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Sample records for acid peptide designated

  1. G-CSF receptor-binding cyclic peptides designed with artificial amino-acid linkers

    SciTech Connect

    Shibata, Kenji . E-mail: kshibata@kyowa.co.jp; Maruyama-Takahashi, Kumiko; Yamasaki, Motoo; Hirayama, Noriaki . E-mail: hirayama@is.icc.u-tokai.ac.jp

    2006-03-10

    Designing small molecules that mimic the receptor-binding local surface structure of large proteins such as cytokines or growth factors is fascinating and challenging. In this study, we designed cyclic peptides that reproduce the receptor-binding loop structures of G-CSF. We found it is important to select a suitable linker to join two or more discontinuous sequences and both termini of the peptide corresponding to the receptor-binding loop. Structural simulations based on the crystallographic structure of KW-2228, a stable and potent analog of human G-CSF, led us to choose 4-aminobenzoic acid (Abz) as a part of the linker. A combination of 4-Abz with {beta}-alanine or glycine, and disulfide bridges between cysteins or homocysteins, gave a structure suitable for receptor binding. In this structure, the side-chains of several amino acids important for the interactions with the receptor are protruding from one side of the peptide ring. This artificial peptide showed G-CSF antagonistic activity in a cell proliferation assay.

  2. Boronic acid functionalized peptidyl synthetic lectins: Combinatorial library design, peptide sequencing, and selective glycoprotein recognition

    PubMed Central

    Bicker, Kevin L.; Sun, Jing; Lavigne, John J.; Thompson, Paul R.

    2011-01-01

    Aberrant glycosylation of cell membrane and secreted glycoproteins is a hallmark of various disease states, including cancer. The natural lectins currently used in the recognition of these glycoproteins are costly, difficult to produce, and unstable towards rigorous use. Herein we describe the design and synthesis of several boronic acid functionalized peptide-based synthetic lectin (SL) libraries, as well as the optimized methodology for obtaining peptide sequences of these SLs. SL libraries were subsequently used to identify SLs with as high as 5-fold selectivity for various glycoproteins. SLs will inevitably find a role in cancer diagnositics, given that they do not suffer from the drawbacks of natural lectins and that the combinatorial nature of these libraries allows for the identification of an SL for nearly any glycosylated biomolecule. PMID:21405093

  3. 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.

  4. Computationally designed β-turn foldamers of γ-peptides based on 2-(aminomethyl)cyclohexanecarboxylic acid.

    PubMed

    Kang, Young Kee; Byun, Byung Jin

    2012-12-01

    The γ-peptide β-turn structures have been designed computationally by the combination of chirospecific γ(2),(3)-residues of 2-(aminomethyl)cyclohexanecarboxylic acid (γAmc(6)) with a cyclohexyl constraint on the C(α) -C(β) bond using density functional methods in water. The chirospecific γAmc(6) dipeptide with the (2S,3S)-(2R,3R) configurations forms a stable turn structure in water, resembling a type II' turn of α-peptides, which can be used as a β-turn motif in β-hairpins of Ala-based α-peptides. The γAmc(6) dipeptide with homochiral (2S,3S)-(2S,3S) configurations but different cyclohexyl puckerings shows the capability to be incorporated into one of two β-turn motifs of gramicidin S. The overall structure of this gramicidin S analogue is quite similar to the native gramicidin S with the same patterns and geometries of hydrogen bonds. Our calculated results and the recently observed results may imply the wider applicability of chirospecific γ-peptides with a cyclohexyl constraint on the backbone to form various peptide foldamers.

  5. Molecular design and genetic optimization of antimicrobial peptides containing unnatural amino acids against antibiotic-resistant bacterial infections.

    PubMed

    He, Yongkang; He, Xiaofeng

    2016-09-01

    Antimicrobial peptides (AMPs) have been the focus of intense research towards the finding of a viable alternative to current small-molecule antibiotics, owing to their commonly observed and naturally occurring resistance against pathogens. However, natural peptides have many problems such as low bioavailability and high allergenicity that largely limit the clinical applications of AMPs. In the present study, an integrative protocol that combined chemoinformatics modeling, molecular dynamics simulations, and in vitro susceptibility test was described to design AMPs containing unnatural amino acids (AMP-UAAs). To fulfill this, a large panel of synthetic AMPs with determined activity was collected and used to perform quantitative structure-activity relationship (QSAR) modeling. The obtained QSAR predictors were then employed to direct genetic algorithm (GA)-based optimization of AMP-UAA population, to which a number of commercially available, structurally diverse unnatural amino acids were introduced during the optimization process. Subsequently, several designed AMP-UAAs were confirmed to have high antibacterial potency against two antibiotic-resistant strains, i.e. multidrug-resistant Pseudomonas aeruginosa (MDRPA) and methicillin-resistant Staphylococcus aureus (MRSA), with minimum inhibitory concentration (MIC) < 10 μg/ml. Structural dynamics characterizations revealed that the most potent AMP-UAA peptide is an amphipathic helix that can spontaneously embed into an artificial lipid bilayer and exhibits a strong destructuring tendency associated with the embedding process. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 746-756, 2016.

  6. Molecular design and genetic optimization of antimicrobial peptides containing unnatural amino acids against antibiotic-resistant bacterial infections.

    PubMed

    He, Yongkang; He, Xiaofeng

    2016-09-01

    Antimicrobial peptides (AMPs) have been the focus of intense research towards the finding of a viable alternative to current small-molecule antibiotics, owing to their commonly observed and naturally occurring resistance against pathogens. However, natural peptides have many problems such as low bioavailability and high allergenicity that largely limit the clinical applications of AMPs. In the present study, an integrative protocol that combined chemoinformatics modeling, molecular dynamics simulations, and in vitro susceptibility test was described to design AMPs containing unnatural amino acids (AMP-UAAs). To fulfill this, a large panel of synthetic AMPs with determined activity was collected and used to perform quantitative structure-activity relationship (QSAR) modeling. The obtained QSAR predictors were then employed to direct genetic algorithm (GA)-based optimization of AMP-UAA population, to which a number of commercially available, structurally diverse unnatural amino acids were introduced during the optimization process. Subsequently, several designed AMP-UAAs were confirmed to have high antibacterial potency against two antibiotic-resistant strains, i.e. multidrug-resistant Pseudomonas aeruginosa (MDRPA) and methicillin-resistant Staphylococcus aureus (MRSA), with minimum inhibitory concentration (MIC) < 10 μg/ml. Structural dynamics characterizations revealed that the most potent AMP-UAA peptide is an amphipathic helix that can spontaneously embed into an artificial lipid bilayer and exhibits a strong destructuring tendency associated with the embedding process. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 746-756, 2016. PMID:27258330

  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. 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

  9. 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

  10. Design of embedded chimeric peptide nucleic acids that efficiently enter and accurately reactivate gene expression in vivo.

    PubMed

    Chen, Joy; Peterson, Kenneth R; Iancu-Rubin, Camelia; Bieker, James J

    2010-09-28

    Pharmacological treatments designed to reactivate fetal γ-globin can lead to an effective and successful clinical outcome in patients with hemoglobinopathies. However, new approaches remain highly desired because such treatments are not equally effective for all patients, and toxicity issues remain. We have taken a systematic approach to develop an embedded chimeric peptide nucleic acid (PNA) that effectively enters the cell and the nucleus, binds to its target site at the human fetal γ-globin promoter, and reactivates this transcript in adult transgenic mouse bone marrow and human primary peripheral blood cells. In vitro and in vivo DNA-binding assays in conjunction with live-cell imaging have been used to establish and optimize chimeric PNA design parameters that lead to successful gene activation. Our final molecule contains a specific γ-promoter-binding PNA sequence embedded within two amino acid motifs: one leads to efficient cell/nuclear entry, and the other generates transcriptional reactivation of the target. These embedded PNAs overcome previous limitations and are generally applicable to the design of in vivo transcriptional activation reagents that can be directed to any promoter region of interest and are of direct relevance to clinical applications that would benefit from such a need.

  11. 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.

  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. Peptide-based delivery of nucleic acids: design, mechanism of uptake and applications to splice-correcting oligonucleotides.

    PubMed

    Abes, S; Moulton, H; Turner, J; Clair, P; Richard, J P; Iversen, P; Gait, M J; Lebleu, B

    2007-02-01

    CPPs (cell-penetrating peptides) have given rise to much interest for the delivery of biomolecules such as peptides, proteins or ONs (oligonucleotides). CPPs and their conjugates were initially thought to translocate through the cell membrane by a non-endocytotic mechanism which has recently been re-evaluated. Basic-amino-acid-rich CPPs first interact with cell-surface proteoglycans before being internalized by endocytosis. Sequestration and degradation in endocytotic vesicles severely limits the cytoplasmic and nuclear delivery of the conjugated biomolecules. Accordingly, splicing correction by CPP-conjugated steric-block ON analogues is inefficient in the absence of endosomolytic agents. New arginine-rich CPPs allowing efficient splicing correction by conjugated PNAs (peptide nucleic acids) or PMO (phosphorodiamidate morpholino oligomer) steric blockers in the absence of endosomolytic agents have recently been defined in our group and are currently being characterized. They offer promising leads for the development of efficient cellular delivery vectors for therapeutic steric-block ON analogues.

  14. CHEMICAL SHIFTS IN AMINO ACIDS, PEPTIDES, AND PROTEINS: From Quantum Chemistry to Drug Design

    NASA Astrophysics Data System (ADS)

    Oldfield, Eric

    2002-10-01

    This chapter discusses recent progress in the investigation and use of 13C, 15N, and 19F nuclear magnetic resonance (NMR) chemical shifts and chemical shift tensors in proteins and model systems primarily using quantum chemical (ab initio Hartree-Fock and density functional theory) techniques. Correlations between spectra and structure are made and the techniques applied to other spectroscopic and electrostatic properties as well, including hydrogen bonding, ligand binding to heme proteins, J-couplings, electric field gradients, and atoms-in-molecules theory, together with a brief review of the use of NMR chemical shifts in drug design.

  15. 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

  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.

  17. 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

  18. 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

  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.

  20. Synthesis of C-linked carbo-β2-amino acids and β2-peptides: design of new motifs for left-handed 12/10- and 10/12-mixed helices.

    PubMed

    Sharma, Gangavaram V M; Reddy, Nelli Yella; Ravi, Rapolu; Sreenivas, Bommagani; Sridhar, Gattu; Chatterjee, Deepak; Kunwar, Ajit C; Hofmann, Hans-Jörg

    2012-12-14

    C-linked carbo-β(2)-amino acids (β(2)-Caa), a new class of β-amino acid with a carbohydrate side chain having d-xylo configuration, were prepared from d-glucose. The main idea behind the design of the new β-amino acids was to move the steric strain of the bulky carbohydrate side chain from the Cβ- to the Cα-carbon atom and to explore its influence on the folding propensities in peptides with alternating (R)- and (S)-β(2)-Caas. The tetra- and hexapeptides derived were studied employing NMR (in CDCl(3)), CD, and molecular dynamics simulations. The β(2)-peptides of the present study form left-handed 12/10- and 10/12-mixed helices independent of the order of the alternating chiral amino acids in the sequence and result in a new motif. These results differ from earlier findings on β(3)-peptides of the same design, containing a carbohydrate side chain with d-xylo configuration, which form exclusively right-handed 12/10-mixed helices. Quantum chemical calculations employing ab initio MO theory suggest the side chain chirality as an important factor for the observed definite left- or right-handedness of the helices in the β(2)- and β(3)-peptides.

  1. 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

  2. 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

  3. 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).

  4. 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.

  5. 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.

  6. Non-peptide angiotensin II receptor antagonists. 2. Design, synthesis, and biological activity of N-substituted (phenylamino)phenylacetic acids and acyl sulfonamides.

    PubMed

    Dhanoa, D S; Bagley, S W; Chang, R S; Lotti, V J; Chen, T B; Kivlighn, S D; Zingaro, G J; Siegl, P K; Patchett, A A; Greenlee, W J

    1993-12-24

    The design, synthesis, and biological activity of a new class of highly potent non-peptide AII receptor antagonists derived from N-substituted (phenylamino)phenylacetic acids and acyl sulfonamides which exhibit a high selectivity for the AT1 receptor are described. A series of N-substituted (phenylamino)phenylacetic acids (9) and acyl sulfonamides (16) and a tetrazole derivative (19) were synthesized and evaluated in the in vitro AT1 (rabbit aorta) and AT2 (rat midbrain) binding assay. The (phenylamino)phenylacetic acids 9c (AT1 IC50 = 4 nM, AT2 IC50 = 0.74 microM), 9d (AT1 IC50 = 5.3 nM, AT2 IC50 = 0.49 microM), and 9e (AT1 IC50 = 5.3 nM, AT2 IC50 = 0.56 microM) were found to be the most potent AT1-selective AII antagonists in the acid series. Incorporation of various substituents in the central and bottom phenyl rings led to a decrease in the AT1 and AT2 binding affinity of the resulting compounds. Replacement of the carboxylic acid (CO2H) in 9c, 9d, and 9e with the bioisostere acyl sulfonamide (CONHSO2Ph) resulted in a (5-7)-fold increase in the AT1 potency of 16a (AT1 IC50 = 0.9 nM, AT2 IC50 = 0.2 microM), 16b (AT1 IC50 = 1 nM, AT2 IC50 = 2.9 microM), and 16c (AT1 IC50 = 0.8 nM, AT2 IC50 = 0.42 microM) and yielded acyl sulfonamides with subnanomolar AT1 activity. Incorporation of the acyl sulfonamide (CONHSO2Ph) for the CO2H of 9c not only enhanced the AT1 potency but also effected a marked increase in the AT2 potency of 16a (AT2 IC50 = 0.74 microM of 9c vs 0.2 microM of 16a) and made it the most potent AT2 antagonist in this study. Replacement of the CO2H of 9b with the bioisostere tetrazole resulted in 19 (AT1 IC50 = 15 nM) with a 2-fold loss in the AT1 and a complete loss in the AT2 binding affinity. (Phenylamino)phenylacetic acid 9c demonstrated good oral activity in AII-infused conscious normotensive rats at an oral dose of 1.0 mg/kg by inhibiting the pressor response for > 6 h. Acyl sulfonamides 16a-c displayed excellent in vivo activity by blocking the

  7. Crystallization of a designed peptide from a molten globule ensemble.

    PubMed

    Betz; Raleigh; DeGrado; Lovejoy; Anderson; Ogihara; Eisenberg

    1995-01-01

    Backgound. The design of amino acid sequences that adopt a desired three-dimensional fold has been of keen interest over the past decade. However, the design of proteins that adopt unique conformations is still a considerable problem. Until very recently, all of the designed proteins that have been extensively characterized possess the hallmarks of the molten globular state. Molten globular intermediates have been observed in both equilibrium and kinetic protein folding/stability studies, and understanding the forces that determine compact non-native states is critical for a comprehensive understanding of proteins. This paper describes the solution and early solid state characterization of peptides that form molten globular ensembles. Results. Crystals diffracting to 3.5Å resolution have been grown of a 16-residue peptide (alpha1A) designed to form a tetramer of alpha-helices. In addition, a closely related peptide, alpha1, has previously been shown to yield crystals that diffract to 1.2Å resolution. The solution properties of these two peptides were examined to determine whether their well defined crystalline conformations were retained in solution. On the basis of an examination of their NMR spectra, sedimentation equilibria, thermal unfolding, and ANS binding, it is concluded that the peptides form alpha-helical aggregates with properties similar to those of the molten globule state. Thus, for these peptides, the process of crystallization bears many similarities to models of protein folding. Upon dissolution, the peptides rapidly assume compact molten globular states similar to the molten globule like intermediates that are formed at short times after refolding is initiated. Following a rate-determining nucleation step, the peptides crystallize into a single or a small number of conformations in a process that mimics the formation of native structure in proteins. PMID:9162140

  8. 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.

  9. Glutamic Acid Selective Chemical Cleavage of Peptide Bonds.

    PubMed

    Nalbone, Joseph M; Lahankar, Neelam; Buissereth, Lyssa; Raj, Monika

    2016-03-01

    Site-specific hydrolysis of peptide bonds at glutamic acid under neutral aqueous conditions is reported. The method relies on the activation of the backbone amide chain at glutamic acid by the formation of a pyroglutamyl (pGlu) imide moiety. This activation increases the susceptibility of a peptide bond toward hydrolysis. The method is highly specific and demonstrates broad substrate scope including cleavage of various bioactive peptides with unnatural amino acid residues, which are unsuitable substrates for enzymatic hydrolysis.

  10. Designer self-assembling peptide materials.

    PubMed

    Zhao, Xiaojun; Zhang, Shuguang

    2007-01-01

    Understanding of macromolecular materials at the molecular level is becoming increasingly important for a new generation of nanomaterials for nanobiotechnology and other disciplines, namely, the design, synthesis, and fabrication of nanodevices at the molecular scale from bottom up. Basic engineering principles for microfabrication can be learned through fully grasping the molecular self-assembly and programmed assembly phenomena. Self- and programmed-assembly phenomena are ubiquitous in nature. Two key elements in molecular macrobiological material productions are chemical complementarity and structural compatibility, both of which require weak and non-covalent interactions that bring building blocks together during self-assembly. Significant advances have been made during the 1990s at the interface of materials chemistry and biology. They include the design of helical ribbons, peptide nanofiber scaffolds for three-dimensional cell cultures and tissue engineering, peptide surfactants for solubilizing and stabilizing diverse types of membrane proteins and their complexes, and molecular ink peptides for arbitrary printing and coating surfaces as well as coiled-coil helical peptides for multi-length scale fractal structures. These designer self-assembling peptides have far reaching implications in a broad spectrum of applications in biology, medicine, nanobiotechnology, and nanobiomedical technology, some of which are beyond our current imaginations. [image: see text

  11. Exploiting protected maleimides to modify oligonucleotides, peptides and peptide nucleic acids.

    PubMed

    Paris, Clément; Brun, Omar; Pedroso, Enrique; Grandas, Anna

    2015-04-10

    This manuscript reviews the possibilities offered by 2,5-dimethylfuran-protected maleimides. Suitably derivatized building blocks incorporating the exo Diels-Alder cycloadduct can be introduced at any position of oligonucleotides, peptide nucleic acids, peptides and peptoids, making use of standard solid-phase procedures. Maleimide deprotection takes place upon heating, which can be followed by either Michael-type or Diels-Alder click conjugation reactions. However, the one-pot procedure in which maleimide deprotection and conjugation are simultaneously carried out provides the target conjugate more quickly and, more importantly, in better yield. This procedure is compatible with conjugates involving oligonucleotides, peptides and peptide nucleic acids. A variety of cyclic peptides and oligonucleotides can be obtained from peptide and oligonucleotide precursors incorporating protected maleimides and thiols.

  12. Fatty acid conjugation enhances the activities of antimicrobial peptides.

    PubMed

    Li, Zhining; Yuan, Penghui; Xing, Meng; He, Zhumei; Dong, Chuanfu; Cao, Yongchang; Liu, Qiuyun

    2013-04-01

    Antimicrobial peptides are small molecules that play a crucial role in innate immunity in multi-cellular organisms, and usually expressed and secreted constantly at basal levels to prevent infection, but local production can be augmented upon an infection. The clock is ticking as rising antibiotic abuse has led to the emergence of many drug resistance bacteria. Due to their broad spectrum antibiotic and antifungal activities as well as anti-viral and anti-tumor activities, efforts are being made to develop antimicrobial peptides into future microbial agents. This article describes some of the recent patents on antimicrobial peptides with fatty acid conjugation. Potency and selectivity of antimicrobial peptide can be modulated with fatty acid tails of variable length. Interaction between membranes and antimicrobial peptides was affected by fatty acid conjugation. At concentrations above the critical miscelle concentration (CMC), propensity of solution selfassembly hampered binding of the peptide to cell membranes. Overall, fatty acid conjugation has enhanced the activities of antimicrobial peptides, and occasionally it rendered inactive antimicrobial peptides to be bioactive. Antimicrobial peptides can not only be used as medicine but also as food additives.

  13. Adding energy minimization strategy to peptide-design algorithm enables better search for RNA-binding peptides: Redesigned λ N peptide binds boxB RNA.

    PubMed

    Xiao, Xingqing; Hung, Michelle E; Leonard, Joshua N; Hall, Carol K

    2016-10-15

    Our previously developed peptide-design algorithm was improved by adding an energy minimization strategy which allows the amino acid sidechains to move in a broad configuration space during sequence evolution. In this work, the new algorithm was used to generate a library of 21-mer peptides which could substitute for λ N peptide in binding to boxB RNA. Six potential peptides were obtained from the algorithm, all of which exhibited good binding capability with boxB RNA. Atomistic molecular dynamics simulations were then conducted to examine the ability of the λ N peptide and three best evolved peptides, viz. Pept01, Pept26, and Pept28, to bind to boxB RNA. Simulation results demonstrated that our evolved peptides are better at binding to boxB RNA than the λ N peptide. Sequence searches using the old (without energy minimization strategy) and new (with energy minimization strategy) algorithms confirm that the new algorithm is more effective at finding good RNA-binding peptides than the old algorithm. © 2016 Wiley Periodicals, Inc.

  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. 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.

  16. A novel antifungal peptide designed from the primary structure of a natural antimicrobial peptide purified from Argopecten purpuratus hemocytes.

    PubMed

    Arenas, Gloria; Guzmán, Fanny; Cárdenas, Constanza; Mercado, Luis; Marshall, Sergio H

    2009-08-01

    We have isolated and purified a natural antimicrobial peptide from Argopecten purpuratus hemocytes. 47 residues were determined from its primary structure representing the N-terminal of the complete sequence. This peptide of 5100.78Da was chemically synthesized and named Ap. The peptide has 25% of hydrophobic amino acids with a net charge of +1, and partial homology with known active antimicrobial peptides. Based on that sequence, a new peptide was designed and modeled to increase hydrophobicity and cationicity. The designed 30-residue peptide was chemically synthesized resulting in a novel 38% hydrophobic molecule named peptide Ap-S, with a net charge of +5 and 3028Da. A secondary structure was shown by circular dichroism, thus exposing a hydrophobic epitope toward the N-terminus and a hydrophilic one toward the C-terminus, improving amphipathicity. Ap-S was much more active than the parental Ap. Ap-S up to 100microM has no cytotoxic effect against fish cell line CHSE-214. We demonstrated that the chemical modification of a natural peptide and the chemical synthesis of derived molecules may be a powerful tool for obtaining substitutes to conventional antibiotics, displaying the many advantages of antimicrobial peptides and overcoming the limitations of natural peptides for large-scale production and application, such as the low specific activity and the minute amounts recovered in vivo. This peptide may have a relevant application in aquaculture by controlling Saprolegna sp., a parasitic pathogen fungus that attacks the culture of fish in different stages of their growth, from egg to adult. PMID:19481126

  17. CycloPs: generating virtual libraries of cyclized and constrained peptides including nonnatural amino acids.

    PubMed

    Duffy, Fergal J; Verniere, Mélanie; Devocelle, Marc; Bernard, Elise; Shields, Denis C; Chubb, Anthony J

    2011-04-25

    We introduce CycloPs, software for the generation of virtual libraries of constrained peptides including natural and nonnatural commercially available amino acids. The software is written in the cross-platform Python programming language, and features include generating virtual libraries in one-dimensional SMILES and three-dimensional SDF formats, suitable for virtual screening. The stand-alone software is capable of filtering the virtual libraries using empirical measurements, including peptide synthesizability by standard peptide synthesis techniques, stability, and the druglike properties of the peptide. The software and accompanying Web interface is designed to enable the rapid generation of large, structurally diverse, synthesizable virtual libraries of constrained peptides quickly and conveniently, for use in virtual screening experiments. The stand-alone software, and the Web interface for evaluating these empirical properties of a single peptide, are available at http://bioware.ucd.ie .

  18. De Novo Peptide Design and Experimental Validation of Histone Methyltransferase Inhibitors

    PubMed Central

    Smadbeck, James; Peterson, Meghan B.; Zee, Barry M.; Garapaty, Shivani; Mago, Aashna; Lee, Christina; Giannis, Athanassios; Trojer, Patrick; Garcia, Benjamin A.; Floudas, Christodoulos A.

    2014-01-01

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

  19. 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

  20. 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.

  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. 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.

  3. 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

  4. Design of amphiphilic oligopeptides as models for fine tuning peptide assembly with plasmid DNA.

    PubMed

    Goparaju, Geetha N; Gupta, Pardeep K

    2014-08-01

    We discuss the design of novel amphiphilic oligopeptides with hydrophobic and cationic amino acids to serve as models to understand peptide-DNA assembly. Biophysical and thermodynamic characterization of interaction of these amphiphilic peptides with plasmid DNA is presented. Peptides with at least +4 charges favor stable complex formation. Surface potential is dependent on the type of hydrophobic amino acid for a certain charge. Thermodynamically it is a spontaneous interaction between most of the peptides and plasmid DNA. Lys(7) and Tyr peptides with +4/+5 charges indicate cooperative binding with pDNA without saturation of interaction while Val(2)-Gly-Lys(4), Val-Gly-Lys(5), and Phe-Gly-Lys(5) lead to saturation of interaction indicating condensed pDNA within the range of N/Ps studied. We show that the biophysical properties of DNA-peptide complexes could be modulated by design and the peptides presented here could be used as building blocks for creating DNA-peptide complexes for various biomedical applications, mainly nucleic acid delivery.

  5. Analysis of Peptides and Conjugates by Amino Acid Analysis.

    PubMed

    Højrup, Peter

    2015-01-01

    Amino acid analysis is a highly accurate method for characterization of the composition of synthetic peptides. Together with mass spectrometry, it gives a reliable control of peptide quality and quantity before conjugation and immunization. Peptides are hydrolyzed, preferably in gas phase, with 6 M HCl at 110 °C for 20-24 h and the resulting amino acids analyzed by ion-exchange chromatography with post-column ninhydrin derivatization. Depending on the hydrolysis conditions, tryptophan is destroyed, and cysteine also, unless derivatized, and the amides, glutamine and asparagine, are deamidated to glutamic acid and aspartic acid, respectively. Three different ways of calculating results are suggested, and taking the above limitations into account, a quantitation better than 5% can usually be obtained. PMID:26424264

  6. 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

  7. 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

  8. 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

  9. 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.

  10. How amino acids and peptides shaped the RNA world.

    PubMed

    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

  11. Peptide modules for overcoming barriers of nucleic acids transport to cells.

    PubMed

    Egorova, Anna A; Kiselev, Anton V

    2016-01-01

    Absence of safe and efficient methods of nucleic acids delivery is one of the major issues which limits the development of human gene therapy. Highly efficient viral vectors raise questions due to safety reasons. Among non-viral vectors peptide-based carriers can be considered as good candidates for the development of "artificial viruses"--multifunctional polyplexes that mimic viruses. Suggested strategy to obtain multifunctionality is to combine several peptide modules into one modular carrier. Different kinds of peptide modules are needed for successful overcoming barriers of nucleic acids transport into the cells. Design of such modules and establishment of structure-function relationships are issues of importance to researchers working in the field of nucleic acids delivery.

  12. Amino Acid and Peptide Requirement of Fusiformis necrophorus

    PubMed Central

    Wahren, Ann; Holme, Tord

    1973-01-01

    Uptake of individual amino acids and peptides by Fusiformis necrophorus was studied in growing cultures and resting cell suspensions. The cells were able to incorporate 16 of 17 14C-labeled amino acids into cell protein, the exception being proline. Proline could neither be formed by the cells from any of the other tested amino acids nor be synthesized from glucose or serine when these were used as energy sources. The addition of di- and tripeptides, the octapeptides vasopressin and oxytocin, and the poly (24) peptide ACTH did not stimulate cell growth, but a marked stimulatory effect was noted after the addition of poly-l-proline (mean molecular weight 2,000). It is concluded that cells of F. necrophorus (i) possess transport systems for most amino acids but not for proline, (ii) are dependent on exogenous proline in the form of proline-containing peptides for growth, and (iii) may be cultivated in a defined amino acid medium provided the proline requirement is met by the addition of a proline-containing peptide. PMID:4745417

  13. 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

  14. Effective Design of Multifunctional Peptides by Combining Compatible Functions.

    PubMed

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

    2016-04-01

    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.

  15. Peptide design for antimicrobial and immunomodulatory applications.

    PubMed

    Haney, Evan F; Hancock, Robert E W

    2013-11-01

    The increasing threat of antibiotic resistance in pathogenic bacteria and the dwindling supply of antibiotics available to combat these infections poses a significant threat to human health throughout the world. Antimicrobial peptides (AMPs) have long been touted as the next generation of antibiotics capable of filling the anti-infective void. Unfortunately, peptide-based antibiotics have yet to realize their potential as novel pharmaceuticals, in spite of the immense number of known AMP sequences and our improved understanding of their antibacterial mechanism of action. Recently, the immunomodulatory properties of certain AMPs have become appreciated. The ability of small synthetic peptides to protect against infection in vivo has demonstrated that modulation of the innate immune response is an effective strategy to further develop peptides as novel anti-infectives. This review focuses on the screening methods that have been used to assess novel peptide sequences for their antibacterial and immunomodulatory properties. It will also examine how we have progressed in our ability to identify and optimize peptides with desired biological characteristics and enhanced therapeutic potential. In addition, the current challenges to the development of peptides as anti-infectives are examined and the strategies being used to overcome these issues are discussed.

  16. Histidine-lysine peptides as carriers of nucleic acids.

    PubMed

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

    2007-03-01

    With their biodegradability and diversity of permutations, peptides have significant potential as carriers of nucleic acids. This review will focus on the sequence and branching patterns of peptide carriers composed primarily of histidines and lysines. While lysines within peptides are important for binding to the negatively charged phosphates, histidines are critical for endosomal lysis enabling nucleic acids to reach the cytosol. Histidine-lysine (HK) polymers by either covalent or ionic bonds with liposomes augment transfection compared to liposome carriers alone. More recently, we have examined peptides as sole carriers of nucleic acids because of their intrinsic advantages compared to the bipartite HK/liposome carriers. With a protocol change and addition of a histidine-rich tail, HK peptides as sole carriers were more effective than liposomes alone in several cell lines. While four-branched polymers with a primary repeating sequence pattern of -HHK- were more effective as carriers of plasmids, eight-branched polymers with a sequence pattern of -HHHK- were more effective as carriers of siRNA. Compared to polyethylenimine, HK carriers of siRNA and plasmids had reduced toxicity. When injected intravenously, HK polymers in complex with plasmids encoding antiangiogenic proteins significantly decreased tumor growth. Furthermore, modification of HK polymers with polyethylene glycol and vascular-specific ligands increased specificity of the polyplex to the tumor by more than 40-fold. Together with further development and insight on the structure of HK polyplexes, HK peptides may prove to be useful as carriers of different forms of nucleic acids both in vitro and in vivo. PMID:17440630

  17. Design and Synthesis of a Novel Cationic Peptide with Potent and Broad-Spectrum Antimicrobial Activity

    PubMed Central

    Liu, Wen-Ping; Chen, Ya-Hui; Ming, Xin; Kong, Yi

    2015-01-01

    Antibacterial and antifungal peptides have increasingly been used to combat the antibiotic-resistant microbes in recent years. KW-13, a novel cationic α-helical antibacterial peptide consisting of 13 amino acid residues, was designed and chemically synthesized. The peptide has a net charge of +6 with a total hydrophobic ratio of 38%. The antibacterial experiments revealed that KW-13 strongly inhibited the growth of human pathogenic bacteria with minimal inhibitory concentrations of 4 and 16 μg/mL for Staphylococcus epidermidis and Staphylococcus aureus, respectively, while the hemolytic assay showed that this peptide did not destroy human red blood cells in vitro. Scanning electron microscopy imaging of Escherichia coli confirmed that KW-13 can damage the membrane of bacterial cells. Thus, this peptide could be a potential candidate for the treatment of infectious diseases. PMID:26688811

  18. Design and Synthesis of a Novel Cationic Peptide with Potent and Broad-Spectrum Antimicrobial Activity.

    PubMed

    Liu, Wen-Ping; Chen, Ya-Hui; Ming, Xin; Kong, Yi

    2015-01-01

    Antibacterial and antifungal peptides have increasingly been used to combat the antibiotic-resistant microbes in recent years. KW-13, a novel cationic α-helical antibacterial peptide consisting of 13 amino acid residues, was designed and chemically synthesized. The peptide has a net charge of +6 with a total hydrophobic ratio of 38%. The antibacterial experiments revealed that KW-13 strongly inhibited the growth of human pathogenic bacteria with minimal inhibitory concentrations of 4 and 16 μg/mL for Staphylococcus epidermidis and Staphylococcus aureus, respectively, while the hemolytic assay showed that this peptide did not destroy human red blood cells in vitro. Scanning electron microscopy imaging of Escherichia coli confirmed that KW-13 can damage the membrane of bacterial cells. Thus, this peptide could be a potential candidate for the treatment of infectious diseases. PMID:26688811

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

    PubMed Central

    Wiedman, Gregory

    2015-01-01

    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 macro-molecular 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

  20. 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

  1. 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

  2. Biological activity and biotechnological aspects of peptide nucleic acid.

    PubMed

    Lundin, Karin E; Good, Liam; Strömberg, Roger; Gräslund, Astrid; Smith, C I Edvard

    2006-01-01

    During the latest decades a number of different nucleic acid analogs containing natural nucleobases on a modified backbone have been synthesized. An example of this is peptide nucleic acid (PNA), a DNA mimic with a noncyclic peptide-like backbone, which was first synthesized in 1991. Owing to its flexible and neutral backbone PNA displays very good hybridization properties also at low-ion concentrations and has subsequently attracted large interest both in biotechnology and biomedicine. Numerous modifications have been made, which could be of value for particular settings. However, the original PNA does so far perform well in many diverse applications. The high biostability makes it interesting for in vivo use, although the very limited diffusion over lipid membranes requires further modifications in order to make it suitable for treatment in eukaryotic cells. The possibility to use this nucleic acid analog for gene regulation and gene editing is discussed. Peptide nucleic acid is now also used for specific genetic detection in a number of diagnostic techniques, as well as for site-specific labeling and hybridization of functional molecules to both DNA and RNA, areas that are also discussed in this chapter.

  3. High Selective Performance of Designed Antibacterial and Anticancer Peptide Amphiphiles.

    PubMed

    Chen, Cuixia; Chen, Yucan; Yang, Cheng; Zeng, Ping; Xu, Hai; Pan, Fang; Lu, Jian Ren

    2015-08-12

    Short designed peptide amphiphiles are attractive at killing bacteria and inhibiting cancer cell growth, and the flexibility in their structural design offers a great potential for improving their potency and biocompatibility to mammalian host cells. Amino acid sequences such as G(IIKK)nI-NH2 (n≥3) have been shown to be membrane lytic, but terminal amino acid modifications could impose a huge influence on their performance. We report in this work how terminal amino acid modifications to G(IIKK)3I-NH2 influence its α-helical structure, membrane penetrating ability, and selective actions against different cell types. Deletion of an N-terminal Gly or a C-terminal Ile did not affect their antibacterial activity much, an observation consistent with their binding behavior to negatively charged membrane lipid monolayers. However, the cytotoxicity against mammalian cells was much worsened by the N-terminal Gly deletion, consistent with an increase in its helical content. Despite little impact on the antibacterial activity of G(IIKK)3I-NH2, deletion of both terminal amino acids greatly reduced its antitumor activity. Cholesterol present in tumor cell membrane-mimic was thought to constrain (IIKK)3-NH2 from penetrating into the cancerous membranes, evident from its lowest surface physical activity at penetrating model lipid membranes. On the other hand, its low toxicity to normal mammalian cells and high antibacterial activity in vitro and in vivo made it an attractive antibacterial agent. Thus, terminal modifications can help rebalance the different interactions involved and are highly effective at manipulating their selective membrane responses.

  4. Bioproduction of highly charged designer peptide surfactants via a chemically cleavable coiled-coil heteroconcatemer.

    PubMed

    Fletcher, Nicholas L; Paquet, Nicolas; Dickinson, Ellyce L; Dexter, Annette F

    2015-02-01

    Designer peptides have recently attracted attention as self-assembling fibrils, hydrogelators and green surfactants with the potential for sustainable bioproduction. Carboxylate-rich peptides in particular have shown potential as salt-resistant emulsifiers; however the expression of highly charged peptides of this kind remains a challenge. To achieve expression of a strongly anionic helical surfactant peptide, we paired the peptide with a cationic helical partner in a coiled-coil miniprotein and optimized the polypeptide sequence for net charge, hydropathy and predicted protease resistance (via the Guruprasad instability index). Our design permitted expression of a soluble concatemer that accumulates to high levels (22% of total protein) in E. coli. The concatemer showed high stability to heat and proteases, allowing isolation by simple heat and pH precipitation steps that yield concatemer at 133 mg per gram of dry cell weight and >99% purity. Aspartate-proline sites were included in the concatemer to allow cleavage with heat and acid to give monomeric peptides. We characterized the acid cleavage pathway of the concatemer by coupled liquid chromatography-mass spectrometry and modeled the kinetic pathways involved. The outcome represents the first detailed kinetic characterization of protein cleavage at aspartate-proline sites, and reveals unexpected cleavage preferences, such as favored cleavage at the C-termini of peptide helices. Chemical denaturation of the concatemer showed an extremely high thermodynamic stability of 38.9 kcal mol(-1) , with cleavage decreasing the stability of the coiled coil to 32.8 kcal mol(-1) . We determined an interfacial pressure of 29 mN m(-1) for both intact and cleaved concatemer at the air-water interface, although adsorption was slightly more rapid for the cleaved peptides. The cleaved peptides could be used to prepare heat-stable emulsions with droplet sizes in the nanometer range.

