protein Interaction for the De Novo Design of Cyclin-Dependent Kinase Peptide Inhibitors.

    PubMed

    Arumugasamy, Karthiga; Tripathi, Sunil Kumar; Singh, Poonam; Singh, Sanjeev Kumar

    2016-01-01

    The homology of the inhibitor binding site regions on the surface of cyclin-dependent kinases (CDKs) makes actual CDK inhibitors unable to bind specifically to their molecular targets. Most of them are ATP competitive inhibitors with low specificity that also affect the phosphorylation mechanisms of other nontarget kinases giving rise to harmful side effects. So, the search of specific and potent inhibitors able to bind to the desired CDK target is still a pending issue. Structure based drug design minimized the erroneous binding and increased the affinity of the inhibitor interaction. In the case of CDKs their activation and regulation mechanisms mainly depend on protein-protein interactions (PPIs). The design of drugs targeting these PPIs makes feasible and promising towards the discovery of new and specific CDK inhibitors. Development of peptide inhibitors for a target protein is an emerging approach in computer aided drug designing. This chapter describes in detail methodology for use of the VitAL-Viterbi algorithm for de novo peptide design of CDK2 inhibitors. PMID:26231708

  11. Marine Antimicrobial Peptides: Nature Provides Templates for the Design of Novel Compounds against Pathogenic Bacteria

    PubMed Central

    Falanga, Annarita; Lombardi, Lucia; Franci, Gianluigi; Vitiello, Mariateresa; Iovene, Maria Rosaria; Morelli, Giancarlo; Galdiero, Massimiliano; Galdiero, Stefania

    2016-01-01

    The discovery of antibiotics for the treatment of bacterial infections brought the idea that bacteria would no longer endanger human health. However, bacterial diseases still represent a worldwide treat. The ability of microorganisms to develop resistance, together with the indiscriminate use of antibiotics, is mainly responsible for this situation; thus, resistance has compelled the scientific community to search for novel therapeutics. In this scenario, antimicrobial peptides (AMPs) provide a promising strategy against a wide array of pathogenic microorganisms, being able to act directly as antimicrobial agents but also being important regulators of the innate immune system. This review is an attempt to explore marine AMPs as a rich source of molecules with antimicrobial activity. In fact, the sea is poorly explored in terms of AMPs, but it represents a resource with plentiful antibacterial agents performing their role in a harsh environment. For the application of AMPs in the medical field limitations correlated to their peptide nature, their inactivation by environmental pH, presence of salts, proteases, or other components have to be solved. Thus, these peptides may act as templates for the design of more potent and less toxic compounds. PMID:27213366

  12. Marine Antimicrobial Peptides: Nature Provides Templates for the Design of Novel Compounds against Pathogenic Bacteria.

    PubMed

    Falanga, Annarita; Lombardi, Lucia; Franci, Gianluigi; Vitiello, Mariateresa; Iovene, Maria Rosaria; Morelli, Giancarlo; Galdiero, Massimiliano; Galdiero, Stefania

    2016-01-01

    The discovery of antibiotics for the treatment of bacterial infections brought the idea that bacteria would no longer endanger human health. However, bacterial diseases still represent a worldwide treat. The ability of microorganisms to develop resistance, together with the indiscriminate use of antibiotics, is mainly responsible for this situation; thus, resistance has compelled the scientific community to search for novel therapeutics. In this scenario, antimicrobial peptides (AMPs) provide a promising strategy against a wide array of pathogenic microorganisms, being able to act directly as antimicrobial agents but also being important regulators of the innate immune system. This review is an attempt to explore marine AMPs as a rich source of molecules with antimicrobial activity. In fact, the sea is poorly explored in terms of AMPs, but it represents a resource with plentiful antibacterial agents performing their role in a harsh environment. For the application of AMPs in the medical field limitations correlated to their peptide nature, their inactivation by environmental pH, presence of salts, proteases, or other components have to be solved. Thus, these peptides may act as templates for the design of more potent and less toxic compounds. PMID:27213366

  13. Targeted disruption of the CCR5 gene in human hematopoietic stem cells stimulated by peptide nucleic acids

    PubMed Central

    Schleifman, Erica B.; Bindra, Ranjit; Leif, Jean; Campo, Jacob del; Rogers, Faye A.; Uchil, Pradeep; Kutsch, Olaf; Shultz, Leonard D.; Kumar, Priti; Greiner, Dale L.; Glazer, Peter M.

    2011-01-01

    SUMMARY Peptide nucleic acids (PNAs) bind duplex DNA in a sequence-specific manner, creating triplex structures that can provoke DNA repair and produce genome modification. CCR5 encodes a chemokine receptor required for HIV-1 entry into human cells and individuals carrying mutations in this gene are resistant to HIV-1 infection. Transfection of human cells with PNAs targeted to the CCR5 gene, plus donor DNAs designed to introduce stop codons mimicking the naturally occurring CCR5-delta32 mutation, produced 2.46% targeted gene modification. CCR5 modification was confirmed at the DNA, RNA and protein levels and was shown to confer resistance to infection with HIV-1. Targeting of CCR5 was achieved in human CD34+ hematopoietic stem cells (HSCs) with subsequent engraftment into mice and persistence of the gene modification more than four months post-transplantation. This work suggests a therapeutic strategy for CCR5 knockout in HSCs from HIV-1-infected individuals, rendering cells resistant to HIV-1 and preserving immune system function. PMID:21944757

  14. Site-directed gene mutation at mixed sequence targets by psoralen-conjugated pseudo-complementary peptide nucleic acids.