  5. 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

  6. 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

  7. Ribosomal Synthesis of Macrocyclic Peptides in Vitro and in Vivo Mediated by Genetically Encoded Amino-Thiol Unnatural Amino Acids

    PubMed Central

    Frost, John R.; Jacob, Nicholas T.; Papa, Louis J.; Owens, Andrew E.

    2015-01-01

    A versatile method for orchestrating the formation of side-chain-to-tail cyclic peptides from ribosomally derived polypeptide precursors is reported. Upon ribosomal incorporation into intein-containing precursor proteins, designer unnatural amino acids bearing side-chain 1,3- or 1,2-aminothiol functionalities are able to promote the cyclization of a downstream target peptide sequence via a C-terminal ligation/ring contraction mechanism. Using this approach, peptide macrocycles of variable size and composition could be generated in a pH-triggered manner in vitro, or directly in living bacterial cells. This methodology furnishes a new platform for the creation and screening of genetically encoded libraries of conformationally constrained peptides. This strategy was applied to identify and isolate a low micromolar streptavidin binder (KD = 1.1 µM) from a library of cyclic peptides produced in E. coli, thereby illustrating its potential toward aiding the discovery of functional peptide macrocycles. PMID:25933125

  8. 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

  9. 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.

  10. A new antifungal peptide from the seeds of Phytolacca americana: characterization, amino acid sequence and cDNA cloning.

    PubMed

    Shao, F; Hu, Z; Xiong, Y M; Huang, Q Z; WangCG; Zhu, R H; Wang, D C

    1999-03-19

    An antifungal peptide from seeds of Phytolacca americana, designated PAFP-s, has been isolated. The peptide is highly basic and consists of 38 residues with three disulfide bridges. Its molecular mass of 3929.0 was determined by mass spectrometry. The complete amino acid sequence was obtained from automated Edman degradation, and cDNA cloning was successfully performed by 3'-RACE. The deduced amino acid sequence of a partial cDNA corresponded to the amino acid sequence from chemical sequencing. PAFP-s exhibited a broad spectrum of antifungal activity, and its activities differed among various fungi. PAFP-s displayed no inhibitory activity towards Escherichia coli. PAFP-s shows significant sequence similarities and the same cysteine motif with Mj-AMPs, antimicrobial peptides from seeds of Mirabilis jalapa belonging to the knottin-type antimicrobial peptide.

  11. Kojic Acid Peptide: A New Compound with Anti-Tyrosinase Potential

    PubMed Central

    Singh, Birendra Kumar; Park, Seok Hoon; Lee, Hyang-Bok; Goo, Young-Aae; Kim, Hyoung Shik; Cho, Seung Hee; Lee, Jeong Hun; Ahn, Ghe Whan; Kim, Jin Pyo; Kang, Su Myoung

    2016-01-01

    Background Kojic acid was used for decades in the cosmetic industry as an antimelanogenic agent. However, there are two major drawbacks of Kojic acid, one is cytotoxicity and second are instability on storage. These limitations led the scientist to synthesize the active Kojic acid peptides. Objective In the present study, we synthesize and investigate the effect of five Kojic acid peptides to overcome the limitation of Kojic acid. Methods The peptide was analyzed and purified by high-performance liquid chromatography and matrix-assisted laser desorption ionization time of flight mass spectroscopy. Further, the tyrosinase activities of the Kojic acid and Kojic acid peptides were compared. The toxicity was measured and the melanin content is recorded in B16F10 mouse melanoma cells. Results Maximum tyrosinase activity was measured by Kojic acid peptides. Therefore, Kojic acid peptides were subjected to melanin assay and cytotoxicity assay and finally the stability of the Kojic acid peptide was measured. Conclusion It was observed that this newly synthesized Kojic acid peptide is stable and potent to inhibit the tyrosinase activity and melanin content of B16F10 mouse melanoma cells without exhibiting cell toxicity. Together, these preliminary results suggest that a further exploration is being needed to establish Kojic acid peptide as antimelanogenic agent. PMID:27746633

  12. 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.

  13. The interaction of amino acids, peptides, and proteins with DNA.

    PubMed

    Solovyev, Andrey Y; Tarnovskaya, Svetlana I; Chernova, Irina A; Shataeva, Larisa K; Skorik, Yury A

    2015-01-01

    Amino acids that carry charges on their side groups can bind to double stranded DNA (dsDNA) and change the strength of the double helix. Measurement of the DNA melting temperature (Tm) confirmed that acidic amino acids (Glu, Asp) weaken the H-bonds between DNA strands, whereas basic amino acids (Arg, Lys) strengthen the interaction between the strands. A rank correlation exists between the amino acid isoelectric points and the observed changes in Tm. A similar dependence of the hyperchromic effect on the isoelectric point of a protein (pepsin, insulin, cortexin, and protamine) was observed for DNA-protein complexes at room temperature. Short peptides (KE, AEDG, and KEDP) containing a mixture of acidic and basic amino acid residues also affect Tm and the stability of the double helix. A model for binding Glu and Lys to dsDNA was explored by a docking simulation. The model shows that Glu, in an untwisted shape, binds to dsDNA in its major groove and disrupts three H-bonds between the strands, thereby destabilizing the double helix. Lys, in an untwisted shape, binds to the external side of the dsDNA and forms two bonds with O atoms of neighboring phosphodiester groups, thereby strengthening the DNA helix.

  14. 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.

  15. Designer antibacterial peptides kill fluoroquinolone-resistant clinical isolates.

    PubMed

    Otvos, Laszlo; Wade, John D; Lin, Feng; Condie, Barry A; Hanrieder, Joerg; Hoffmann, Ralf

    2005-08-11

    A significant number of Escherichia coli and Klebsiella pneumoniae bacterial strains in urinary tract infections are resistant to fluoroquinolones. Peptide antibiotics are viable alternatives although these are usually either toxic or insufficiently active. By applying multiple alignment and sequence optimization steps, we designed multifunctional proline-rich antibacterial peptides that maintained their DnaK-binding ability in bacteria and low toxicity in eukaryotes, but entered bacterial cells much more avidly than earlier peptide derivatives. The resulting chimeric and statistical analogues exhibited 8-32 microg/mL minimal inhibitory concentration efficacies in Muller-Hinton broth against a series of clinical pathogens. Significantly, the best peptide, compound 5, A3-APO, retained full antibacterial activity in the presence of mouse serum. Across a set of eight fluoroquinolone-resistant clinical isolates, peptide 5 was 4 times more potent than ciprofloxacin. On the basis of the in vitro efficacy, toxicity, and pharmacokinetics data, we estimate that peptide 5 will be suitable for treating infections in the 3-5 mg/kg dose range.

  16. Facile Analysis and Sequencing of Linear and Branched Peptide Boronic Acids by MALDI Mass Spectrometry

    PubMed Central

    Crumpton, Jason; Zhang, Wenyu; Santos, Webster

    2011-01-01

    Interest in peptides incorporating boronic acid moieties is increasing due to their potential as therapeutics/diagnostics for a variety of diseases such as cancer. The utility of peptide boronic acids may be expanded with access to vast libraries that can be deconvoluted rapidly and economically. Unfortunately, current detection protocols using mass spectrometry are laborious and confounded by boronic acid trimerization, which requires time consuming analysis of dehydration products. These issues are exacerbated when the peptide sequence is unknown, as with de novo sequencing, and especially when multiple boronic acid moieties are present. Thus, a rapid, reliable and simple method for peptide identification is of utmost importance. Herein, we report the identification and sequencing of linear and branched peptide boronic acids containing up to five boronic acid groups by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Protocols for preparation of pinacol boronic esters were adapted for efficient MALDI analysis of peptides. Additionally, a novel peptide boronic acid detection strategy was developed in which 2,5-dihydroxybenzoic acid (DHB) served as both matrix and derivatizing agent in a convenient, in situ, on-plate esterification. Finally, we demonstrate that DHB-modified peptide boronic acids from a single bead can be analyzed by MALDI-MSMS analysis, validating our approach for the identification and sequencing of branched peptide boronic acid libraries. PMID:21449540

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

    PubMed

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

    2013-12-28

    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 0.2-4mM octreotide or leuprolide acetate salts in a 0.1M HEPES buffer, pH7.4, with polymer particles or films at 4-37°C for 24h. The extent of absorption/loading of peptides in PLGA particles/films was assayed by two-phase extraction and amino acid analysis. Confocal Raman microspectroscopy, stimulated Raman scattering (SRS) and laser scanning confocal imaging, and microtome sectioning techniques were used to examine peptide penetration into 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 also can be 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.

  18. 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

  19. Molecular modeling in the design of peptidomimetics and peptide surrogates.

    PubMed

    Perez, Juan J; Corcho, Francesc; Llorens, Oriol

    2002-12-01

    The most important natural sources of new leads are plant extracts, bacterial broths, animal venoms and peptides isolated from living organisms. However, only the three first have been used extensively in the development of new therapeutic agents. This is probably due to the low pharmacological profile exhibited by peptides, that requires a lengthy transformation to make them suitable as new leads. In contrast, bioactive compounds isolated from the other sources are regularly closer to be used as lead compounds. Nevertheless, the sources for compounds of this category are nowadays scarce. In contrast, there are new bioactive peptides discovered quite often and reported as ligands for different receptors. Under these circumstances peptides appear as an attractive source of prospective new leads. In order to reduce the time involved in the design of a potential lead from a peptide, molecular modeling tools have been developed in the last few years. The purpose of the present work is to review the different techniques available and to report various successful examples of design of new peptidomimetics published in the literature.

  20. 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

  1. [Cellular delivery of modified peptide nucleic acids: a review].

    PubMed

    Liu, Chundong; Wang, Jianhua; Zeng, Fang

    2016-03-01

    Peptide nucleic acid (PNA) is a DNA surrogate in which the phosphate deoxyribose backbone of DNA is replaced by repeating N-(2-aminoethyl)glycine units. PNA can hybridize to the complementary DNA and RNA with higher affinity than their oligonucleotide counterparts. This character of PNA not only makes it a new tool for the studies of molecular biology but also the potential candidate for gene-targeting drugs. The non-ionic backbone of PNA leads to stable hybrids with the nucleic acids, but at the same time, the neutral backbone results in poor cellular uptake. To address this problem, studies on modified PNA progress rapidly in recent years. We reviewed literature reports combined with our study about the delivery methods, including backbone modified PNA and PNA-ligand conjugates, and the cellular uptake of modified PNA. In addition, we summarized the problems and future prospect of the cellular delivery of modified PNA.

  2. Peptide nucleic acid films and capsules: assembly and enzymatic degradation.

    PubMed

    Becker, Alisa L; Johnston, Angus P R; Caruso, Frank

    2010-05-14

    Sequence-directed hybridization of nucleic acids provides a high level of control for the bottom-up assembly of nanostructured materials. Altering the DNA sequence affords control and versatility over the film structure, but is limited by the chemical and physical properties of DNA. Here, we use DNA analogues, peptide nucleic acids (PNAs), to introduce new properties to multilayered thin films and retain the advantages of sequence-directed assembly. Thin films, formed by the layer-by-layer (LbL) assembly of PNA strands, were assembled from short PNA sequences on planar and colloidal substrates. In the case of PNA-coated particles, hollow capsules were obtained following removal of the sacrificial particle template. The PNA films were stable to both nuclease and protease degradation, and the nuclease degradation rate could be tuned by varying the amount of DNA incorporated into the films. These thin films may find use in biomedical applications.

  3. 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.

  4. Improving surface functional properties of tofu whey-derived peptides by chemical modification with fatty acids.

    PubMed

    Matemu, Athanasia Oswald; Katayama, Shigeru; Kayahara, Hisataka; Murasawa, Hisashi; Nakamura, Soichiro

    2012-04-01

    Effect of acylation with saturated fatty acids on surface functional properties of tofu whey-derived peptides was investigated. Tofu whey (TW) and soy proteins (7S, 11S, and acid-precipitated soy protein [APP]) were hydrolyzed by Protease M 'Amano' G, and resulting peptide mixtures were acylated with esterified fatty acids of different chain length (6C to 18C) to form a covalent linkage between the carboxyl group of fatty acid and the free amino groups of peptide. Acylation significantly (P < 0.05) increased emulsifying properties of 7S, 11S, and APP peptides independent of fatty acid chain length. Acylation decreased water binding capacity although oil binding capacity of acylated tofu whey ultra filtered fraction (UFTW < 3 kDa), 7S- and 11S-peptides were improved compared to native peptides. 7S peptides acylated with long chain fatty acids had shown significant higher surface hydrophobicity as in contrast with acylated UFTW < 3 kDa and APP peptides. Fluorescence spectra studies revealed structural conformation of acylated soy peptides as compared to native peptides. This study shows that chemical modification with fatty acids can further affect functional properties of soy proteins.

  5. Design of Responsive Peptide-based Hydrogels as Therapeutics

    NASA Astrophysics Data System (ADS)

    Schneider, Joel

    2008-03-01

    Hydrogels composed of self-assembled peptides have been designed to allow minimally invasive delivery of cells in-vivo. These peptides undergo sol-gel phase transitions in response to biological media enabling the three-dimensional encapsulation of cells. Peptides are designed such that when dissolved in aqueous solution, exist in an ensemble of random coil conformations rendering them fully soluble. The addition of an exogenous stimulus results in peptide folding into beta-hairpin conformation. This folded structure undergoes rapid self-assembly into a highly crosslinked hydrogel network whose nanostructure is defined and controllable. This mechanism, which links intramolecular peptide folding to self-assembly, allows temporally resolved material formation. In general, peptides can be designed to fold and assemble affording hydrogel in response to changes in pH or ionic strength, the addition of heat or even light. In addition to these stimuli, DMEM cell culture media is able to initiate folding and consequent self-assembly. DMEM-induced gels are cytocompatible towards NIH 3T3 murine fibroblasts, mesenchymal stem cells, hepatocytes, osteoblasts and chondrocytes. As an added bonus, many of these hydrogels possess broad spectrum antibacterial activity suggesting that adventitious bacterial infections that may occur during surgical manipulations and after implantation can be greatly reduced. Lastly, when hydrogelation is triggered in the presence of cells, gels become impregnated and can serve as a delivery vehicle. A unique characteristic of these gels is that when an appropriate shear stress is applied, the gel will shear-thin, becoming an injectable low viscosity gel. However, after the application of shear has stopped, the material quickly self-heals producing a gel with mechanical rigidity nearly identical to the original hydrogel. This attribute allows cell-impregnated gels to be delivered to target tissues via syringe where they quickly recover complementing

  6. 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

  7. 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.

  8. 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

  9. 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.

  10. Metal Stabilization of Collagen and de Novo Designed Mimetic Peptides.

    PubMed

    Parmar, Avanish S; Xu, Fei; Pike, Douglas H; Belure, Sandeep V; Hasan, Nida F; Drzewiecki, Kathryn E; Shreiber, David I; Nanda, Vikas

    2015-08-18

    We explore the design of metal binding sites to modulate triple-helix stability of collagen and collagen-mimetic peptides. Globular proteins commonly utilize metals to connect tertiary structural elements that are well separated in sequence, constraining structure and enhancing stability. It is more challenging to engineer structural metals into fibrous protein scaffolds, which lack the extensive tertiary contacts seen in globular proteins. In the collagen triple helix, the structural adjacency of the carboxy-termini of the three chains makes this region an attractive target for introducing metal binding sites. We engineered His3 sites based on structural modeling constraints into a series of designed homotrimeric and heterotrimeric peptides, assessing the capacity of metal binding to improve stability and in the case of heterotrimers, affect specificity of assembly. Notable enhancements in stability for both homo- and heteromeric systems were observed upon addition of zinc(II) and several other metal ions only when all three histidine ligands were present. Metal binding affinities were consistent with the expected Irving-Williams series for imidazole. Unlike other metals tested, copper(II) also bound to peptides lacking histidine ligands. Acetylation of the peptide N-termini prevented copper binding, indicating proline backbone amide metal-coordination at this site. Copper similarly stabilized animal extracted Type I collagen in a metal-specific fashion, highlighting the potential importance of metal homeostasis within the extracellular matrix.

  11. 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

  12. 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

  13. Design of Cyclic Peptides That Bind Protein Surfaces with Antibody-Like Affinity

    PubMed Central

    Millward, Steven W.; Fiacco, Stephen; Austin, Ryan J.; Roberts, Richard W.

    2012-01-01

    There is a pressing need for new molecular tools to target protein surfaces with high affinity and specificity. Here, we describe cyclic messenger RNA display with a trillion-member covalent peptide macrocycle library. Using this library, we have designed a number of high-affinity, redox-insensitive, cyclic peptides that target the signaling protein Gαi1. In addition to cyclization, our library construction took advantage of an expanded genetic code, utilizing nonsense suppression to insert N-methylphenylalanine as a 21st amino acid. The designed macrocycles exhibit several intriguing features. First, the core motif seen in all of the selected variants is the same and shares an identical context with respect to the macrocyclic scaffold, consistent with the idea that selection simultaneously optimizes both the cyclization chemistry and the structural placement of the binding epitope. Second, detailed characterization of one molecule, cyclic Gαi binding peptide (cycGiBP), demonstrates substantially enhanced proteolytic stability relative to that of the parent linear molecule. Third and perhaps most important, the cycGiBP peptide binds the target with very high affinity (Ki ≈ 2.1 nM), similar to those of many of the best monoclonal antibodies and higher than that of the βγ heterodimer, an endogenous Gαi1 ligand. Overall the work provides a general route to design novel, low-molecular-weight, high-affinity ligands that target protein surfaces. PMID:17894440

  14. Design of cyclic peptides that bind protein surfaces with antibody-like affinity.

    PubMed

    Millward, Steven W; Fiacco, Stephen; Austin, Ryan J; Roberts, Richard W

    2007-09-21

    There is a pressing need for new molecular tools to target protein surfaces with high affinity and specificity. Here, we describe cyclic messenger RNA display with a trillion-member covalent peptide macrocycle library. Using this library, we have designed a number of high-affinity, redox-insensitive, cyclic peptides that target the signaling protein G alpha i1. In addition to cyclization, our library construction took advantage of an expanded genetic code, utilizing nonsense suppression to insert N-methylphenylalanine as a 21st amino acid. The designed macrocycles exhibit several intriguing features. First, the core motif seen in all of the selected variants is the same and shares an identical context with respect to the macrocyclic scaffold, consistent with the idea that selection simultaneously optimizes both the cyclization chemistry and the structural placement of the binding epitope. Second, detailed characterization of one molecule, cyclic G alpha i binding peptide (cycGiBP), demonstrates substantially enhanced proteolytic stability relative to that of the parent linear molecule. Third and perhaps most important, the cycGiBP peptide binds the target with very high affinity ( K i approximately 2.1 nM), similar to those of many of the best monoclonal antibodies and higher than that of the betagamma heterodimer, an endogenous G alpha i1 ligand. Overall the work provides a general route to design novel, low-molecular-weight, high-affinity ligands that target protein surfaces.

  15. New mechanisms that regulate Saccharomyces cerevisiae short peptide transporter achieve balanced intracellular amino acid concentrations.

    PubMed

    Melnykov, Artem V

    2016-01-01

    The budding yeast Saccharomyces cerevisiae is able to take up large quantities of amino acids in the form of di- and tripeptides via a short peptide transporter, Ptr2p. It is known that PTR2 can be induced by certain peptides and amino acids, and the mechanisms governing this upregulation are understood at the molecular level. We describe two new opposing mechanisms of regulation that emphasize potential toxicity of amino acids: the first is upregulation of PTR2 in a population of cells, caused by amino acid secretion that accompanies peptide uptake; the second is loss of Ptr2p activity, due to transporter internalization following peptide uptake. Our findings emphasize the importance of proper amino acid balance in the cell and extend understanding of peptide import regulation in yeast.

  16. 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.

  17. 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

  18. 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.

  19. 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

  20. 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.

  1. The amino-acid sequences of sculpin islet somatostatin-28 and peptide YY.

    PubMed

    Cutfield, S M; Carne, A; Cutfield, J F

    1987-04-01

    Two pancreatic peptides, somatostatin-28 and peptide YY, have been isolated from the Brockmann bodies of the teleost fish Cottus scorpius (daddy sculpin). Following purification by reverse-phase HPLC, each peptide was sequenced completely through to the carboxyl-terminus by gas-phase Edman degradation. Somatostatin-28 was the major form of somatostatin detected and is similar to the gene II product from anglerfish. Peptide YY (36 amino acids) more closely resembles porcine neuropeptide YY and intestinal peptide YY than it does the pancreatic polypeptides. PMID:2883025

  2. 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

  3. Bioprocess-centered molecular design (BMD) for the efficient production of an interfacially active peptide.

    PubMed

    Morreale, Giacomo; Lee, Eun Gyo; Jones, Daniel B; Middelberg, Anton P J

    2004-09-30

    The efficient expression and purification of an interfacially active peptide (mLac21) was achieved by using bioprocess-centered molecular design (BMD), wherein key bioprocess considerations are addressed during the initial molecular biology work. The 21 amino acid mLac21 peptide sequence is derived from the lac repressor protein and is shown to have high affinity for the oil-water interface, causing a substantial reduction in interfacial tension following adsorption. The DNA coding for the peptide sequence was cloned into a modified pET-31(b) vector to permit the expression of mLac21 as a fusion to ketosteroid isomerase (KSI). Rational iterative molecular design, taking into account the need for a scaleable bioprocess flowsheet, led to a simple and efficient bioprocess yielding mLac21 at 86% purity following ion exchange chromatography (and >98% following chromatographic polishing). This case study demonstrates that it is possible to produce acceptably pure peptide for potential commodity applications using common scaleable bioprocess unit operations. Moreover, it is shown that BMD is a powerful strategy that can be deployed to reduce bioseparation complexity.

  4. 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.

  5. Mutual Amino Acid Catalysis in Salt-Induced Peptide Formation Supports this Mechanism's Role in Prebiotic Peptide Evolution

    NASA Astrophysics Data System (ADS)

    Suwannachot, Yuttana; Rode, Bernd M.

    1999-10-01

    The presence of some amino acids and dipeptides under the conditions of the salt-induced peptide formation reaction (aqueous solution at 85 °C, Cu(II) and NaCl) has been found to catalyze the formation of homopeptides of other amino acids, which are otherwise produced only in traces or not at all by this reaction. The condensation of Val, Leu and Lys to form their homodipeptides can occur to a considerable extent due to catalytic effects of other amino acids and related compounds, among which glycine, histidine, diglycine and diketopiperazine exhibit the most remarkable activity. These findings also lead to a modification of the table of amino acid sequences preferentially formed by the salt-induced peptide formation (SIPF) reaction, previously used for a comparison with the sequence preferences in membrane proteins of primitive organisms

  6. Peptide immobilization onto radiation grafted PVDF-g-poly(acrylic acid) films

    NASA Astrophysics Data System (ADS)

    Clochard, M.-C.; Betz, N.; Goncalves, M.; Bittencourt, C.; Pireaux, J.-J.; Gionnet, K.; Déléris, G.; Moël, A. Le

    2005-07-01

    Introducing hydrophilic functions on poly(vinylidene fluoride) (PVDF) films surface allows the covalent immobilization of peptides. Therefore radiation grafting of acrylic acid (AA) in pre-irradiated PVDF films was achieved to allow surface functionalization with linear and cyclic peptides. Peptides were bound via spacer molecules using EDC as a coupling agent. The reactions were followed by Fourier Transform Infrared (FTIR) spectroscopy in attenuated total reflection (ATR) mode. The amount of immobilized peptides was determined by UV spectroscopy. As well, an uncommon method for PVDF characterization and reactions quantification was used: high-resolution-magic angle spinning nuclear mass spectroscopy (HR-MAS NMR). Spacer saturation of the film surface corresponded to 25 mol% yield meaning that one spacer on 4 carboxylic acids was covalently bound. XPS experiments were also performed to deepen analysis of the surface composition. Peptide density is governed by steric hindrance. ELISA tests showed that the peptides' activity is maintained.

  7. Convergent evolution-guided design of antimicrobial peptides derived from influenza A virus hemagglutinin.

    PubMed

    Zhu, Shunyi; Aumelas, André; Gao, Bin

    2011-02-24

    Antimicrobial activity and solution structures of four 13-amino acid peptides derived from the fusion domain of viral hemagglutinin proteins are presented. The results show that carboxyl-terminal amidation is a key factor to switch a viral fusion domain-derived sequence into an antimicrobial peptide. Optimization of amphiphilic balance on the amidated analogue largely improves efficacy and enlarges antimicrobial spectra of these peptides. Our work indicates that viral fusion domains have potential to be engineered into potent antimicrobial peptides.

  8. Self-assembling properties of all γ-cyclic peptides containing sugar amino acid residues.

    PubMed

    Guerra, Arcadio; Brea, Roberto J; Amorín, Manuel; Castedo, Luis; Granja, Juan R

    2012-11-28

    In this study, a novel dimer-forming cyclic peptide composed exclusively by cyclic γ-amino acids with a saccharide-like outer surface is described. The antiparallel β-sheet type hydrogen bonding interactions responsible for the large association constant in non-polar solvents constitute a suitable model for a novel class of self-assembling peptide nanotubes.

  9. Self-assembling properties of all γ-cyclic peptides containing sugar amino acid residues.

    PubMed

    Guerra, Arcadio; Brea, Roberto J; Amorín, Manuel; Castedo, Luis; Granja, Juan R

    2012-11-28

    In this study, a novel dimer-forming cyclic peptide composed exclusively by cyclic γ-amino acids with a saccharide-like outer surface is described. The antiparallel β-sheet type hydrogen bonding interactions responsible for the large association constant in non-polar solvents constitute a suitable model for a novel class of self-assembling peptide nanotubes. PMID:23060041

  10. 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.

  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. Controlling resistant bacteria with a novel class of β-lactamase inhibitor peptides: from rational design to in vivo analyses

    PubMed Central

    Mandal, Santi M.; Migliolo, Ludovico; Silva, Osmar N.; Fensterseifer, Isabel C. M.; Faria-Junior, Celio; Dias, Simoni C.; Basak, Amit; Hazra, Tapas K.; Franco, Octávio L.

    2014-01-01

    Peptide rational design was used here to guide the creation of two novel short β-lactamase inhibitors, here named dBLIP-1 and -2, with length of five amino acid residues. Molecular modeling associated with peptide synthesis improved bactericidal efficacy in addition to amoxicillin, ampicillin and cefotaxime. Docked structures were consistent with calorimetric analyses against bacterial β-lactamases. These two compounds were further tested in mice. Whereas commercial antibiotics alone failed to cure mice infected with Staphylococcus aureus and Escherichia coli expressing β-lactamases, infection was cleared when treated with antibiotics in combination with dBLIPs, clearly suggesting that peptides were able to neutralize bacterial resistance. Moreover, immunological assays were also performed showing that dBLIPs were unable to modify mammalian immune response in both models, reducing the risks of collateral effects. In summary, the unusual peptides here described provide leads to overcome β-lactamase-based resistance, a remarkable clinical challenge. PMID:25109311

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

    PubMed

    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.

  14. 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.

  15. 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

  16. Effective delivery of a rationally designed intracellular peptide drug with gold nanoparticle-peptide hybrids

    NASA Astrophysics Data System (ADS)

    Lee, Daiyoon; Zhao, Jinbo; Yang, Hong; Xu, Shuyun; Kim, Hyunhee; Pacheco, Shaun; Keshavjee, Shaf; Liu, Mingyao

    2015-07-01

    A novel gold nanoparticle-peptide hybrid strategy was developed to intracellularly deliver a potent PKCδ inhibitor peptide for the treatment of acute lung injury. The gold nanoparticle-peptide hybrids showed good stability with high uptake, and demonstrated in vitro and in vivo efficacy. Our formulation strategy shows great promise in intracellular delivery of peptides.A novel gold nanoparticle-peptide hybrid strategy was developed to intracellularly deliver a potent PKCδ inhibitor peptide for the treatment of acute lung injury. The gold nanoparticle-peptide hybrids showed good stability with high uptake, and demonstrated in vitro and in vivo efficacy. Our formulation strategy shows great promise in intracellular delivery of peptides. Electronic supplementary information (ESI) available: Materials and methods section and additional experiments to support the results in the main text. See DOI: 10.1039/c5nr02377g

  17. β-Amino acids containing peptides and click-cyclized peptide as β-turn mimics: a comparative study with 'conventional' lactam- and disulfide-bridged hexapeptides.

    PubMed

    Larregola, Maud; Lequin, Olivier; Karoyan, Philippe; Guianvarc'h, Dominique; Lavielle, Solange

    2011-09-01

    The increasing interest in click chemistry and its use to stabilize turn structures led us to compare the propensity for β-turn stabilization of different analogs designed as mimics of the β-turn structure found in tendamistat. The β-turn conformation of linear β-amino acid-containing peptides and triazole-cyclized analogs were compared to 'conventional' lactam- and disulfide-bridged hexapeptide analogs. Their 3D structures and their propensity to fold in β-turns in solution, and for those not structured in solution in the presence of α-amylase, were analyzed by NMR spectroscopy and by restrained molecular dynamics with energy minimization. The linear tetrapeptide Ac-Ser-Trp-Arg-Tyr-NH(2) and both the amide bond-cyclized, c[Pro-Ser-Trp-Arg-Tyr-D-Ala] and the disulfide-bridged, Ac-c[Cys-Ser-Trp-Arg-Tyr-Cys]-NH(2) hexapeptides adopt dominantly in solution a β-turn conformation closely related to the one observed in tendamistat. On the contrary, the β-amino acid-containing peptides such as Ac-(R)-β(3) -hSer-(S)-Trp-(S)-β(3) -hArg-(S)-β(3) -hTyr-NH(2) , and the triazole cyclic peptide, c[Lys-Ser-Trp-Arg-Tyr-βtA]-NH(2) , both specifically designed to mimic this β-turn, do not adopt stable structures in solution and do not show any characteristics of β-turn conformation. However, these unstructured peptides specifically interact in the active site of α-amylase, as shown by TrNOESY and saturation transfer difference NMR experiments performed in the presence of the enzyme, and are displaced by acarbose, a specific α-amylase inhibitor. Thus, in contrast to amide-cyclized or disulfide-bridged hexapeptides, β-amino acid-containing peptides and click-cyclized peptides may not be regarded as β-turn stabilizers, but can be considered as potential β-turn inducers.

  18. Probing the Efficacy of Peptide-Based Inhibitors against Acid- and Zinc-Promoted Oligomerization of Amyloid-β Peptide via Single-Oligomer Spectroscopy

    PubMed Central

    Powell, Lyndsey R.; Dukes, Kyle D.; Lammi, Robin K.

    2011-01-01

    One avenue for prevention and treatment of Alzheimer's disease involves inhibiting the aggregation of amyloid-β peptide (Aβ). Given the deleterious effects reported for Aβ dimers and trimers, it is important to investigate inhibition of the earliest association steps. We have employed quantized photobleaching of dye-labeled Aβ peptides to characterize four peptide-based inhibitors of fibrillogenesis and/or cytotoxicity, assessing their ability to inhibit association in the smallest oligomers (n = 2–5). Inhibitors were tested at acidic pH and in the presence of zinc, conditions that may promote oligomerization in vivo. Distributions of peptide species were constructed by examining dozens of surface-tethered monomers and oligomers, one at a time. Results show that all four inhibitors shift the distribution of Aβ species toward monomers; however, efficacies vary for each compound and sample environment. Collectively, these studies highlight promising design strategies for future oligomerization inhibitors, affording insight into oligomer structures and inhibition mechanisms in two physiologically significant environments. PMID:21945664

  19. 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.

  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. 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.

  2. Bioplex technology: novel synthetic gene delivery pharmaceutical based on peptides anchored to nucleic acids.

    PubMed

    Simonson, Oscar E; Svahn, Mathias G; Törnquist, Elisabeth; Lundin, Karin E; Smith, C I E

    2005-01-01

    Non-viral gene delivery is an important approach in order to establish safe in vivo gene therapy in the clinic. Although viral vectors currently exhibit superior gene transfer efficacy, the safety aspect of viral gene delivery is a concern. In order to improve non-viral in vivo gene delivery we have designed a pharmaceutical platform called Bioplex (biological complex). The concept of Bioplex is to link functional entities via hybridising anchors, such as Peptide Nucleic Acids (PNA), directly to naked DNA. In order to promote delivery functional entities consisting of biologically active peptides or carbohydrates, are linked to the PNA anchor. The PNA acts as genetic glue and hybridises with DNA in a sequence specific manner. By using functional entities, which elicit receptor-mediated endocytosis, improved endosomal escape and enhance nuclear entry we wish to improve the transfer of genetic material into the cell. An important aspect is that the functional entities should also have tissue-targeting properties in vivo. Examples of functional entities investigated to date are the Simian virus 40 nuclear localisation signal to improve nuclear uptake and different carbohydrate ligands in order to achieve receptor specific uptake. The delivery system is also endowed with regulatory capability, since the release of functional entities can be controlled. The aim is to create a safe, pharmaceutically defined and stable delivery system for nucleic acids with enhanced transfection properties that can be used in the clinic.

  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. 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

  6. 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.

  7. 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

  8. 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.

  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. 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

  11. 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

  12. Designing injectable beta-hairpin peptide hydrogels for cartilage tissue engineering application

    NASA Astrophysics Data System (ADS)

    Sinthuvanich, Chomdao

    In this work, it was demonstrated that peptide-based gels having different electrostatic network character but similar mechanical properties can be designed by modulating the primary sequence of the peptides used for self-assembly. As a result, HLT2 and HET1 peptides, having formal charge states of +5 per monomer, were designed using MAX8, a peptide with a charge state of +7 per monomer, as a template. Using gels prepared from all three peptides (MAX8, HLT2, and HET1), it was shown that the electropositive character of the network influences chondrocyte behavior. Specifically, the less electropositive gel (HLT2) is able to maintain chondrocyte viability and phenotype. In contrast, chondrocytes encapsulated in the more positively charged gel (MAX8) are more prone to dedifferentiation, resulting in tissue constructs with inferior mechanical properties. Gels prepared from peptides having the same net charge but differing only in their primary sequences (HLT2 and HET1) were also shown to influence cell behavior, but only during the early period of culturing. If constructs derived from these two different peptide gels are allowed to culture for extended times, their mechanical properties become similar. This suggests that the amino acid composition and sequence of the peptides used to make the gels also influences cell behavior, but perhaps not to the extent that network electrostatics plays. Supplementation of bioactive factors in the culturing media, as opposed to being encapsulated directly in the network, was shown to adversely affect the cellular response resulting in tissue constructs where extracellular matrix (ECM) components are non-uniformly distributed. When bioactive factors were encapsulated and co-delivered with cells, positive results were observed, particularly when cells were co-encapsulated with the growth factor, TGF-β1. The effect of TGF-β1 on cellular response and the mechanical properties of the tissue-engineered constructs is largely governed by

  13. Radical stability directs electron capture and transfer dissociation of β-amino acids in peptides.

    PubMed

    Ben Hamidane, Hisham; Vorobyev, Aleksey; Larregola, Maud; Lukaszuk, Aneta; Tourwé, Dirk; Lavielle, Solange; Karoyan, Philippe; Tsybin, Yury O

    2010-04-19

    We report on the characteristics of the radical-ion-driven dissociation of a diverse array of β-amino acids incorporated into α-peptides, as probed by tandem electron-capture and electron-transfer dissociation (ECD/ETD) mass spectrometry. The reported results demonstrate a stronger ECD/ETD dependence on the nature of the amino acid side chain for β-amino acids than for their α-form counterparts. In particular, only aromatic (e.g., β-Phe), and to a substantially lower extent, carbonyl-containing (e.g., β-Glu and β-Gln) amino acid side chains, lead to N-Cβ bond cleavage in the corresponding β-amino acids. We conclude that radical stabilization must be provided by the side chain to enable the radical-driven fragmentation from the nearby backbone carbonyl carbon to proceed. In contrast with the cleavage of backbones derived from α-amino acids, ECD of peptides composed mainly of β-amino acids reveals a shift in cleavage priority from the N-Cβ to the Cα-C bond. The incorporation of CH2 groups into the peptide backbone may thus drastically influence the backbone charge solvation preference. The characteristics of radical-driven β-amino acid dissociation described herein are of particular importance to methods development, applications in peptide sequencing, and peptide and protein modification (e.g., deamidation and isomerization) analysis in life science research.

  14. Proteolytically stable peptides by incorporation of alpha-Tfm amino acids.

    PubMed

    Koksch, B; Sewald, N; Hofmann, H J; Burger, K; Jakubke, H D

    1997-01-01

    A series of model peptides containing alpha-trifluoromethyl-substituted amino acids in five different positions relative to the predominant cleavage site of the serine protease alpha-chymotrypsin was synthesized by solution methods to investigate the influence of alpha-Tfm substitution on the proteolytic stability of peptides. Proteolysis studies demonstrated absolute stability of peptides substituted to the P1 position and still considerable proteolytic stability for peptides substituted at the P2 and P'2 positions compared with the corresponding unsubstituted model peptide. Comparison with peptides containing the fluorine-free disubstituted amino acid alpha-aminoisobutyric acid allowed to separate electronic from steric effects. Furthermore, the absolute configuration of the alpha-Tfm-substituted amino acid was found to exert considerable effects on the proteolytic stability, especially in P'1 substituted peptides. Investigations of this phenomenon using empirical force field calculations revealed that in the (S,R,S)-diasteromer the steric constraints exhibited by the alpha-Tfm group can be outweighed by an advantageous interaction of the flourine atoms with the serine side chain of the enzyme. In contrast, a favourable interaction between substrate and enzyme is impossible for the (S,S,S)-diastereomer. PMID:9230481

  15. Discriminating D-amino acid-containing peptide epimers by radical-directed dissociation mass spectrometry.

    PubMed

    Tao, Yuanqi; Quebbemann, Neil R; Julian, Ryan R

    2012-08-01

    The presence of a single D-amino acid in a peptide is very difficult to detect. Mass spectrometry-based approaches rely on differences in fragmentation between all L-amino acid-containing peptides and single D-amino acid-containing peptides (which are epimers) for identification. The success of this approach is dependent on the structural sensitivity of the fragmentation method. Recently, experiments have demonstrated that fragmentation initiated by radical chemistry, or radical-directed dissociation (RDD), is particularly sensitive to the structure of the ion being fragmented. Herein, RDD is used to identify the presence of D-serine, D-alanine, or D-aspartic acid in eight biologically relevant peptides. It is demonstrated that chiral disambiguation by RDD is dependent on both the initial radical site and subsequent radical migration. Fortuitously, RDD can be initiated by a variety of different radical precursors which can be associated with the peptide via covalent or noncovalent means, and RDD can be examined in all observable charge states (both positive and negative). This diversity enables numerous initial radical sites and migration pathways to be explored. For all but one of the peptides that were examined, RDD provides significantly better chiral discrimination than CID. Quantitation of peptide epimers by RDD is also described.

  16. Effect of N-terminal glutamic acid and glutamine on fragmentation of peptide ions.

    PubMed

    Godugu, Bhaskar; Neta, Pedatsur; Simón-Manso, Yamil; Stein, Stephen E

    2010-07-01

    A prominent dissociation path for electrospray generated tryptic peptide ions is the dissociation of the peptide bond linking the second and third residues from the amino-terminus. The formation of the resulting b(2) and y(n-2) fragments has been rationalized by specific facile mechanisms. An examination of spectral libraries shows that this path predominates in diprotonated peptides composed of 12 or fewer residues, with the notable exception of peptides containing glutamine or glutamic acid at the N-terminus. To elucidate the mechanism by which these amino acids affect peptide fragmentation, we synthesized peptides of varying size and composition and examined their MS/MS spectra as a function of collision voltage in a triple quadrupole mass spectrometer. Loss of water from N-terminal glutamic acid and glutamine is observed at a lower voltage than any other fragmentation, leading to cyclization of the terminal residue. This cyclization results in the conversion of the terminal amine group to an imide, which has a lower proton affinity. As a result, the second proton is not localized at the N-terminus but is readily transferred to other sites, leading to fragmentation near the center of the peptide. Further confirmation was obtained by examining peptides with N-terminal pyroglutamic acid and N-acetyl peptides. Peptides with N-terminal proline maintain the trend of forming b(2) and y(n-2) because their ring contains an imine rather than imide and has sufficient proton affinity to retain the proton at the N-terminus.

  17. 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.

  18. Between peptides and bile acids: self-assembly of phenylalanine substituted cholic acids.

    PubMed

    Travaglini, Leana; D'Annibale, Andrea; di Gregorio, Maria Chiara; Schillén, Karin; Olsson, Ulf; Sennato, Simona; Pavel, Nicolae V; Galantini, Luciano

    2013-08-01

    Biocompatible molecules that undergo self-assembly are of high importance in biological and medical applications of nanoscience. Peptides and bile acids are among the most investigated due to their ability to self-organize into many different, often stimuli-sensitive, supramolecular structures. With the aim of preparing molecules mixing the aggregation properties of bile acid and amino acid-based molecules, we report on the synthesis and self-association behavior of two diastereomers obtained by substituting a hydroxyl group of cholic acid with a l-phenylalanine residue. The obtained molecules are amphoteric, and we demonstrate that they show a pH-dependent self-assembly. Both molecules aggregate in globular micelles at high pH, whereas they form tubular superstructures under acid conditions. Unusual narrow nanotubes with outer and inner cross-section diameters of about 6 and 3 nm are formed by the derivatives. The diasteroisomer with α orientation of the substituent forms in addition a wider tubule (17 nm cross-section diameter). The ability to pack in supramolecular tubules is explained in terms of a wedge-shaped bola-form structure of the derivatives. Parallel or antiparallel face-to-face dimers are hypothesized as fundamental building blocks for the formation of the narrow and wide nanotubes, respectively.

  19. Serology in the Digital Age: Using Long Synthetic Peptides Created from Nucleic Acid Sequences as Antigens in Microarrays

    PubMed Central

    Rizwan, Muhammad; Rönnberg, Bengt; Cistjakovs, Maksims; Lundkvist, Åke; Pipkorn, Rudiger; Blomberg, Jonas

    2016-01-01

    Background: Antibodies to microbes, or to autoantigens, are important markers of disease. Antibody detection (serology) can reveal both past and recent infections. There is a great need for development of rational ways of detecting and quantifying antibodies, both for humans and animals. Traditionally, serology using synthetic antigens covers linear epitopes using up to 30 amino acid peptides. Methods: We here report that peptides of 100 amino acids or longer (“megapeptides”), designed and synthesized for optimal serological performance, can successfully be used as detection antigens in a suspension multiplex immunoassay (SMIA). Megapeptides can quickly be created just from pathogen sequences. A combination of rational sequencing and bioinformatic routines for definition of diagnostically-relevant antigens can, thus, rapidly yield efficient serological diagnostic tools for an emerging infectious pathogen. Results: We designed megapeptides using bioinformatics and viral genome sequences. These long peptides were tested as antigens for the presence of antibodies in human serum to the filo-, herpes-, and polyoma virus families in a multiplex microarray system. All of these virus families contain recently discovered or emerging infectious viruses. Conclusion: Long synthetic peptides can be useful as serological diagnostic antigens, serving as biomarkers, in suspension microarrays. PMID:27600087

  20. 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.

  1. Butelase-Mediated Macrocyclization of d-Amino-Acid-Containing Peptides.

    PubMed

    Nguyen, Giang K T; Hemu, Xinya; Quek, Jun-Ping; Tam, James P

    2016-10-01

    Macrocyclic compounds have received increasing attention in recent years. With their large surface area, they hold promise for inhibiting protein-protein interactions, a chemical space that was thought to be undruggable. Although many chemical methods have been developed for peptide macrocyclization, enzymatic methods have emerged as a promising new economical approach. Thus far, most enzymes have been shown to act on l-peptides; their ability to cyclize d-amino-acid-containing peptides has rarely been documented. Herein we show that macrocycles consisting of d-amino acids, except for the Asn residue at the ligating site, were efficiently synthesized by butelase 1, an Asn/Asp-specific ligase. Furthermore, by using a peptide-library approach, we show that butelase 1 tolerates most of the d-amino acid residues at the P1'' and P2'' positions. PMID:27624217

  2. Amino acids, peptides, and proteins as chemically bonded stationary phases--A review.

    PubMed

    Bocian, Szymon; Skoczylas, Magdalena; Buszewski, Bogusław

    2016-01-01

    The selectivity of chromatographic separation depends mostly on the stationary phase and mobile phase composition. Despite being a material with bonded simple organic molecule, a wide group of stationary phases contain immobilized compound that possesses biological activity. Stationary phases that contain amino acids and peptides as well as enzymes and proteins are alternative materials that may be used for liquid chromatographic separations and are reviewed in this work. In the case of peptide-bonded stationary phases, most of these types of materials were elaborated in the 1970s and 1980s; however, over the last few years a growing interest has been observed which is connected with hydrophilic interaction liquid chromatography. The most important application of amino acid and peptide-bonded stationary phases is connected with separation of amino acids, their derivatives, and peptides. The main advantage of such materials is the ability for chiral separations.

  3. 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.

  4. 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

  5. Incorporation of extra amino acids in peptide recognition probe to improve specificity and selectivity of an electrochemical peptide-based sensor.

    PubMed

    Zaitouna, Anita J; Maben, Alex J; Lai, Rebecca Y

    2015-07-30

    We investigated the effect of incorporating extra amino acids (AA) at the n-terminus of the thiolated and methylene blue-modified peptide probe on both specificity and selectivity of an electrochemical peptide-based (E-PB) HIV sensor. The addition of a flexible (SG)3 hexapeptide is, in particular, useful in improving sensor selectivity, whereas the addition of a highly hydrophilic (EK)3 hexapeptide has shown to be effective in enhancing sensor specificity. Overall, both E-PB sensors fabricated using peptide probes with the added AA (SG-EAA and EK-EAA) showed better specificity and selectivity, especially when compared to the sensor fabricated using a peptide probe without the extra AA (EAA). For example, the selectivity factor recorded in the 50% saliva was ∼2.5 for the EAA sensor, whereas the selectivity factor was 7.8 for both the SG-EAA and EK-EAA sensors. Other sensor properties such as the limit of detection and dynamic range were minimally affected by the addition of the six AA sequence. The limit of detection was 0.5 nM for the EAA sensor and 1 nM for both SG-EAA and EK-EAA sensors. The saturation target concentration was ∼200 nM for all three sensors. Unlike previously reported E-PB HIV sensors, the peptide probe functions as both the recognition element and antifouling passivating agent; this modification eliminates the need to include an additional antifouling diluent, which simplifies the sensor design and fabrication protocol.

  6. 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

  7. Rational Design of Cancer-Targeted Benzoselenadiazole by RGD Peptide Functionalization for Cancer Theranostics.

    PubMed

    Yang, Liye; Li, Wenying; Huang, Yanyu; Zhou, Yangliang; Chen, Tianfeng

    2015-09-01

    A cancer-targeted conjugate of the selenadiazole derivative BSeC (benzo[1,2,5] selenadiazole-5-carboxylic acid) with RGD peptide as targeting molecule and PEI (polyethylenimine) as a linker is rationally designed and synthesized in the present study. The results show that RGD-PEI-BSeC forms nanoparticles in aqueous solution with a core-shell nanostructure and high stability under physiological conditions. This rational design effectively enhances the selective cellular uptake and cellular retention of BSeC in human glioma cells, and increases its selectivity between cancer and normal cells. The nanoparticles enter the cells through receptor-mediated endocytosis via clathrin-mediated and nystatin-dependent lipid raft-mediated pathways. Internalized nanoparticles trigger glioma cell apoptosis by activation of ROS-mediated p53 phosphorylation. Therefore, this study provides a strategy for the rational design of selenium-containing cancer-targeted theranostics.

  8. 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

  9. Design of cationic microspheres based on aminated gelatin for controlled release of peptide and protein drugs.

    PubMed

    Morimoto, Kazuhiro; Chono, Sumio; Kosai, Tadashi; Seki, Toshinobu; Tabata, Yasuhiko

    2008-02-01

    Two different types of cationized microspheres based on a native cationic gelatin (NGMS) and aminated gelatin with ethylendiamine (CGMS) were investigated for the controlled release of three model acidic peptide/protein drugs with different molecular weights (MWs) and isoelectric points (IEPs). Recombinant human (rh)-insulin (MW: 5.8 kDa, IEP: 5.3), bovine milk lactoalbumin, BMLA (MW: 14 kDa, IEP: 4.3), and bovine serum albumin (BSA MW: 67 kDa, IEP: 4.9) were used as model acidic peptide/protein drugs. The in vitro release profiles of these acidic peptide/protein drugs from NGMS and CGMS were compared and different periods of cross-linking were obtained. The slower release of these acidic peptide/protein drugs from CGMS compared with those from NGMS with cross-linking for 48 hr. was caused by the suppression of burst release during the initial phase. The degree of suppression of burst release of the three peptide/protein drugs during the initial phase by CGMS was in the following order: (rh)-insulin > BMLA > BSA. The release of insulin with a lower molecular weight from CGMS was particularly suppressed compared with the other two drugs with higher molecular weights in the initial phase. The control of the release rate of acidic peptide/protein drugs from gelatin microsphere can be achieved by amination of gelatin. Therefore, CGMS is useful for the controlled release of acidic peptide/ protein drugs.

  10. Protease-resistant peptide design-empowering nature's fragile warriors against HIV.

    PubMed

    Weinstock, Matthew T; Francis, J Nicholas; Redman, Joseph S; Kay, Michael S

    2012-01-01

    Peptides have great potential as therapeutic agents, but their use is often limited by susceptibility to proteolysis and their resulting in vivo fragility. In this review, we focus on peptidomimetic approaches to produce protease-resistant peptides with the potential for greatly improved clinical utility. We focus on the use of mirror-image (D-peptide) and ß-peptides as two leading approaches with distinct design principles and challenges. Application to the important and difficult problem of inhibiting HIV entry illustrates the current state-of-the-art in peptidomimetic technologies. We also summarize future directions for this field and highlight remaining obstacles to widespread use of protease-resistant peptides.

  11. 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.

  12. 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.

  13. Effect of environment on the free and peptide amino acids in rice, wheat, and soybeans.

    PubMed

    Ahn, D J; Adeola, O; Nielsen, S S

    2001-01-01

    Controlled environments (CE) in which light, carbon dioxide, and nutrients are regulated are known to affect the chemical composition of plants. Controlled Ecological Life Support System (CELSS) environments are required for a Mars or lunar base where food resupply is both impractical and risky. Astronauts in a CELSS would need to grow and process edible biomass into foods. The complete nature of the changes in chemical composition of CE-grown plants is unknown but must be determined to ensure a safe and nutritionally adequate diet. In this article, we report the changes that occur in free and peptide-bound amino acids (AA) of select CELSS crops (rice, wheat, and soybean) grown in the field or in CE. The nonnitrate nonprotein nitrogen fraction was extracted and then analyzed for free and peptide AA. For grain or seeds, AA levels tended to increase from field to CE conditions; however, for vegetative material, AA levels remained the same or decreased from field to CE conditions. As such compositional changes are identified, researchers will be better able to design safe and nutritious diets for astronauts while minimizing needed energy and other resources.

  14. Effect of environment on the free and peptide amino acids in rice, wheat, and soybeans.

    PubMed

    Ahn, D J; Adeola, O; Nielsen, S S

    2001-01-01

    Controlled environments (CE) in which light, carbon dioxide, and nutrients are regulated are known to affect the chemical composition of plants. Controlled Ecological Life Support System (CELSS) environments are required for a Mars or lunar base where food resupply is both impractical and risky. Astronauts in a CELSS would need to grow and process edible biomass into foods. The complete nature of the changes in chemical composition of CE-grown plants is unknown but must be determined to ensure a safe and nutritionally adequate diet. In this article, we report the changes that occur in free and peptide-bound amino acids (AA) of select CELSS crops (rice, wheat, and soybean) grown in the field or in CE. The nonnitrate nonprotein nitrogen fraction was extracted and then analyzed for free and peptide AA. For grain or seeds, AA levels tended to increase from field to CE conditions; however, for vegetative material, AA levels remained the same or decreased from field to CE conditions. As such compositional changes are identified, researchers will be better able to design safe and nutritious diets for astronauts while minimizing needed energy and other resources. PMID:11676459

  15. Use of Peptide Nucleic Acids to Manipulate Gene Expression in the Malaria Parasite Plasmodium falciparum

    PubMed Central

    Naik, Shankar; Yavin, Eylon; Dzikowski, Ron

    2014-01-01

    One of the major concerns in treating malaria by conventional small drug molecules is the rapid emergence of drug resistance. Specific silencing of essential genes by antisense oliogomers has been proposed as an alternative approach that may result in antimalarial activity which is not associated with drug resistance. In addition, such an approach could be an important biological tool for studying many genes' function by reverse genetics. Here we present a novel methodology of using peptide nucleic acids (PNAs) as a useful tool for gene silencing in Plasmodium falciparum. PNAs, designed as specific antisense molecules, were conjugated to a cell penetrating peptide (CPP); namely, octa-D-lysine via the C-terminus, to allow facile delivery through cell membranes. PNAs added to P. falciparum cultures were found exclusively in infected erythrocytes and were eventually localized in nuclei of the parasites at all stages of intra erythrocytic development. We show that these PNAs specifically down regulated both a stably expressed transgene as well as an endogenous essential gene, which significantly reduced parasites' viability. This study paves the way for a simple approach to silence a variety of P. falciparum genes as means of deciphering their function and potentially to develop highly specific and potent antimalarial agents. PMID:24466246

  16. 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.

  17. 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

  18. ATP selection in a random peptide library consisting of prebiotic amino acids.

    PubMed

    Kang, Shou-Kai; Chen, Bai-Xue; Tian, Tian; Jia, Xi-Shuai; Chu, Xin-Yi; Liu, Rong; Dong, Peng-Fei; Yang, Qing-Yong; Zhang, Hong-Yu

    2015-10-23

    Based upon many theoretical findings on protein evolution, we proposed a ligand-selection model for the origin of proteins, in which the most ancient proteins originated from ATP selection in a pool of random peptides. To test this ligand-selection model, we constructed a random peptide library consisting of 15 types of prebiotic amino acids and then used cDNA display to perform six rounds of in vitro selection with ATP. By means of next-generation sequencing, the most prevalent sequence was defined. Biochemical and biophysical characterization of the selected peptide showed that it was stable and foldable and had ATP-hydrolysis activity as well.

  19. Synthesis and application of acid labile anchor groups for the synthesis of peptide amides by Fmoc-solid-phase peptide synthesis.

    PubMed

    Breipohl, G; Knolle, J; Stüber, W

    1989-10-01

    The preparation and application of a new linker for the synthesis of peptide amides using a modified Fmoc-method is described. The new anchor group was developed based on our experience with 4,4'-dimethoxybenzhydryl (Mbh)-protecting group for amides. Lability towards acid treatment was increased dramatically and results in an easy cleavage procedure for the preparation of peptide amides. The synthesis of N-9-fluorenylmethoxycarbonyl- ([5-carboxylatoethyl-2.4-dimethoxyphenyl)- 4'-methoxyphenyl]-methylamin is reported in detail. This linker was coupled to a commercially available aminomethyl polystyrene resin. Peptide synthesis proceeded smoothly using HOOBt esters of Fmoc-amino acids. Release of the peptide amide and final cleavage of the side chain protecting groups was accomplished by treatment with trifluoroacetic acid-dichloromethane mixtures in the presence of scavengers. The synthesis of peptide amides such as LHRH and C-terminal hexapeptide of secretin are given as examples.

  20. 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.

  1. Predicting three-dimensional conformations of peptides constructed of only glycine, alanine, aspartic acid, and valine.

    PubMed

    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.

  2. HPLC monitoring of spontaneous non-linear peptidization dynamics of selected amino acids in solution.

    PubMed

    Godziek, Agnieszka; Maciejowska, Anna; Sajewicz, Mieczysław; Kowalska, Teresa

    2015-03-01

    This is our new study in a series of publications devoted to exploration of applicability of high-performance liquid chromatography (HPLC) to providing answers to difficult questions from the area of the reaction kinetics and mechanisms with non-linear reactions. Although an excellent analytical performance of HPLC is an indisputable fact, so far its performance as a tool in the kinetic and mechanistic studies has been tested to a lesser extent. In our earlier studies, spontaneous peptidization dynamics of amino acids in solution was demonstrated by means of HPLC upon a few amino acid examples, and on that basis a theoretical model has been developed, anticipating an interdependence of dynamics on chemical structures of amino acids involved. In order to expand the spectrum of experimentally investigated amino acid cases, in this study we present the results valid for three novel amino acids of significant life sciences importance, which differ in terms of peptidization dynamics. Experimental evidence originates from the achiral HPLC with the evaporative light scattering detection and MS detection. A conclusion is drawn that different spontaneous peptidization dynamics of amino acids may significantly influence chemical composition of proteins encountered in living organisms. Hence, a need emerges for systematic physicochemical studies on spontaneous non-linear peptidization dynamics of proteinogenic amino acids in liquid abiotic (but also in the biotic) systems.

  3. 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

  4. 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

  5. Disrupting protein expression with Peptide Nucleic Acids reduces infection by obligate intracellular Rickettsia.

    PubMed

    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.

  6. Epitope-based peptide vaccine design and target site depiction against Ebola viruses: an immunoinformatics study.

    PubMed

    Khan, M A; Hossain, M U; Rakib-Uz-Zaman, S M; Morshed, M N

    2015-07-01

    Ebola viruses (EBOVs) have been identified as an emerging threat in recent year as it causes severe haemorrhagic fever in human. Epitope-based vaccine design for EBOVs remains a top priority because a mere progress has been made in this regard. Another reason is the lack of antiviral drug and licensed vaccine although there is a severe outbreak in Central Africa. In this study, we aimed to design an epitope-based vaccine that can trigger a significant immune response as well as to prognosticate inhibitor that can bind with potential drug target sites using various immunoinformatics and docking simulation tools. The capacity to induce both humoral and cell-mediated immunity by T cell and B cell was checked for the selected protein. The peptide region spanning 9 amino acids from 42 to 50 and the sequence TLASIGTAF were found as the most potential B and T cell epitopes, respectively. This peptide could interact with 12 HLAs and showed high population coverage up to 80.99%. Using molecular docking, the epitope was further appraised for binding against HLA molecules to verify the binding cleft interaction. In addition with this, the allergenicity of the epitopes was also evaluated. In the post-therapeutic strategy, docking study of predicted 3D structure identified suitable therapeutic inhibitor against targeted protein. However, this computational epitope-based peptide vaccine designing and target site prediction against EBOVs open up a new horizon which may be the prospective way in Ebola viruses research; the results require validation by in vitro and in vivo experiments.

  7. 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

  8. Sequence-based design and discovery of peptide inhibitors of HIV-1 integrase: insight into the binding mode of the enzyme.

    PubMed

    Li, Hui-Yuan; Zawahir, Zahrah; Song, Lai-Dong; Long, Ya-Qiu; Neamati, Nouri

    2006-07-27

    Integration of viral DNA into the host chromosome is an essential step in the HIV life cycle. This process is mediated by integrase (IN), a 32 kDa viral enzyme that has no mammalian counterpart, rendering it an attractive target for antiviral drug design. Herein, we present a novel approach toward elucidating "hot spots" of protein-protein or protein-nucleic acid interactions of IN through the design of peptides that encompass conserved amino acids and residues known to be important for enzymatic activity. We designed small peptides (7-17 residues) containing at least one amino acid residue that is important for IN catalytic activities (3'-processing and strand transfer) or viral replication. All these peptides were synthesized on solid phase by fluorenylmethoxycarbonyl (Fmoc) chemistry and evaluated for their inhibition of IN catalytic activities. Such specific sites of interest (i.e., protein-DNA or protein-drug interactions) could potentially be used as drug targets. This novel "sequence walk" strategy across the entire 288 residues of IN has allowed the identification of two peptides NL-6 and NL-9 with 50% inhibitory concentration (IC50) values of 2.7 and 56 microM for strand transfer activity, respectively. Amino acid substitution analysis on these peptides revealed essential residues for activity, and the rational truncation of NL-6 produced a novel hexapeptide (peptide NL6-5) with inhibitory potency equal to that of the parent dodecapeptide (peptide NL-6). More significantly, the retroinverso analogue of NL-6 (peptide RDNL-6) in which the direction of the sequence is reversed and the chirality of each amino acid residue is inverted displayed improved inhibitory potency against 3'-processing of HIV-1 IN by 6-fold relative to the parent NL-6, serving as a metabolically stable derivative for further in vitro and in vivo analyses.

  9. Targeting pre-miRNA by Peptide Nucleic Acids

    PubMed Central

    Avitabile, Concetta; Saviano, Michele; D'Andrea, Luca; Bianchi, Nicoletta; Fabbri, Enrica; Brognara, Eleonora; Gambari, Roberto; Romanelli, Alessandra

    2012-01-01

    PNAs conjugated to carrier peptides have been employed for the targeting of miRNA precursor, with the aim to develop molecules able to interfere in the pre-miRNA processing. The capability of the molecules to bind pre-miRNA has been tested in vitro by fluorescence assayes on Thiazole Orange labeled molecules and in vivo, in K562 cells, evaluating the amount of miRNA produced after treatment of cells with two amounts of PNAs. PMID:22699795

  10. Design of Protein-Peptide Interaction Modules for Assembling Supramolecular Structures in Vivo and in Vitro.

    PubMed

    Speltz, Elizabeth B; Nathan, Aparna; Regan, Lynne

    2015-09-18

    Synthetic biology and protein origami both require protein building blocks that behave in a reliable, predictable fashion. In particular, we require protein interaction modules with known specificity and affinity. Here, we describe three designed TRAP (Tetratricopeptide Repeat Affinity Protein)-peptide interaction pairs that are functional in vivo. We show that each TRAP binds to its cognate peptide and exhibits low cross-reactivity with the peptides bound by the other TRAPs. In addition, we demonstrate that the TRAP-peptide interactions are functional in many cellular contexts. In extensions of these designs, we show that the binding affinity of a TRAP-peptide pair can be systematically varied. The TRAP-peptide pairs we present thus represent a powerful set of new building blocks that are suitable for a variety of applications.

  11. Intercalation of amino acids and peptides into Mg-Al layered double hydroxide by reconstruction method.

    PubMed

    Nakayama, Hirokazu; Wada, Natsuko; Tsuhako, Mitsutomo

    2004-01-28

    The intercalation of amino acids and some peptides into Mg-Al layered double hydroxide known as hydrotalcite was examined. Although the intercalation by ion-exchange method was unsuccessful, all the amino acids except for Lys and Arg, and peptides examined could be intercalated into the layered double hydroxide by reconstruction method using Mg-Al oxide precursor. The uptake amounts of amino acids and peptides were 0.9-2.7 mmol per 1 g of LDH. Intercalation compounds were examined by using XRD and solid-state NMR. For Gly, Ala, Ser, Thr, Pro, Asn, Gln, Asp, Glu, and aspartame the intercalation accompanied the expansion of interlayer distance of the solid products, whereas the other amino acids and oligoglycine showed no expansion. The intercalation mechanism and release profile in K(2)CO(3) aqueous solution were also investigated. And the cointercalation of amino acids and peptides into Mg-Al LDH and easy release of amino acids from the LDH layer were found.

  12. De novo design of self-assembled hexapeptides as β-amyloid (Aβ) peptide inhibitors.

    PubMed

    Wang, Qiuming; Liang, Guizhao; Zhang, Mingzhen; Zhao, Jun; Patel, Kunal; Yu, Xiang; Zhao, Chao; Ding, Binrong; Zhang, Ge; Zhou, Feimeng; Zheng, Jie

    2014-10-15

    The ability of peptides to construct specific secondary structures provides a useful function for biomaterial design that cannot be achieved with traditional organic molecules and polymers. Inhibition of amyloid formation is a promising therapeutic approach for the treatment of neurodegenerative diseases. Existing peptide-based inhibitors are mainly derived from original amyloid sequences, which have very limited sequence diversity and activity. It is highly desirable to explore other peptide-based inhibitors that are not directly derived from amyloid sequences. Here, we develop a hybrid high-throughput computational method to efficiently screen and design hexapeptide inhibitors against amyloid-β (Aβ) aggregation and toxicity from the first principle. Computationally screened/designed inhibitors are then validated for their inhibition activity using biophysical experiments. We propose and demonstrate a proof-of-concept of the "like-interacts-like" design principle that the self-assembling peptides are able to interact strongly with conformationally similar motifs of Aβ peptides and to competitively reduce Aβ-Aβ interactions, thus preventing Aβ aggregation and Aβ-induced toxicity. Such a de novo design can also be generally applicable to design new peptide inhibitors against other amyloid diseases, beyond traditional peptide inhibitors with homologous sequences to parent amyloid peptides.

  13. 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.

  14. [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

  15. 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

  16. Design and synthesis of peptides with hybrid helix-turn-helix (HTH) motif and their conformational study.

    PubMed

    Sharma, Gangavaram V M; Thodupunuri, Prashanth; Sirisha, Katukuri; Basha, Shaik Jeelani; Gurava Reddy, Pottireddygari; Sarma, Akella V S

    2014-09-19

    The present study is aimed at the design and synthesis of peptides with hybrid helix-turn-helix (HTH) motif and their conformational analysis (NMR, MD, and CD studies). The requisite peptides with heterogeneous backbones were prepared from β-, γ-, and δ-amino acids with carbohydrate side chains and α-amino acid, L-Ala. The α/β-peptides were prepared from (S)-β-Caa(l) (C-linked carbo-β-amino acid with D-lyxo furanoside side chain) and L-Ala with a 1:1 alternation. The α/β-peptides with "helix-turn" motif displayed a 11/9-helix nucleating a 13-atom H-bonding turn. The α/β-octapeptides showed the presence of HTH structures with bifurcated 11/15-H-bonded turn. Further, the α/β-hexapeptide with HT motif, independently on coupling with γ/α/γ/α- and δ/α/δ/α-tetrapeptides at the C-terminus provided access to the decapeptides with "hybrid HTH" motifs. The decapeptide ("α-β-α-β-α-β-γ-α-γ-α") showed a hybrid HTH with "11/9/11/9/11/16/9/12/10" H-bonding, while the decapeptide ("α-β-α-β-α-β-δ-α-δ-α") revealed the presence of a "11/9/11/9/11/17/9/13/11" helical pattern. The above peptides thus have shown compatibility between different types of helices and serendipitous bifurcated 11/16- and 11/17-turns. The present study thus provided the first opportunity for the design and study of "hybrid HTH" motifs with more than one kind of helical structures in them.

  17. Design and synthesis of peptides with hybrid helix-turn-helix (HTH) motif and their conformational study.

    PubMed

    Sharma, Gangavaram V M; Thodupunuri, Prashanth; Sirisha, Katukuri; Basha, Shaik Jeelani; Gurava Reddy, Pottireddygari; Sarma, Akella V S

    2014-09-19

    The present study is aimed at the design and synthesis of peptides with hybrid helix-turn-helix (HTH) motif and their conformational analysis (NMR, MD, and CD studies). The requisite peptides with heterogeneous backbones were prepared from β-, γ-, and δ-amino acids with carbohydrate side chains and α-amino acid, L-Ala. The α/β-peptides were prepared from (S)-β-Caa(l) (C-linked carbo-β-amino acid with D-lyxo furanoside side chain) and L-Ala with a 1:1 alternation. The α/β-peptides with "helix-turn" motif displayed a 11/9-helix nucleating a 13-atom H-bonding turn. The α/β-octapeptides showed the presence of HTH structures with bifurcated 11/15-H-bonded turn. Further, the α/β-hexapeptide with HT motif, independently on coupling with γ/α/γ/α- and δ/α/δ/α-tetrapeptides at the C-terminus provided access to the decapeptides with "hybrid HTH" motifs. The decapeptide ("α-β-α-β-α-β-γ-α-γ-α") showed a hybrid HTH with "11/9/11/9/11/16/9/12/10" H-bonding, while the decapeptide ("α-β-α-β-α-β-δ-α-δ-α") revealed the presence of a "11/9/11/9/11/17/9/13/11" helical pattern. The above peptides thus have shown compatibility between different types of helices and serendipitous bifurcated 11/16- and 11/17-turns. The present study thus provided the first opportunity for the design and study of "hybrid HTH" motifs with more than one kind of helical structures in them. PMID:25180942

  18. 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

  19. 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.

  20. Oxidative diversification of amino acids and peptides by small-molecule iron catalysis

    NASA Astrophysics Data System (ADS)

    Osberger, Thomas J.; Rogness, Donald C.; Kohrt, Jeffrey T.; Stepan, Antonia F.; White, M. Christina

    2016-09-01

    Secondary metabolites synthesized by non-ribosomal peptide synthetases display diverse and complex topologies and possess a range of biological activities. Much of this diversity derives from a synthetic strategy that entails pre- and post-assembly oxidation of both the chiral amino acid building blocks and the assembled peptide scaffolds. The vancomycin biosynthetic pathway is an excellent example of the range of oxidative transformations that can be performed by the iron-containing enzymes involved in its biosynthesis. However, because of the challenges associated with using such oxidative enzymes to carry out chemical transformations in vitro, chemical syntheses guided by these principles have not been fully realized in the laboratory. Here we report that two small-molecule iron catalysts are capable of facilitating the targeted C-H oxidative modification of amino acids and peptides with preservation of α-centre chirality. Oxidation of proline to 5-hydroxyproline furnishes a versatile intermediate that can be transformed to rigid arylated derivatives or flexible linear carboxylic acids, alcohols, olefins and amines in both monomer and peptide settings. The value of this C-H oxidation strategy is demonstrated in its capacity for generating diversity: four ‘chiral pool’ amino acids are transformed to twenty-one chiral unnatural amino acids representing seven distinct functional group arrays; late-stage C-H functionalizations of a single proline-containing tripeptide furnish eight tripeptides, each having different unnatural amino acids. Additionally, a macrocyclic peptide containing a proline turn element is transformed via late-stage C-H oxidation to one containing a linear unnatural amino acid.

  1. Oxidative diversification of amino acids and peptides by small-molecule iron catalysis

    NASA Astrophysics Data System (ADS)

    Osberger, Thomas J.; Rogness, Donald C.; Kohrt, Jeffrey T.; Stepan, Antonia F.; White, M. Christina

    2016-09-01

    Secondary metabolites synthesized by non-ribosomal peptide synthetases display diverse and complex topologies and possess a range of biological activities. Much of this diversity derives from a synthetic strategy that entails pre- and post-assembly oxidation of both the chiral amino acid building blocks and the assembled peptide scaffolds. The vancomycin biosynthetic pathway is an excellent example of the range of oxidative transformations that can be performed by the iron-containing enzymes involved in its biosynthesis. However, because of the challenges associated with using such oxidative enzymes to carry out chemical transformations in vitro, chemical syntheses guided by these principles have not been fully realized in the laboratory. Here we report that two small-molecule iron catalysts are capable of facilitating the targeted C–H oxidative modification of amino acids and peptides with preservation of α-centre chirality. Oxidation of proline to 5-hydroxyproline furnishes a versatile intermediate that can be transformed to rigid arylated derivatives or flexible linear carboxylic acids, alcohols, olefins and amines in both monomer and peptide settings. The value of this C–H oxidation strategy is demonstrated in its capacity for generating diversity: four ‘chiral pool’ amino acids are transformed to twenty-one chiral unnatural amino acids representing seven distinct functional group arrays; late-stage C–H functionalizations of a single proline-containing tripeptide furnish eight tripeptides, each having different unnatural amino acids. Additionally, a macrocyclic peptide containing a proline turn element is transformed via late-stage C–H oxidation to one containing a linear unnatural amino acid.

  2. Identification of potent 11mer glucagon-like peptide-1 receptor agonist peptides with novel C-terminal amino acids: Homohomophenylalanine analogs.

    PubMed

    Haque, Tasir S; Lee, Ving G; Riexinger, Douglas; Lei, Ming; Malmstrom, Sarah; Xin, Li; Han, Songping; Mapelli, Claudio; Cooper, Christopher B; Zhang, Ge; Ewing, William R; Krupinski, John

    2010-05-01

    We report the identification of potent agonists of the Glucagon-Like Peptide-1 Receptor (GLP-1R). These compounds are short, 11 amino acid peptides containing several unnatural amino acids, including (in particular) analogs of homohomophenylalanine (hhPhe) at the C-terminal position. Typically the functional activity of the more potent peptides in this class is in the low picomolar range in an in vitro cAMP assay, with one example demonstrating excellent in vivo activity in an ob/ob mouse model of diabetes.

  3. 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.

  4. 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

  5. Synthetic amphibian peptides and short amino-acids derivatives against planktonic cells and mature biofilm of Providencia stuartii clinical strains.

    PubMed

    Ostrowska, Kinga; Kamysz, Wojciech; Dawgul, Małgorzata; Różalski, Antoni

    2014-01-01

    Over the last decade, the growing number of multidrug resistant strains limits the use of many of the currently available chemotherapeutic agents. Furthermore, bacterial biofilm, due to its complex structure, constitutes an effective barrier to conventional antibiotics. The in vitro activities of naturally occurring peptide (Citropin 1.1), chemically engineered analogue (Pexiganan), newly-designed, short amino-acid derivatives (Pal-KK-NH2, Pal-KKK-NH2, Pal-RRR-NH2) and six clinically used antimicrobial agents (Gatifloxacin, Ampicilin, Cefotaxime, Ceftriaxone, Cefuroxime and Cefalexin) were investigated against planktonic cells and mature biofilm of multidrug-resistant Providencia stuartii strains, isolated from urological catheters. The MICs, MBCs values were determined by broth microdilution technique. Inhibition of biofilm formation by antimicrobial agents as well as biofilm susceptibility assay were tested using a surrogate model based on the Crystal Violet method. The antimicrobial activity of amino-acids derivatives and synthetic peptides was compared to that of clinically used antibiotics. For planktonic cells, MICs of peptides and antibiotics ranged between 1 and 256 μg/ml and 256 and ≥ 2048 μg/ml, respectively. The MBCs values of Pexiganan, Citropin 1.1 and amino-acids derivatives were between 16 and 256 μg/ml, 64 and 256 μg/ml and 16 and 512 μg/ml, respectively. For clinically used antibiotics the MBCs values were above 2048 μg/ml. All of the tested peptides and amino-acids derivatives, showed inhibitory activity against P. stuartii biofilm formation, in relation to their concentrations. Pexiganan and Citropin 1.1 in concentration range 32 and 256 μg/ml caused both strong and complete suppression of biofilm formation. None of the antibiotics caused complete inhibition of biofilm formation process. The biofilm susceptibility assay verified the extremely poor antibiofilm activity of conventional antibiotics compared to synthetic peptides. The

  6. Synthetic amphibian peptides and short amino-acids derivatives against planktonic cells and mature biofilm of Providencia stuartii clinical strains.