    PubMed

    Kim, Ki-Hyun; Nielsen, Peter E; Glazer, Peter M

    2007-01-01

    Sequence-specific DNA-binding molecules such as triple helix-forming oligonucleotides (TFOs) provide a means for inducing site-specific mutagenesis and recombination at chromosomal sites in mammalian cells. However, the utility of TFOs is limited by the requirement for homopurine stretches in the target duplex DNA. Here, we report the use of pseudo-complementary peptide nucleic acids (pcPNAs) for intracellular gene targeting at mixed sequence sites. Due to steric hindrance, pcPNAs are unable to form pcPNA-pcPNA duplexes but can bind to complementary DNA sequences by Watson-Crick pairing via double duplex-invasion complex formation. We show that psoralen-conjugated pcPNAs can deliver site-specific photoadducts and mediate targeted gene modification within both episomal and chromosomal DNA in mammalian cells without detectable off-target effects. Most of the induced psoralen-pcPNA mutations were single-base substitutions and deletions at the predicted pcPNA-binding sites. The pcPNA-directed mutagenesis was found to be dependent on PNA concentration and UVA dose and required matched pairs of pcPNAs. Neither of the individual pcPNAs alone had any effect nor did complementary PNA pairs of the same sequence. These results identify pcPNAs as new tools for site-specific gene modification in mammalian cells without purine sequence restriction, thereby providing a general strategy for designing gene targeting molecules. PMID:17977869

  15. Investigation on natural diets of larval marine animals using peptide nucleic acid-directed polymerase chain reaction clamping.

    PubMed

    Chow, Seinen; Suzuki, Sayaka; Matsunaga, Tadashi; Lavery, Shane; Jeffs, Andrew; Takeyama, Haruko

    2011-04-01

    The stomach contents of the larvae of marine animals are usually very small in quantity and amorphous, especially in invertebrates, making morphological methods of identification very difficult. Nucleotide sequence analysis using polymerase chain reaction (PCR) is a likely approach, but the large quantity of larval (host) DNA present may mask subtle signals from the prey genome. We have adopted peptide nucleic acid (PNA)-directed PCR clamping to selectively inhibit amplification of host DNA for this purpose. The Japanese spiny lobster (Panulirus japonicus) and eel (Anguilla japonica) were used as model host and prey organisms, respectively. A lobster-specific PNA oligomer (20 bases) was designed to anneal to the sequence at the junction of the 18 S rDNA gene and the internal transcribed spacer 1 (ITS1) of the lobster. PCR using eukaryote universal primers for amplifying the ITS1 region used in conjunction with the lobster-specific PNA on a mixed DNA template of lobster and eel demonstrated successful inhibition of lobster ITS1 amplification while allowing efficient amplification of eel ITS1. This method was then applied to wild-caught lobster larvae of P. japonicus and P. longipes bispinosus collected around Ryukyu Archipelago, Japan. ITS1 sequences of a wide variety of animals (Ctenophora, Cnidaria, Crustacea, Teleostei, Mollusca, and Chaetognatha) were detected. PMID:20535520

  16. An equation to estimate the difference between theoretically predicted and SDS PAGE-displayed molecular weights for an acidic peptide.

    PubMed

    Guan, Yihong; Zhu, Qinfang; Huang, Delai; Zhao, Shuyi; Jan Lo, Li; Peng, Jinrong

    2015-01-01

    The molecular weight (MW) of a protein can be predicted based on its amino acids (AA) composition. However, in many cases a non-chemically modified protein shows an SDS PAGE-displayed MW larger than its predicted size. Some reports linked this fact to high content of acidic AA in the protein. However, the exact relationship between the acidic AA composition and the SDS PAGE-displayed MW is not established. Zebrafish nucleolar protein Def is composed of 753 AA and shows an SDS PAGE-displayed MW approximately 13 kDa larger than its predicted MW. The first 188 AA in Def is defined by a glutamate-rich region containing ~35.6% of acidic AA. In this report, we analyzed the relationship between the SDS PAGE-displayed MW of thirteen peptides derived from Def and the AA composition in each peptide. We found that the difference between the predicted and SDS PAGE-displayed MW showed a linear correlation with the percentage of acidic AA that fits the equation y = 276.5x - 31.33 (x represents the percentage of acidic AA, 11.4% ≤ x ≤ 51.1%; y represents the average ΔMW per AA). We demonstrated that this equation could be applied to predict the SDS PAGE-displayed MW for thirteen different natural acidic proteins. PMID:26311515

  17. Highly sensitive detection of influenza virus by boron-doped diamond electrode terminated with sialic acid-mimic peptide.