    PubMed

    Ostrowska, Kinga; Kamysz, Wojciech; Dawgul, Małgorzata; Różalski, Antoni

    2014-01-01

    Over the last decade, the growing number of multidrug resistant strains limits the use of many of the currently available chemotherapeutic agents. Furthermore, bacterial biofilm, due to its complex structure, constitutes an effective barrier to conventional antibiotics. The in vitro activities of naturally occurring peptide (Citropin 1.1), chemically engineered analogue (Pexiganan), newly-designed, short amino-acid derivatives (Pal-KK-NH2, Pal-KKK-NH2, Pal-RRR-NH2) and six clinically used antimicrobial agents (Gatifloxacin, Ampicilin, Cefotaxime, Ceftriaxone, Cefuroxime and Cefalexin) were investigated against planktonic cells and mature biofilm of multidrug-resistant Providencia stuartii strains, isolated from urological catheters. The MICs, MBCs values were determined by broth microdilution technique. Inhibition of biofilm formation by antimicrobial agents as well as biofilm susceptibility assay were tested using a surrogate model based on the Crystal Violet method. The antimicrobial activity of amino-acids derivatives and synthetic peptides was compared to that of clinically used antibiotics. For planktonic cells, MICs of peptides and antibiotics ranged between 1 and 256 μg/ml and 256 and ≥ 2048 μg/ml, respectively. The MBCs values of Pexiganan, Citropin 1.1 and amino-acids derivatives were between 16 and 256 μg/ml, 64 and 256 μg/ml and 16 and 512 μg/ml, respectively. For clinically used antibiotics the MBCs values were above 2048 μg/ml. All of the tested peptides and amino-acids derivatives, showed inhibitory activity against P. stuartii biofilm formation, in relation to their concentrations. Pexiganan and Citropin 1.1 in concentration range 32 and 256 μg/ml caused both strong and complete suppression of biofilm formation. None of the antibiotics caused complete inhibition of biofilm formation process. The biofilm susceptibility assay verified the extremely poor antibiofilm activity of conventional antibiotics compared to synthetic peptides. The

  7. Designing functional materials using the hydrophobic face of a self-assembling amphiphilic beta-hairpin peptide

    NASA Astrophysics Data System (ADS)

    Micklitsch, Christopher M.

    The ability of proteins to adopt discrete structures, as well as the ability of folded proteins to undergo supramolecular assembly, is based on the display of chemical functionality of the protein. Proteins fold and assemble according to the influence of intra- and intermolecular forces that act upon it to form ordered structures. By identifying these forces and how they influence assembly, they, in turn, can be exploited to design and synthesize materials from protein-based building blocks. By fabricating materials in this manner, the bulk material properties can be tuned by engineering at the molecular level. This study explores how modulation of the hydrophobic face of a de novo designed self-assembling beta-hairpin peptide affects its ability to fold and self-assemble to form a hydrogel, as well as on the resulting hydrogel's nanoscale structure and bulk properties. Initially, sequence modifications using amino acids of varying hydrophobicity were used to modify the hydrophobic face of the amphiphilic peptide. These modifications demonstrate how important hydrophobic regions of the peptide are to its ability to fold, self-assemble and form a hydrogel. The knowledge acquired from these studies was then used in the de novo design of a zinc-triggered peptide hydrogel, employing a nonnatural metal-binding amino acid on the hydrophobic face to instill metal-sensitivity in the peptide. Finally, aromatic interactions were incorporated on the topologically smooth hydrophobic face to direct self-assembly so as to impede the formation of interfibril junctions that lead to crosslinking of the fibrils that comprise the hydrogel scaffold.

  8. Self-assembling tryptophan-based designer peptides as intracellular delivery vehicles.

    PubMed

    Bhardwaj, Ishanki; Jha, Divya; Admane, Prasad; Panda, Amulya K; Haridas, V

    2016-01-15

    A series of tryptophan-based peptides W1a, b-W4a, b, with diverse architectures were designed and synthesized. These tryptophan containing peptides can self-assemble to spherical particle. This self-assembled system was demonstrated to encapsulate rhodamine B and penetrate the cell membrane.

  9. Discovery and structural characterization of a phospholamban-binding cyclic peptide and design of novel inhibitors of phospholamban.

    PubMed

    Tilgmann, Carola; Pollesello, Piero; Ovaska, Martti; Kaivola, Juha; Pystynen, Jarmo; Tiainen, Eija; Yliperttula, Marjo; Annila, Arto; Levijoki, Jouko

    2013-04-01

    The interplay between cardiac sarcoplasmic Ca(2+)ATPase and phospholamban is a key regulating factor of contraction and relaxation in the cardiac muscle. In heart failure, aberrations in the inhibition of sarcoplasmic Ca(2+)ATPase by phospholamban are associated with anomalies in cardiac functions. In experimental heart failure models, modulation of the interaction between these two proteins has been shown to be a potential therapeutic approach. The aim of our research was to find molecules able to interfere with the inhibitory activity of phospholamban on sarcoplasmic Ca(2+)ATPase. For this purpose, a portion of phospholamban was synthesized and used as target for a phage-display peptide library screening. The cyclic peptide C-Y-W-E-L-E-W-L-P-C-A was found to bind to phospholamban (1-36) with high specificity. Its functional activity was tested in Ca(2+)uptake assays utilizing preparations from cardiac sarcoplasmic reticulum. By synthesizing and testing a series of alanine point-mutated cyclic peptides, we identified which amino acid was important for the inhibition of the phospholamban function. The structures of active and inactive alanine-mutated cyclic peptides, and of phospholamban (1-36), were determined by NMR. This structure-activity analysis allowed building a model of phospholamban -cyclic peptide complex. Thereafter, a simple pharmacophore was defined and used for the design of small molecules. Finally, examples of such molecules were synthesized and characterized as phospholamban inhibitors.

  10. 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.

  11. Design of Embedded-Hybrid Antimicrobial Peptides with Enhanced Cell Selectivity and Anti-Biofilm Activity

    PubMed Central

    Xu, Wei; Zhu, Xin; Tan, Tingting; Li, Weizhong; Shan, Anshan

    2014-01-01

    Antimicrobial peptides have attracted considerable attention because of their broad-spectrum antimicrobial activity and their low prognostic to induce antibiotic resistance which is the most common source of failure in bacterial infection treatment along with biofilms. The method to design hybrid peptide integrating different functional domains of peptides has many advantages. In this study, we designed an embedded-hybrid peptide R-FV-I16 by replacing a functional defective sequence RR7 with the anti-biofilm sequence FV7 embedded in the middle position of peptide RI16. The results demonstrated that the synthetic hybrid the peptide R-FV-I16 had potent antimicrobial activity over a wide range of Gram-negative and Gram-positive bacteria, as well as anti-biofilm activity. More importantly, R-FV-I16 showed lower hemolytic activity and cytotoxicity. Fluorescent assays demonstrated that R-FV-I16 depolarized the outer and the inner bacterial membranes, while scanning electron microscopy and transmission electron microscopy further indicated that this peptide killed bacterial cells by disrupting the cell membrane, thereby damaging membrane integrity. Results from SEM also provided evidence that R-FV-I16 inherited anti-biofilm activity from the functional peptide sequence FV7. Embedded-hybrid peptides could provide a new pattern for combining different functional domains and showing an effective avenue to screen for novel antimicrobial agents. PMID:24945359

  12. 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

  13. Development of a novel composite material with carbon nanotubes assisted by self-assembled peptides designed in conjunction with β-sheet formation.

    PubMed

    Hashida, Yasuhiko; Umeyama, Tomokazu; Mihara, Junya; Imahori, Hiroshi; Tsujimoto, Masahiko; Isoda, Seiji; Takano, Mikio; Hashida, Mitsuru

    2012-09-01

    A novel composite material is developed with single-walled carbon nanotubes (SWCNTs) and artificially designed peptides, and its chemical and physicochemical characteristics are evaluated with an aim toward biomedical application. The peptides were designed to form a β-sheet structure that would be suitable for wrapping SWCNTs. The complex of SWCNTs and peptide (SWCNT-peptide) showed good dispersibility in aqueous media and was considerably stable even in the absence of an excess amount of peptide in the media. The formation of SWCNT-peptide was confirmed by its performance in water, atomic force microscopy and transmission electron microscopy observation, and molecular modeling. The possibility of introducing various functions to SWCNT-peptide was also demonstrated by several methods, such as introduction of special amino acids, chemical modification, and additional complex formation based on electrostatic interaction. These results suggest the potential of the SWCNT-peptide complex as a molecular platform on which a desirable structure and/or function can be constructed for biomedical and industrial application.

  14. 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

  15. Distinguishing Aspartic and Isoaspartic Acids in Peptides by Several Mass Spectrometric Fragmentation Methods

    NASA Astrophysics Data System (ADS)

    DeGraan-Weber, Nick; Zhang, Jun; Reilly, James P.

    2016-09-01

    Six ion fragmentation techniques that can distinguish aspartic acid from its isomer, isoaspartic acid, were compared. MALDI post-source decay (PSD), MALDI 157 nm photodissociation, tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP) charge tagging in PSD and photodissociation, ESI collision-induced dissociation (CID), electron transfer dissociation (ETD), and free-radical initiated peptide sequencing (FRIPS) with CID were applied to peptides containing either aspartic or isoaspartic acid. Diagnostic ions, such as the y-46 and b+H2O, are present in PSD, photodissociation, and charge tagging. c•+57 and z-57 ions are observed in ETD and FRIPS experiments. For some molecules, aspartic and isoaspartic acid yield ion fragments with significantly different intensities. ETD and charge tagging appear to be most effective at distinguishing these residues.

  16. 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.

  17. 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

  18. In vitro activities of native and designed peptide antibiotics against drug sensitive and resistant tumor cell lines.

    PubMed

    Kim, Sunkyu; Kim, Sukwon S; Bang, Yung Jue; Kim, Seong Jin; Lee, Byeong Jae

    2003-07-01

    In order to develop peptide agents with reduced length and enhanced tumoricidal activity, we have designed gaegurin 6 (GGN6) derivatives through deletions and/or substitutions of amino acids. The deletion of hydrophobic amino terminal region completely abolished antitumor activity whereas the deletion of carboxy terminal region had little influence on antitumor activity. Antitumor activity of the PTP peptides did not correlate with antibacterial activity. PTP7, the most potent derivative, was found to have comparable antitumor activity to GGN6 in spite of reduced number of amino acids which is about half the size of gaegurin 6; furthermore, it showed little cytotoxicity on PBMCs and RBCs. GGN6 and PTP7 also showed equivalent cytotoxicity against drug sensitive (MCF-7) and multidrug-resistant cell lines (MCF-7/DOX). Plasma membrane blebbing and DNA fragmentation of peptide-treated tumor cells indicated that the peptides could induce apoptosis in tumor cells. These results suggest that GGN6 and its derivatives can be developed as new anticancer agents and may provide a new strategy for overcoming MDR which is a major problem in cancer therapy.

  19. 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 ...

  20. 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

  1. 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.

  2. Can natural proteins designed with 'inverted' peptide sequences adopt native-like protein folds?

    PubMed

    Sridhar, Settu; Guruprasad, Kunchur

    2014-01-01

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

  3. 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

  4. A systems biology perspective on rational design of peptide vaccine against virus infections.

    PubMed

    Chen, Jiajia; Wang, Ying; Guo, Deyin; Shen, Bairong

    2012-01-01

    With the recent onset of influenza A (H1N1) pandemic, the need for improved vaccines against virus infections has become an international priority. Strategies for vaccine development have changed over time, from whole-virus to immunogenic proteins and further to antigenic viral peptides. Various algorithms and bioinformatics tools have been developed to predict immunogenic peptide regions in an antigenic protein sequence. Recent advances in next-generation sequencing technologies, as represented by real time DNA sequencing, provide increased throughput and yield of data on viral pathogens and host cells. This enables us to 'mine' the genomic sequence for putative vaccine candidates or targets, allowing a more rational approach to the peptide vaccine design. This review first describes current computational tools available for the rational design of peptide vaccines and then addresses recent attempts to define pathogenic peptides at '- omics' level. As there are interplay between antibody and T cells, as well as intersection between viruses and hosts, the vaccine-mediated immunity are orchestrated by multiple factors within an interaction network. Therefore, single viral peptide alone fails to provide optimal immunity. Systems biology offers a systems-level perspective of how the various arms of the immune response are integrated to give immune response, as well as how host and virus interact, thereby providing an integrated approach to select the most promising candidates for peptide vaccines development. We highlight in this article the system-level application of rational peptide vaccine design, which may be a general paradigm for future viral vaccine development.

  5. 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

  6. Alkylating potency of nitrosated amino acids and peptides.

    PubMed

    Shephard, S E; Meier, I; Lutz, W K

    1991-01-01

    The alkylating potency of unstable N-nitrosamino acids and N-nitrosopeptides was investigated in vitro using 4-(para-nitrobenzyl)pyridine (NBP) as nucleophile. Of the amino acids, Met and those with an aromatic side chain were the most potent. The relative overall alkylating potency was 23:10:5:4:2:1: for Trp, Met, His, Tyr, Phe and Gly, respectively. The homo-dipeptides were much more potent than the amino acids, with relative potencies of 400:110:100:8:3:1, for Trp-Trp, Tyr-Tyr, Met-Met, Asp-Asp, Phe-Phe and Gly, respectively. In the one-phase reaction system (in which NBP is already present during the nitrosation reaction at acidic pH), all amino acids tested showed a second-order reaction for nitrite. In the two-phase system (in which NBP is added only after bringing the nitrosation reaction mixture to neutrality), all amino acids tested except one again showed a second-order reaction for nitrite (Phe, His, Asp and the dipeptide artificial sweetener aspartame); only Met under these conditions had a reaction order of one for nitrite. This could mean that nitrosation of the side chain of Met produces a second N-nitroso product which is relatively stable in acid but reacts with NBP under neutral conditions. In the human stomach, this side-chain nitrosation might become more important than the reactions at the primary amino group, firstly because of the greater stability of the product(s) in acid and secondly because of the first-order reaction rate for nitrite.(ABSTRACT TRUNCATED AT 250 WORDS)

  7. Intracellular delivery of peptide nucleic acid and organic molecules using zeolite-L nanocrystals.

    PubMed

    Bertucci, Alessandro; Lülf, Henning; Septiadi, Dedy; Manicardi, Alex; Corradini, Roberto; De Cola, Luisa

    2014-11-01

    The design and synthesis of smart nanomaterials can provide interesting potential applications for biomedical purposes from bioimaging to drug delivery. Manufacturing multifunctional systems in a way to carry bioactive molecules, like peptide nucleic acids able to recognize specific targets in living cells, represents an achievement towards the development of highly selective tools for both diagnosis and therapeutics. This work describes a very first example of the use of zeolite nanocrystals as multifunctional nanocarriers to deliver simultaneously PNA and organic molecules into living cells. Zeolite-L nanocrystals are functionalized by covalently attaching the PNA probes onto the surface, while the channel system is filled with fluorescent guest molecules. The cellular uptake of the PNA/Zeolite-L hybrid material is then significantly increased by coating the whole system with a thin layer of biodegradable poly-L-lysine. The delivery of DAPI as a model drug molecule, inserted into the zeolite pores, is also demonstrated to occur in the cells, proving the multifunctional ability of the system. Using this zeolite nanosystem carrying PNA probes designed to target specific RNA sequences of interest in living cells could open new possibilities for theranostic and gene therapy applications. PMID:24789252

  8. Intracellular delivery of peptide nucleic acid and organic molecules using zeolite-L nanocrystals.

    PubMed

    Bertucci, Alessandro; Lülf, Henning; Septiadi, Dedy; Manicardi, Alex; Corradini, Roberto; De Cola, Luisa

    2014-11-01

    The design and synthesis of smart nanomaterials can provide interesting potential applications for biomedical purposes from bioimaging to drug delivery. Manufacturing multifunctional systems in a way to carry bioactive molecules, like peptide nucleic acids able to recognize specific targets in living cells, represents an achievement towards the development of highly selective tools for both diagnosis and therapeutics. This work describes a very first example of the use of zeolite nanocrystals as multifunctional nanocarriers to deliver simultaneously PNA and organic molecules into living cells. Zeolite-L nanocrystals are functionalized by covalently attaching the PNA probes onto the surface, while the channel system is filled with fluorescent guest molecules. The cellular uptake of the PNA/Zeolite-L hybrid material is then significantly increased by coating the whole system with a thin layer of biodegradable poly-L-lysine. The delivery of DAPI as a model drug molecule, inserted into the zeolite pores, is also demonstrated to occur in the cells, proving the multifunctional ability of the system. Using this zeolite nanosystem carrying PNA probes designed to target specific RNA sequences of interest in living cells could open new possibilities for theranostic and gene therapy applications.

  9. 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.

  10. Hydrophobic amino acids in the hinge region of the 5A apolipoprotein mimetic peptide are essential for promoting cholesterol efflux by the ABCA1 transporter.

    PubMed

    Sviridov, Denis O; Andrianov, Alexander M; Anishchenko, Ivan V; Stonik, John A; Amar, Marcelo J A; Turner, Scott; Remaley, Alan T

    2013-01-01

    The bihelical apolipoprotein mimetic peptide 5A effluxes cholesterol from cells and reduces inflammation and atherosclerosis in animal models. We investigated how hydrophobic residues in the hinge region between the two helices are important in the structure and function of this peptide. By simulated annealing analysis and molecular dynamics modeling, two hydrophobic amino acids, F-18 and W-21, in the hinge region were predicted to be relatively surface-exposed and to interact with the aqueous solvent. Using a series of 5A peptide analogs in which F-18 or W-21 was changed to either F, W, A, or E, only peptides with hydrophobic amino acids in these two positions were able to readily bind and solubilize phospholipid vesicles. Compared with active peptides containing F or W, peptides containing E in either of these two positions were more than 10-fold less effective in effluxing cholesterol by the ABCA1 transporter. Intravenous injection of 5A in C57BL/6 mice increased plasma-free cholesterol (5A: 89.9 ± 13.6 mg/dl; control: 38.7 ± 4.3 mg/dl (mean ± S.D.); P < 0.05) and triglycerides (5A: 887.0 ± 172.0 mg/dl; control: 108.9 ± 9.9 mg/dl; P < 0.05), whereas the EE peptide containing E in both positions had no effect. Finally, 5A increased cholesterol efflux approximately 2.5-fold in vivo from radiolabeled macrophages, whereas the EE peptide was inactive. These results provide a rationale for future design of therapeutic apolipoprotein mimetic peptides and provide new insights into the interaction of hydrophobic residues on apolipoproteins with phospholipids in the lipid microdomain created by the ABCA1 transporter during the cholesterol efflux process.

  11. Antimicrobial peptides targeting Gram-negative pathogens, produced and delivered by lactic acid bacteria.

    PubMed

    Volzing, Katherine; Borrero, Juan; Sadowsky, Michael J; Kaznessis, Yiannis N

    2013-11-15

    We present results of tests with recombinant Lactococcus lactis that produce and secrete heterologous antimicrobial peptides with activity against Gram-negative pathogenic Escherichia coli and Salmonella . In an initial screening, the activities of numerous candidate antimicrobial peptides, made by solid state synthesis, were assessed against several indicator pathogenic E. coli and Salmonella strains. Peptides A3APO and Alyteserin were selected as top performers based on high antimicrobial activity against the pathogens tested and on significantly lower antimicrobial activity against L. lactis . Expression cassettes containing the signal peptide of the protein Usp45 fused to the codon-optimized sequence of mature A3APO and Alyteserin were cloned under the control of a nisin-inducible promoter PnisA and transformed into L. lactis IL1403. The resulting recombinant strains were induced to express and secrete both peptides. A3APO- and Alyteserin-containing supernatants from these recombinant L. lactis inhibited the growth of pathogenic E. coli and Salmonella by up to 20-fold, while maintaining the host's viability. This system may serve as a model for the production and delivery of antimicrobial peptides by lactic acid bacteria to target Gram-negative pathogenic bacteria populations.

  12. 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.

  13. 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

  14. 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.

  15. 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

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

    PubMed Central

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

    2016-01-01

    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

  17. Entropy reduction in unfolded peptides (and proteins) due to conformational preferences of amino acid residues.

    PubMed

    Schweitzer-Stenner, Reinhard; Toal, Siobhan E

    2014-11-01

    As established by several groups over the last 20 years, amino acid residues in unfolded peptides and proteins do not exhibit the unspecific random distribution as assumed by the classical random coil model. Individual amino acid residues in small peptides were found to exhibit different conformational preferences. Here, we utilize recently obtained conformational distributions of guest amino acid residues in GxG peptides to estimate their conformational entropy, which we find to be significantly lower than the entropy of an assumed random coil like distribution. Only at high temperature do backbone entropies approach random coil like values. We utilized the obtained backbone entropies of the investigated amino acid residues to estimate the loss of conformational entropy caused by a coil → helix transition and identified two subsets of amino acid residues for which the thus calculated entropy losses correlate well with the respective Gibbs energy of helix formation obtained for alanine based host-guest systems. Calculated and experimentally derived entropic losses were found to be in good agreement. For most of the amino acid residues investigated entropic losses derived from our GxG distributions correlate very well with corresponding values recently obtained from MD simulations biased by conformational propensities derived from truncated coil libraries. Both, conformational entropy and the entropy of solvation exhibit a strong, residue specific temperature dependence, which can be expected to substantially affect the stability of unfolded states. Altogether, our results provide strong evidence for the notion that conformational preferences of amino acid residues matter with regard to the thermodynamics of peptide and protein folding.

  18. 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.

  19. 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.

  20. 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.

  1. 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

  2. Question 1: Peptide nucleic acids and the origin and homochirality of life.

    PubMed

    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.

  3. 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.

  4. Applications of hydrophilic interaction chromatography to amino acids, peptides, and proteins.

    PubMed

    Periat, Aurélie; Krull, Ira S; Guillarme, Davy

    2015-02-01

    This review summarizes the recent advances in the analysis of amino acids, peptides, and proteins using hydrophilic interaction chromatography. Various reports demonstrate the successful analysis of amino acids under such conditions. However, a baseline resolution of the 20 natural amino acids has not yet been published and for this reason, there is often a need to use mass spectrometry for detection to further improve selectivity. Hydrophilic interaction chromatography is also recognized as a powerful technique for peptide analysis, and there are a lot of papers showing its applicability for proteomic applications (peptide mapping). It is expected that its use for peptide mapping will continue to grow in the future, particularly because this analytical strategy can be combined with reversed-phase liquid chromatography, in a two-dimensional setup, to reach very high resolving power. Finally, the interest in hydrophilic interaction chromatography for intact proteins analysis is less evident due to possible solubility issues and a lack of suitable hydrophilic interaction chromatography stationary phases. To date, it has been successfully employed only for the characterization of membrane proteins, histones, and the separation of glycosylated isoforms of an intact glycoprotein. From our point of view, the number of hydrophilic interaction chromatography columns compatible with intact proteins (higher upper temperature limit, large pore size, etc.) is still too limited.

  5. 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

  6. Photomodulation of conformational states. II. Mono- and bicyclic peptides with (4-aminomethyl)phenylazobenzoic acid as backbone constituent.

    PubMed

    Renner, C; Cramer, J; Behrendt, R; Moroder, L

    2000-12-01

    It has been reported that backbone cyclization of octapeptides with the photoresponsive (4-aminomethyl)phenylazobenzoic acid imparts sufficient restraints to induce and stabilize ordered conformations of the peptide backbone in both the cis- and trans-azo-isomers (L. Ulysse, J. Cubillos, and J. Chmielewski, Journal of the American Chemical Society, 1995, Vol. 117, pp. 8466-8467). Correspondingly, the active-site octapeptide fragment H-Ala-Cys-Ala-Thr-Cys-Asp-Gly-Phe-OH [134-141] of thioredoxin reductase, with its high preference for a 3(10)-helix turn conformation centered on the Thr-Cys sequence, was backbone cyclized with this azobenzene moiety in the attempt to design a photoresponsive system where the conformational states of the peptide backbone are dictated by the configuration of the azobenzene and can be further modulated by the disulfide bridge. Nuclear magnetic resonance conformational analysis of the monocyclic compound clearly revealed the presence of two conformational families in both the cis- and trans-azo configuration. Of the higher populated conformational families, the structure of the trans-isomer seems like a pretzel-like folding, while the cis-isomer relaxes into a significantly less defined conformational state that does not exhibit any regular structural elements. Further restrictions imparted by disulfide bridging of the peptide moiety leads to an even better defined conformation for the trans-azo-isomer, whereas the cis-isomer can be described as a frustrated system without pronounced energy minima and thus with little conformational preferences. Our findings would suggest that this photoresponsive peptide template may not be of general usefulness for light-induced conformational transitions between two well-defined conformational states at least under the experimental conditions employed, even in the bicyclic form. However, trans --> cis isomerization of the bicyclic peptide is accompanied by a switch from a well-defined conformation to

  7. 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

  8. Synthesis and evaluation of antineurotoxicity properties of an amyloid-β peptide targeting ligand containing a triamino acid.

    PubMed

    Honcharenko, Dmytro; Bose, Partha Pratim; Maity, Jyotirmoy; Kurudenkandy, Firoz Roshan; Juneja, Alok; Flöistrup, Erik; Biverstål, Henrik; Johansson, Jan; Nilsson, Lennart; Fisahn, André; Strömberg, Roger

    2014-09-14

    Peptide-like compounds containing an arginine have been shown to bind and stabilize the central helix of the Alzheimer's disease related amyloid-β peptide (Aβ) in an α-helical conformation, thereby delaying its aggregation into cytotoxic species. Here we study a novel Aβ targeting ligand AEDabDab containing the triamino acid, N(γ)-(2-aminoethyl)-2,4-diaminobutanoic (AEDab) acid. The new AEDab triamino acid carries an extra positive charge in the side chain and is designed to be incorporated into a ligand AEDabDab where the AEDab replaces an arginine moiety in a previously developed ligand Pep1b. This is done in order to increase the Aβ-ligand interaction, and molecular dynamics (MD) simulation of the stability of the Aβ central helix in the presence of the AEDabDab ligand shows further stabilization of the helical conformation of Aβ compared to the previously reported Pep1b as well as compared to the AEOrnDab ligand containing an N(δ)-(2-aminoethyl)-2,5-diaminopentanoic acid unit which has an additional methylene group. To evaluate the effect of the AEDabDab ligand on the Aβ neurotoxicity the AEDab triamino acid building block is synthesized by reductive alkylation of N-protected-glycinal with α-amino-protected diaminobutanoic acid, and the Aβ targeting ligand AEDabDab is prepared by solid-phase synthesis starting with attachment of glutarate to the Wang support. Replacement of the arginine residue by the AEDab triamino acid resulted in an improved capability of the ligand to prevent the Aβ1-42 induced reduction of gamma (γ) oscillations in hippocampal slice preparation.

  9. Electroporation-based delivery of cell-penetrating peptide conjugates of peptide nucleic acids for antisense inhibition of intracellular bacteria.

    PubMed

    Ma, Sai; Schroeder, Betsy; Sun, Chen; Loufakis, Despina Nelie; Cao, Zhenning; Sriranganathan, Nammalwar; Lu, Chang

    2014-10-01

    Cell penetrating peptides (CPPs) have been used for a myriad of cellular delivery applications and were recently explored for delivery of antisense agents such as peptide nucleic acids (PNAs) for bacterial inhibition. Although these molecular systems (i.e. CPP-PNAs) have shown ability to inhibit growth of bacterial cultures in vitro, they show limited effectiveness in killing encapsulated intracellular bacteria in mammalian cells such as macrophages, presumably due to difficulty involved in the endosomal escape of the reagents. In this report, we show that electroporation delivery dramatically increases the bioavailability of CPP-PNAs to kill Salmonella enterica serovar Typhimurium LT2 inside macrophages. Electroporation delivers the molecules without involving endocytosis and greatly increases the antisense effect. The decrease in the average number of Salmonella per macrophage under a 1200 V cm(-1) and 5 ms pulse was a factor of 9 higher than that without electroporation (in an experiment with a multiplicity of infection of 2 : 1). Our results suggest that electroporation is an effective approach for a wide range of applications involving CPP-based delivery. The microfluidic format will allow convenient functional screening and testing of PNA-based reagents for antisense applications.

  10. Insights on peptide backbone N-H acidity: Structure of anions, hydration effects

    NASA Astrophysics Data System (ADS)

    Oliva, Antoni; Henry, Bernard; Ruiz-López, Manuel F.

    2013-03-01

    Despite the key role played by deamidation reactions in biochemical phenomena such as aging processes, knowledge of factors determining peptide backbone N-H acidities is scarce. We report a theoretical study on this topic by means of quantum-chemical calculations. Gas-phase acidities and pKa's in water have been estimated. The results agree reasonably well with available experimental data. Further analysis suggests that the secondary peptide structure, in addition to hydration effects, is the main factor determining pKa. In particular, we predict N-H protons to be more acidic in β-turns than in α-helices, a finding that may have broad biological implications.

  11. 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

  12. Redesigning Channel-Forming Peptides: Amino Acid Substitutions that Enhance Rates of Supramolecular Self-Assembly and Raise Ion Transport Activity

    PubMed Central

    Shank, Lalida P.; Broughman, James R.; Takeguchi, Wade; Cook, Gabriel; Robbins, Ashley S.; Hahn, Lindsey; Radke, Gary; Iwamoto, Takeo; Schultz, Bruce D.; Tomich, John M.

    2006-01-01

    Three series of 22-residue peptides derived from the transmembrane M2 segment of the glycine receptor α1-subunit (M2GlyR) have been designed, synthesized, and tested to determine the plasticity of a channel-forming sequence and to define whether channel pores with enhanced conductive properties could be created. Sixteen sequences were examined for aqueous solubility, solution-association tendency, secondary structure, and half-maximal concentration for supramolecular assembly, channel activity, and ion transport properties across epithelial monolayers. All peptides interact strongly with membranes: associating with, inserting across, and assembling to form homooligomeric bundles when in micromolar concentrations. Single and double amino acid replacements involving arginine and/or aromatic amino acids within the final five C-terminal residues of the peptide cause dramatic effects on the concentration dependence, yielding a range of K1/2 values from 36 ± 5 to 390 ± 220 μM for transport activity. New water/lipid interfacial boundaries were established for the transmembrane segment using charged or aromatic amino acids, thus limiting the peptides' ability to move perpendicularly to the plane of the bilayer. Formation of discrete water/lipid interfacial boundaries appears to be necessary for efficient supramolecular assembly and high anion transport activity. A peptide sequence is identified that may show efficacy in channel replacement therapy for channelopathies such as cystic fibrosis. PMID:16387776

  13. Synthesis of peptide nucleic acids containing a crosslinking agent and evaluation of their reactivities.

    PubMed

    Akisawa, Takuya; Ishizawa, Yuki; Nagatsugi, Fumi

    2015-03-13

    Peptide nucleic acids (PNAs) are structural mimics of nucleic acids that form stable hybrids with DNA and RNA. In addition, PNAs can invade double-stranded DNA. Due to these characteristics, PNAs are widely used as biochemical tools, for example, in antisense/antigene therapy. Interstrand crosslink formation in nucleic acids is one of the strategies for preparing a stable duplex by covalent bond formation. In this study, we have synthesized PNAs incorporating 4-amino-6-oxo-2-vinylpyrimidine (AOVP) as a crosslinking agent and evaluated their reactivities for targeting DNA and RNA.

  14. Rational design and synthesis of an orally bioavailable peptide guided by NMR amide temperature coefficients

    PubMed Central

    Wang, Conan K.; Northfield, Susan E.; Colless, Barbara; Chaousis, Stephanie; Hamernig, Ingrid; Lohman, Rink-Jan; Nielsen, Daniel S.; Schroeder, Christina I.; Liras, Spiros; Price, David A.; Fairlie, David P.; Craik, David J.

    2014-01-01

    Enhancing the oral bioavailability of peptide drug leads is a major challenge in drug design. As such, methods to address this challenge are highly sought after by the pharmaceutical industry. Here, we propose a strategy to identify appropriate amides for N-methylation using temperature coefficients measured by NMR to identify exposed amides in cyclic peptides. N-methylation effectively caps these amides, modifying the overall solvation properties of the peptides and making them more membrane permeable. The approach for identifying sites for N-methylation is a rapid alternative to the elucidation of 3D structures of peptide drug leads, which has been a commonly used structure-guided approach in the past. Five leucine-rich peptide scaffolds are reported with selectively designed N-methylated derivatives. In vitro membrane permeability was assessed by parallel artificial membrane permeability assay and Caco-2 assay. The most promising N-methylated peptide was then tested in vivo. Here we report a novel peptide (15), which displayed an oral bioavailability of 33% in a rat model, thus validating the design approach. We show that this approach can also be used to explain the notable increase in oral bioavailability of a somatostatin analog. PMID:25416591

  15. Tritium labeling of amino acids and peptides with liquid and solid tritium

    SciTech Connect

    Peng, C.T.; Hua, R.L.; Souers, P.C.; Coronado, P.R.

    1988-01-01

    Amino acids and peptides were labeled with liquid and solid tritium at 21 K and 9 K. At these low temperatures radiation degradation is minimal, and tritium incorporation increases with tritium concentration and exposure time. Ring saturation in L-phenyl-alanine does not occur. Peptide linkage in oligopeptides is stable toward tritium. Deiodination in 3-iodotyrosine and 3,5-diiodotyrosine occurs readily and proceeds in steps by losing one iodine atom at a time. Nickel and noble metal supported catalysts when used as supports for dispersion of the substrate promote tritium labeling at 21 K. Our study shows that both liquid and solid tritium are potentially useful agents for labeling peptides and proteins. 11 refs., 1 fig., 3 tabs.

  16. Tritium labeling of amino acids and peptides with liquid and solid tritium

    SciTech Connect

    Souers, P.C.; Coronado, P.R.; Peng, C.T.; Hua, R.L.

    1988-01-01

    Amino acids and peptides were labeled with liquid and solid tritium at 21/degree/K and 9/degree/K. At these low temperatures radiation degradation is minimal, and tritium incorporation increases with tritium concentration and exposure time. Ring saturation in L-phenylalanine does not occur. Peptide linkage in oligopeptides is stable toward tritium. Deiodination in 3-iodotyrosine and 3,5-diiodotyrosine occurs readily and proceeds in steps by losing one iodine atom at a time. Nickel and noble metal supported catalysts when used as supports for dispersion of the substrate promote tritium labeling at 21 K. Our study shows that both liquid and solid tritiums are potentially useful agents for labeling peptides and proteins.

  17. Tritium labeling of amino acids and peptides with liquid and solid tritium

    SciTech Connect

    Peng, C.T.; Hua, R.L.; Souers, P.C.; Coronado, P.R.

    1988-09-01

    Amino acids and peptides were labeled with liquid and solid tritium at 21 K and 9 K. At these low temperatures radiation degradation is minimal, and tritium incorporation increases with tritium concentration and exposure time. Ring saturation in L-phenyl-alanine does not occur. Peptide linkage in oligopeptides is stable toward tritium. Deiodination in 3-iodotyrosine and 3,5-diiodotyrosine occurs readily and proceeds in steps by losing one iodine atom at a time. Nickel and noble metal supported catalysts when sued as supports for dispersion of the substrate promote tritium labeling at 21 K. The authors study shows that both liquid and solid tritium are potentially useful agents for labeling peptides and proteins.

  18. Selective rhodium-catalyzed reduction of tertiary amides in amino acid esters and peptides.

    PubMed

    Das, Shoubhik; Li, Yuehui; Bornschein, Christoph; Pisiewicz, Sabine; Kiersch, Konstanze; Michalik, Dirk; Gallou, Fabrice; Junge, Kathrin; Beller, Matthias

    2015-10-12

    Efficient reduction of the tertiary amide bond in amino acid derivatives and peptides is described. Functional group selectivity has been achieved by applying a commercially available rhodium precursor and bis(diphenylphosphino)propane (dppp) ligand together with phenyl silane as a reductant. This methodology allows for specific reductive derivatization of biologically interesting peptides and offers straightforward access to a variety of novel peptide derivatives for chemical biology studies and potential pharmaceutical applications. The catalytic system tolerates a variety of functional groups including secondary amides, ester, nitrile, thiomethyl, and hydroxy groups. This convenient hydrosilylation reaction proceeds at ambient conditions and is operationally safe because no air-sensitive reagents or highly reactive metal hydrides are needed. PMID:26189442

  19. Selective rhodium-catalyzed reduction of tertiary amides in amino acid esters and peptides.

    PubMed

    Das, Shoubhik; Li, Yuehui; Bornschein, Christoph; Pisiewicz, Sabine; Kiersch, Konstanze; Michalik, Dirk; Gallou, Fabrice; Junge, Kathrin; Beller, Matthias

    2015-10-12

    Efficient reduction of the tertiary amide bond in amino acid derivatives and peptides is described. Functional group selectivity has been achieved by applying a commercially available rhodium precursor and bis(diphenylphosphino)propane (dppp) ligand together with phenyl silane as a reductant. This methodology allows for specific reductive derivatization of biologically interesting peptides and offers straightforward access to a variety of novel peptide derivatives for chemical biology studies and potential pharmaceutical applications. The catalytic system tolerates a variety of functional groups including secondary amides, ester, nitrile, thiomethyl, and hydroxy groups. This convenient hydrosilylation reaction proceeds at ambient conditions and is operationally safe because no air-sensitive reagents or highly reactive metal hydrides are needed.

  20. Tailor-made designer helical peptides that induce mitochondrion-mediated cell death without necrosis.

    PubMed

    Nogami, Kagayaki; Takahama, Kentaro; Okushima, Ayako; Oyoshi, Takanori; Fujimoto, Kazuhisa; Inouye, Masahiko

    2014-11-24

    Managing protein-protein interactions is essential for resolving unknown biological events at the molecular level and developing drugs. We have designed and synthesized a side-chain-crosslinked helical peptides based on the binding domain of a pro-apoptotic protein (Bad) that induces programmed cell death. The peptide showed high helical content and bound to its target, Bcl-XL, more strongly than its non-crosslinked counterparts. When HeLa cells were incubated with the crosslinked peptide, the peptide entered the cytosol across the plasma membrane. The peptide formed a stable complex with Bcl-XL localized at the outer mitochondrial membrane, and this binding event caused the release of cytochrome c from the intermembrane space of mitochondria into the cytosol. This activated the caspase cascade: 70% of HeLa cells died by the apoptosis pathway (without evidence of necrosis).

  1. 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

  2. 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.

  3. Peptide nucleic acids (PNAs) patterning by an automated microarray synthesis system through photolithography.

    PubMed

    Wu, Yan-Qi; Yang, Fei-Peng; Wang, Hong-Yin; Liu, Jian-Xin; Liu, Zheng-Chun

    2013-03-01

    Peptide nucleic acids (PNA) microarray assembled with hundreds of unique PNA oligomers has been regarded as a new and mighty competitor of DNA chip in gene analyzing. However, PNA microarray is still a luxury art due to the difficult and laborious chemical synthesis. Herein, we have developed a fully-automated synthesizer for PNA microarray through photolithography. A preactivation mixer was designed and integrated into the synthesizer in order to get rid of the annoying manual process and increase the coupling efficiency of PNA monomers. The PNA patterning model was carried out to check the performance of the automated synthesizer, revealing that an exposure time of 3 min was sufficient for the complete removal of o-nitroveratryloxycarbonyl (NVOC) groups from the synthetic sites with the help of photosensitizer isopropylthioxanthone and the stepwise yield was measured to be about 98.0%, which is comparable with that from conventional fluorenyl-methyloxycarbonyl (FMOC) chemistry. Those results have definitely demonstrated the possibility and capability of this fully-automated synthesizer to fabricate high-quality PNA microarrays.

  4. Application of Peptide Nucleic Acid-based Assays Toward Detection of Somatic Mosaicism.

    PubMed

    Hong, Christopher S; Yang, Chunzhang; Zhuang, Zhengping

    2016-04-26

    Peptide nucleic acids (PNAs) are synthetic oligonucleotides with many applications. Compared with DNA, PNAs bind their complementary DNA strand with higher specificity and strength, an attribute that can make it an effective polymerase chain reaction clamp. A growing body of work has demonstrated the utility of PNAs in detecting low levels of mutant DNA, particularly in the detection of circulating mutated tumor cells in the peripheral blood. The PNA-based assay has greater sensitivity than direct sequencing and is significantly more affordable and rapid than next-generation deep sequencing. We have previously demonstrated that PNAs can successfully detect somatic mosaicism in patients with suspected disease phenotypes. In this report, we detail our methodology behind PNA design and application. We describe our protocol for optimizing the PNA for sequencing use and for determining the sensitivity of the PNA-based assay. Lastly, we discuss the potential applications of our assay for future laboratory and clinical purposes and highlight the role of PNAs in the detection of somatic mosaicism.

  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. 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

  7. 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

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

    PubMed

    Layek, Buddhadev; Lipp, Lindsey; Singh, Jagdish

    2015-12-04

    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.

  9. End invasion of peptide nucleic acids (PNAs) with mixed-base composition into linear DNA duplexes.

    PubMed

    Smolina, Irina V; Demidov, Vadim V; Soldatenkov, Viatcheslav A; Chasovskikh, Sergey G; Frank-Kamenetskii, Maxim D

    2005-01-01

    Peptide nucleic acid (PNA) is a synthetic DNA mimic with valuable properties and a rapidly growing scope of applications. With the exception of recently introduced pseudocomplementary PNAs, binding of common PNA oligomers to target sites located inside linear double-stranded DNAs (dsDNAs) is essentially restricted to homopurine-homopyrimidine sequence motifs, which significantly hampers some of the PNA applications. Here, we suggest an approach to bypass this limitation of common PNAs. We demonstrate that PNA with mixed composition of ordinary nucleobases is capable of sequence-specific targeting of complementary dsDNA sites if they are located at the very termini of DNA duplex. We then show that such targeting makes it possible to perform capturing of designated dsDNA fragments via the DNA-bound biotinylated PNA as well as to signal the presence of a specific dsDNA sequence, in the case a PNA beacon is employed. We also examine the PNA-DNA conjugate and prove that it can initiate the primer-extension reaction starting from the duplex DNA termini when a DNA polymerase with the strand-displacement ability is used. We thus conclude that recognition of duplex DNA by mixed-base PNAs via the end invasion has a promising potential for site-specific and sequence-unrestricted DNA manipulation and detection.

  10. Quantitative rRNA-targeted solution-based hybridization assay using peptide nucleic acid molecular beacons.

    PubMed

    Li, Xu; Morgenroth, Eberhard; Raskin, Lutgarde

    2008-12-01

    The potential of a solution-based hybridization assay using peptide nucleic acid (PNA) molecular beacon (MB) probes to quantify 16S rRNA of specific populations in RNA extracts of environmental samples was evaluated by designing PNA MB probes for the genera Dechloromonas and Dechlorosoma. In a kinetic study with 16S rRNA from pure cultures, the hybridization of PNA MB to target 16S rRNA exhibited a higher final hybridization signal and a lower apparent rate constant than the hybridizations to nontarget 16S rRNAs. A concentration of 10 mM NaCl in the hybridization buffer was found to be optimal for maximizing the difference between final hybridization signals from target and nontarget 16S rRNAs. Hybridization temperatures and formamide concentrations in hybridization buffers were optimized to minimize signals from hybridizations of PNA MB to nontarget 16S rRNAs. The detection limit of the PNA MB hybridization assay was determined to be 1.6 nM of 16S rRNA. To establish proof for the application of PNA MB hybridization assays in complex systems, target 16S rRNA from Dechlorosoma suillum was spiked at different levels to RNA isolated from an environmental (bioreactor) sample, and the PNA MB assay enabled effective quantification of the D. suillum RNA in this complex mixture. For another environmental sample, the quantitative results from the PNA MB hybridization assay were compared with those from clone libraries.

  11. Application of Peptide Nucleic Acid-based Assays Toward Detection of Somatic Mosaicism

    PubMed Central

    Hong, Christopher S; Yang, Chunzhang; Zhuang, Zhengping

    2016-01-01

    Peptide nucleic acids (PNAs) are synthetic oligonucleotides with many applications. Compared with DNA, PNAs bind their complementary DNA strand with higher specificity and strength, an attribute that can make it an effective polymerase chain reaction clamp. A growing body of work has demonstrated the utility of PNAs in detecting low levels of mutant DNA, particularly in the detection of circulating mutated tumor cells in the peripheral blood. The PNA-based assay has greater sensitivity than direct sequencing and is significantly more affordable and rapid than next-generation deep sequencing. We have previously demonstrated that PNAs can successfully detect somatic mosaicism in patients with suspected disease phenotypes. In this report, we detail our methodology behind PNA design and application. We describe our protocol for optimizing the PNA for sequencing use and for determining the sensitivity of the PNA-based assay. Lastly, we discuss the potential applications of our assay for future laboratory and clinical purposes and highlight the role of PNAs in the detection of somatic mosaicism. PMID:27115839

  12. Fluorine containing amino acids: synthesis and peptide coupling of amino acids containing the all-cis tetrafluorocyclohexyl motif.

    PubMed

    Ayoup, Mohammed Salah; Cordes, David B; Slawin, Alexandra M Z; O'Hagan, David

    2015-05-28

    A synthesis of two (S)-phenylalanine derivatives is described which have the all-cis, 2,3,5,6-tetrafluorocyclohexyl motif attached to the aromatic ring at the meta and para positions; the para substituted isomer is elaborated into illustrative dipeptides via the free amine and carboxylate respectively demonstrating its utility as a novel amino acid for peptide synthesis and offering a vehicle for incorporation of this unique and facially polarized ring system into bioactive compounds.

  13. 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).

  14. Transporters for ammonium, amino acids and peptides are expressed in pitchers of the carnivorous plant Nepenthes.

    PubMed

    Schulze, W; Frommer, W B; Ward, J M

    1999-03-01

    Insect capture and digestion contribute substantially to the nitrogen budget of carnivorous plants. In Nepenthes, insect-derived nitrogenous compounds are imported from the pitcher fluid and transported throughout the plant via the vascular tissue to support growth. Import and distribution of nutrients may require transmembrane nitrogen transporters. Representatives of three classes of genes encoding transporters for the nitrogenous compounds ammonium, amino acids and peptides were identified in Nepenthes pitchers. The expression at the cellular level of an ammonium transporter gene, three amino acid transporter genes, and one peptide transporter gene were investigated in the insect trapping organs of Nepenthes. Expression of the ammonium transporter gene NaAMT1 was detected in the head cells of digestive glands in the lower part of the pitcher where NaAMT1 may function in ammonium uptake from the pitcher fluid. One amino acid transporter gene, NaAAP1, was expressed in bundle sheath cells surrounding the vascular tissue. To understand the locations where transmembrane transport could be required within the pitcher, symplasmic and apoplasmic continuity was probed using fluorescent dyes. Symplasmic connections were not found between cortical cells and vascular bundles. Therefore, the amino acid transporter encoded by NaAAP1 may be involved in transport of amino acids into the vascular tissue. In contrast, expression of the peptide transporter gene NaNTR1 was detected in phloem cells of the vascular tissue within pitchers. NaNTR1 may function in the export of nitrogen from the pitcher by loading peptides into the phloem. PMID:10230062

  15. A General Synthetic Approach for Designing Epitope Targeted Macrocyclic Peptide Ligands.

    PubMed

    Das, Samir; Nag, Arundhati; Liang, JingXin; Bunck, David N; Umeda, Aiko; Farrow, Blake; Coppock, Matthew B; Sarkes, Deborah A; Finch, Amethist S; Agnew, Heather D; Pitram, Suresh; Lai, Bert; Yu, Mary Beth; Museth, A Katrine; Deyle, Kaycie M; Lepe, Bianca; Rodriguez-Rivera, Frances P; McCarthy, Amy; Alvarez-Villalonga, Belen; Chen, Ann; Heath, John; Stratis-Cullum, Dimitra N; Heath, James R

    2015-11-01

    We describe a general synthetic strategy for developing high-affinity peptide binders against specific epitopes of challenging protein biomarkers. The epitope of interest is synthesized as a polypeptide, with a detection biotin tag and a strategically placed azide (or alkyne) presenting amino acid. This synthetic epitope (SynEp) is incubated with a library of complementary alkyne or azide presenting peptides. Library elements that bind the SynEp in the correct orientation undergo the Huisgen cycloaddition, and are covalently linked to the SynEp. Hit peptides are tested against the full-length protein to identify the best binder. We describe development of epitope-targeted linear or macrocycle peptide ligands against 12 different diagnostic or therapeutic analytes. The general epitope targeting capability for these low molecular weight synthetic ligands enables a range of therapeutic and diagnostic applications, similar to those of monoclonal antibodies.

  16. 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

  17. Antimicrobial activity of de novo designed cationic peptides against multi-resistant clinical isolates.

    PubMed

    Faccone, Diego; Veliz, Omar; Corso, Alejandra; Noguera, Martin; Martínez, Melina; Payes, Cristian; Semorile, Liliana; Maffía, Paulo Cesar

    2014-01-01

    Antibiotic resistance is one of the main problems concerning public health or clinical practice. Antimicrobial peptides appear as good candidates for the development of new therapeutic drugs. In this study we de novo designed a group of cationic antimicrobial peptides, analyzed its physicochemical properties, including its structure by circular dichroism and studied its antimicrobial properties against a panel of clinical isolates expressing different mechanisms of resistance. Three cationic alpha helical peptides exhibited antimicrobial activity comparable to, or even better than the comparator omiganan (MBI-226).

  18. 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

  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. Self-assembly of short peptides to form hydrogels: design of building blocks, physical properties and technological applications.

    PubMed

    Fichman, Galit; Gazit, Ehud

    2014-04-01

    Hydrogels are unique supramolecular solid-like assemblies composed mainly of water molecules that are held by molecular networks. Physical hydrogels that are formed by a set of non-covalent interactions to establish a well-ordered scaffold devoid of any chemical cross-linking are especially intriguing for various biotechnological and medical applications. Peptides are particularly interesting building blocks of physical gels because of the role of polypeptides as structural elements in biological systems, the extensive ability for their chemical and biological decoration and functionalization, and the facile synthesis of natural and modified peptides. This review describes the assembly and properties of physical hydrogels that have been formed by the self-association of very simple peptide building blocks. Natural short peptides, as short as dipeptides, can form ordered gel assemblies. Moreover, in the case of N-terminal protection, even a protected amino acid can serve as an efficient hydrogelator. Further elucidation of hydrogelators' assembly, as well as the characterization of their physical properties, can guide the rational design of building blocks for a desired application. The possible mechanism of self-assembly is discussed in line with the chemical nature of the short peptides. Different methods have been used to induce hydrogel assembly, which may significantly affect the mechanical characteristics of the resulting gels. Here, special emphasis is given to methods that allow either spatial control of hydrogel formation or modulation of physical properties of the gel. Finally, the parameters that influence hydrogelation are described, and insights for their design are provided. PMID:23958781

  1. Effects of salt concentration on the reaction rate of Glc with amino acids, peptides, and proteins.

    PubMed

    Yamaguchi, Keiko; Noumi, Yuri; Nakajima, Katsumi; Nagatsuka, Chiharu; Aizawa, Haruko; Nakawaki, Rie; Mizude, Eri; Otsuka, Yuzuru; Homma, Takeshi; Chuyen, Nguyen Van

    2009-11-01

    The reaction between the amino group and the carbonyl group is important in food quality control. Furthermore, advanced glycation end products from foods are considered to relate to aging and diabetes. Thus, it is important to control this reaction. In this study, we investigated the effects of salt concentration on the rates of browning reaction of amino acid, peptides, and proteins. A high concentration of sodium chloride retarded the reaction rate of Glc with amino acids as measured with the absorbance at 470 nm, but did not change the browning rate of Glc with peptides. On the other hand, sodium chloride retarded the browning reaction rate of proteins as measured with polymerization degree or by the loss of Lys. It is hoped that the results of this study will be applied in the control of amino-carbonyl reaction rates in the food industry. PMID:19897911

  2. Kinetics of interaction of vanillin with amino acids and peptides in model systems.

    PubMed

    Chobpattana, W; Jeon, I J; Smith, J S

    2000-09-01

    Model systems were used to study the reaction kinetics of vanillin and pentalysine, lysine, glutathione, cysteine, aspartame, or phenylalanine (molar ratio 1:1) in phosphate buffer. The buffer pH was adjusted to the pK(a)(2) of the available alpha-amino group of each amino acid or peptide. Reductions of vanillin followed first-order kinetics at 55, 65, and 75 degrees C in the presence of each of the amino acids or peptides used. The reaction rates were accelerated as the temperature increased. The rate constants were highest for pentalysine followed by lysine, phenylalanine, glutathione/cysteine, and aspartame. The reduction of phenylalanine followed first-order kinetics, whereas the formation of its reaction product followed zero-order kinetics. The activation energy (E(a)) for the reaction ranged from 5.6 to 14.5 kcal/mol.

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

    PubMed Central

    Pearson, C. Seth; Kloos, Zachary; Murray, Brian; Tabe, Ebot; Gupta, Monica; Kwak, Jun Ha; Karande, Pankaj

    2016-01-01

    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

  4. 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.

  5. A molecular peptide beacon for the ratiometric sensing of nucleic acids.

    PubMed

    Wu, Junchen; Zou, Ying; Li, Chunyan; Sicking, Wilhelm; Piantanida, Ivo; Yi, Tao; Schmuck, Carsten

    2012-02-01

    A pyrene-functionalized cationic oligopeptide 1 efficiently binds to double-stranded DNA, as shown by different spectrophotochemical studies. Upon binding, the conformation of 1 changes from a folded to an extended form, which leads to a distinct change in the fluorescence properties. Thus, 1 functions as a molecular peptide beacon, and as it is easily taken up by cells, 1 can also be used for imaging of nucleic acids within cells.

  6. Development of SI-traceable C-peptide certified reference material NMIJ CRM 6901-a using isotope-dilution mass spectrometry-based amino acid analyses.

    PubMed

    Kinumi, Tomoya; Goto, Mari; Eyama, Sakae; Kato, Megumi; Kasama, Takeshi; Takatsu, Akiko

    2012-07-01

    A certified reference material (CRM) is a higher-order calibration material used to enable a traceable analysis. This paper describes the development of a C-peptide CRM (NMIJ CRM 6901-a) by the National Metrology Institute of Japan using two independent methods for amino acid analysis based on isotope-dilution mass spectrometry. C-peptide is a 31-mer peptide that is utilized for the evaluation of β-cell function in the pancreas in clinical testing. This CRM is a lyophilized synthetic peptide having the human C-peptide sequence, and contains deamidated and pyroglutamylated forms of C-peptide. By adding water (1.00 ± 0.01) g into the vial containing the CRM, the C-peptide solution in 10 mM phosphate buffer saline (pH 6.6) is reconstituted. We assigned two certified values that represent the concentrations of total C-peptide (mixture of C-peptide, deamidated C-peptide, and pyroglutamylated C-peptide) and C-peptide. The certified concentration of total C-peptide was determined by two amino acid analyses using pre-column derivatization liquid chromatography-mass spectrometry and hydrophilic chromatography-mass spectrometry following acid hydrolysis. The certified concentration of C-peptide was determined by multiplying the concentration of total C-peptide by the ratio of the relative area of C-peptide to that of the total C-peptide measured by liquid chromatography. The certified value of C-peptide (80.7 ± 5.0) mg/L represents the concentration of the specific entity of C-peptide; on the other hand, the certified value of total C-peptide, (81.7 ± 5.1) mg/L can be used for analyses that does not differentiate deamidated and pyroglutamylated C-peptide from C-peptide itself, such as amino acid analyses and immunochemical assays.

  7. 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

  8. 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

  9. 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.

  10. 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

  11. Binding of acylated peptides and fatty acids to phospholipid vesicles: pertinence to myristoylated proteins.

    PubMed

    Peitzsch, R M; McLaughlin, S

    1993-10-01

    We studied the binding of fatty acids and acylated peptides to phospholipid vesicles by making electrophoretic mobility and equilibrium dialysis measurements. The binding energies of the anionic form of the fatty acids and the corresponding acylated glycines were identical; the energies increased by 0.8 kcal/mol per number of carbons in the acyl chain (Ncarbon = 10, 12, 14, 16), a value identical to that for the classical entropy-driven hydrophobic effect discussed by Tanford [The Hydrophobic Effect (1980) Wiley, New York]. The unitary Gibbs free binding energy, delta Gou, of myristoylated glycine, 8 kcal/mol, is independent of the nature of the electrically neutral lipids used to form the vesicles. Similar binding energies were obtained with other myristoylated peptides (e.g., Gly-Ala, Gly-Ala-Ala). The 8 kcal/mol, which corresponds to an effective dissociation constant of 10(-4) M for myristoylated peptides with lipids, provides barely enough energy to attach a myristoylated protein in the cytoplasm to the plasma membrane. Thus, other factors that reduce (e.g., hydrophobic interaction of myristate with the covalently attached protein) or enhance (e.g., electrostatic interactions of basic residues with acidic lipids; protein-protein interactions with intrinsic receptor proteins) the interaction of myristoylated proteins with membranes are likely to be important and may cause reversible translocation of these proteins to the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

  12. Acid-base titration of melanocortin peptides: evidence of Trp rotational conformers interconversion.

    PubMed

    Fernandez, Roberto M; Vieira, Renata F F; Nakaie, Clóvis R; Lamy, M Teresa; Ito, Amando S

    2005-01-01

    Tryptophantime-resolved fluorescence was used to monitor acid-base titration properties of alpha-melanocyte stimulating hormone (alpha-MSH) and the biologically more potent analog [Nle4, D-Phe7]alpha -MSH (NDP-MSH), labeled or not with the paramagnetic amino acid probe 2,2,6,6-tetramthylpiperidine-N-oxyl-4-amino-4-carboxylic acid (Toac). Global analysis of fluorescence decay profiles measured in the pH range between 2.0 and 11.0 showed that, for each peptide, the data could be well fitted to three lifetimes whose values remained constant. The less populated short lifetime component changed little with pH and was ascribed to Trp g+ chi1 rotamer, in which electron transfer deactivation predominates over fluorescence. The long and intermediate lifetime preexponential factors interconverted along that pH interval and the result was interpreted as due to interconversion between Trp g- and trans chi1 rotamers, driven by conformational changes promoted by modifications in the ionization state of side-chain residues. The differences in the extent of interconversion in alpha-MSH and NDP-MSH are indicative of structural differences between the peptides, while titration curves suggest structural similarities between each peptide and its Toac-labeled species, in aqueous solution. Though less sensitive than fluorescence, the Toac electron spin resonance (ESR) isotropic hyperfine splitting parameter can also monitor the titration of side-chain residues located relatively far from the probe.

  13. Reaction of chlorine dioxide with amino acids and peptides: kinetics and mutagenicity studies.

    PubMed

    Tan, H K; Wheeler, W B; Wei, C I

    1987-08-01

    Chlorine dioxide (ClO2) is currently being considered as an alternate to chlorine as a disinfectant for water treatment. Many organic compounds present in water and food treated with ClO2 are subject to oxidation. 21 amino acids and 3 peptides (L-aspartyl-L-phenylalanine methyl ester (aspartame), L-glycyl-L-tryptophan and L-tryptophylglycine) were studied for their reactivity with ClO2. Chlorine dioxide reacted only with 6 amino acids in 0.1 M sodium phosphate buffer, pH 6.0. The reaction with cysteine, tryptophan and tyrosine was too rapid to be monitored either iodometrically or spectrophotometrically. The reaction with histidine, hydroxyproline and proline was found to be pseudo-first order. ClO2 readily reacted with L-glycyl-L-tryptophan and L-tryptophylglycine but not with aspartame. Mutagenicity studies with the Salmonella microsome assay of the reaction mixtures of ClO2 with those 6 reactive amino acids and the 3 peptides indicated that the reaction products of the 3 peptides, hydroxyproline, and tyrosine exerted mutagenic activity toward both tester strains of TA98 and TA100 in the presence and absence of rat-liver S9 mix.

  14. Application of hydrophilic interaction chromatography retention coefficients for predicting peptide elution with TFA and methanesulfonic acid ion-pairing reagents.

    PubMed

    Wujcik, Chad E; Tweed, Joseph; Kadar, Eugene P

    2010-03-01

    Hydrophilic retention coefficients for 17 peptides were calculated based on retention coefficients previously published for TSKgel silica-60 and were compared with the experimental elution profile on a Waters Atlantis HILIC silica column using TFA and methanesulfonic acid (MSA) as ion-pairing reagents. Relative peptide retention could be accurately determined with both counter-ions. Peptide retention and chromatographic behavior were influenced by the percent acid modifier used with increases in both retention and peak symmetry observed at increasing modifier concentrations. The enhancement of net peptide polarity through MSA pairing shifted retention out by nearly five-fold for the earliest eluting peptide, compared with TFA. Despite improvements in retention and efficiency (N(eff)) for MSA over TFA, a consistent reduction in calculated selectivity (alpha) was observed. This result is believed to be attributed to the stronger polar contribution of MSA masking and diminishing the underlying influence of the amino acid residues of each associated peptide. Finally, post-column infusion of propionic acid and acetic acid was evaluated for their potential to recover signal intensity for TFA and MSA counter-ions for LC-ESI-MS applications. Acetic acid generally yielded more substantial signal improvements over propionic acid on the TFA system while minimal benefits and some further reductions were noted with MSA.

  15. Formation pathways and opioid activity data for 3-hydroxypyridinium compounds derived from glucuronic acid and opioid peptides by Maillard processes.

    PubMed

    Horvat, Stefica; Roscić, Maja; Lemieux, Carole; Nguyen, Thi M-D; Schiller, Peter W

    2007-07-01

    The kinetics of formation and identity of the reaction products of the glucuronic acid with three representative opioid peptides were investigated in vitro. Peptides were conjugated with glucuronic acid either in solution or under dry-heating conditions. From the incubations performed in solution N-(1-deoxy-D-fructofuranos-1-yluronic acid)-peptide derivatives (Amadori compounds) were isolated, whereas from the dry-heated reactions products containing the 3-hydroxypyridinium moiety at the N-terminal of the peptide chain were obtained. Experiments performed under mild dry-heating conditions (40 degrees C) in model systems based on Leu-enkephalin and glucuronic acid, and in environment of either 40% or 75% relative humidity, revealed that the higher level of humidity promoted a process that enhanced 3-hydroxypyridinium compound generation. The mechanism of 3-hydroxypyridinium formation is discussed. In comparison with their respective parent peptides, the N-(1-deoxy-D-fructofuranosyl-uronic acid) derivatives of the opioid peptides showed three- to 11-fold lower mu- and delta-receptor-binding affinities and agonist potencies in the functional assays, likely as a consequence of the steric bulk introduced at the N-terminal amino group. The further decrease in opioid activity observed with the 3-hydroxypyridinium-containing peptides may be due to the lower pK(a) of the 3-hydroxypyridinium moiety and to delocalization of the positive charge in the pyridinium ring system. PMID:17630992

  16. De novo design of peptide scaffolds as novel preorganized ligands for metal-ion coordination.

    PubMed

    Gamble, Aimee J; Peacock, Anna F A

    2014-01-01

    This chapter describes how de novo designed peptides can be used as novel preorganized ligands for metal ion coordination. The focus is on the design of peptides which are programmed to spontaneously self-assemble into α-helical coiled coils in aqueous solution, and how metal ion binding sites can be engineered onto and into these structures. In addition to describing the various design principles, some key examples are covered illustrating the success of this approach, including a more detailed example in the case study.

  17. Design of Peptide and Peptidomimetic Ligands with Novel Pharmacological Activity Profiles

    PubMed Central

    Hruby, Victor J.; Cai, Minying

    2016-01-01

    Peptide hormones and neurotransmitters are of central importance in most aspects of intercellular communication and are involved in virtually all degenerative diseases. In this review, we discuss physicochemical approaches to the design of novel peptide and peptidomimetic agonists, antagonists, inverse agonists, and related compounds that have unique biological activity profiles, reduced toxic side effects, and, if desired, the ability to cross the blood-brain barrier. Designing ligands for specific biological and medical needs is emphasized, as is the close collaboration of chemists and biologists to maximize the chances for success. Special emphasis is placed on the use of conformational (φ-ψ space) and topographical (χ space) considerations in design. PMID:23294313

  18. Conformational study of N-methylated alanine peptides and design of Abeta inhibitor.

    PubMed

    Nandel, Fateh S; Jaswal, Radhika R

    2014-02-01

    N-Methylation increases the proteolytic stability of peptides and leads to improved pharmacological and increased nematicidal property against plant pathogens. In this study, the quantum mechanical and molecular dynamic simulation approaches were used to investigate conformational behavior of peptides containing only N-methylated alanine (NMeAla) residues and N-methylated alanine and alanine residues at alternate positions. The amide bond geometry was found to be trans and the poly NMeAla peptides were shown to populate in the helical structure without hydrogen bond with phi, psi values of - 0, 90 degrees stabilized by carbonyl-carbonyl interactions. Molecular dynamic simulations in water/methanol revealed the formation of beta-strand structure, irrespective of the starting geometry due to the interaction of solvent molecules with the carbonyl groups of peptide backbone. Analysis of simulation results as a function of time suggested that the opening of helical structure without hydrogen bond started from C-terminal. Conformational behavior of peptides containing N-MeAla and Ala was used to design Abeta peptide inhibitor and the model tetrapeptide Ac-Ala-NMeAla-Ala-NHMe in the beta-strand structure was shown to interact with the hydrophobic stretch of Abeta15-42 peptide.

  19. 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

  20. Osmotic Pressure Simulations of Amino Acids and Peptides Highlight Potential Routes to Protein Force Field Parameterization.

    PubMed

    Miller, Mark S; Lay, Wesley K; Elcock, Adrian H

    2016-08-25

    Recent molecular dynamics (MD) simulations of proteins have suggested that common force fields overestimate the strength of amino acid interactions in aqueous solution. In an attempt to determine the causes of these effects, we have measured the osmotic coefficients of a number of amino acids using the AMBER ff99SB-ILDN force field with two popular water models, and compared the results with available experimental data. With TIP4P-Ew water, interactions between aliphatic residues agree well with experiment, but interactions of the polar residues serine and threonine are found to be excessively attractive. For all tested amino acids, the osmotic coefficients are lower when the TIP3P water model is used. Additional simulations performed on charged amino acids indicate that the osmotic coefficients are strongly dependent on the parameters assigned to the salt ions, with a reparameterization of the sodium/carboxylate interaction reported by the Aksimentiev group significantly improving description of the osmotic coefficient for glutamate. For five neutral amino acids, we also demonstrate a decrease in solute-solute attractions using the recently reported TIP4P-D water model and using the KBFF force field. Finally, we show that for four two-residue peptides improved agreement with experiment can be achieved by rederiving the partial charges for each peptide. PMID:27052117

  1. 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

  2. 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

  3. Update of PROFEAT: a web server for computing structural and physicochemical features of proteins and peptides from amino acid sequence.

    PubMed

    Rao, H B; Zhu, F; Yang, G B; Li, Z R; Chen, Y Z

    2011-07-01

    Sequence-derived structural and physicochemical features have been extensively used for analyzing and predicting structural, functional, expression and interaction profiles of proteins and peptides. PROFEAT has been developed as a web server for computing commonly used features of proteins and peptides from amino acid sequence. To facilitate more extensive studies of protein and peptides, numerous improvements and updates have been made to PROFEAT. We added new functions for computing descriptors of protein-protein and protein-small molecule interactions, segment descriptors for local properties of protein sequences, topological descriptors for peptide sequences and small molecule structures. We also added new feature groups for proteins and peptides (pseudo-amino acid composition, amphiphilic pseudo-amino acid composition, total amino acid properties and atomic-level topological descriptors) as well as for small molecules (atomic-level topological descriptors). Overall, PROFEAT computes 11 feature groups of descriptors for proteins and peptides, and a feature group of more than 400 descriptors for small molecules plus the derived features for protein-protein and protein-small molecule interactions. Our computational algorithms have been extensively tested and used in a number of published works for predicting proteins of specific structural or functional classes, protein-protein interactions, peptides of specific functions and quantitative structure activity relationships of small molecules. PROFEAT is accessible free of charge at http://bidd.cz3.nus.edu.sg/cgi-bin/prof/protein/profnew.cgi.

  4. 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

  5. Differentiating N-terminal aspartic and isoaspartic acid residues in peptides.

    PubMed

    Sargaeva, Nadezda P; Lin, Cheng; O'Connor, Peter B

    2011-09-01

    Formation of isoaspartic acid (isoAsp) is a common modification of aspartic acid (Asp) or asparagine (Asn) residue in proteins. Differentiation of isoAsp and Asp residues is a challenging task owing to their similar properties and identical molecular mass. It was recently shown that they can be differentiated using ion-electron or ion-ion interaction fragmentation methods (ExD) because these methods provide diagnostic fragments c + 57 and z(•) - 57 specific to the isoAsp residue. To date, however, the presence of such fragments has not been explored on peptides with an N-terminal isoAsp residue. To address this question, several N-terminal isoAsp-containing peptides were analyzed using ExD methods alone or combined with chromatography. A diagnostic fragment [M + 2H - 74](+•) was observed for the doubly charged precursor ions with N-terminal isoAsp residues. For some peptides, identification of the N-terminal isoAsp residue was challenging because of the low diagnostic ion peak intensity and the presence of interfering peaks. Supplemental activation was used to improve diagnostic ion detection. Further, N-terminal acetylation was offered as a means to overcome the interference problem by shifting the diagnostic fragment peak to [M + 2H - 116](+•).

  6. The Perseus Exobiology mission on MIR: behaviour of amino acids and peptides in Earth orbit.

    PubMed

    Boillot, F; Chabin, A; Buré, C; Venet, M; Belsky, A; Bertrand-Urbaniak, M; Delmas, A; Brack, A; Barbier, B

    2002-08-01

    Leucine, alpha-methyl leucine and two peptides were exposed to space conditions on board the MIR station during the Perseus-Exobiology mission. This long duration space mission was aimed at testing the delivery of prebiotic building blocks. During this mission, two amino acids (leucine and alpha-methyl leucine) and two peptides (leucine-diketopiperazine and trileucine thioethylester) were exposed in Earth orbit for three months. Basalt, clay and meteorite powder were also mixed with the samples in order to simulate the effects of potential meteorite protection. Analysis of the material after the flight did not reveal any racemization or polymerisation but did provide information regarding photochemical pathways for the degradation of leucine and of the tripeptide. Amino acids appeared to be more sensitive to UV radiation than peptides, the cyclic dipeptide being found to be as particularly resistant. Meteorite powder which exhibits the highest absorption in Vacuum UltraViolet (VUV) afforded the best protection to the organic molecules whereas montmorillonite clay, almost transparent in VUV, was the least efficient. By varying the thickness of the meteorite, we found that the threshold for efficient protection against radiation was about 5 microm. The possible exogenous origin of biological building blocks is discussed with respect to the stability to the molecules and the nature of the associated minerals.

  7. The Perseus Exobiology Mission on MIR: Behaviour of Amino Acids and Peptides in Earth Orbit

    NASA Astrophysics Data System (ADS)

    Boillot, F.; Chabin, A.; Buré, C.; Venet, M.; Belsky, A.; Bertrand-Urbaniak, M.; Delmas, A.; Brack, A.; Barbier, B.

    2002-08-01

    Leucine, α-methyl leucine and two peptides were exposed to space conditions on board the MIR station during the Perseus-Exobiology mission. This long duration space mission was aimed at testing the delivery of prebiotic building blocks. During this mission, two amino acids (leucine and α-methyl leucine) and two peptides (leucine-diketopiperazine and trileucine thioethylester) were exposed in Earth orbit for three months. Basalt, clay and meteorite powder were also mixed with the samples in order to simulate the effects of potential meteorite protection. Analysis of the material after the flight did not reveal any racemization or polymerisation but did provide information regarding photochemical pathways for the degradation of leucine and of the tripeptide. Amino acids appeared to be more sensitive to UV radiation than peptides, the cyclic dipeptide being found to be as particularly resistant. Meteorite powder which exhibits the highest absorption in Vacuum UltraViolet (VUV) afforded the best protection to the organic molecules whereas montmorillonite clay, almost transparent in VUV, was the least efficient. By varying the thickness of the meteorite, we found that the threshold for efficient protection against radiation was about 5 μm. The possible exogenous origin of biological building blocks is discussed with respect to the stability to the molecules and the nature of the associated minerals.

  8. 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

  9. 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.

  10. Peptide nucleic acid (PNA) binding-mediated induction of human gamma-globin gene expression.

    PubMed

    Wang, G; Xu, X; Pace, B; Dean, D A; Glazer, P M; Chan, P; Goodman, S R; Shokolenko, I

    1999-07-01

    Peptide nucleic acids (PNAs) can bind to homopurine/homopyrimidine sequences of double-stranded DNA targets in a sequence-specific manner and form [PNA]2/DNA triplexes with single-stranded DNA D-loop structures at the PNA binding sites. These D-loop structures have been found to have a capacity to initiate transcription in vitro. If this strategy can be used to induce transcription of endogenous genes, it may provide a novel approach for gene therapy of many human diseases. Human [beta] globin disorders such as sickle cell anemia and beta-thalassemia are very common genetic diseases that are caused by mutations in the beta-globin gene. When gamma-globin genes are highly expressed in sickle cell patients, the presence of high levels of fetal hemoglobin (HbF, alpha2gamma2) can compensate for the defective beta-globin gene product and such patients have much improved symptoms or are free of disease. However, the gamma-globin genes are developmentally regulated and normally expressed at very low levels (>1%) in adult blood cells. We have investigated the possibility of inducing gamma-globin gene expression with PNAs. Using PNAs designed to bind to the 5' flanking region of the gamma-globin gene, induction of expression of a reporter gene construct was demonstrated both in vitro and in vivo. More importantly, PNA-mediated induction of endogenous gamma-globin gene expression was also demonstrated in K562 human erythroleukemia cells. This result suggests that induction of gamma-globin gene expression with PNAs might provide a new approach for the treatment of sickle cell disease. PNA-induced gene expression strategy also may have implications in gene therapy of other diseases such as genetic diseases, cancer and infectious diseases.

  11. Protein fatty acid acylation: enzymatic synthesis of an N-myristoylglycyl peptide

    SciTech Connect

    Towler, D.; Glaser, L.

    1986-05-01

    Incubation of Saccharomyces cerevisiae strain JR153 with either (/sup 3/H)myristate or (/sup 3/H)palmitate demonstrates the synthesis of proteins that contain covalently bound fatty acids. A unique set of proteins is labeled by each fatty acid. Detailed analysis of a 20-kDa protein labeled with myristic acid demonstrates that myristate is linked to the amino-terminal glycine. We describe an enzymatic activity in yeast that will transfer myristic acid to the amino terminus of the octapeptide Gly-Asn-Ala-Ala-Ala-Ala-Arg-Arg, whose sequence was derived from a known N-myristoylated acyl protein, the catalytic subunit of cAMP-dependent protein kinase of bovine cardiac muscle. The acylation reaction is dependent on ATP and CoA, is enriched in a crude membrane fraction, and will use myristate but not palmitate as the acyl donor. Specificity of the glycyl peptide substrate is demonstrated by the observation that other glycyl peptides do not competitively inhibit myristoylation of Gly-Asn-Ala-Ala-Ala-Ala-Arg-Arg.

  12. 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

  13. Interactions of a designed peptide with lipopolysaccharide: Bound conformation and anti-endotoxic activity

    SciTech Connect

    Bhunia, Anirban; Chua, Geok Lin; Domadia, Prerna N.; Warshakoon, Hemamali; Cromer, Jens R.; David, Sunil A.; Bhattacharjya, Surajit

    2008-05-09

    Designed peptides that would selectively interact with lipopolysaccharide (LPS) or endotoxin and fold into specific conformations could serve as important scaffolds toward the development of antisepsis compounds. Here, we describe solution structure of a designed amphipathic peptide, H{sub 2}N-YVKLWRMIKFIR-CONH{sub 2} (YW12D) in complex with endotoxin as determined by transferred nuclear Overhauser effect spectroscopy. The conformation of the isolated peptide is highly flexible, but undergoes a dramatic structural stabilization in the presence of LPS. Structure calculations reveal that the peptide presents two amphipathic surfaces in its bound state to LPS whereby each surface is characterized by two positive charges and a number of aromatic and/or aliphatic residues. ITC data suggests that peptide interacts with two molecules of lipid A. In activity assays, YW12D exhibits neutralization of LPS toxicity with very little hemolysis of red blood cells. Structural and functional properties of YW12D would be applicable in designing low molecular weight non-toxic antisepsis molecules.