    PubMed

    Matsubara, Teruhiko; Ujie, Michiko; Yamamoto, Takashi; Akahori, Miku; Einaga, Yasuaki; Sato, Toshinori

    2016-08-01

    The progression of influenza varies according to age and the presence of an underlying disease; appropriate treatment is therefore required to prevent severe disease. Anti-influenza therapy, such as with neuraminidase inhibitors, is effective, but diagnosis at an early phase of infection before viral propagation is critical. Here, we show that several dozen plaque-forming units (pfu) of influenza virus (IFV) can be detected using a boron-doped diamond (BDD) electrode terminated with a sialic acid-mimic peptide. The peptide was used instead of the sialyloligosaccharide receptor, which is the common receptor of influenza A and B viruses required during the early phase of infection, to capture IFV particles. The peptide, which was previously identified by phage-display technology, was immobilized by click chemistry on the BDD electrode, which has excellent electrochemical characteristics such as low background current and weak adsorption of biomolecules. Electrochemical impedance spectroscopy revealed that H1N1 and H3N2 IFVs were detectable in the range of 20-500 pfu by using the peptide-terminated BDD electrode. Our results demonstrate that the BDD device integrated with the receptor-mimic peptide has high sensitivity for detection of a low number of virus particles in the early phase of infection. PMID:27457924

  18. Primary structure of a histidine-rich proteolytic fragment of human ceruloplasmin. II. Amino acid sequence of the tryptic peptides.

    PubMed

    Kingston, I B; Kingston, B L; Putnam, F W

    1980-04-10

    Amino acid sequence studies of tryptic peptides isolated from a histidine-rich fragment (Cp F5) of human ceruloplasmin are described. Nineteen tryptic peptides were isolated from unmodified Cp F5 and five tryptic peptides were isolated from citraconylated Cp F5. These peptides, together with the cyanogen bromide fragments reported previously, allowed the assembly of the complete sequence of Cp F5. The fragment has 159 residues and a molecular weight of 18,650; it lacks carbohydrate, is rich in histidine, and contains 1 free cysteine that may be part of a copper-binding site. Human ceruloplasmin is a single polypeptide chain with a molecular weight of about 130,000 that is readily cleaved to large fragments by proteolytic enzymes; the relationships of Cp F5 to intact ceruloplasmin and to structural subunits earlier proposed is described. Cp F5 probably is an intact globular domain that is attached to the COOH-terminal end of ceruloplasmin by a labile interdomain peptide bond. PMID:6987230

  19. Chemical vectors for gene delivery: a current review on polymers, peptides and lipids containing histidine or imidazole as nucleic acids carriers

    PubMed Central

    Midoux, Patrick; Pichon, Chantal; Yaouanc, Jean-Jacques; Jaffrès, Paul-Alain

    2009-01-01

    DNA/cationic lipid (lipoplexes), DNA/cationic polymer (polyplexes) and DNA/cationic polymer/cationic lipid (lipopolyplexes) electrostatic complexes are proposed as non-viral nucleic acids delivery systems. These DNA-nanoparticles are taken up by the cells through endocytosis processes, but the low capacity of DNA to escape from endosomes is regarded as the major limitations of their transfection efficiency. Here, we present a current report on a particular class of carriers including the polymers, peptides and lipids, which is based on the exploitation of the imidazole ring as an endosome destabilization device to favour the nucleic acids delivery in the cytosol. The imidazole ring of histidine is a weak base that has the ability to acquire a cationic charge when the pH of the environment drops bellow 6. As it has been demonstrated for poly(histidine), this phenomena can induce membrane fusion and/or membrane permeation in an acidic medium. Moreover, the accumulation of histidine residues inside acidic vesicles can induce a proton sponge effect, which increases their osmolarity and their swelling. The proof of concept has been shown with polylysine partially substituted with histidine residues that has caused a dramatic increase by 3–4.5 orders of magnitude of the transfection efficiency of DNA/polylysine polyplexes. Then, several histidine-rich polymers and peptides as well as lipids with imidazole, imidazolinium or imidazolium polar head have been reported to be efficient carriers to deliver nucleic acids including genes, mRNA or SiRNA in vitro and in vivo. More remarkable, histidylated carriers are often weakly cytotoxic, making them promising chemical vectors for nucleic acids delivery. This article is part of a themed section on Vector Design and Drug Delivery. For a list of all articles in this section see the end of this paper, or visit: http://www3.interscience.wiley.com/journal/121548564/issueyear?year=2009 PMID:19459843

  20. MALDI TOF/TOF-Based Approach for the Identification of d- Amino Acids in Biologically Active Peptides and Proteins.

    PubMed

    Koehbach, Johannes; Gruber, Christian W; Becker, Christian; Kreil, David P; Jilek, Alexander