  14. Thioacetic acid/NaSH-mediated synthesis of N-protected amino thioacids and their utility in peptide synthesis.

    PubMed

    Mali, Sachitanand M; Gopi, Hosahudya N

    2014-03-21

    Thioacids are recently gaining momentum due to their versatile reactivity. The reactivity of thioacids has been widely explored in the selective amide/peptide bond formation. Thioacids are generally synthesized from the reaction between activated carboxylic acids such as acid chlorides, active esters, etc., and Na2S, H2S, or NaSH. We sought to investigate whether the versatile reactivity of the thioacids can be tuned for the conversion of carboxylic acids into corresponding thioacids in the presence of NaSH. Herein, we report that thioacetic acid- and NaSH-mediated synthesis of N-protected amino thioacids from the corresponding N-protected amino acids, oxidative dimerization of thioacids, crystal conformations of thioacid oxidative dimers, and the utility of thioacids and oxidative dimers in peptide synthesis. Our results suggest that peptides can be synthesized without using standard coupling agents.

  15. 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.

  16. Design, synthesis and biological evaluation of new peptide-based ureas and thioureas as potential antagonists of the thrombin receptor PAR1.

    PubMed

    Ventosa-Andrés, Pilar; Valdivielso, Angel M; Pappos, Ioannis; García-López, M Teresa; Tsopanoglou, Nikos E; Herranz, Rosario

    2012-12-01

    By applying a diversity oriented synthesis strategy for the search of new antagonists of the thrombin receptor PAR1, a series of peptide-based ureas and thioureas, including analogues of the PAR1 reference antagonist RWJ-58259, has been designed and synthesized. The general synthetic scheme involves reduction of basic amino acid-derived amino nitriles by hydrogen transfer from hydrazine monohydrate in the presence of Raney Ni, followed by reaction with diverse isocyanates and isothiocyanates, and protecting group removal. All new compounds have been evaluated as inhibitors of human platelet aggregation induced by the PAR1 agonist SFLLRN. Some protected peptide-based ureas displayed significant antagonist activity. PMID:23123726

  17. Synthesis of stable C-linked ferrocenyl amino acids and their use in solution-phase peptide synthesis.

    PubMed

    Philip, Anijamol T; Chacko, Shibin; Ramapanicker, Ramesh

    2015-12-01

    Incorporation of ferrocenyl group to peptides is an efficient method to alter their hydrophobicity. Ferrocenyl group can also act as an electrochemical probe when incorporated onto functional peptides. Most often, ferrocene is incorporated onto peptides post-synthesis via amide, ester or triazole linkages. Stable amino acids containing ferrocene as a C-linked side chain are potentially useful building units for the synthesis of ferrocene-containing peptides. We report here an efficient route to synthesize ferrocene-containing amino acids that are stable and can be used in peptide synthesis. Coupling of 2-ferrocenyl-1,3-dithiane and iodides derived from aspartic acid or glutamic acid using n-butyllithium leads to the incorporation of a ferrocenyl unit to the δ-position or ε-position of an α-amino acid. The reduction or hydrolysis of the dithiane group yields an alkyl or an oxo derivative. The usability of the synthesized amino acids is demonstrated by incorporating one of the amino acids in both C-terminus and N-terminus of tripeptides in solution phase.

  18. 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

  19. 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

  20. 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.

  1. Rationally designed coiled-coil DNA looping peptides control DNA topology.

    PubMed

    Gowetski, Daniel B; Kodis, Erin J; Kahn, Jason D

    2013-09-01

    Artificial DNA looping peptides were engineered to study the roles of protein and DNA flexibility in controlling the geometry and stability of protein-mediated DNA loops. These LZD (leucine zipper dual-binding) peptides were derived by fusing a second, C-terminal, DNA-binding region onto the GCN4 bZip peptide. Two variants with different coiled-coil lengths were designed to control the relative orientations of DNA bound at each end. Electrophoretic mobility shift assays verified formation of a sandwich complex containing two DNAs and one peptide. Ring closure experiments demonstrated that looping requires a DNA-binding site separation of 310 bp, much longer than the length needed for natural loops. Systematic variation of binding site separation over a series of 10 constructs that cyclize to form 862-bp minicircles yielded positive and negative topoisomers because of two possible writhed geometries. Periodic variation in topoisomer abundance could be modeled using canonical DNA persistence length and torsional modulus values. The results confirm that the LZD peptides are stiffer than natural DNA looping proteins, and they suggest that formation of short DNA loops requires protein flexibility, not unusual DNA bendability. Small, stable, tunable looping peptides may be useful as synthetic transcriptional regulators or components of protein-DNA nanostructures.

  2. Peptide hairpins with strand segments containing alpha- and beta-amino acid residues: cross-strand aromatic interactions of facing Phe residues.

    PubMed

    Roy, Rituparna S; Gopi, Hosahudya N; Raghothama, S; Gilardi, Richard D; Karle, Isabella L; Balaram, Padmanabhan

    2005-01-01

    The incporation of beta-amino acid residues into the strand segments of designed beta-hairpin leads to the formation of polar sheets, since in the case of beta-peptide strands, all adjacent carbonyl groups point in one direction and the amide groups orient in the opposite direction. The conformational analysis of two designed peptide hairpins composed of alpha/beta-hybrid segments are described: Boc-Leu-betaPhe-Val-(D)-Pro-Gly-Leu-betaPhe-Val-OMe (1) and Boc-betaLeu-Phe-betaVal-D-Pro-Gly-betaLeu-Phe-betaVal-OMe (2). A 500-MHz 1H-NMR (nuclear magnetic resonance) analysis in methanol supports a significant population of hairpin conformations in both peptides. Diagnostic nuclear Overhauser effects (NOEs) are observed in both cases. X-ray diffraction studies on single crystals of peptide 1 reveal a beta-hairpin conformation in both the molecules, which constitute the crystallographic asymmetric unit. Three cross-strand hydrogen bonds and a nucleating type II' beta-turn at the D-Pro-Gly segment are observed in the two independent molecules. In peptide 1, the betaPhe residues at positions 2 and 7 occur at the nonhydrogen-bonding position, with the benzyl side chains pointing on opposite faces of the beta-sheet. The observed aromatic centroid-to-centroid distances are 8.92 A (molecule A) and 8.94 A (molecule B). In peptide 2, the aromatic rings must occupy facing positions in antiparallel strands, in the NMR-derived structure. Peptide 1 yields a normal "hairpin-like" CD spectrum in methanol with a minimum at 224 nm. The CD spectrum of peptide 2 reveals a negative band at 234 nm and a positive band at 221 nm, suggestive of an exciton split doublet. Modeling of the facing Phe side chains at the hydrogen-bonding position of a canonical beta-hairpin suggests that interring separation is approximately 4.78 A for the gauche+ gauche- (g+ g-) rotamer. A previously reported peptide beta-hairpin composed of only alpha-amino acids, Boc-Leu-Phe-Val-D-Pro-Gly-Leu-Phe-Val-OMe also

  3. 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.

  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. Machine learning assisted design of highly active peptides for drug discovery.

    PubMed

    Giguère, Sébastien; Laviolette, François; Marchand, Mario; Tremblay, Denise; Moineau, Sylvain; Liang, Xinxia; Biron, Éric; Corbeil, Jacques

    2015-04-01

    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/.

  6. 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

  7. 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.

  8. Detection of peptidic sequences in the ancient acidic sediments of Río Tinto, Spain.

    PubMed

    Colín-García, María; Kanawati, Basem; Harir, Mourad; Schmitt-Kopplin, Phillippe; Amils, Ricardo; Parro, Victor; García, Miriam; Fernández-Remolar, David

    2011-12-01

    Biomarkers are molecules that are produced by or can be associated with biological activities. They can be used as tracers that give us an idea of the ancient biological communities that produced them, the paleoenvironmental conditions where they lived, or the mechanism involved in their transformation and preservation. As a consequence, the preservation potential of molecules over time depends largely on their nature, but also on the conditions of the environment, which controls the decomposition kinetics. In this context, proteins and nucleic acids, which are biomolecules bearing biological information, are among the most labile molecules. In this research, we report the presence of short-chained peptides obtained from extracts of ferruginous sedimentary deposits that have been produced under the acidic and oxidizing solutions of Río Tinto, Spain. These preliminary results go against the paradigmatic idea that considers the acidic and oxidizing environments inappropriate for the preservation of molecular information.

  9. Interfacial self-assembly of amino acids and peptides: Scanning tunneling microscopy investigation

    NASA Astrophysics Data System (ADS)

    Xu, Li-Ping; Liu, Yibiao; Zhang, Xueji

    2011-12-01

    Proteins play important roles in human daily life. To take advantage of the lessons learned from nature, it is essential to investigate the self-assembly of subunits of proteins, i.e., amino acids and polypeptides. Due to its high resolution and versatility of working environment, scanning tunneling microscopy (STM) has become a powerful tool for studying interfacial molecular assembly structures. This review is intended to reflect the progress in studying interfacial self-assembly of amino acids and peptides by STM. In particular, we focus on environment-induced polymorphism, chiral recognition, and coadsorption behavior with molecular templates. These studies would be highly beneficial to research endeavors exploring the mechanism and nanoscale-controlling molecular assemblies of amino acids and polypeptides on surfaces, understanding the origin of life, unravelling the essence of disease at the molecular level and deeming what is necessary for the ``bottom-up'' nanofabrication of molecular devices and biosensors being constructed with useful properties and desired performance.

  10. Folic acid administration inhibits amyloid β-peptide accumulation in APP/PS1 transgenic mice.

    PubMed

    Li, Wen; Liu, Huan; Yu, Min; Zhang, Xumei; Zhang, Meilin; Wilson, John X; Huang, Guowei

    2015-08-01

    Alzheimer's disease (AD) is associated with malnutrition, altered one-carbon metabolism and increased hippocampal amyloid-β peptide (Aβ) accumulation. Aberrant DNA methylation may be an epigenetic mechanism that underlies AD pathogenesis. We hypothesized that folic acid acts through an epigenetic gene silencing mechanism to lower Aβ levels in the APP/PS1 transgenic mouse model of AD. APP/PS1 mice were fed either folate-deficient or control diets and gavaged daily with 120 μg/kg folic acid, 13.3mg/kg S-adenosylmethionine (SAM) or both. Examination of the mice after 60 days of treatment showed that serum folate concentration increased with intake of folic acid but not SAM. Folate deficiency lowered endogenous SAM concentration, whereas neither intervention altered S-adenosylhomocysteine concentration. DNA methyltransferase (DNMT) activity increased with intake of folic acid raised DNMT activity in folate-deficient mice. DNA methylation rate was stimulated by folic acid in the amyloid precursor protein (APP) promoter and in the presenilin 1 (PS1) promoter. Folate deficiency elevated hippocampal APP, PS1 and Aβ protein levels, and these rises were prevented by folic acid. In conclusion, these findings are consistent with a mechanism in which folic acid increases methylation potential and DNMT activity, modifies DNA methylation and ultimately decreases APP, PS1 and Aβ protein levels.

  11. Stereochemical Sequence Ion Selectivity: Proline versus Pipecolic-acid-containing Protonated Peptides

    NASA Astrophysics Data System (ADS)

    Abutokaikah, Maha T.; Guan, Shanshan; Bythell, Benjamin J.

    2016-10-01

    Substitution of proline by pipecolic acid, the six-membered ring congener of proline, results in vastly different tandem mass spectra. The well-known proline effect is eliminated and amide bond cleavage C-terminal to pipecolic acid dominates instead. Why do these two ostensibly similar residues produce dramatically differing spectra? Recent evidence indicates that the proton affinities of these residues are similar, so are unlikely to explain the result [Raulfs et al., J. Am. Soc. Mass Spectrom. 25, 1705-1715 (2014)]. An additional hypothesis based on increased flexibility was also advocated. Here, we provide a computational investigation of the "pipecolic acid effect," to test this and other hypotheses to determine if theory can shed additional light on this fascinating result. Our calculations provide evidence for both the increased flexibility of pipecolic-acid-containing peptides, and structural changes in the transition structures necessary to produce the sequence ions. The most striking computational finding is inversion of the stereochemistry of the transition structures leading to "proline effect"-type amide bond fragmentation between the proline/pipecolic acid-congeners: R (proline) to S (pipecolic acid). Additionally, our calculations predict substantial stabilization of the amide bond cleavage barriers for the pipecolic acid congeners by reduction in deleterious steric interactions and provide evidence for the importance of experimental energy regime in rationalizing the spectra.

  12. 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.

  13. 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

  14. Design of peptides with α,β-dehydro-residues: syntheses, crystal structures and molecular conformations of two ΔPhe-Trp containing peptides

    NASA Astrophysics Data System (ADS)

    Vijayaraghavan, R.; Makker, J.; Kumar, P.; Dey, S.; Singh, T. P.

    2003-06-01

    The ΔPhe-Trp is a newly designed moiety that was found inducing a unique conformation in peptides. The peptides Boc-L-Val-ΔPhe-L-Trp-OCH 3 (I) and Boc-L-Leu-ΔPhe-L-Trp-OCH 3 (II) were synthesized by azlactone method in solution phase. The peptide (I) was crystallized from its solution in ethanol-water mixture in orthorhombic space group P2 12 12 1 with a=10.663(3) Å, b=11.204(3) Å, c=26.516(10) Å and peptide (II) was crystallized from its solution in acetone in a monoclinic space group P2 1 with a=9.354(1)Å, b=11.218(4)Å, c=15.633(1)Å and β=101.83(1)°. The structures were determined by direct methods. Peptide (I) was refined to an R value of 0.059 for 1554 observed reflections [ I≥2 σ (I)] and peptide (II) was refined to an R value of 0.043 for 2920 observed reflections [ I≥2 σ (I)]. The structures of peptides (I) and (II) were found to be identical. They formed an unusual type VIa β-turn conformation which is observed for the first time with a ΔPhe residue at ( i+2) position indicating a unique influence of ΔPhe-Trp moiety in inducing a reproducible new structure in peptides.

  15. 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.

  16. Cα-C bond cleavage of the peptide backbone in MALDI in-source decay using salicylic acid derivative matrices.

    PubMed

    Asakawa, Daiki; Takayama, Mitsuo

    2011-07-01

    The use of 5-formylsalicylic acid (5-FSA) and 5-nitrosalicylic acid (5-NSA) as novel matrices for in-source decay (ISD) of peptides in matrix-assisted laser desorption/ionization (MALDI) is described. The use of 5-FSA and 5-NSA generated a- and x-series ions accompanied by oxidized peptides [M - 2 H + H](+). The preferential formation of a- and x-series ions was found to be dependent on the hydrogen-accepting ability of matrix. The hydrogen-accepting ability estimated from the ratio of signal intensity of oxidized product [M - 2 H + H](+) to that of non-oxidized protonated molecule [M + H](+) of peptide was of the order 5-NSA > 5-FSA > 5-aminosalicylic acid (5-ASA) ≒ 2,5-dihydroxyl benzoic acid (2,5-DHB) ≒ 0. The results suggest that the hydrogen transfer reaction from peptide to 5-FSA and 5-NSA occurs during the MALDI-ISD processes. The hydrogen abstraction from peptides results in the formation of oxidized peptides containing a radical site on the amide nitrogen with subsequent radical-induced cleavage at the Cα-C bond, leading to the formation of a- and x-series ions. The most significant feature of MALDI-ISD with 5-FSA and 5-NSA is the specific cleavage of the Cα-C bond of the peptide backbone without degradation of side-chain and post-translational modifications (PTM). The matrix provides a useful complementary method to conventional MALDI-ISD for amino acid sequencing and site localization of PTMs in peptides.

  17. Synthesis, characterization, and evaluation of radiometal-containing peptide nucleic acids.

    PubMed

    Stephan, Holger; Foerster, Christian; Gasser, Gilles

    2014-01-01

    Peptide nucleic acids (PNAs) have very attractive properties for applications in nuclear medicine. Because PNAs have high selectivity for DNA/RNA recognition, resistance to nuclease/protease degradation, and high thermal and radiolytic stabilities, PNA bioconjugates could transform the areas of diagnostic and therapeutic nuclear medicine. In this book chapter, we report on the current developments towards the preparation of radiometal-containing PNA constructs and summarize the protocols for labeling these probes with (99m)Tc, (111)In, (64)Cu, (90)Y, and (177)Lu.

  18. 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.

  19. Binding of Eu(III) to 1,2-hydroxypyridinone-modified peptide nucleic acids.

    PubMed

    de Leon, Arnie R; Olatunde, Abiola O; Morrow, Janet R; Achim, Catalina

    2012-12-01

    Substitution of a nucleobase pair with a pair of 1,2-hydroxypyridinone (1,2-HOPO) ligands in the center of a 10-base-pair peptide nucleic acid (PNA) duplex provides a strong binding site for Eu(III) as evidenced by UV thermal melting curves, UV titrations, and luminescence spectroscopy. Eu(III) excitation spectra and luminescence lifetime data are consistent with Eu(III) bound to both 1,2 HOPO ligands in a PNA-HOPO duplex as the major species present in solution.

  20. 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

  1. Sequence selective recognition of double-stranded RNA using triple helix-forming peptide nucleic acids.

    PubMed

    Zengeya, Thomas; Gupta, Pankaj; Rozners, Eriks

    2014-01-01

    Noncoding RNAs are attractive targets for molecular recognition because of the central role they play in gene expression. Since most noncoding RNAs are in a double-helical conformation, recognition of such structures is a formidable problem. Herein, we describe a method for sequence-selective recognition of biologically relevant double-helical RNA (illustrated on ribosomal A-site RNA) using peptide nucleic acids (PNA) that form a triple helix in the major grove of RNA under physiologically relevant conditions. Protocols for PNA preparation and binding studies using isothermal titration calorimetry are described in detail.

  2. Sequence selective double strand DNA cleavage by peptide nucleic acid (PNA) targeting using nuclease S1.

    PubMed Central

    Demidov, V; Frank-Kamenetskii, M D; Egholm, M; Buchardt, O; Nielsen, P E

    1993-01-01

    A novel method for sequence specific double strand DNA cleavage using PNA (peptide nucleic acid) targeting is described. Nuclease S1 digestion of double stranded DNA gives rise to double strand cleavage at an occupied PNA strand displacement binding site, and under optimized conditions complete cleavage can be obtained. The efficiency of this cleavage is more than 10 fold enhanced when a tandem PNA site is targeted, and additionally enhanced if this site is in trans rather than in cis orientation. Thus in effect, the PNA targeting makes the single strand specific nuclease S1 behave like a pseudo restriction endonuclease. Images PMID:8502550

  3. Introduction of multiphosphonate ligand to peptide nucleic acid for metal ion conjugation

    PubMed Central

    Aiba, Yuichiro; Honda, Yuta; Han, Yue; Komiyama, Makoto

    2012-01-01

    Peptide nucleic acid (PNA) is one of the most widely used synthetic DNA analogs. Conjugation of functional molecules to PNA is very effective to further widen its potential applications. For this purpose, here we report the synthesis of several ligand monomers and introduced them to PNA. These ligand-modified PNAs attract cerium ion and are useful for site-selective DNA hydrolysis. It should be noted that these ligands on PNA are also effective even under the conditions of invasion complex. PMID:22772037

  4. Inducible Expression of the De-Novo Designed Antimicrobial Peptide SP1-1 in Tomato Confers Resistance to Xanthomonas campestris pv. vesicatoria

    PubMed Central

    Herrera Diaz, Areli; Kovacs, Izabella; Lindermayr, Christian

    2016-01-01

    Antimicrobial peptides (AMPs) are small peptides with less than 50 amino acids and are part of the innate immune response in almost all organisms, including bacteria, vertebrates, invertebrates and plants. AMPs are active against a broad-spectrum of pathogens. The inducible expression of AMPs in plants is a promising approach to combat plant pathogens with minimal negative side effects, such as phytotoxicity or infertility. In this study, inducible expression of the de-novo designed AMP SP1-1 in Micro Tom tomato protected tomato fruits against bacterial spot disease caused by Xanthomonas campestris pv. vesicatoria. The peptide SP1-1 was targeted to the apoplast which is the primary infection site for plant pathogens, by fusing SP1-1 peptide to the signal peptide RsAFP1 of radish (Raphanus sativus). The pathogen inducibility of the expression was enabled by using an optimized inducible 4XW2/4XS promoter. As a result, the tomato fruits of independently generated SP1-1 transgenic lines were significantly more resistant to X. campestris pv. vesicatoria than WT tomato fruits. In transgenic lines, bacterial infection was reduced up to 65% in comparison to the infection of WT plants. Our study demonstrates that the combination of the 4XW2/4XS cis-element from parsley with the synthetic antimicrobial peptide SP1-1 is a good alternative to protect tomato fruits against infections with X. campestris pv. vesicatoria. PMID:27706237

  5. Synthesis and antimicrobial activity in vitro of new amino acids and peptides containing thiazole and oxazole moieties.

    PubMed

    Stanchev, M; Tabakova, S; Videnov, G; Golovinsky, E; Jung, G

    1999-09-01

    2-(Pyrrolidinyl)thiazole-4-carboxylic acid 5d, 2-(1-aminoalkyl)thiazole-4-carboxamides and hydrazides 8, 10 have been synthesized using alanine, valine, and proline as educts. In addition oxazole amino acids derived from leucine 20a and alanine 20b and some peptides 13, 14, 16 containing the 5-ring heterocyclic backbone modifications have been prepared. The thiazole and oxazole containing amino acids and peptides showed moderate antibacterial activity in vitro against various Gram-positive (Staphylococcus aureus, Bacillus cereus, etc.) and Gram-negative (Escherichia coli, Protens vulgaris, etc.) bacteria, fungi (Candida albicans), and yeast (Saccharomyces cerevisae, etc.). PMID:10520298

  6. 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

  7. Differentiating amino acid residues and side chain orientations in peptides using scanning tunneling microscopy.

    PubMed

    Claridge, Shelley A; Thomas, John C; Silverman, Miles A; Schwartz, Jeffrey J; Yang, Yanlian; Wang, Chen; Weiss, Paul S

    2013-12-11

    Single-molecule measurements of complex biological structures such as proteins are an attractive route for determining structures of the large number of important biomolecules that have proved refractory to analysis through standard techniques such as X-ray crystallography and nuclear magnetic resonance. We use a custom-built low-current scanning tunneling microscope to image peptide structures at the single-molecule scale in a model peptide that forms β sheets, a structural motif common in protein misfolding diseases. We successfully differentiate between histidine and alanine amino acid residues, and further differentiate side chain orientations in individual histidine residues, by correlating features in scanning tunneling microscope images with those in energy-optimized models. Beta sheets containing histidine residues are used as a model system due to the role histidine plays in transition metal binding associated with amyloid oligomerization in Alzheimer's and other diseases. Such measurements are a first step toward analyzing peptide and protein structures at the single-molecule level.

  8. 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

  9. Effect of D-amino acid substitutions on Ni(II)-assisted peptide bond hydrolysis.

    PubMed

    Ariani, Hanieh H; Polkowska-Nowakowska, Agnieszka; Bal, Wojciech

    2013-03-01

    Previously we demonstrated the sequence-specific hydrolysis of the R1-(Ser/Thr)-peptide bond in Ni(II) complexes of peptides with a general R1-(Ser/Thr)-Xaa-His-Zaa-R2 sequence (R1 and R2 being any sequences) (Kopera, E.; Krezel, A.; Protas, A. M.; Belczyk, A.; Bonna, A.; Wyslouch-Cieszynska, A.; Poznanski, J.; Bal, W. Inorg. Chem. 2010, 49, 6636). In order to refine our understanding of the mechanism of this reaction and to find ways to accelerate it, we undertook a systematic study of effects of d-amino acid substitutions in the template Ac-Gly-Ala-Ser-Arg-His-Trp-Lys-Phe-Leu-NH2 peptide on the hydrolysis rate constants. We found that all stereochemical alterations made around the Ni(II) chelate plane resulted in the decrease of the reaction rate. However, the Ni(II) coordination, a prerequisite to the reaction, was not compromised by these substitutions. We demonstrated that the reaction is only possible when either the side chain of the crucial Ser (or Thr) residue is on the same part of the chelate plane as the next residue in the sequence (Arg), or the side chain of the residue following His (Trp) resides on the opposite side of the plane. The rate of reaction is the fastest when both these conditions are fulfilled. Another novel effect is the strong dependence of the rate of the acyl shift step on the character of the leaving group. PMID:23427909

  10. 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

  11. 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

  12. Computational Design of the Sequence and Structure of a Protein-Binding Peptide

    SciTech Connect

    Sammond, Deanne W.; Bosch, Dustin E.; Butterfoss, Glenn L.; Purbeck, Carrie; Machius, Mischa; Siderovski, David P.; Kuhlman, Brian

    2012-08-10

    The de novo design of protein-binding peptides is challenging because it requires the identification of 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 G{alpha}{sub i1}. An X-ray crystal structure of the redesigned complex closely matches the computational model, with a backbone root-mean-square deviation of 1.1 {angstrom}.

  13. 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

  14. 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.

  15. 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

  16. D-Amino acids incorporation in the frog skin-derived peptide esculentin-1a(1-21)NH2 is beneficial for its multiple functions.

    PubMed

    Di Grazia, Antonio; Cappiello, Floriana; Cohen, Hadar; Casciaro, Bruno; Luca, Vincenzo; Pini, Alessandro; Di, Y Peter; Shai, Yechiel; Mangoni, Maria Luisa

    2015-12-01

    Naturally occurring antimicrobial peptides (AMPs) represent promising future antibiotics. We have previously isolated esculentin-1a(1-21)NH2, a short peptide derived from the frog skin AMP esculentin-1a, with a potent anti-Pseudomonal activity. Here, we investigated additional functions of the peptide and properties responsible for these activities. For that purpose, we synthesized the peptide, as well as its structurally altered analog containing two D-amino acids. The peptides were then biophysically and biologically investigated for their cytotoxicity and immunomodulating activities. The data revealed that compared to the wild-type, the diastereomer: (1) is significantly less toxic towards mammalian cells, in agreement with its lower α-helical structure, as determined by circular dichroism spectroscopy; (2) is more effective against the biofilm form of Pseudomonas aeruginosa (responsible for lung infections in cystic fibrosis sufferers), while maintaining a high activity against the free-living form of this important pathogen; (3) is more stable in serum; (4) has a higher activity in promoting migration of lung epithelial cells, and presumably in healing damaged lung tissue, and (5) disaggregates and detoxifies the bacterial lipopolysaccharide (LPS), albeit less than the wild-type. Light scattering studies revealed a correlation between anti-LPS activity and the ability to disaggregate the LPS. Besides shedding light on the multifunction properties of esculentin-1a(1-21)NH2, the D-amino acid containing isomer may serve as an attractive template for the development of new anti-Pseudomonal compounds with additional beneficial properties. Furthermore, together with other studies, incorporation of D-amino acids may serve as a general approach to optimize the future design of new AMPs.

  17. 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

  18. 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

  19. Stapled BH3 Peptides against MCL-1: Mechanism and Design Using Atomistic Simulations

    PubMed Central

    Joseph, Thomas L.; Lane, David P.; Verma, Chandra S.

    2012-01-01

    Atomistic simulations of a set of stapled alpha helical peptides derived from the BH3 helix of MCL-1 (Stewart et al. (2010) Nat Chem Biol 6: 595–601) complexed to a fragment (residues 172–320) of MCL-1 revealed that the highest affinity is achieved when the staples engage the surface of MCL-1 as has also been demonstrated for p53-MDM2 (Joseph et al. (2010) Cell Cycle 9: 4560–4568; Baek et al. (2012) J Am Chem Soc 134: 103–106). Affinity is also modulated by the ability of the staples to pre-organize the peptides as helices. Molecular dynamics simulations of these stapled BH3 peptides were carried out followed by determination of the energies of interactions using MM/GBSA methods. These show that the location of the staple is a key determinant of a good binding stapled peptide from a bad binder. The good binder derives binding affinity from interactions between the hydrophobic staple and a hydrophobic patch on MCL-1. The position of the staple was varied, guiding the design of new stapled peptides with higher affinities. PMID:22952838

  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. 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

  2. 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

  3. 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

  4. 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.

  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.

  6. 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

  7. Separation of amino acids, peptides and corresponding Amadori compounds on a silica column at elevated temperature.

    PubMed

    Hao, Zhigang; Lu, Chih-Ying Joey; Xiao, Baiming; Weng, Naidong; Parker, Barry; Knapp, Michael; Ho, Chi-Tang

    2007-04-20

    Maillard reaction of glucose with amino acids and peptides has become a very important experimental model in the food flavor and pharmaceutical industries for better understanding the mechanism of food flavor generation and drug stability. Because of the amino acid and sugar functional groups present in their structures, most of the reaction components formed during the initial stages of Maillard reaction as well as the substrates are relatively polar. These compounds are poorly retained on a conventional reversed phase column. While polar stationary phases like HILIC column do provide better retention for these polar components, method selectivity could still be a challenge due to the structural similarity between these analytes. In this report, parameters such as pH, mobile phase composition and temperature were investigated using different brands of bare silica columns in order to separate glycine (G), diglycine (DG), triglycine (TG), and the corresponding Amadori compounds of glucose-glycine (GG), glucose-diglycine (GDG) and glucose-triglycine (GTG). An excellent separation for glycine, glycine peptides and their Amadori compounds was obtained on a bare silica column at an elevated temperature. PMID:17350634

  8. In situ synthesis of peptide nucleic acids in porous silicon for drug delivery and biosensing.

    PubMed

    Beavers, Kelsey R; Mares, Jeremy W; Swartz, Caleb M; Zhao, Yiliang; Weiss, Sharon M; Duvall, Craig L

    2014-07-16

    Peptide nucleic acids (PNA) are a unique class of synthetic molecules that have a peptide backbone and can hybridize with nucleic acids. Here, a versatile method has been developed for the automated, in situ synthesis of PNA from a porous silicon (PSi) substrate for applications in gene therapy and biosensing. Nondestructive optical measurements were performed to monitor single base additions of PNA initiated from (3-aminopropyl)triethoxysilane attached to the surface of PSi films, and mass spectrometry was conducted to verify synthesis of the desired sequence. Comparison of in situ synthesis to postsynthesis surface conjugation of the full PNA molecules showed that surface mediated, in situ PNA synthesis increased loading 8-fold. For therapeutic proof-of-concept, controlled PNA release from PSi films was characterized in phosphate buffered saline, and PSi nanoparticles fabricated from PSi films containing in situ grown PNA complementary to micro-RNA (miR) 122 generated significant anti-miR activity in a Huh7 psiCHECK-miR122 cell line. The applicability of this platform for biosensing was also demonstrated using optical measurements that indicated selective hybridization of complementary DNA target molecules to PNA synthesized in situ on PSi films. These collective data confirm that we have established a novel PNA-PSi platform with broad utility in drug delivery and biosensing.

  9. Peptide and amino acid transporters are differentially regulated during seed development and germination in faba bean.

    PubMed

    Miranda, Manoela; Borisjuk, Ljudmilla; Tewes, Annegret; Dietrich, Daniela; Rentsch, Doris; Weber, Hans; Wobus, Ulrich

    2003-08-01

    Two peptide transporter (PTR) homologs have been isolated from developing seeds of faba bean (Vicia faba). VfPTR1 was shown to be a functional peptide transporter through complementation of a yeast mutant. Expression patterns of VfPTR1 and VfPTR2 as well as of the amino acid permease VfAAP1 (Miranda et al., 2001) were compared throughout seed development and germination. In developing seeds, the highest levels of VfPTR1 transcripts were reached during midcotyledon development, whereas VfAAP1 transcripts were most abundant during early cotyledon development, before the appearance of storage protein gene transcripts, and were detectable until late cotyledon development. During early germination, VfPTR1 mRNA appeared first in cotyledons and later, during seedling growth, also in axes and roots. Expression of VfPTR2 and VfAAP1 was delayed compared with VfPTR1, and was restricted to the nascent organs of the seedlings. Localization of VfPTR1 transcripts showed that this PTR is temporally and spatially regulated during cotyledon development. In germinating seeds, VfPTR1 mRNA was localized in root hairs and root epidermal cells, suggesting a role in nutrient uptake from the soil. In seedling roots, VfPTR1 was repressed by a dipeptide and by an amino acid, whereas nitrate was without influence.

  10. Design of potent inhibitors of human RAD51 recombinase based on BRC motifs of BRCA2 protein: modeling and experimental validation of a chimera peptide.

    PubMed

    Nomme, Julian; Renodon-Cornière, Axelle; Asanomi, Yuya; Sakaguchi, Kazuyasu; Stasiak, Alicja Z; Stasiak, Andrzej; Norden, Bengt; Tran, Vinh; Takahashi, Masayuki

    2010-08-12

    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.

  11. 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.

  12. A Statistical Analysis of the PPII Propensity of Amino Acid Guests in Proline-Rich Peptides

    PubMed Central

    Moradi, Mahmoud; Babin, Volodymyr; Sagui, Celeste; Roland, Christopher

    2011-01-01

    There has been considerable debate about the intrinsic PPII propensity of amino-acid residues in denatured polypeptides. Experimentally, the propensity scale is based on the behavior of guest amino-acid residues placed in the middle of polyproline hosts. We have used classical molecular dynamics simulations, with state-of-the-art force fields to carry out a comprehensive analysis of the conformational equilibria of the proline-based host oligopeptides with single guests. The tracked structural characteristics include the PPII content, the cis/trans isomerization of the prolyl bonds, the puckering of the pyrrolidine rings of the proline residues, and the secondary structural motifs. We find no evidence for an intrinsic PPII propensity in any of the guest amino acids other than proline. Instead, the PPII content as derived from experiments may be explained in terms of: 1), a local correlation between the dihedral angles of the guest amino acid and the proline residue immediately preceding it; and 2), a nonlocal correlation between the cis/trans states of the peptide bonds. In terms of the latter, we find that the presence of a guest (other than proline, tyrosine, or tryptophan) increases the trans content of most of the prolyl bonds, which results in an effective increase of the peptide PPII content. With respect to the local dihedral correlations, we find that these are well described in terms of the so-called odds-ratio statistic. Expressed in terms of free energy language, the PPII content based on the odds-ratio of the relevant residues correlate well with the experimentally measured PPII content. PMID:21320454

  13. Factors that restrict the cell permeation of cyclic prodrugs of an opioid peptide, part 3: Synthesis of analogs designed to have improved stability to oxidative metabolism.

    PubMed

    Nofsinger, Rebecca; Fuchs-Knotts, Tarra; Borchardt, Ronald T

    2012-09-01

    Previously, our laboratory reported that cyclic peptide prodrugs of the opioid peptide H-Tyr-D-Ala-Gly-Phe-D-Leu-OH (DADLE) are metabolized by cytochrome P450 (CYP450) enzymes, which limits their systemic exposure after oral dosing to animals. In an attempt to design more metabolically stable cyclic prodrugs of DADLE, we synthesized analogs of DADLE cyclized with a coumarinic acid linker (CA; CA-DADLE), which contained modifications in the amino acid residues known to be susceptible to CYP450 oxidation. Metabolic stability and metabolite identification studies of CA-DADLE and its analogs were then compared using rat liver microsomes (RLM), guinea pig liver microsomes (GPLM), and human liver microsomes (HLM), as well as recombinant human recombinant cytochrome P450 3A4 (hCYP3A4). Similar to the results observed for CA-DADLE, incubation of its analogs with RLM, GPLM, and HLM resulted in monohydroxylation of an amino acid side chain on these cyclic prodrugs. When CA-DADLE was incubated with hCYP3A4, similar oxidative metabolism of the peptide was observed. In contrast, incubation of the CA-DADLE analogs with hCYP3A4 showed that these amino-acid-modified analogs are not substrates for this CYP450 isozyme. These results suggest that the amino-acid-modified analogs of CA-DADLE prepared in this study could be stable to metabolic oxidation by CYP3A4 expressed in human intestinal mucosal cells.

  14. 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

  15. 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.

  16. Isolation of alligator gar (Lepisosteus spatula) glucagon, oxyntomodulin, and glucagon-like peptide: amino acid sequences of oxyntomodulin and glucagon-like peptide.

    PubMed

    Pollock, H G; Kimmel, J R; Ebner, K E; Hamilton, J W; Rouse, J B; Lance, V; Rawitch, A B

    1988-01-01

    Oxyntomodulin, glucagon, and a glucagon-like peptide (GLP) have been isolated from the endocrine pancreas of the alligator gar (Lepisosteus spatula), a ganoid fish. The three peptides were isolated by gel filtration and HPLC and were identified by size, composition, and glucagon-like immunoreactivity. The amino acid sequences of the oxyntomodulin and GLP were determined. The oxyntomodulin contains 36 amino acid residues and its sequence is H S Q G T F T N D Y S K Y L D T R R A Q D F V Q W L M S T K R S G G I T. The composition of the glucagon is identical to the N-terminal 29 residues of the gar oxyntomodulin. The single form of GLP found contains 34 amino acid residues in the following sequence: H A D G T Y T S D V S S Y L Q D Q A A K K F V T W L K Q G Q D R R E. These findings suggest that all three peptides are derived from a common precursor. PMID:3282974

  17. Isolation of alligator gar (Lepisosteus spatula) glucagon, oxyntomodulin, and glucagon-like peptide: amino acid sequences of oxyntomodulin and glucagon-like peptide.