    2016-05-01

    Several biologically active peptides contain a d- amino acid in a well-defined position, which is position 2 in all peptide epimers isolated to date from vertebrates and also some from invertebrates. The detection of such D- residues by standard analytical techniques is challenging. In tandem mass spectrometric (MS) analysis, although fragment masses are the same for all stereoisomers, peak intensities are known to depend on chirality. Here, we observe that the effect of a d- amino acid in the second N-terminal position on the fragmentation pattern in matrix assisted laser desorption time-of-flight spectrometry (MALDI-TOF/TOF MS) strongly depends on the peptide sequence. Stereosensitive fragmentation (SF) is correlated to a neighborhood effect, but the d- residue also exerts an overall effect influencing distant bonds. In a fingerprint analysis, multiple peaks can thus serve to identify the chirality of a sample in short time and potentially high throughput. Problematic variations between individual spots could be successfully suppressed by cospotting deuterated analogues of the epimers. By identifying the [d-Leu2] isomer of the predicted peptide GH-2 (gene derived bombininH) in skin secretions of the toad Bombina orientalis, we demonstrated the analytical power of SF-MALDI-TOF/TOF measurements. In conclusion, SF-MALDI-TOF/TOF MS combines high sensitivity, versatility, and the ability to complement other methods. PMID:26985971

  1. Killing of Mycobacterium avium by Lactoferricin Peptides: Improved Activity of Arginine- and d-Amino-Acid-Containing Molecules

    PubMed Central

    Silva, Tânia; Magalhães, Bárbara; Maia, Sílvia; Gomes, Paula; Nazmi, Kamran; Bolscher, Jan G. M.; Rodrigues, Pedro N.; Bastos, Margarida

    2014-01-01

    Mycobacterium avium causes respiratory disease in susceptible individuals, as well as disseminated infections in immunocompromised hosts, being an important cause of morbidity and mortality among these populations. Current therapies consist of a combination of antibiotics taken for at least 6 months, with no more than 60% overall clinical success. Furthermore, mycobacterial antibiotic resistance is increasing worldwide, urging the need to develop novel classes of antimicrobial drugs. One potential and interesting alternative strategy is the use of antimicrobial peptides (AMP). These are present in almost all living organisms as part of their immune system, acting as a first barrier against invading pathogens. In this context, we investigated the effect of several lactoferrin-derived AMP against M. avium. Short peptide sequences from both human and bovine lactoferricins, namely, hLFcin1-11 and LFcin17-30, as well as variants obtained by specific amino acid substitutions, were evaluated. All tested peptides significantly inhibited the axenic growth of M. avium, the bovine peptides being more active than the human. Arginine residues were found to be crucial for the display of antimycobacterial activity, whereas the all-d-amino-acid analogue of the bovine sequence displayed the highest mycobactericidal activity. These findings reveal the promising potential of lactoferricins against mycobacteria, thus opening the way for further research on their development and use as a new weapon against mycobacterial infections. PMID:24709266

  2. MALDI TOF/TOF-Based Approach for the Identification of d- Amino Acids in Biologically Active Peptides and Proteins

    PubMed Central

    2016-01-01

    Several biologically active peptides contain a d- amino acid in a well-defined position, which is position 2 in all peptide epimers isolated to date from vertebrates and also some from invertebrates. The detection of such D- residues by standard analytical techniques is challenging. In tandem mass spectrometric (MS) analysis, although fragment masses are the same for all stereoisomers, peak intensities are known to depend on chirality. Here, we observe that the effect of a d- amino acid in the second N-terminal position on the fragmentation pattern in matrix assisted laser desorption time-of-flight spectrometry (MALDI-TOF/TOF MS) strongly depends on the peptide sequence. Stereosensitive fragmentation (SF) is correlated to a neighborhood effect, but the d- residue also exerts an overall effect influencing distant bonds. In a fingerprint analysis, multiple peaks can thus serve to identify the chirality of a sample in short time and potentially high throughput. Problematic variations between individual spots could be successfully suppressed by cospotting deuterated analogues of the epimers. By identifying the [d-Leu2] isomer of the predicted peptide GH-2 (gene derived bombininH) in skin secretions of the toad Bombina orientalis, we demonstrated the analytical power of SF-MALDI-TOF/TOF measurements. In conclusion, SF-MALDI-TOF/TOF MS combines high sensitivity, versatility, and the ability to complement other methods. PMID:26985971

  3. Analysis of peptide-protein binding using amino acid descriptors: prediction and experimental verification for human histocompatibility complex HLA-A0201.

    PubMed

    Guan, Pingping; Doytchinova, Irini A; Walshe, Valerie A; Borrow, Persephone; Flower, Darren R

    2005-11-17

    Amino acid descriptors are often used in quantitative structure-activity relationship (QSAR) analysis of proteins and peptides. In the present study, descriptors were used to characterize peptides binding to the human MHC allele HLA-A0201. Two sets of amino acid descriptors were chosen: 93 descriptors taken from the amino acid descriptor database AAindex and the z descriptors defined by Wold and Sandberg. Variable selection techniques (SIMCA, genetic algorithm, and GOLPE) were applied to remove redundant descriptors. Our results indicate that QSAR models generated using five z descriptors had the highest predictivity and explained variance (q2 between 0.6 and 0.7 and r2 between 0.6 and 0.9). Further to the QSAR analysis, 15 peptides were synthesized and tested using a T2 stabilization assay. All peptides bound to HLA-A0201 well, and four peptides were identified as high-affinity binders. PMID:16279801

  4. Rationally Designed Interfacial Peptides Are Efficient In Vitro Inhibitors of HIV-1 Capsid Assembly with Antiviral Activity

    PubMed Central

    Bocanegra, Rebeca; Nevot, María; Doménech, Rosa; López, Inmaculada; Abián, Olga; Rodríguez-Huete, Alicia; Cavasotto, Claudio N.; Velázquez-Campoy, Adrián; Gómez, Javier; Martínez, Miguel Ángel; Neira, José Luis; Mateu, Mauricio G.