    PubMed

    Pollock, H G; Kimmel, J R; Ebner, K E; Hamilton, J W; Rouse, J B; Lance, V; Rawitch, A B

    1988-01-01

    Oxyntomodulin, glucagon, and a glucagon-like peptide (GLP) have been isolated from the endocrine pancreas of the alligator gar (Lepisosteus spatula), a ganoid fish. The three peptides were isolated by gel filtration and HPLC and were identified by size, composition, and glucagon-like immunoreactivity. The amino acid sequences of the oxyntomodulin and GLP were determined. The oxyntomodulin contains 36 amino acid residues and its sequence is H S Q G T F T N D Y S K Y L D T R R A Q D F V Q W L M S T K R S G G I T. The composition of the glucagon is identical to the N-terminal 29 residues of the gar oxyntomodulin. The single form of GLP found contains 34 amino acid residues in the following sequence: H A D G T Y T S D V S S Y L Q D Q A A K K F V T W L K Q G Q D R R E. These findings suggest that all three peptides are derived from a common precursor.

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. Design of peptide substrates for nanosecond time-resolved fluorescence assays of proteases: 2,3-diazabicyclo[2.2.2]oct-2-ene as a noninvasive fluorophore.

    PubMed

    Hennig, Andreas; Florea, Mara; Roth, Doris; Enderle, Thilo; Nau, Werner M

    2007-01-15

    Fluorescence protease assays were investigated with peptide substrates containing a 2,3-diazabicyclo[2.2.2]oct-2-ene-labeled asparagine (Dbo) as a fluorescent amino acid. The special characteristic of the fluorophore Dbo is its exceedingly long fluorescence lifetime (ca. 300 ns in water under air), which allows the use of nanosecond time-resolved fluorescence (Nano-TRF) detection to efficiently suppress shorter-lived background emission. In addition, the natural amino acids tryptophan and tyrosine can be employed as intramolecular fluorescence quenchers, which facilitates substrate design. Fourteen synthetic peptide substrates (composed of 2-19 amino acids) and five enzymes (trypsin, pepsin, carboxypeptidase A, leucine aminopeptidase, and chymotrypsin) were investigated and, in all 28 examined combinations, enzymatic activity was detected by monitoring the increase in steady state fluorescence with time and determining the reaction rates as kcat/Km values, which ranged from 0.2 to 80x10(6) M-1 min-1. The results suggest an excellent compatibility of the very small and hydrophilic fluorescent probe Dbo with solid-phase peptide synthesis and the investigated proteases. For all 14 peptides the fluorescence lifetimes before and after enzymatic cleavage were measured and Nano-TRF measurements were performed in 384-well microplates. The fluorescence lifetimes of the different peptides provide the basis for the rational design of Dbo-based fluorescent substrates for protease assays. Measurements in Nano-TRF mode revealed, in addition to efficient suppression of background fluorescence, an increased differentiation between cleaved and uncleaved substrate. The Dbo-based assays can be adapted for high-throughput screening.

  4. 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.

  5. Peptide nucleic acids inhibit growth of Brucella suis in pure culture and in infected murine macrophages

    PubMed Central

    Rajasekaran, Parthiban; Alexander, Jeffry C.; Seleem, Mohamed N.; Jain, Neeta; Sriranganathan, Nammalwar; Wattam, Alice R.; Setubal, João C.; Boyle, Stephen M.

    2012-01-01

    Peptide nucleic acids (PNAs) are single-stranded, synthetic nucleic acid analogues containing a pseudopeptide backbone in place of the phosphodiester sugar–phosphate. When PNAs are covalently linked to cell-penetrating peptides (CPPs) they readily penetrate the bacterial cell envelope, inhibit expression of targeted genes and cause growth inhibition both of Gram-positive and Gram-negative bacteria. However, the effectiveness of PNAs against Brucella, a facultative intracellular bacterial pathogen, was unknown. The susceptibility of a virulent Brucella suis strain to a variety of PNAs was assessed in pure culture as well as in murine macrophages. The studies showed that some of the PNAs targeted to Brucella genes involved in DNA (polA, dnaG, gyrA), RNA (rpoB), cell envelope (asd), fatty acid (kdtA, acpP) and protein (tsf) synthesis inhibit the growth of B. suis in culture and in macrophages after 24 h of treatment. PNA treatment inhibited Brucella growth by interfering with gene expression in a sequence-specific and dose-dependent manner at micromolar concentrations. The most effective PNA in broth culture was that targeting polA at ca. 12 μM. In contrast, in B. suis-infected macrophages, the most effective PNAs were those targeting asd and dnaG at 30 μM; both of these PNAs had little inhibitory effect on Brucella in broth culture. The polA PNA that inhibits wild-type B. suis also inhibits the growth of wild-type Brucella melitensis 16M and Brucella abortus 2308 in culture. This study reveals the potential usefulness of antisense PNA constructs as novel therapeutic agents against intracellular Brucella. PMID:23305655

  6. Light-up probes: thiazole orange-conjugated peptide nucleic acid for detection of target nucleic acid in homogeneous solution.

    PubMed

    Svanvik, N; Westman, G; Wang, D; Kubista, M

    2000-05-15

    We have constructed light-up probes for nucleic acid detection. The light-up probe is a peptide nucleic acid (PNA) oligonucleotide to which the asymmetric cyanine dye thiazole orange (TO) is tethered. It combines the excellent hybridization properties of PNA and the large fluorescence enhancement of TO upon binding to DNA. When the PNA hybridizes to target DNA, the dye binds and becomes fluorescent. Free probes have low fluorescence, which may increase almost 50-fold upon hybridization to complementary nucleic acid. This makes the light-up probes particularly suitable for homogeneous hybridization assays, where separation of the bound and free probe is not necessary. We find that the fluorescence enhancement upon hybridization varies among different probes, which is mainly due to variations in free probe fluorescence. For eight probes studied the fluorescence quantum yield at 25 degrees C in the unbound state ranged from 0.0015 to 0.08 and seemed to depend mainly on the PNA sequence. The binding of the light-up probes to target DNA is highly sequence specific and a single mismatch in a 10-mer target sequence was readily identified.

  7. Femtomolar Zn(II) affinity in a peptide-based ligand designed to model thiolate-rich metalloprotein active sites.

    PubMed

    Petros, Amy K; Reddi, Amit R; Kennedy, Michelle L; Hyslop, Alison G; Gibney, Brian R

    2006-12-11

    Metal-ligand interactions are critical components of metalloprotein assembly, folding, stability, electrochemistry, and catalytic function. Research over the past 3 decades on the interaction of metals with peptide and protein ligands has progressed from the characterization of amino acid-metal and polypeptide-metal complexes to the design of folded protein scaffolds containing multiple metal cofactors. De novo metalloprotein design has emerged as a valuable tool both for the modular synthesis of these complex metalloproteins and for revealing the fundamental tenets of metalloprotein structure-function relationships. Our research has focused on using the coordination chemistry of de novo designed metalloproteins to probe the interactions of metal cofactors with protein ligands relevant to biological phenomena. Herein, we present a detailed thermodynamic analysis of Fe(II), Co(II), Zn(II), and[4Fe-4S]2(+/+) binding to IGA, a 16 amino acid peptide ligand containing four cysteine residues, H2N-KLCEGG-CIGCGAC-GGW-CONH2. These studies were conducted to delineate the inherent metal-ion preferences of this unfolded tetrathiolate peptide ligand as well as to evaluate the role of the solution pH on metal-peptide complex speciation. The [4Fe-4S]2(+/+)-IGA complex is both an excellent peptide-based synthetic analogue for natural ferredoxins and is flexible enough to accommodate mononuclear metal-ion binding. Incorporation of a single ferrous ion provides the FeII-IGA complex, a spectroscopic model of a reduced rubredoxin active site that possesses limited stability in aqueous buffers. As expected based on the Irving-Williams series and hard-soft acid-base theory, the Co(II) and Zn(II) complexes of IGA are significantly more stable than the Fe(II) complex. Direct proton competition experiments, coupled with determinations of the conditional dissociation constants over a range of pH values, fully define the thermodynamic stabilities and speciation of each MII-IGA complex. The

  8. In silico design of discontinuous peptides representative of B and T-cell epitopes from HER2-ECD as potential novel cancer peptide vaccines.

    PubMed

    Manijeh, Mahdavi; Mehrnaz, Keyhanfar; Violaine, Moreau; Hassan, Mohabatkar; Abbas, Jafarian; Mohammad, Rabbani

    2013-01-01

    At present, the most common cause of cancer-related death in women is breast cancer. In a large proportion of breast cancers, there is the overexpression of human epidermal growth factor receptor 2 (HER2). This receptor is a 185 KDa growth factor glycoprotein, also known as the first tumor-associated antigen for different types of breast cancers. Moreover, HER2 is an appropriate cell-surface specific antigen for passive immunotherapy, which relies on the repeated application of monoclonal antibodies that are transferred to the patient. However, vaccination is preferable because it would stimulate a patient's own immune system to actively respond to a disease. In the current study, several bioinformatics tools were used for designing synthetic peptide vaccines. PEPOP was used to predict peptides from HER2 ECD subdomain III in the form of discontinuous-continuous B-cell epitopes. Then, T-cell epitope prediction web servers MHCPred, SYFPEITHI, HLA peptide motif search, Propred, and SVMHC were used to identify class-I and II MHC peptides. In this way, PEPOP selected 12 discontinuous peptides from the 3D structure of the HER2 ECD subdomain III. Furthermore, T-cell epitope prediction analyses identified four peptides containing the segments 77 (384-391) and 99 (495-503) for both B and T-cell epitopes. This work is the only study to our knowledge focusing on design of in silico potential novel cancer peptide vaccines of the HER2 ECD subdomain III that contain epitopes for both B and T-cells. These findings based on bioinformatics analyses may be used in vaccine design and cancer therapy; saving time and minimizing the number of tests needed to select the best possible epitopes.

  9. 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

  10. 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

  11. 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

  12. Functional mimicry of a discontinuous antigenic site by a designed synthetic peptide.

    PubMed

    Villén, Judit; Borràs, Eva; Schaaper, Wim M M; Meloen, Rob H; Dávila, Mercedes; Domingo, Esteban; Giralt, Ernest; Andreu, David

    2002-03-01

    Functional reproduction of the discontinuous antigenic site D of foot-and-mouth disease virus (FMDV) has been achieved by means of synthetic peptide constructions that integrate each of the three protein loops that define the antigenic site into a single molecule. The site D mimics were designed on the basis of the X-ray structure of FMDV type C-S8c1 with the aid of molecular dynamics, so that the five residues assumed to be involved in antigenic recognition are located on the same face of the molecule, exposed to solvent and defining a set of native-like distances and angles. The designed site D mimics are disulfide-linked heterodimers that consist of a larger unit containing VP2(71-84), followed by a polyproline module and by VP3(52-62), and a smaller unit corresponding to VP1(188-194) (VP=viral protein). Guinea pig antisera to the peptides recognized the viral particle and competed with site D-specific monoclonal antibodies, while inoculation with a simple (not covalently joined to one another) admixture of the three VP1-VP3 sequences yielded no detectable virus-specific serum conversion. Similar results have been reproduced in two bovines. Antisera to the peptides also moderately neutralize FMDV in cell cultures and partially protect guinea pigs against challenge with the virus. These results demonstrate functional mimicry of the discontinuous site D by the peptides, which are therefore obvious candidates for a multicomponent, peptide-based vaccine against FMDV. PMID:11921395

  13. Amino Acid and Peptide Utilization Profiles of the Fluoroacetate-Degrading Bacterium Synergistetes Strain MFA1 Under Varying Conditions.

    PubMed

    Leong, Lex E X; Denman, Stuart E; Hugenholtz, Philip; McSweeney, Christopher S

    2016-02-01

    Synergistetes strain MFA1 is an asaccharolytic ruminal bacterium isolated based on its ability to degrade fluoroacetate, a plant toxin. The amino acid and peptide requirements of the bacterium were investigated under different culturing conditions. The growth of strain MFA1 and its fluoroacetate degradation rate were enhanced by peptide-rich protein hydrolysates (tryptone and yeast extract) compared to casamino acid, an amino acid-rich protein hydrolysate. Complete utilization and preference for arginine, asparagine, glutamate, glycine, and histidine as free amino acids from yeast extract were observed, while the utilization of serine, threonine, and lysine in free form and peptide-bound glutamate was stimulated during growth on fluoroacetate. A predominant peptide in yeast extract preferentially utilized by strain MFA1 was partially characterized by high-liquid performance chromatography-mass spectrometry as a hepta-glutamate oligopeptide. Similar utilization profiles of amino acids were observed between the co-culture of strain MFA1 with Methanobrevibacter smithii without fluoroacetate and pure strain MFA1 culture with fluoroacetate. This suggests that growth of strain MFA1 could be enhanced by a reduction of hydrogen partial pressure as a result of hydrogen removal by a methanogen or reduction of fluoroacetate.

  14. Gamma Peptide Nucleic Acids: As Orthogonal Nucleic Acid Recognition Codes for Organizing Molecular Self-Assembly.

    PubMed

    Sacui, Iulia; Hsieh, Wei-Che; Manna, Arunava; Sahu, Bichismita; Ly, Danith H

    2015-07-01

    Nucleic acids are an attractive platform for organizing molecular self-assembly because of their specific nucleobase interactions and defined length scale. Routinely employed in the organization and assembly of materials in vitro, however, they have rarely been exploited in vivo, due to the concerns for enzymatic degradation and cross-hybridization with the host's genetic materials. Herein we report the development of a tight-binding, orthogonal, synthetically versatile, and informationally interfaced nucleic acid platform for programming molecular interactions, with implications for in vivo molecular assembly and computing. The system consists of three molecular entities: the right-handed and left-handed conformers and a nonhelical domain. The first two are orthogonal to each other in recognition, while the third is capable of binding to both, providing a means for interfacing the two conformers as well as the natural nucleic acid biopolymers (i.e., DNA and RNA). The three molecular entities are prepared from the same monomeric chemical scaffold, with the exception of the stereochemistry or lack thereof at the γ-backbone that determines if the corresponding oligo adopts a right-handed or left-handed helix, or a nonhelical motif. These conformers hybridize to each other with exquisite affinity, sequence selectivity, and level of orthogonality. Recognition modules as short as five nucleotides in length are capable of organizing molecular assembly.

  15. Pipa carvalhoi skin secretion profiling: absence of peptides and identification of kynurenic acid as the major constitutive component.

    PubMed

    Mariano, Douglas Oscar Ceolin; Yamaguchi, Lydia Fumiko; Jared, Carlos; Antoniazzi, Marta Maria; Sciani, Juliana Mozer; Kato, Massuo Jorge; Pimenta, Daniel Carvalho

    2015-01-01

    The presence of peptides has been identified in all African pipid genera; nevertheless, little is known about skin secretion of South American frog genus Pipa. Skin secretion from captive and wild Pipa carvalhoi were obtained in the presence or absence of norepinephrine stimulation. The <10 kDa fraction was analyzed by liquid chromatography and mass spectrometry, searching for peptides. Chromatographic profiles show the presence of a major component in this secretion, regardless of the stimulation method (norepinephrine or mechanical stimulation) and the origin of the animal (captivity or wild), as well as in the absence of any stimulus. The general mass distribution profile in P. carvalhoi skin secretion shows numerous components below 800 Da. Moreover, no peptide could be identified, regardless of the chromatographic approach. The major component was purified and identified as kynurenic acid, an L-tryptophan derivative. P. carvalhoi does not secrete peptides as toxins in its skin. In addition, we here report that kynurenic acid is the main component of P. carvalhoi skin secretion. Although no biological activity was associated with kynurenic acid, we propose that this molecule is a pheromone that signals the presence of a co-specific in the shady environment in which this animal lives. In this study we demonstrate the absence of peptidic toxins in the skin secretion of P. carvalhoi, a break of paradigm in the pipid family.

  16. Rhizobins, a Group of Peptides in the Free-Amino-Acid Pool of the Soybean-Rhizobium System †

    PubMed Central

    Garay, Andrew S.; Ahlgren, Joy A.; Gonzalez, Mark A.; Stasney, Mark A.; Madtes, Paul C.

    1986-01-01

    Free-living Rhizobium (according to Bergey's Manual of Systematic Bacteriology, [1984, The Williams & Wilkins Co., Baltimore], Bradyrhizobium) japonicum was found to release a peptide into the nutrient media. Soybean nodules contained this peptide and exuded it into the soil. The name “rhizobin A” is suggested for this peptide. Nodules also contained another peptide, rhizobin B, as well as an unidentified, ninhydrin-positive compound, rhizobin C. The three peptides were confined to the free-amino-acid pool of the soluble fraction and eluted consecutively from a cation-exchange column. Rhizobin A was isolated in a highly purified form; its molecular mass was approximately 1,600 daltons as determined by Sephadex gel filtration and mass spectrometry. The amino-acid composition could be determined only approximately, because a long time was necessary for acid hydrolysis, possibly due to unusual linkages. The rhizobin concentration in soybean nodules continually increased during 50 days of growth, from 2 to approximately 400 μg/g (fresh weight). When combined nitrogen was added to nodulated soybean and subsequently removed, nitrogenase activity, nodulation, and nodule growth first decreased and then recovered. The relative amount of rhizobin A followed a similar pattern. Rhizobins were not detected in the roots, stems, and leaves of nodulated soybean plants. They were present in Lupinus nodules, but absent in alder nodules. PMID:16347004

  17. 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

  18. Diphenylarsinic acid increased the synthesis and release of neuroactive and vasoactive peptides in rat cerebellar astrocytes.

    PubMed

    Negishi, Takayuki; Takahashi, Masaki; Matsunaga, Yuki; Hirano, Seishiro; Tashiro, Tomoko

    2012-06-01

    An incident of poisoning occurred in Japan in 2003 when high-level contamination with arsenic, mainly diphenylarsinic acid (DPAA), was found in well water. People using this water particularly experienced cerebellar symptoms. In the present study, we investigated the adverse effects of DPAA on the cerebellum in vitro and in vivo to understand the biological mechanisms that cause cerebellar symptoms. Comprehensive gene expression analyses in primary cultured ratcerebellar cells exposed to 10 μM DPAA for 24 hours indicated significant alterations in the mRNA expression of genes encoding antioxidative stress proteins (heme oxigenase 1 and heat shock protein72) and neuroactive and vasoactive peptides (neuropeptide Y, adrenomedullin, monocyte chemoattractant protein 1, and fibroblast growth factor 2). Further analyses of proteins revealed that cultured cerebellar astrocytes expressed these antioxidative stress proteins and peptides in response to exposure to DPAA. In addition, these adverseeffects were also observed in the cerebellum exposed in vivo to DPAA (100 mg/L) for 21 days. These results suggested that cerebellarastrocytes irregularly secrete neuroactive and vasoactive peptidesagainst DPAA-induced oxidative stress, which leads to abnormal neural functions and disrupted cerebellar autoregulation dynamics and results in the onset of cerebellar symptoms.

  19. 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.

  20. 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

  1. Cryo-transmission electron microscopy structure of a gigadalton peptide fiber of de novo design.

    PubMed

    Sharp, Thomas H; Bruning, Marc; Mantell, Judith; Sessions, Richard B; Thomson, Andrew R; Zaccai, Nathan R; Brady, R Leo; Verkade, Paul; Woolfson, Derek N

    2012-08-14

    Nature presents various protein fibers that bridge the nanometer to micrometer regimes. These structures provide inspiration for the de novo design of biomimetic assemblies, both to address difficulties in studying and understanding natural systems, and to provide routes to new biomaterials with potential applications in nanotechnology and medicine. We have designed a self-assembling fiber system, the SAFs, in which two small α-helical peptides are programmed to form a dimeric coiled coil and assemble in a controlled manner. The resulting fibers are tens of nm wide and tens of μm long, and, therefore, comprise millions of peptides to give gigadalton supramolecular structures. Here, we describe the structure of the SAFs determined to approximately 8 Å resolution using cryotransmission electron microscopy. Individual micrographs show clear ultrastructure that allowed direct interpretation of the packing of individual α-helices within the fibers, and the construction of a 3D electron density map. Furthermore, a model was derived using the cryotransmission electron microscopy data and side chains taken from a 2.3 Å X-ray crystal structure of a peptide building block incapable of forming fibers. This was validated using single-particle analysis techniques, and was stable in prolonged molecular-dynamics simulation, confirming its structural viability. The level of self-assembly and self-organization in the SAFs is unprecedented for a designed peptide-based material, particularly for a system of considerably reduced complexity compared with natural proteins. This structural insight is a unique high-resolution description of how α-helical fibrils pack into larger protein fibers, and provides a basis for the design and engineering of future biomaterials.

  2. 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

  3. 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

  4. Development of a peptide nucleic acid array platform for the detection of genetically modified organisms in food.

    PubMed

    Germini, Andrea; Rossi, Stefano; Zanetti, Alessandro; Corradini, Roberto; Fogher, Corrado; Marchelli, Rosangela

    2005-05-18

    Two previously developed platforms, a multiplex polymerase chain reaction (PCR) and a peptide nucleic acid (PNA) array, the former allowing for the simultaneous detection of five transgenes and two endogenous controls in food and feed matrices and the latter for the assessment of the identity of amplified PCR products, were combined in order to develop a PNA array device for the screening of genetically modified organisms (GMOs) in food. PNA probes were opportunely designed, synthesized, and deposited on commercial slides. The length of the probes as well as the distance of the probes from the surface were evaluated and found to be critical points. The most suitable probes were found to be 15-mer PNAs linked to the slide surface by means of two 2-(2-aminoethoxy)ethoxyacetic acids as spacers. The device was tested on a model system constituted by flour samples containing a mixture of standards at known concentrations of transgenic material, in particular Roundup Ready soybean and Bt11, Bt176, Mon810, and GA21 maize: The DNA was amplified using the specific multiplex PCR method and tested on the PNA array. The method proposed was found to be able to correctly identify every GMO present in the tested samples.

  5. 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

  6. Design and biophysical characterization of novel polycationic epsilon-peptides for DNA compaction and delivery.

    PubMed

    Huang, Dandan; Korolev, Nikolay; Eom, Khee Dong; Tam, James P; Nordenskiöld, Lars

    2008-01-01

    Design and solid-phase synthesis of novel and chemically defined linear and branched -oligo( l-lysines) (denoted -K n, where n is the number of lysine residues) and their alpha-substituted homologues (epsilon-(R)K10, epsilon-(Y)K10, epsilon-(L)K10, epsilon-(YR)K10, and epsilon-(LYR)K10) for DNA compaction and delivery are reported. The ability to condense viral (T2 and T4) and plasmid DNA as well as the size of -peptide DNA complexes under different conditions was investigated with static and dynamic light scattering, isothermal titration calorimetry, and fluorescence microscopy. Nanoparticle diameters varied from 100 to 150 and 375 to 550 nm for plasmid and T4 DNA peptide complexes, respectively. Smaller sizes were observed for oligo(L-lysines) compared to alpha-poly( L-lysine). The linear -oligo-lysines are less toxic and epsilon-(LYR)K10 showed higher transfection efficiency in HeLa cells than corresponding controls. The results also demonstrate that with a branched design having pendent groups of short alpha-oligopeptides, improved transfection can be achieved. This study supports the hypothesis that available alpha-oligolysine derived systems would potentially have more favorable delivery properties if they are based instead on epsilon-oligo( L-lysines). The flexible design and unambiguous synthesis that enables variation of pendent groups holds promise for optimization of such -peptides to achieve improved DNA compaction and delivery.

  7. Computational Design of Hypothetical New Peptides Based on a Cyclotide Scaffold as HIV gp120 Inhibitor.

    PubMed

    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.

  8. Rational design of peptide-based building blocks for nanoscience and synthetic biology.

    PubMed

    Armstrong, Craig T; Boyle, Aimee L; Bromley, Elizabeth H C; Mahmoud, Zahra N; Smith, Lisa; Thomson, Andrew R; Woolfson, Derek N

    2009-01-01

    The rational design of peptides that fold to form discrete nanoscale objects, and/ or self-assemble into nanostructured materials is an exciting challenge. Such efforts test and extend our understanding of sequence-to-structure relationships in proteins, and potentially provide materials for applications in bionanotechnology. Over the past decade or so, rules for the folding and assembly of one particular protein-structure motif--the alpha-helical coiled coil have advanced sufficiently to allow the confident design of novel peptides that fold to prescribed structures. Coiled coils are based on interacting alpha-helices, and guide and cement many protein-protein interactions in nature. As such, they present excellent starting points for building complex objects and materials that span the nano-to-micron scales from the bottom up. Along with others, we have translated and extended our understanding of coiled-coil folding and assembly to develop novel peptide-based biomaterials. Herein, we outline briefly the rules for the folding and assembly of coiled-coil motifs, and describe how we have used them in de novo design of discrete nanoscale objects and soft synthetic biomaterials. Moreover, we describe how the approach can be extended to other small, independently folded protein motifs--such as zinc fingers and EF-hands--that could be incorporated into more complex, multi-component synthetic systems and new hybrid and responsive biomaterials.

  9. Enhanced lubrication on tissue and biomaterial surfaces through peptide-mediated binding of hyaluronic acid.

    PubMed

    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.

  10. 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

  11. Evidence for extensive non-endocytotic translocation of peptide nucleic acids across mammalian plasma membranes.

    PubMed

    Oehlke, Johannes; Turner, Yvonne; Pritz, Stephan; Bienert, Michael

    2011-09-01

    The ability of peptide nucleic acids (PNA) to enter and to cross filter-grown MDCK, HEK and CHO cells was studied by means of a protocol based on capillary electrophoresis combined with laser-induced fluorescence detection. The used approach avoided possible errors encountered in protocols based on confocal laserscanning microscopy and FACS analysis. In contradiction to the commonly anticipated unability of PNA to cross biomembranes, extensive translocation of unmodified PNA into and across the investigated cell types was found. The transport mode comprised a variety of energy dependent and -independent as well as temperature sensitive mechanisms being probably destined to natural substrates and hijacked by PNA. The presented results suggest active as well as passive export mechanisms rather than poor penetration into cells to be responsible for the only weak biological activity of unmodified PNA.

  12. Dimerization of a PACAP peptide analogue in DMSO via asparagine and aspartic acid residues.

    PubMed

    Severs, Joanne C; Froland, Wayne A

    2008-03-01

    To optimize the stability of a peptide development candidate for the treatment of type II diabetes, formulation studies were initiated in organic solvents and compared to results obtained in aqueous solutions. Stability was assessed by reversed phase liquid chromatography (RPLC) and electrospray ionization mass spectrometry (ESI-MS). Previous studies had shown deamidation and hydrolysis to be the primary mechanisms of degradation in aqueous formulations. Surprisingly, the use of an organic solvent did not decrease the rate of degradation and, as presented here, produced degradation products including dimers. We propose here that deamidation can readily occur in polar anhydrous organic solvents such as DMSO and that the dimer forms through intermolecular nucleophilic attack of an amino acid side chain on a stabilized cyclic imide intermediate.

  13. Development of Peptide Nucleic Acid Probes for Detection of the HER2 Oncogene

    PubMed Central

    Song, Young K.; Evangelista, Jennifer; Aschenbach, Konrad; Johansson, Peter; Wen, Xinyu; Chen, Qingrong; Lee, Albert; Hempel, Heidi; Gheeya, Jinesh S.; Getty, Stephanie; Gomez, Romel; Khan, Javed

    2013-01-01

    Peptide nucleic acids (PNAs) have gained much interest as molecular recognition tools in biology, medicine and chemistry. This is due to high hybridization efficiency to complimentary oligonucleotides and stability of the duplexes with RNA or DNA. We have synthesized 15/16-mer PNA probes to detect the HER2 mRNA. The performance of these probes to detect the HER2 target was evaluated by fluorescence imaging and fluorescence bead assays. The PNA probes have sufficiently discriminated between the wild type HER2 target and the mutant target with single base mismatches. Furthermore, the probes exhibited excellent linear concentration dependence between 0.4 to 400 fmol for the target gene. The results demonstrate potential application of PNAs as diagnostic probes with high specificity for quantitative measurements of amplifications or over-expressions of oncogenes. PMID:23593123

  14. Stability analysis of glutamic acid linked peptides coupled to NOTA through different chemical linkages.

    PubMed

    Lang, Lixin; Ma, Ying; Kiesewetter, Dale O; Chen, Xiaoyuan

    2014-11-01

    Glutamic acid is a commonly used linker to form dimeric peptides with enhanced binding affinity than their corresponding monomeric counterparts. We have previously labeled NOTA-Bn-NCS-PEG3-E[c(RGDyK)]2 (NOTA-PRGD2) [1] with [(18)F]AlF and (68)Ga for imaging tumor angiogenesis. The p-SCN-Bn-NOTA was attached to E[c(RGDyK)]2 [2] through a mini-PEG with a thiourea linkage, and the product [1] was stable at radiolabeling condition of 100 °C and pH 4.0 acetate buffer. However, when the same p-SCN-Bn-NOTA was directly attached to the α-amine of E[c(RGDfK)]2 [3], the product NOTA-Bn-NCS-E[c(RGDfK)]2 [4] became unstable under similar conditions and the release of monomeric c(RGDfK) [5] was observed. The purpose of this work was to use HPLC and LC-MS to monitor the decomposition of glutamic acid linked dimeric peptides and their NOTA derivatives. A c(RGDyK) [6] and bombesin (BBN) [7] heterodimer c(RGDyK)-E-BBN [8], and a dimeric bombesin E(BBN)2 [9], both with a glutamic acid as the linker, along with a model compound PhSCN-E[c(RGDfK)] [10] were also studied. All the compounds were dissolved in 0.5 M pH 4.0 acetate buffer at the concentration of 1 mg/mL, and 0.1 mL of each sample was heated at 100 °C for 10 min and the more stable compounds were heated for another 30 min. The samples at both time points were analyzed with analytical HPLC to monitor the decomposition of the heated samples. The samples with decomposition were further analyzed by LC-MS to determine the mass of products from the decomposition for possible structure elucidation. After 10 min heating, the obvious release of c(RGDfK) [5] was observed for NOTA-Bn-NCS-E[c(RGDfK)]2 [4] and Ph-SCN-E[c(RGDfK)] [10]. Little or no release of monomers was observed for the remaining samples at this time point. After further heating, the release of monomers was clearly observed for E[c(RGDyK)]2 [2], E[c(RGDfK)]2 [3], c(RGDyK)-E-BBN [8], and E(BBN)2 [9]. No decomposition or little decomposition was observed for NOTA

  15. 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

  16. 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.

  17. DNA detection using water-soluble conjugated polymers and peptide nucleic acid probes

    NASA Astrophysics Data System (ADS)

    Gaylord, Brent S.; Heeger, Alan J.; Bazan, Guillermo C.

    2002-08-01

    The light-harvesting properties of cationic conjugated polymers are used to sensitize the emission of a dye on a specific peptide nucleic acid (PNA) sequence for the purpose of homogeneous, "real-time" DNA detection. Signal transduction is controlled by hybridization of the neutral PNA probe and the negative DNA target. Electrostatic interactions bring the hybrid complex and cationic polymer within distances required for Förster energy transfer. Conjugated polymer excitation provides fluorescein emission >25 times higher than that obtained by exciting the dye, allowing detection of target DNA at concentrations of 10 pM with a standard fluorometer. A simple and highly sensitive assay with optical amplification that uses the improved hybridization behavior of PNA/DNA complexes is thus demonstrated.

  18. Quantification of glycated N-terminal peptide of hemoglobin using derivatization for multiple functional groups of amino acids followed by liquid chromatography/tandem mass spectrometry.

    PubMed

    Sakaguchi, Yohei; Kinumi, Tomoya; Yamazaki, Taichi; Takatsu, Akiko

    2016-02-01

    A novel method of amino acid analysis using derivatization of multiple functional groups (amino, carboxyl, and phenolic hydroxyl groups) was applied to measure glycated amino acids in order to quantify glycated peptides and evaluate the degree of glycation of peptide. Amino and carboxyl groups of amino acids were derivatized with 1-bromobutane so that the hydrophobicities and basicities of the amino acids, including glycated amino acids, were improved. These derivatized amino acids could be detected with high sensitivity using LC-MS/MS. In this study, 1-deoxyfructosyl-VHLTPE and VHLTPE, which are N-terminal peptides of the β-chains of hemoglobin, were selected as target compounds. After reducing the peptide sample solution with sodium borohydride, the obtained peptides were hydrolyzed with hydrochloric acid. The released amino acids were then derivatized with 1-bromobutane and analyzed with LC-MS/MS. The derivatized amino acids, including glycated amino acids, could be separated using an octadecyl silylated silica column and good sharp peaks were detected. We show a confirmatory experiment that the proposed method can be applied to evaluate the degree of glycation of peptides, using mixtures of glycated and non-glycated peptide.

  19. 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

  20. N- and C-capping preferences for all 20 amino acids in alpha-helical peptides.

    PubMed Central

    Doig, A. J.; Baldwin, R. L.

    1995-01-01

    We have determined the N- and C-capping preferences of all 20 amino acids by substituting residue X in the peptides NH2-XAKAAAAKAAAAKAAGY-CONH2 and in Ac-YGAAKAAAAKAAAAKAX-CO2H. Helix contents were measured by CD spectroscopy to obtain rank orders of capping preferences. The data were further analyzed by our modified Lifson-Roig helix-coil theory, which includes capping parameters (n and c), to find free energies of capping (-RT ln n and -RT ln c), relative to Ala. Results were obtained for charged and uncharged termini and for different charged states of titratable side chains. N-cap preferences varied from Asn (best) to Gln (worst). We find, as expected, that amino acids that can accept hydrogen bonds from otherwise free backbone NH groups, such as Asn, Asp, Ser, Thr, and Cys generally have the highest N-cap preference. Gly and acetyl group are favored, as are negative charges in side chains and at the N-terminus. Our N-cap preference scale agrees well with preferences in proteins. In contrast, we find little variation when changing the identity of the C-cap residue. We find no preference for Gly at the C-cap in contrast to the situation in proteins. Both N-cap and C-cap results for Tyr and Trp are inaccurate because their aromatic groups affect the CD spectrum. The data presented here are of value in rationalizing mutations at capping sites in proteins and in predicting the helix contents of peptides. PMID:7670375

  1. 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.

  2. 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

  3. 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...

  4. How mono-valent cations bend peptide turns and a first-principles database of amino acids and dipeptides

    NASA Astrophysics Data System (ADS)

    Baldauf, Carsten; Ropo, Matti; Blum, Volker; Scheffler, Matthias

    2014-10-01

    In this contribution we detail our efforts to investigate the structural effects of cations binding to peptides and amino acids. We perform first-principles studies employing long-range dispersion-corrected approximate density-functional theory and compare to gas-phase experiments.

  5. Peptide Antibodies: Past, Present, and Future.

    PubMed

    Houen, Gunnar

    2015-01-01

    Peptide antibodies recognize epitopes with amino acid residues adjacent in sequence ("linear" epitopes). Such antibodies can be made to virtually any sequence and have been immensely important in all areas of molecular biology and diagnostics due to their versatility and to the rapid growth in protein sequence information. Today, peptide antibodies can be routinely and rapidly made to large numbers of peptides, including peptides with posttranslationally modified residues, and are used for immunoblotting, immunocytochemistry, immunohistochemistry, and immunoassays. In the future, peptide antibodies will continue to be immensely important for molecular biology, TCR- and MHC-like peptide antibodies may be produced routinely, peptide antibodies with predetermined conformational specificities may be designed, and peptide-based vaccines may become part of vaccination programs.

  6. Design and synthesis of a potent peptide containing both specific and non-specific cell-adhesion motifs.

    PubMed

    Lai, Yuxiao; Xie, Cao; Zhang, Zheng; Lu, Weiyue; Ding, Jiandong

    2010-06-01

    This article reports a potent chemical to promote cell adhesion on a substrate by combination of both moieties for specific and non-specific adhesion. The cyclic (-RGDfK-) (R: arginine, G: glycine, D: aspartic acid, f: D-phenylalanine, K: lysine) is employed to trigger specific cell adhesion, and a linear tripeptide KKK is introduced to enhance early non-specific cell adhesion. A series of cyclic and linear peptides with different charges were synthesized and then functionalized with thiol end-group. All the peptides were immobilized on gold layers, which were later passivated by bovine serum albumin. The coverage of NIH/3T3 fibroblast cells on the substrate modified by the linker containing both cyclic (-RGDfK-) and linear KKK is, surprisingly, significantly better than the summation using one of them, which reveals the strong cooperativity of specific and non-specific cell adhesions. The resultant cell adhesion on the substrates modified by appropriate linkers was much better than on tissue-culture plates. The cooperativity principle and the design strategy of the combined linker might be helpful for fundamental research of cell-material or cell-extracellular matrix interactions, and for modification of new biomaterials in regenerative medicine and targeted drug delivery.

  7. Inhibition of Influenza A Virus Infection In Vitro by Peptides Designed In Silico

    PubMed Central

    López-Martínez, Rogelio; Ramírez-Salinas, G. Lizbeth; Correa-Basurto, José; Barrón, Blanca L.

    2013-01-01

    Influenza A viruses are enveloped, segmented negative single-stranded RNA viruses, capable of causing severe human respiratory infections. Currently, only two types of drugs are used to treat influenza A infections, the M2 H+ ion channel blockers (amantadine and rimantadine) and the neuraminidase inhibitors (NAI) (oseltamivir and zanamivir). Moreover, the emergence of drug-resistant influenza A virus strains has emphasized the need to develop new antiviral agents to complement or replace the existing drugs. Influenza A virus has on the surface a glycoprotein named hemagglutinin (HA) which due to its important role in the initial stage of infection: receptor binding and fusion activities of viral and endosomal membranes, is a potential target for new antiviral drugs. In this work we designed nine peptides using several bioinformatics tools. These peptides were derived from the HA1 and HA2 subunits of influenza A HA with the aim to inhibit influenza A virus infection. The peptides were synthetized and their antiviral activity was tested in vitro against several influenza A viral strains: Puerto Rico/916/34 (H1N1), (H1N1)pdm09, swine (H1N1) and avian (H5N2). We found these peptides were able to inhibit the influenza A viral strains tested, without showing any cytotoxic effect. By docking studies we found evidence that all the peptides were capable to bind to the viral HA, principally to important regions on the viral HA stalk, thus could prevent the HA conformational changes required to carry out its membranes fusion activity. PMID:24146939

  8. Oostatic peptides containing D-amino acids: synthesis, oostatic activity, degradation, accumulation in ovaries and NMR study.