    2011-01-01

    Virus capsid assembly constitutes an attractive target for the development of antiviral therapies; a few experimental inhibitors of this process for HIV-1 and other viruses have been identified by screening compounds or by selection from chemical libraries. As a different, novel approach we have undertaken the rational design of peptides that could act as competitive assembly inhibitors by mimicking capsid structural elements involved in intersubunit interfaces. Several discrete interfaces involved in formation of the mature HIV-1 capsid through polymerization of the capsid protein CA were targeted. We had previously designed a peptide, CAC1, that represents CA helix 9 (a major part of the dimerization interface) and binds the CA C-terminal domain in solution. Here we have mapped the binding site of CAC1, and shown that it substantially overlaps with the CA dimerization interface. We have also rationally modified CAC1 to increase its solubility and CA-binding affinity, and designed four additional peptides that represent CA helical segments involved in other CA interfaces. We found that peptides CAC1, its derivative CAC1M, and H8 (representing CA helix 8) were able to efficiently inhibit the in vitro assembly of the mature HIV-1 capsid. Cocktails of several peptides, including CAC1 or CAC1M plus H8 or CAI (a previously discovered inhibitor of CA polymerization), or CAC1M+H8+CAI, also abolished capsid assembly, even when every peptide was used at lower, sub-inhibitory doses. To provide a preliminary proof that these designed capsid assembly inhibitors could eventually serve as lead compounds for development of anti-HIV-1 agents, they were transported into cultured cells using a cell-penetrating peptide, and tested for antiviral activity. Peptide cocktails that drastically inhibited capsid assembly in vitro were also able to efficiently inhibit HIV-1 infection ex vivo. This study validates a novel, entirely rational approach for the design of capsid assembly

  5. Computer-Aided Virtual Screening and Designing of Cell-Penetrating Peptides.

    PubMed

    Gautam, Ankur; Chaudhary, Kumardeep; Kumar, Rahul; Raghava, Gajendra Pal Singh

    2015-01-01

    Cell-penetrating peptides (CPPs) have proven their potential as versatile drug delivery vehicles. Last decade has witnessed an unprecedented growth in CPP-based research, demonstrating the potential of CPPs as therapeutic candidates. In the past, many in silico algorithms have been developed for the prediction and screening of CPPs, which expedites the CPP-based research. In silico screening/prediction of CPPs followed by experimental validation seems to be a reliable, less time-consuming, and cost-effective approach. This chapter describes the prediction, screening, and designing of novel efficient CPPs using "CellPPD," an in silico tool. PMID:26202262

  6. Design of an α-helical antimicrobial peptide with improved cell-selective and potent anti-biofilm activity.

    PubMed

    Zhang, Shi-Kun; Song, Jin-Wen; Gong, Feng; Li, Su-Bo; Chang, Hong-Yu; Xie, Hui-Min; Gao, Hong-Wei; Tan, Ying-Xia; Ji, Shou-Ping

    2016-01-01

    AR-23 is a melittin-related peptide with 23 residues. Like melittin, its high α-helical amphipathic structure results in strong bactericidal activity and cytotoxicity. In this study, a series of AR-23 analogues with low amphipathicity were designed by substitution of Ala1, Ala8 and Ile17 with positively charged residues (Arg or Lys) to study the effect of positively charged residue distribution on the biological viability of the antimicrobial peptide. Substitution of Ile17 on the nonpolar face with positively charged Lys dramatically altered the hydrophobicity, amphipathicity, helicity and the membrane-penetrating activity against human cells as well as the haemolytic activity of the peptide. However, substitution on the polar face only slightly affected the peptide biophysical properties and biological activity. The results indicate that the position rather than the number of positively charged residue affects the biophysical properties and selectivity of the peptide. Of all the analogues, A(A1R, A8R, I17K), a peptide with Ala1-Arg, Ala8-Arg and Ile17-Lys substitutions, exhibited similar bactericidal activity and anti-biofilm activity to AR-23 but had much lower haemolytic activity and cytotoxicity against mammalian cells compared with AR-23. Therefore, the findings reported here provide a rationalization for peptide design and optimization, which will be useful for the future development of antimicrobial agents. PMID:27271216

  7. Design and analysis of structure-activity relationship of novel antimicrobial peptides derived from the conserved sequence of cecropin.

    PubMed

    Hao, Gang; Shi, Yong-Hui; Han, Jing-Hui; Li, Qi-Hui; Tang, Ya-Li; Le, Guo-Wei

    2008-03-01

    We have de novo designed four antimicrobial peptides AMP-A/B/C/D, the 51-residues peptides, which are based on the conserved sequence of cecropin. In the present study, the four peptides were chemically synthesized and their activities assayed. Their secondary structure, amphipathic property, electric field distribution and transmembrane domain were subsequently predicted by bioinformatics tools. Finally, the structure-activity relationship was analyzed from the results of activity experiments and prediction. The results of activity experiments indicated that AMP-B/C/D clearly possessed excellent broad-spectrum activity against bacteria, whereas AMP-A was almost inactive against most of the bacterial strains tested. AMP-B/C/D showed more potent activity against Gram-positive bacteria than against Gram-negative bacteria. By utilizing bioinformatics analysis tools, we found that the secondary structure of the four cation peptides was mainly alpha-helix, and the result of CD spectrum also displayed that all the peptides had considerable alpha-helix in the presence of either 50% TFE or SDS micelles. AMP-C showed much better activity than other peptides against most of the bacteria tested, owing to its remarkable cation property and the amphipathic character of its N-terminal. The study of structure-activity relationship of the designed peptides confirmed that amphipathic structure and high net positive charge were prerequisites for maintaining their activities. PMID:17929330

  8. Design of an α-helical antimicrobial peptide with improved cell-selective and potent anti-biofilm activity

    PubMed Central

    Zhang, Shi-Kun; Song, Jin-wen; Gong, Feng; Li, Su-Bo; Chang, Hong-Yu; Xie, Hui-Min; Gao, Hong-Wei; Tan, Ying-Xia; Ji, Shou-Ping

    2016-01-01

    AR-23 is a melittin-related peptide with 23 residues. Like melittin, its high α-helical amphipathic structure results in strong bactericidal activity and cytotoxicity. In this study, a series of AR-23 analogues with low amphipathicity were designed by substitution of Ala1, Ala8 and Ile17 with positively charged residues (Arg or Lys) to study the effect of positively charged residue distribution on the biological viability of the antimicrobial peptide. Substitution of Ile17 on the nonpolar face with positively charged Lys dramatically altered the hydrophobicity, amphipathicity, helicity and the membrane-penetrating activity against human cells as well as the haemolytic activity of the peptide. However, substitution on the polar face only slightly affected the peptide biophysical properties and biological activity. The results indicate that the position rather than the number of positively charged residue affects the biophysical properties and selectivity of the peptide. Of all the analogues, A(A1R, A8R, I17K), a peptide with Ala1-Arg, Ala8-Arg and Ile17-Lys substitutions, exhibited similar bactericidal activity and anti-biofilm activity to AR-23 but had much lower haemolytic activity and cytotoxicity against mammalian cells compared with AR-23. Therefore, the findings reported here provide a rationalization for peptide design and optimization, which will be useful for the future development of antimicrobial agents. PMID:27271216

  9. Prediction of calcite morphology from computational and experimental studies of mutations of a de novo-designed peptide.

    PubMed

    Schrier, Sarah B; Sayeg, Marianna K; Gray, Jeffrey J

    2011-09-20

    Many organisms use macromolecules, often proteins or peptides, to control the growth of inorganic crystals into complex materials. The ability to model peptide-mineral interactions accurately could allow for the design of novel peptides to produce materials with desired properties. Here, we tested a computational algorithm developed to predict the structure of peptides on mineral surfaces. Using this algorithm, we analyzed energetic and structural differences between a 16-residue peptide (bap4) designed to interact with a calcite growth plane and single- and double-point mutations of the charged residues. Currently, no experimental method is available to resolve the structures of proteins on solid surfaces, which precludes benchmarking for computational models. Therefore, to test the models, we chemically synthesized each peptide and analyzed its effects on calcite crystal growth. Whereas bap4 affected the crystal growth by producing heavily stepped corners and edges, point mutants had variable influences on morphology. Calculated residue-specific binding energies correlated with experimental observations; point mutations of residues predicted to be crucial to surface interactions produced morphologies most similar to unmodified calcite. These results suggest that peptide conformation plays a role in mineral interactions and that the computational model supplies valid energetic and structural data that can provide information about expected crystal morphology. PMID:21797243

  10. Sequence dependent N-terminal rearrangement and degradation of peptide nucleic acid (PNA) in aqueous solution

    NASA Technical Reports Server (NTRS)

    Eriksson, M.; Christensen, L.; Schmidt, J.; Haaima, G.; Orgel, L.; Nielsen, P. E.

    1998-01-01

    The stability of the PNA (peptide nucleic acid) thymine monomer inverted question markN-[2-(thymin-1-ylacetyl)]-N-(2-aminoaminoethyl)glycine inverted question mark and those of various PNA oligomers (5-8-mers) have been measured at room temperature (20 degrees C) as a function of pH. The thymine monomer undergoes N-acyl transfer rearrangement with a half-life of 34 days at pH 11 as analyzed by 1H NMR; and two reactions, the N-acyl transfer and a sequential degradation, are found by HPLC analysis to occur at measurable rates for the oligomers at pH 9 or above. Dependent on the amino-terminal sequence, half-lives of 350 h to 163 days were found at pH 9. At pH 12 the half-lives ranged from 1.5 h to 21 days. The results are discussed in terms of PNA as a gene therapeutic drug as well as a possible prebiotic genetic material.