    PubMed

    Hlaváček, Jan; Tykva, Richard; Holík, Josef; Bennettová, Blanka; Buděšínský, Miloš; Vlasáková, Věra; Cerný, Bohuslav; Slaninová, Jiřina

    2012-05-01

    Analogs of the H-Tyr-Asp-Pro-Ala-Pro-OH pentapeptide with D-amino acid residues either in differing or in all of the positions of the sequences were prepared and their oostatic potency was compared with that of the parent pentapeptide. The D-amino acid residue containing analogs exhibited an equal or even higher oostatic effect in the flesh fly Neobellieria bullata than the parent peptide. Contrary to the rapid incorporation of radioactivity from the labeled H-Tyr-Asp-[3H]Pro-Ala-Pro-OH pentapeptide into the ovaries of N. bullata in vitro, the radioactivity incorporation from the labeled pentapeptides with either D-aspartic acid or D-alanine was significantly delayed. As compared to the parent pentapeptide, also the degradation of both the D-amino acid-containing analogs mentioned above proceeded at a significantly lower rate. The decreased intake of radioactivity, the lower degradation and finally also the high oostatic effect may be ascribed to the decreased enzymatic degradation of the peptide bonds neighboring the D-amino acid residues in the corresponding peptides. The introduction of the non-coded D: -amino acids thus enhances the oostatic effect in N. bullata owing to the prolonged half-life of the corresponding pentapeptides, which can thus affect more ovarian cells.

  9. Design of a RANK-Mimetic Peptide Inhibitor of Osteoclastogenesis with Enhanced RANKL-Binding Affinity.

    PubMed

    Hur, Jeonghwan; Ghosh, Ambarnil; Kim, Kabsun; Ta, Hai Minh; Kim, Hyunju; Kim, Nacksung; Hwang, Hye-Yeon; Kim, Kyeong Kyu

    2016-04-30

    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.

  10. 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

  11. 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

  12. Improved synthesis strategy for peptide nucleic acids (PNA) appropriate for cell-specific fluorescence imaging.

    PubMed

    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.

  13. Evolving serodiagnostics by rationally designed peptide arrays: the Burkholderia paradigm in Cystic Fibrosis

    PubMed Central

    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

  14. Evolving serodiagnostics by rationally designed peptide arrays: the Burkholderia paradigm in Cystic Fibrosis

    NASA Astrophysics Data System (ADS)

    Peri, Claudio; Gori, Alessandro; Gagni, Paola; Sola, Laura; Girelli, Daniela; Sottotetti, Samantha; Cariani, Lisa; Chiari, Marcella; Cretich, Marina; Colombo, Giorgio

    2016-09-01

    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.

  15. 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

  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. 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.

  18. Rational design of alpha-helical antimicrobial peptides: do's and don'ts.

    PubMed

    Uggerhøj, Lars Erik; Poulsen, Tanja Juul; Munk, Jens Kristian; Fredborg, Marlene; Sondergaard, Teis Esben; Frimodt-Moller, Niels; Hansen, Paul Robert; Wimmer, Reinhard

    2015-01-19

    Antimicrobial peptides (AMPs) are promising candidates for battling multiresistant bacteria. Despite extensive research, structure-activity relationships of AMPs are not fully understood, and there is a lack of structural data relating to AMPs in lipids. Here we present the NMR structure of anoplin (GLLKRIKTLL-NH2 ) in a micellar environment. A vast library of substitutions was designed and tested for antimicrobial and hemolytic activity, as well as for changes in structure and lipid interactions. This showed that improvement of antimicrobial activity without concomitant introduction of strong hemolytic activity can be achieved through subtle increases in the hydrophobicity of the hydrophobic face or through subtle increases in the polarity of the hydrophilic face of the helix, or-most efficiently-a combination of both. A set of guidelines based on the results is given, for assistance in how to modify cationic α-helical AMPs in order to control activity and selectivity. The guidelines are finally tested on a different peptide.

  19. 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

  20. 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

  1. 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

  2. Exogenous loading of a tapasin-dependent peptide onto HLA-B*44:02 can be restored by acid treatment or fixation of target cells

    PubMed Central

    Stroobant, Vincent; Demotte, Nathalie; Luiten, Rosalie M.; Leonhardt, Ralf M.; Cresswell, Peter; Bonehill, Aude; Michaux, Alexandre; Ma, Wenbin; Mulder, Arend; Van den Eynde, Benoît J.; van der Bruggen, Pierre; Vigneron, Nathalie

    2013-01-01

    Anti-tumor CTLs recognize peptides derived from cellular proteins and presented on MHC class I. One category of peptides recognized by these CTLs is derived from proteins encoded by “cancer-germline” genes, which are specifically expressed in tumors, and therefore represent optimal targets for cancer immunotherapy. Here, we identify an antigenic peptide, which is derived from the MAGE-A1-encoded protein (160-169) and presented to CTLs by HLA-B*44:02. Although this peptide is encoded by MAGE-A1, processed endogenously and presented by tumor cells, the corresponding synthetic peptide is hardly able to sensitize target cells to CTL recognition when pulsed exogenously. Endogenous processing and presentation of this peptide is strictly dependent on the presence of tapasin, which is believed to help peptide loading by stabilizing a peptide-receptive form of HLA-B*44:02. Exogenous loading of the peptide can be dramatically improved by paraformaldehyde fixation of surface molecules or by peptide loading at acidic pH. Either strategy allows efficient exogenous loading of the peptide, presumably by generating or stabilizing a peptide-receptive, empty conformation of the HLA. Altogether, our results indicate a potential drawback of short peptide-based vaccination strategies and offer possible solutions regarding the use of problematic epitopes such as the one described here. PMID:22678898

  3. On the activation of bovine plasma factor XIII. Amino acid sequence of the peptide released by thrombin and the terminal residues of the subunit polypeptides.

    PubMed

    Nakamura, S; Iwanaga, S; Suzuki, T

    1975-12-01

    A blood coagulation factor, Factor XIII, was highly purified from bovine fresh plasma by a method similar to those used for human plasma Factor XIII. The isolated Factor XIII consisted of two subunit polypeptides, a and b chains, with molecular weights of 79,000 +/- 2,000 and 75,000 +/- 2,000, respectively. In the conversion of Factor XIII to the active enzyme, Factor XIIIa, by bovine thrombin [EC 3.4.21.5], a peptide was liberated. This peptide, designated tentatively as "activation peptide," was isolated by gel-filtration on a Sephadex G-75 column. It contained a total of 37 amino acid residues with a masked N-terminal residue and C-terminal arginine. The whole amino acid sequence of "Activation peptide" was established by the dansyl-Edman method and standard enzymatic techniques, and the masked N-terminal residue was identified as N-acetylserine by using a rat liver acylamino acid-releasing enzyme. This enzyme specifically cleaved the N-acetylserylglutamyl peptide bond serine and the remaining peptide, which was now reactive to 1-dimethylamino-naphthalene-5-sulfonyl chloride. A comparison of the sequences of human and bovine "Activation peptide" revealed five amino acids replacements, Ser-3 to Thr; Gly-5 to Arg; Ile-14 to Val; Thr-18 to Asn, and Pro-26 to Leu. Another difference was the deletion of Leu-34 in the human peptide. Adsorption chromatography on a hydroxylapatite column in the presence of 0.1% sodium dodecyl sulfate was developed as a preparative procedure for the resolution of the two subunit polypeptides, a or a' chain and b chain, constituting the protein molecule of Factor XIII or Factor XIIIa. End group analyses on the isolated pure chains revealed that the structural change of Factor XIII during activation with thrombin occurs only in the N-terminal portion of the a chain, not in the N-terminal end of the b chain or in the C-terminal ends of the a and b chains. From these results, it was concluded that the activation of bovine plasma Factor XIII

  4. 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.

  5. Label-free potentiometry for detecting DNA hybridization using peptide nucleic acid and DNA probes.

    PubMed

    Goda, Tatsuro; Singi, Ankit Balram; Maeda, Yasuhiro; Matsumoto, Akira; Torimura, Masaki; Aoki, Hiroshi; Miyahara, Yuji

    2013-02-07

    Peptide nucleic acid (PNA) has outstanding affinity over DNA for complementary nucleic acid sequences by forming a PNA-DNA heterodimer upon hybridization via Watson-Crick base-pairing. To verify whether PNA probes on an electrode surface enhance sensitivity for potentiometric DNA detection or not, we conducted a comparative study on the hybridization of PNA and DNA probes on the surface of a 10-channel gold electrodes microarray. Changes in the charge density as a result of hybridization at the solution/electrode interface on the self-assembled monolayer (SAM)-formed microelectrodes were directly transformed into potentiometric signals using a high input impedance electrometer. The charge readout allows label-free, reagent-less, and multi-parallel detection of target oligonucleotides without any optical assistance. The differences in the probe lengths between 15- to 22-mer dramatically influenced on the sensitivity of the PNA and DNA sensors. Molecular type of the capturing probe did not affect the degree of potential shift. Theoretical model for charged rod-like duplex using the Gouy-Chapman equation indicates the dominant effect of electrostatic attractive forces between anionic DNA and underlying electrode at the electrolyte/electrode interface in the potentiometry.

  6. 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.

  7. Synthesis and optical properties of pyrrolidinyl peptide nucleic acid carrying a clicked Nile red label

    PubMed Central

    Yotapan, Nattawut; Charoenpakdee, Chayan; Wathanathavorn, Pawinee; Ditmangklo, Boonsong

    2014-01-01

    Summary DNA or its analogues with an environment-sensitive fluorescent label are potentially useful as a probe for studying the structure and dynamics of nucleic acids. In this work, pyrrolidinyl peptide nucleic acid (acpcPNA) was labeled at its backbone with Nile red, a solvatochromic benzophenoxazine dye, by means of click chemistry. The optical properties of the Nile red-labeled acpcPNA were investigated by UV–vis and fluorescence spectroscopy in the absence and in the presence of DNA. In contrast to the usual quenching observed in Nile red-labeled DNA, the hybridization with DNA resulted in blue shifting and an enhanced fluorescence regardless of the neighboring bases. More pronounced blue shifts and fluorescence enhancements were observed when the DNA target carried a base insertion in close proximity to the Nile red label. The results indicate that the Nile red label is located in a more hydrophobic environment in acpcPNA–DNA duplexes than in the single-stranded acpcPNA. The different fluorescence properties of the acpcPNA hybrids of complementary DNA and DNA carrying a base insertion are suggestive of different interactions between the Nile red label and the duplexes. PMID:25246975

  8. Continuous β-turn fold of an alternating alanyl/homoalanyl peptide nucleic acid.

    PubMed

    Cuesta-Seijo, Jose A; Zhang, Jinhua; Diederichsen, Ulf; Sheldrick, George M

    2012-08-01

    The crystal structure of the PNA (peptide nucleic acid) oligomer H-Lys-HalG-AlaG-HalC-AlaG-HalC-AlaC-Lys-NH(2) (PNA1, amino acids with D-configuration are underlined, Ala = alanyl, Hal = homoalanyl) has been determined by ab initio direct methods and refined against 1.0 Å data. The asymmetric unit consists of a tetrameric cage with almost ideal Watson-Crick C-G base pairing of all the guanine and cytosine side-chain substituents. Each PNA strand has a 90° β-turn every second residue, stabilized by three hydrogen bonds between the backbone amides. The first, second, fifth and sixth bases stack on one side of the monomer and pair with the corresponding complementary bases of a second monomer to form a dimer. The two remaining bases on each side of the resulting dimer form Watson-Crick pairs with the complementary bases of a second dimer, leading to a unique cage structure. The extra methylene groups in the homoalanyl residues enable stacking of the bases with an optimal distance between base-planes but also with an appreciable lateral displacement (slide).

  9. Peptides and peptidomimetics as immunomodulators

    PubMed Central

    Gokhale, Ameya S; Satyanarayanajois, Seetharama

    2014-01-01

    Peptides and peptidomimetics can function as immunomodulating agents by either blocking the immune response or stimulating the immune response to generate tolerance. Knowledge of B- or T-cell epitopes along with conformational constraints is important in the design of peptide-based immunomodulating agents. Work on the conformational aspects of peptides, synthesis and modified amino acid side chains have contributed to the development of a new generation of therapeutic agents for autoimmune diseases and cancer. The design of peptides/peptidomimetics for immunomodulation in autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, systemic lupus and HIV infection is reviewed. In cancer therapy, peptide epitopes are used in such a way that the body is trained to recognize and fight the cancer cells locally as well as systemically. PMID:25186605

  10. 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

  11. Unraveling the Mechanisms of Peptide-Mediated Delivery of Nucleic Acids Using Electron Microscopy.

    PubMed

    Margus, Helerin; Juks, Carmen; Pooga, Margus

    2015-01-01

    Cell-penetrating peptides (CPPs) are efficient non-viral delivery vectors for bioactive cargos, both in vitro and in vivo. Cargo molecules can be attached to CPPs either via covalent conjugation or by complex formation using co-incubation, which is typically used for charged molecules such as nucleic acids. The latter technique is efficiently used in case of CADY, MPG, Pep peptides, NickFects and PepFects that condense oligonucleotides (ONs) into nanoparticles, which efficiently enter cells and induce biological effects. Despite being highly promising candidates for developing new-generation medicines, CPPs' internalization mechanisms and intracellular trafficking are still far from being well-understood, and obtained data are often controversial. Transmission electron microscopy (TEM) is an informative and valuable tool for examining the mechanisms of CPP-ON nanoparticles. TEM enables to visualize nanoparticles or single molecules labeled with Nanogold™ tag, and follow their association with cells and intracellular localization. In this chapter, we present methods for preparation of CPP-ON nanoparticles for TEM analysis and for examination of their interactions with the plasma membrane, and subsequent cellular uptake either by direct translocation or endocytosis. In case of endocytosis, ONs have to be released from endosomes and reach their target site in nucleus or cytoplasm to reveal their activity. TEM enables to estimate when the endosomal escape begins, from which type of endosomal vesicles it occurs, whether the vesicles are broken, or nanocomplexes translocate across the membrane into cytosol. Since single ONs could be followed, the time-frame that is necessary for the splice-switching nucleotides to translocate into cell nucleus can be analyzed by TEM.

  12. Laccase-catalyzed cross-linking of amino acids and peptides with dihydroxylated aromatic compounds.

    PubMed

    Mikolasch, Annett; Hahn, Veronika; Manda, Katrin; Pump, Judith; Illas, Nicole; Gördes, Dirk; Lalk, Michael; Gesell Salazar, Manuela; Hammer, Elke; Jülich, Wolf-Dieter; Rawer, Stephan; Thurow, Kerstin; Lindequist, Ulrike; Schauer, Frieder

    2010-08-01

    In order to design potential biomaterials, we investigated the laccase-catalyzed cross-linking between L-lysine or lysine-containing peptides and dihydroxylated aromatics. L-Lysine is one of the major components of naturally occurring mussel adhesive proteins (MAPs). Dihydroxylated aromatics are structurally related to 3,4-dihydroxyphenyl-L-alanine, another main component of MAPs. Mass spectrometry and nuclear magnetic resonance analyses show that the epsilon-amino group of L-lysine is able to cross-link dihydroxylated aromatics. Additional oligomer and polymer cross-linked products were obtained from di- and oligopeptides containing L-lysine. Potential applications in medicine or industry for biomaterials synthesised via the three component system consisting of the oligopeptide [Tyr-Lys]10, dihydroxylated aromatics and laccase are discussed.

  13. Biosynthesis of a D-amino acid in peptide linkage by an enzyme from frog skin secretions

    PubMed Central

    Jilek, Alexander; Mollay, Christa; Tippelt, Christa; Grassi, Jacques; Mignogna, Giuseppina; Müllegger, Johannes; Sander, Veronika; Fehrer, Christine; Barra, Donatella; Kreil, Günther

    2005-01-01

    d-amino acids are present in some peptides from amphibian skin. These residues are derived from the corresponding l-amino acids present in the respective precursors. From skin secretions of Bombinae, we have isolated an enzyme that catalyzes the isomerization of an l-Ile in position 2 of a model peptide to d-allo-Ile. In the course of this reaction, which proceeds without the addition of a cofactor, radioactivity from tritiated water is incorporated into the second position of the product. The amino acid sequence of this isomerase could be deduced from cloned cDNA and genomic DNA. After expression of this cDNA in oocytes of Xenopus laevis, isomerase activity could be detected. Polypeptides related to the frog skin enzyme are present in several vertebrate species, including humans. PMID:15758070

  14. Structure-Based Design and Synthesis of Potent Cyclic Peptides Inhibiting the YAP-TEAD Protein-Protein Interaction.

    PubMed

    Zhang, Zhisen; Lin, Zhaohu; Zhou, Zheng; Shen, Hong C; Yan, S Frank; Mayweg, Alexander V; Xu, Zhiheng; Qin, Ning; Wong, Jason C; Zhang, Zhenshan; Rong, Yiping; Fry, David C; Hu, Taishan

    2014-09-11

    The YAP-TEAD protein-protein interaction (PPI) mediates the oncogenic function of YAP, and inhibitors of this PPI have potential usage in treatment of YAP-involved cancers. Here we report the design and synthesis of potent cyclic peptide inhibitors of the YAP-TEAD interaction. A truncation study of YAP interface 3 peptide identified YAP(84-100) as a weak peptide inhibitor (IC50 = 37 μM), and an alanine scan revealed a beneficial mutation, D94A. Subsequent replacement of a native cation-π interaction with an optimized disulfide bridge for conformational constraint and synergistic effect between macrocyclization and modification at positions 91 and 93 greatly boosted inhibitory activity. Peptide 17 was identified with an IC50 of 25 nM, and the binding affinity (K d = 15 nM) of this 17mer peptide to TEAD1 proved to be stronger than YAP(50-171) (K d = 40 nM).

  15. 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

  16. 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.

  17. d-Amino Acids Modulate the Cellular Response of Enzymatic-Instructed Supramolecular Nanofibers of Small Peptides

    PubMed Central

    2015-01-01

    Peptides made of d-amino acids, as the enantiomer of corresponding l-peptides, are able to resist proteolysis. It is, however, unclear or much less explored whether or how d-amino acids affect the cellular response of supramolecular nanofibers formed by enzyme-triggered self-assembly of d-peptides. In this work, we choose a cell compatible molecule, Nap-l-Phe-l-Phe-l-pTyr (LLL-1P), and systematically replace the l-amino acids in this tripeptidic precursor or its hydrogelator by the corresponding d-amino acid(s). The replacement of even one d-amino acid in this tripeptidic precursor increases its proteolytic resistance. The results of static light scattering and TEM images show the formation of nanostructures upon the addition of alkaline phosphatase, even at concentrations below the minimum gelation concentration (mgc). All these isomers are able to form ordered nanostructures and exhibit different morphologies. According to the cell viability assay on these stereochemical isomers, cells exhibit drastically different responses to the enantiomeric precursors, but almost same responses to the enantiomeric hydrogelators. Furthermore, the different cellular responses of LLL-1P and DDD-1P largely originate from the ecto-phosphatases catalyzed self-assembly of DDD-1 on the surface of cells. Therefore, this report not only illustrates a new way for tailoring the properties of supramolecular assemblies, but also provides new insights to answering the fundamental question of how mammalian cells respond to enzymatic formation of nanoscale supramolecular assemblies (e.g., nanofibers) of d-peptides. PMID:25230147

  18. 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

  19. 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

  20. SUPERFICIAL – Surface mapping of proteins via structure-based peptide library design

    PubMed Central

    Goede, Andrean; Jaeger, Ines S; Preissner, Robert

    2005-01-01

    Background The determination of protein surfaces and the detection of binding sites are essential to our understanding of protein-protein interactions. Such binding sites can be characterised as linear and non-linear, the non-linear sites being prevailant. Conventional mapping techniques with arrays of synthetic peptides have limitations with regard to the location of discontinuous or non-linear binding sites of proteins. Results We present a structure-based approach to the design of peptide libraries that mimic the whole surface or a particular region of a protein. Neighbouring sequence segments are linked by short spacers to conserve local conformation. To this end, we have developed SUPERFICIAL, a program that uses protein structures as input and generates library proposals consisting of linear and non-linear peptides. This process can be influenced by a graphical user interface at different stages, from the surface computation up to the definition of spatial regions. Conclusion Based on 3D structures, SUPERFICIAL may help to negotiate some of the existing limitations, since binding sites consisting of several linear pieces can now be detected. PMID:16153304

  1. PChopper: high throughput peptide prediction for MRM/SRM transition design

    PubMed Central

    2011-01-01

    Background The use of selective reaction monitoring (SRM) based LC-MS/MS analysis for the quantification of phosphorylation stoichiometry has been rapidly increasing. At the same time, the number of sites that can be monitored in a single LC-MS/MS experiment is also increasing. The manual processes associated with running these experiments have highlighted the need for computational assistance to quickly design MRM/SRM candidates. Results PChopper has been developed to predict peptides that can be produced via enzymatic protein digest; this includes single enzyme digests, and combinations of enzymes. It also allows digests to be simulated in 'batch' mode and can combine information from these simulated digests to suggest the most appropriate enzyme(s) to use. PChopper also allows users to define the characteristic of their target peptides, and can automatically identify phosphorylation sites that may be of interest. Two application end points are available for interacting with the system; the first is a web based graphical tool, and the second is an API endpoint based on HTTP REST. Conclusions Service oriented architecture was used to rapidly develop a system that can consume and expose several services. A graphical tool was built to provide an easy to follow workflow that allows scientists to quickly and easily identify the enzymes required to produce multiple peptides in parallel via enzymatic digests in a high throughput manner. PMID:21838934

  2. 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

  3. 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

  4. Structure-Based Design of Peptidic Inhibitors of the Interaction between CC Chemokine Ligand 5 (CCL5) and Human Neutrophil Peptides 1 (HNP1).

    PubMed

    Wichapong, Kanin; Alard, Jean-Eric; Ortega-Gomez, Almudena; Weber, Christian; Hackeng, Tilman M; Soehnlein, Oliver; Nicolaes, Gerry A F

    2016-05-12

    Protein-protein interactions (PPIs) are receiving increasing interest, much sparked by the realization that they represent druggable targets. Recently, we successfully developed a peptidic inhibitor, RRYGTSKYQ ("SKY" peptide), that shows high potential in vitro and in vivo to interrupt a PPI between the platelet-borne chemokine CCL5 and the neutrophil-derived granule protein HNP1. This PPI plays a vital role in monocyte adhesion, representing a key mechanism in acute and chronic inflammatory diseases. Here, we present extensive and detailed computational methods applied to develop the SKY peptide. We combined experimentally determined binding affinities (KD) of several orthologs of CCL5 with HNP1 with in silico studies to identify the most likely heterodimeric CCL5-HNP1 complex which was subsequently used as a starting structure to rationally design peptidic inhibitors. Our method represents a fast and simple approach that can be widely applied to determine other protein-protein complexes and moreover to design inhibitors or stabilizers of protein-protein interaction.

  5. The importance of peptide detectability for protein identification, quantification, and experiment design in MS/MS proteomics

    PubMed Central

    Li, Yong Fuga; Arnold, Randy J.; Tang, Haixu

    2010-01-01

    Peptide detectability is defined as the probability that a peptide is identified in an LC-MS/MS experiment and has been useful in providing solutions to protein inference and label-free quantification. Previously, predictors for peptide detectability trained on standard or complex samples were proposed. Although the models trained on complex samples may benefit from the large training data sets, it is unclear to what extent they are affected by the unequal abundances of identified proteins. To address this challenge and improve detectability prediction, we present a new algorithm for the iterative learning of peptide detectability from complex mixtures. We provide evidence that the new method approximates detectability with useful accuracy and, based on its design, can be used to interpret the outcome of other learning strategies. We studied the properties of peptides from the bacterium Deinococcus radiodurans and found that at standard quantities, its tryptic peptides can be roughly classified as either detectable or undetectable, with a relatively small fraction having medium detectability. We extend the concept of detectability from peptides to proteins and apply the model to predict the behavior of a replicate LC-MS/MS experiment from a single analysis. Finally, our study summarizes a theoretical framework for peptide/protein identification and label-free quantification. PMID:21067214

  6. 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

  7. Targeting the S1 and S3 subsite of trypsin with unnatural cationic amino acids generates antimicrobial peptides with potential for oral administration.

    PubMed

    Karstad, Rasmus; Isaksen, Geir; Wynendaele, Evelien; Guttormsen, Yngve; De Spiegeleer, Bart; Brandsdal, Bjørn-Olav; Svendsen, John Sigurd; Svenson, Johan

    2012-07-26

    This study investigates how the S1 and S3 site of trypsin can be challenged with cationic amino acid analogues to yield active antimicrobial peptides with stability toward tryptic degradation. It is shown that unnatural analogues can be incorporated to generate stable peptides with maintained bioactivity to allow for a potential oral uptake. Selected peptides were studied using isothermal calorimetry and computational methods. Both stable and unstable peptides were found to bind stoichiometrically to trypsin with dissociation constants ranging 2-60 μM, suggesting several different binding modes. The stability of selected peptides was analyzed in whole organ extracts and the incorporation of homoarginine and 2-amino-(3-guanidino)propanoic acid resulted in a 14- and 50-fold increase in duodenal stability. In addition, a 40- and 70-fold increase in stomach stability is also reported. Overall, these results illustrate how the incorporation of cationic side chains can be employed to generate bioactive peptides with significant systemic stability.

  8. Site-directed gene mutation at mixed sequence targets by psoralen-conjugated pseudo-complementary peptide nucleic acids

    PubMed Central

    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

  9. Real time RNA transcription monitoring by Thiazole Orange (TO)-conjugated Peptide Nucleic Acid (PNA) probes: norovirus detection.

    PubMed

    Tonelli, Alessandro; Tedeschi, Tullia; Germini, Andrea; Sforza, Stefano; Corradini, Roberto; Medici, Maria Cristina; Chezzi, Carlo; Marchelli, Rosangela

    2011-05-01

    Thiazole Orange (TO)-conjugated Peptide Nucleic Acid (PNA) probes have been reported as a valuable strategy for DNA analysis; however, no investigations targeting RNA molecules and no comparisons between different derivatization approaches have been reported so far. In this work, two TO-conjugated PNAs for genogroup II noroviruses (NoV GII) detection were designed and synthesized. Both the probes target the most conserved stretch of nucleotides identified in the open reading frame 1-2 (ORF1-ORF2) junction region and differ for the dye conjugation strategy: one PNA is end-labelled with the TO molecule tethered by a linker; the other probe bears the TO molecule directly linked to the PNA backbone, replacing a conventional nucleobase. The spectroscopic properties of the two PNA probes were studied and their applicability to NoVs detection, using an isothermal assay, was investigated. Both probes showed good specificity and high fluorescence enhancement upon hybridization, especially targeting RNA molecules. Moreover, the two probes were successfully employed for NoVs detection from stool specimens in an isothermal-based amplification assay targeting RNA 'amplicons'. The probes showed to be specific even in the presence of high concentrations of non-target RNA.

  10. Identification in pituitary tissue of a peptide alpha-amidation activity that acts on glycine-extended peptides and requires molecular oxygen, copper, and ascorbic acid.

    PubMed

    Eipper, B A; Mains, R E; Glembotski, C C

    1983-08-01

    An enzymatic activity capable of producing an alpha-amidated peptide product from its glycine-extended precursor has been identified in secretory granules of rat anterior, intermediate, and neural pituitary and bovine intermediate pituitary. High levels of endogenous inhibitors of this alpha-amidation activity have also been found in tissue homogenates. The alpha-amidation activity is totally inhibited by addition of divalent metal ion chelators such as diethyldithiocarbamate, o-phenanthroline, and EDTA; alpha-amidation activity is restored to above control levels upon addition of copper. The alpha-amidation reaction requires the presence of molecular oxygen. Of the various cofactors tested, ascorbic acid was the most potent stimulator of alpha-amidation. The alpha-amidation activity has a neutral pH optimum and is primarily soluble following several cycles of freezing and thawing. Kinetic studies with the bovine intermediate pituitary granule-associated activity demonstrated a linear Lineweaver-Burk plot when D-Tyr-Val-Gly was the varied substrate; the apparent Km and Vmax varied with the concentration of ascorbic acid. The substrate specificity of the alpha-amidation activity appears to be quite broad; the conversion of D-Tyr-Val-Gly into D-Tyr-Val-NH2 is inhibited by the addition of a variety of glycine-extended peptides.

  11. 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.

  12. 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

  13. 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

  14. 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

  15. Molecular self-assembly: Design, synthesis, and characterization of peptidic materials for bio- and nano-technologies

    NASA Astrophysics Data System (ADS)

    Lamm, Matthew S.

    The research presented in this dissertation focuses on the design, synthesis, and characterization of amphiphilic peptides capable of self-assembling into beta-sheet fibrils under specific aqueous solution conditions. The peptide design consists of two beta-sheet forming strands of alternating valine and lysine residues, flanking a central tetrapeptide sequence that contains a diproline. Depending on the chirality of the prolines the peptide can assume either an intramolecularly folded or an extended conformation in the self-assembled state. For the peptide where intramolecular folding is designed against, the self-assembled nanostructure was found to exhibit a unique, nontwisted laminated morphology. Experimental techniques including transmission electron microscopy, atomic force microscopy, circular dichroism spectroscopy, Fourier transform infra-red spectroscopy and x-ray diffraction were employed to characterize the self-assembled structure and kinetics. With the understanding of the self-assembly process gained from these first peptides, other peptide sequences were rationally designed to assemble with a desired nanostructure. For example, the effect of peptide strand length in conserving the laminated morphology and controlling the fibril height was investigated. In addition, other peptides were designed so as to affect the self-assembled nanostructure by enforcing a parallel versus anti-parallel beta-sheet or by disrupting the registry of laminating beta-sheet filaments. In some cases, the peptides assembled into structures predicted by the initial design while other peptides assembled into unexpected fibril morphologies. The major conclusions from the research on these peptides is that the diproline sequence plays an important role in disrupting the beta-strand twist thereby resulting in a nontwisted laminated morphology. However, when the flanking beta-strands become long enough, the effect of the diproline sequence becomes diminished and the fibrils do

  16. A role for phosphorylation in the regulation of the barley scutellar peptide transporter HvPTR1 by amino acids.

    PubMed

    Waterworth, Wanda M; Ashley, Merewyn K; West, Christopher E; Sunderland, Paul A; Bray, Clifford M

    2005-06-01

    Protein reserves in the cereal endosperm are sequentially degraded to small peptides and amino acids during germination and these are translocated across the scutellum to support growth of the embryo. Peptide transport in the germinating barley grain is mediated by specific carriers localized to the plasma membrane of the scutellar epithelium. In isolated barley embryos peptide transport is rapidly inhibited by amino acid concentrations comparable with those found in the post-germination barley grain. However, this inhibition of HvPTR1 activity is not effected at either the transcriptional or translational level. The protein phosphatase inhibitor okadaic acid repressed transport of Ala-[14C]Phe, but not [14C]Ala, into the barley scutellar epithelium. In vivo [32P]orthophosphate labelling studies of barley scutellar tissue in combination with immunoprecipitation studies using antiserum raised to HvPTR1 showed that HvPTR1 (66 kDa) is phosphorylated in the presence of amino acids. Immunopurified HvPTR1 was further demonstrated to be phosphorylated on serine residues. Digestion with the N-glycosidase enzyme PNGase F results in a shift in the molecular mass of the protein by 10 kDa, indicating that HvPTR1 is an N-linked glycoprotein. These results provide strong circumstantial evidence that HvPTR1 peptide transport activity in the germinating barley grain is regulated at the post-translational level by phosphorylation in response to rising levels of amino acids emanating from the endosperm as a result of storage protein breakdown and mobilization. This is potentially an important element in balancing the flux of organic nitrogen and carbon from the endosperm to embryo during germination and seedling establishment.

  17. 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

  18. Identification of New Snake Venom Metalloproteinase Inhibitors Using Compound Screening and Rational Peptide Design

    PubMed Central

    2012-01-01

    The majority of snakebite envenomations in Central America are caused by the viperid species Bothrops asper, whose venom contains a high proportion of zinc-dependent metalloproteinases that play a relevant role in the pathogenesis of hemorrhage characteristic of these envenomations. Broad metalloproteinase inhibitors, such as the peptidomimetic hydroxamate Batimastat, have been shown to inhibit snake venom metalloproteinases (SVMP). However, the difficulty in having open public access to Batimastat and similar molecules highlights the need to design new inhibitors of SVMPs that could be applied in the treatment of snakebite envenomations. We have chosen the SVMP BaP1 as a model to search for new inhibitors using different strategies, that is, screening of the Prestwick Chemical Library and rational peptide design. Results from these approaches provide clues on the structural requirements for efficient BaP1 inhibition and pave the way for the design of new inhibitors of SVMP. PMID:24900507

  19. Identification of new snake venom metalloproteinase inhibitors using compound screening and rational Peptide design.

    PubMed

    Villalta-Romero, Fabián; Gortat, Anna; Herrera, Andrés E; Arguedas, Rebeca; Quesada, Javier; de Melo, Robson Lopes; Calvete, Juan J; Montero, Mavis; Murillo, Renato; Rucavado, Alexandra; Gutiérrez, José María; Pérez-Payá, Enrique

    2012-07-12

    The majority of snakebite envenomations in Central America are caused by the viperid species Bothrops asper, whose venom contains a high proportion of zinc-dependent metalloproteinases that play a relevant role in the pathogenesis of hemorrhage characteristic of these envenomations. Broad metalloproteinase inhibitors, such as the peptidomimetic hydroxamate Batimastat, have been shown to inhibit snake venom metalloproteinases (SVMP). However, the difficulty in having open public access to Batimastat and similar molecules highlights the need to design new inhibitors of SVMPs that could be applied in the treatment of snakebite envenomations. We have chosen the SVMP BaP1 as a model to search for new inhibitors using different strategies, that is, screening of the Prestwick Chemical Library and rational peptide design. Results from these approaches provide clues on the structural requirements for efficient BaP1 inhibition and pave the way for the design of new inhibitors of SVMP. PMID:24900507

  20. Molecular dynamics simulations for designing biomimetic pores based on internally functionalized self-assembling α,γ-peptide nanotubes.

    PubMed

    Calvelo, Martín; Vázquez, Saulo; García-Fandiño, Rebeca

    2015-11-21

    A molecular dynamics study on internally functionalized peptide nanotubes composed of α- and γ-amino acids self-assembled in lipid bilayers is presented. One of the main advantages of peptide nanotubes composed of γ-amino acids is that the properties of their inner cavities can be tuned by introducing different functions on β-carbon of the γ-amino acid. In the work described here we studied the effect of the presence of different numbers of hydroxyl groups in different positions in the lumen of these channels when they are inserted into a lipid bilayer and assessed how they affect the structural and dynamic behavior of the modified peptide nanotubes as well as the transmembrane transport of different ions. The results provided atomic information about the effect of polar groups on the dynamic, structural and transport properties of this type of peptidic channel upon insertion into lipid bilayers, projecting a promising future for their use as biomimetic channels when properly inner-derivatized. Furthermore, the chemical versatility of the hydroxyl groups in the lumen of the peptide nanotubes would enable appealing applications for these channels, such as a controlled method for the activation/inactivation of the transmembrane transport along the nanopore.

  1. 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

  2. Omega-3 fatty acids regulate the interaction of the Alzheimer's aβ(25-35) peptide with lipid membranes.

    PubMed

    Vitiello, Giuseppe; Di Marino, Sara; D'Ursi, Anna Maria; D'Errico, Gerardino

    2013-11-19

    Polyunsaturated omega-3 fatty acids are increasingly proposed as dietary supplements able to reduce the risk of development or progression of the Alzheimer's disease (AD). To date, the molecular mechanism through which these lipids act has not been yet univocally identified. In this work, we investigate whether omega-3 fatty acids could interfere with the fate of the Alzheimer-related amyloid peptide by tuning the microstructural and dynamical properties of the neuronal membrane. To this aim, the influence of the omega-3 lipid, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine [22:6(cis)PC] on the biophysical properties of lipid bilayers, and on their interaction with the amyloid peptide fragment Aβ(25-35) has been investigated by Electron Spin Resonance (ESR), using spin-labeled phospholipids. The results show that the peptide selectively interacts with bilayers enriched in cholesterol (Chol) and sphingomyelin (SM). [22:6(cis)PC] enhances the Aβ(25-35)/membrane interaction, favoring a deeper internalization of the peptide among the lipid acyl chains and, consequently, hindering its pathogenic self-aggregation. PMID:24144219

  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. Design and synthesis of cyclic and linear peptide-agarose tools for baiting interacting protein partners of GPCRs.

    PubMed

    Granier, Sébastien; Jean-Alphonse, Frédéric; Déméné, Hélène; Guillon, Gilles; Pascal, Robert; Mendre, Christiane

    2006-02-01

    A ligation strategy for the synthesis of cyclic and linear peptides covalently linked to agarose beads designed as baits to identify new interacting partners of intracellular loops of the V2 vasopressin receptor, a member of the G-protein-coupled receptor family, is reported. The peptide-resin conjugates were subsequently shown to interact specifically with a fraction of proteins present in cellular lysates.

  5. 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.

  6. 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

  7. 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.

  8. 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

  9. 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 an