  11. Peptide Nucleic Acid Promotes Systemic Dystrophin Expression and Functional Rescue in Dystrophin-deficient mdx Mice

    PubMed Central

    Gao, Xianjun; Shen, Xiaoyong; Dong, Xue; Ran, Ning; Han, Gang; Cao, Limin; Gu, Ben; Yin, HaiFang

    2015-01-01

    Antisense oligonucleotide (AO)-mediated exon-skipping therapeutics shows great promise for Duchenne muscular dystrophy (DMD) patients. However, recent failure with drisapersen, an AO candidate drug in phase 3 trial, highlights the importance of exploring other effective AO chemistries for DMD. Previously, we demonstrated the appreciable biological activity of peptide nucleic acid (PNA) AOs in restoring dystrophin expression in dystrophin-deficient mdx mice intramuscularly. Here, we further explore the systemic potential and feasibility of PNA AOs in mediating exon skipping in mdx mice as a comprehensive systemic evaluation remains lacking. Systemic delivery of PNA AOs resulted in therapeutic level of dystrophin expression in body-wide peripheral muscles and improved dystrophic pathology in mdx mice without any detectable toxicity. Up to 40% of dystrophin restoration was achieved in gastrocnemius, to a less extent with other skeletal muscles, with no dystrophin in heart. Notably, comparable systemic activity was obtained between PNA AOs and phosphorodiamidate morpholino oligomer, a DMD AO chemistry in phase 3 clinical trial, under an identical dosing regimen. Overall, our data demonstrate that PNA is viable for DMD exon-skipping therapeutics with 20 mer showing the best combination of activity, solubility, and safety and further modifications to increase PNA aqueous solubility can enable longer, more effective therapeutics without the associated toxicity. PMID:26440599

  12. Targeted gene correction using psoralen, chlorambucil and camptothecin conjugates of triplex forming peptide nucleic acid (PNA)

    PubMed Central

    Birkedal, Henrik

    2011-01-01

    Gene correction activation effects of a small series of triplex forming peptide nucleic acid (PNA) covalently conjugated to the DNA interacting ligands psoralen, chlorambucil and camptothecin targeted proximal to a stop codon mutation in an EGFP reporter gene were studied. A 15-mer homopyrimidine PNA conjugated to the topoisomerase I inhibitor camptothecin was found to increase the frequency of repair domain mediated gene correctional events of the EGFP reporter in an in vitro HeLa cell nuclear extract assay, whereas PNA psoralen or chlorambucil conjugates both of which form covalent and also interstrand crosslinked adducts with dsDNA dramatically decreased the frequency of targeted repair/correction. The PNA conjugates were also studied in mammalian cell lines upon transfection of PNA bound EGFP reporter vector and scoring repair of the EGFP gene by FACS analysis of functional EGFP expression. Consistent with the extract experiments, treatment with adduct forming PNA conjugates (psoralen and chlorambucil) resulted in a decrease in background correction frequencies in transiently transfected cells, whereas unmodified PNA or the PNA-camptothecin conjugate had little or no effect. These results suggest that simple triplex forming PNAs have little effect on proximal gene correctional events whereas PNA conjugates capable of forming DNA adducts and interstrand crosslinks are strong inhibitors. Most interestingly the PNA conjugated to the topoisomerase inhibitor, camptothecin enhanced repair in nuclear extract. Thus the effects and use of camptothecin conjugates in gene targeted repair merit further studies. PMID:21686249

  13. Isodesmic reaction for accurate theoretical pKa calculations of amino acids and peptides.

    PubMed

    Sastre, S; Casasnovas, R; Muñoz, F; Frau, J

    2016-04-20

    Theoretical and quantitative prediction of pKa values at low computational cost is a current challenge in computational chemistry. We report that the isodesmic reaction scheme provides semi-quantitative predictions (i.e. mean absolute errors of 0.5-1.0 pKa unit) for the pKa1 (α-carboxyl), pKa2 (α-amino) and pKa3 (sidechain groups) of a broad set of amino acids and peptides. This method fills the gaps of thermodynamic cycles for the computational pKa calculation of molecules that are unstable in the gas phase or undergo proton transfer reactions or large conformational changes from solution to the gas phase. We also report the key criteria to choose a reference species to make accurate predictions. This method is computationally inexpensive and makes use of standard density functional theory (DFT) and continuum solvent models. It is also conceptually simple and easy to use for researchers not specialized in theoretical chemistry methods. PMID:27052591

  14. Diagnosis of bacterial vaginosis by a new multiplex peptide nucleic acid fluorescence in situ hybridization method

    PubMed Central

    Machado, António; Castro, Joana; Cereija, Tatiana; Almeida, Carina

    2015-01-01

    Bacterial vaginosis (BV) is one of most common vaginal infections. However, its diagnosis by classical methods reveals low specificity. Our goal was to evaluate the accuracy diagnosis of 150 vaginal samples with research gold standard methods and our Peptide Nucleic Acid (PNA) probes by Fluorescence in situ Hybridization (FISH) methodology. Also, we described the first PNA-FISH methodology for BV diagnosis, which provides results in approximately 3 h. The results showed a sensitivity of 84.6% (95% confidence interval (CI), from 64.3 to 95.0%) and a specificity of 97.6% (95% CI [92.6–99.4%]), demonstrating the higher specificity of the PNA-FISH method and showing false positive results in BV diagnosis commonly obtained by the classical methods. This methodology combines the specificity of PNA probes for Lactobacillus species and G. vaginalis visualization and the calculation of the microscopic field by Nugent score, allowing a trustful evaluation of the bacteria present in vaginal microflora and avoiding the occurrence of misleading diagnostics. Therefore, the PNA-FISH methodology represents a valuable alternative for BV diagnosis. PMID:25737820

  15. Caffeic acid phenethyl ester (CAPE), an active component of propolis, inhibits Helicobacter pylori peptide deformylase activity.

    PubMed

    Cui, Kunqiang; Lu, Weiqiang; Zhu, Lili; Shen, Xu; Huang, Jin

    2013-05-31

    Helicobacter pylori (H. pylori) is a major causative factor for gastrointestinal illnesses, H. pylori peptide deformylase (HpPDF) catalyzes the removal of formyl group from the N-terminus of nascent polypeptide chains, which is essential for H. pylori survival and is considered as a promising drug target for anti-H. pylori therapy. Propolis, a natural antibiotic from honeybees, is reported to have an inhibitory effect on the growth of H. pylori in vitro. In addition, previous studies suggest that the main active constituents in the propolis are phenolic compounds. Therefore, we evaluated a collection of phenolic compounds derived from propolis for enzyme inhibition against HpPDF. Our study results show that Caffeic acid phenethyl ester (CAPE), one of the main medicinal components of propolis, is a competitive inhibitor against HpPDF, with an IC50 value of 4.02 μM. Furthermore, absorption spectra and crystal structural characterization revealed that different from most well known PDF inhibitors, CAPE block the substrate entrance, preventing substrate from approaching the active site, but CAPE does not have chelate interaction with HpPDF and does not disrupt the metal-dependent catalysis. Our study provides valuable information for understanding the potential anti-H. pylori mechanism of propolis, and CAPE could be served as a lead compound for further anti-H. pylori drug discovery. PMID:23611786

  16. Peptide Nucleic Acid Promotes Systemic Dystrophin Expression and Functional Rescue in Dystrophin-deficient mdx Mice.

    PubMed

    Gao, Xianjun; Shen, Xiaoyong; Dong, Xue; Ran, Ning; Han, Gang; Cao, Limin; Gu, Ben; Yin, HaiFang

    2015-01-01

    Antisense oligonucleotide (AO)-mediated exon-skipping therapeutics shows great promise for Duchenne muscular dystrophy (DMD) patients. However, recent failure with drisapersen, an AO candidate drug in phase 3 trial, highlights the importance of exploring other effective AO chemistries for DMD. Previously, we demonstrated the appreciable biological activity of peptide nucleic acid (PNA) AOs in restoring dystrophin expression in dystrophin-deficient mdx mice intramuscularly. Here, we further explore the systemic potential and feasibility of PNA AOs in mediating exon skipping in mdx mice as a comprehensive systemic evaluation remains lacking. Systemic delivery of PNA AOs resulted in therapeutic level of dystrophin expression in body-wide peripheral muscles and improved dystrophic pathology in mdx mice without any detectable toxicity. Up to 40% of dystrophin restoration was achieved in gastrocnemius, to a less extent with other skeletal muscles, with no dystrophin in heart. Notably, comparable systemic activity was obtained between PNA AOs and phosphorodiamidate morpholino oligomer, a DMD AO chemistry in phase 3 clinical trial, under an identical dosing regimen. Overall, our data demonstrate that PNA is viable for DMD exon-skipping therapeutics with 20 mer showing the best combination of activity, solubility, and safety and further modifications to increase PNA aqueous solubility can enable longer, more effective therapeutics without the associated toxicity. PMID:26440599

  17. Diagnosis of bacterial vaginosis by a new multiplex peptide nucleic acid fluorescence in situ hybridization method.

    PubMed

    Machado, António; Castro, Joana; Cereija, Tatiana; Almeida, Carina; Cerca, Nuno

    2015-01-01

    Bacterial vaginosis (BV) is one of most common vaginal infections. However, its diagnosis by classical methods reveals low specificity. Our goal was to evaluate the accuracy diagnosis of 150 vaginal samples with research gold standard methods and our Peptide Nucleic Acid (PNA) probes by Fluorescence in situ Hybridization (FISH) methodology. Also, we described the first PNA-FISH methodology for BV diagnosis, which provides results in approximately 3 h. The results showed a sensitivity of 84.6% (95% confidence interval (CI), from 64.3 to 95.0%) and a specificity of 97.6% (95% CI [92.6-99.4%]), demonstrating the higher specificity of the PNA-FISH method and showing false positive results in BV diagnosis commonly obtained by the classical methods. This methodology combines the specificity of PNA probes for Lactobacillus species and G. vaginalis visualization and the calculation of the microscopic field by Nugent score, allowing a trustful evaluation of the bacteria present in vaginal microflora and avoiding the occurrence of misleading diagnostics. Therefore, the PNA-FISH methodology represents a valuable alternative for BV diagnosis. PMID:25737820

  18. A peptide nucleic