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Sample records for acid peptide fragment

  1. 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.

  2. 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.

  3. Fragmentation pathways of protonated peptides.

    PubMed

    Paizs, Béla; Suhai, Sándor

    2005-01-01

    The fragmentation pathways of protonated peptides are reviewed in the present paper paying special attention to classification of the known fragmentation channels into a simple hierarchy defined according to the chemistry involved. It is shown that the 'mobile proton' model of peptide fragmentation can be used to understand the MS/MS spectra of protonated peptides only in a qualitative manner rationalizing differences observed for low-energy collision induced dissociation of peptide ions having or lacking a mobile proton. To overcome this limitation, a deeper understanding of the dissociation chemistry of protonated peptides is needed. To this end use of the 'pathways in competition' (PIC) model that involves a detailed energetic and kinetic characterization of the major peptide fragmentation pathways (PFPs) is proposed. The known PFPs are described in detail including all the pre-dissociation, dissociation, and post-dissociation events. It is our hope that studies to further extend PIC will lead to semi-quantative understanding of the MS/MS spectra of protonated peptides which could be used to develop refined bioinformatics algorithms for MS/MS based proteomics. Experimental and computational data on the fragmentation of protonated peptides are reevaluated from the point of view of the PIC model considering the mechanism, energetics, and kinetics of the major PFPs. Evidence proving semi-quantitative predictability of some of the ion intensity relationships (IIRs) of the MS/MS spectra of protonated peptides is presented. PMID:15389847

  4. Energetics and dynamics of the fragmentation reactions of protonated peptides containing methionine sulfoxide or aspartic acid via energy- and time-resolved surface induced dissociation.

    PubMed

    Lioe, Hadi; Laskin, Julia; Reid, Gavin E; O'Hair, Richard A J

    2007-10-25

    The surface-induced dissociation (SID) of six model peptides containing either methionine sulfoxide or aspartic acid (GAILM(O)GAILR, GAILM(O)GAILK, GAILM(O)GAILA, GAILDGAILR, GAILDGAILK, and GAILDGAILA) have been studied using a specially configured Fourier transform ion-cyclotron resonance mass spectrometer (FT-ICR MS). In particular, we have investigated the energetics and dynamics associated with (i) preferential cleavage of the methionine sulfoxide side chain via the loss of CH3SOH (64 Da), and (ii) preferential cleavage of the amide bond C-terminal to aspartic acid. The role of proton mobility in these selective bond cleavage reactions was examined by changing the C-terminal residue of the peptide from arginine (nonmobile proton conditions) to lysine (partially mobile proton conditions) to alanine (mobile proton conditions). Time- and energy-resolved fragmentation efficiency curves (TFECs) reveal that selective cleavages due to the methionine sulfoxide and aspartic acid residues are characterized by slow fragmentation kinetics. RRKM modeling of the experimental data suggests that the slow kinetics is associated with large negative entropy effects and these may be due to the presence of rearrangements prior to fragmentation. It was found that the Arrhenius pre-exponential factor (A) for peptide fragmentations occurring via selective bond cleavages are 1-2 orders of magnitude lower than nonselective peptide fragmentation reactions, while the dissociation threshold (E0) is relatively invariant. This means that selective bond cleavage is kinetically disfavored compared to nonselective amide bond cleavage. It was also found that the energetics and dynamics for the preferential loss of CH3SOH from peptide ions containing methionine sulfoxide are very similar to selective C-terminal amide bond cleavage at the aspartic acid residue. These results suggest that while preferential cleavage can compete with amide bond cleavage energetically, dynamically, these processes

  5. Energetics and Dynamics of the Fragmentation Reactions of Protonated Peptides Containing Methionine Sulfoxide or Aspartic Acid via Energy- and Time-Resolved Surface Induced Dissociation

    SciTech Connect

    Lioe, Hadi; Laskin, Julia; Reid, Gavin E.; O'Hair, Richard Aj

    2007-10-25

    The surface-induced dissociation (SID) of six model peptides containing either methionine sulfoxide or aspartic acid (GAILM(O)GAILR, GAILM(O)GAILK, GAILM(O)GAILA, GAILDGAILR, GAILDGAILK, and GAILDGAILA) have been studied using a specially configured Fourier transform ion-cyclotron resonance mass spectrometer (FT-ICR MS). In particular, we have investigated the energetics and dynamics associated with (i) preferential cleavage of the methionine sulfoxide side chain via the loss of CH3SOH (64Da), and (ii) preferential cleavage of the amide bond C-terminal to aspartic acid. The role of proton mobility on these selective bond cleavage reactions was examined by changing the C-terminal residue of the peptide from arginine (non-mobile proton conditions) to lysine (partially-mobile proton conditions) to alanine (mobile proton conditions). Time- and energy-resolved fragmentation efficiency curves (TFEC) reveals that selective cleavages due to the methionine sulfoxide and aspartic acid residues are characterized by slow fragmentation kinetics. RRKM modeling of the experimental data suggests that the slow kinetics is associated with large negative entropy effects and these may be due to the presence of rearrangements prior to fragmentation. It was found that the Arrhenius pre-exponential factor (A) for peptide fragmentations occurring via selective bond cleavages are 1–2 orders of magnitude lower than non-selective peptide fragmentation reactions, while the dissociation threshold (E0) is relatively invariant. This means that selective bond cleavage is kinetically disfavored compared to non-selective amide bond cleavage. It was also found that the energetics and dynamics for the preferential loss of CH3SOH from peptide ions containing methionine sulfoxide are very similar to selective C-terminal amide bond cleavage at the aspartic acid residue. These results suggest that while preferential cleavage can compete with amide bond cleavage energetically, dynamically, these

  6. New aspects in fragmentation of peptide nucleic acids: comparison of positive and negative ions by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.

    PubMed

    Ziehe, Matthias; Grossmann, Tom N; Seitz, Oliver; Linscheid, Michael W

    2009-04-01

    The use of peptide nucleic acids (PNAs) is steadily increasing in biochemistry and diagnostics. So far, PNAs have mostly been investigated using cationic conditions in mass spectrometry. Furthermore, the use of fragmentation techniques developed for peptides and proteins like infrared multiphoton dissociation (IRMPD) and electron capture dissociation (ECD) has barely been examined. However, especially the fragmentation behavior of PNA oligomers in negative ion mode is of high importance, due to the ability to interact with nucleic acids which are almost exclusively analyzed in the negatively charged state. In the current study PNA fragmentations under cationic and anionic conditions were investigated and different fragmentation techniques like collision-induced dissociation (CID), IRMPD and ECD were applied. Especially when using CID and IRMPD, amide bonds were broken, whereas ECD resulted in the elimination of nucleobases. Differences were also observed between positive and negative ionization, while the sequence coverage for the negative ions was superior to positive ions. The fragmentation behavior using IRMPD led to almost complete sequence coverage. Additionally, in anions the interesting effect of multiple eliminations of HNCO was found. PMID:19280610

  7. Peptide Dimethylation: Fragmentation Control via Distancing the Dimethylamino Group

    NASA Astrophysics Data System (ADS)

    McShane, Adam J.; Shen, Yuanyuan; Castillo, Mary Joan; Yao, Xudong

    2014-10-01

    Direct reductive methylation of peptides is a common method for quantitative proteomics. It is an active derivatization technique; with participation of the dimethylamino group, the derivatized peptides preferentially release intense a1 ions. The advantageous generation of a1 ions for quantitative proteomic profiling, however, is not desirable for targeted proteomic quantitation using multiple reaction monitoring mass spectrometry; this mass spectrometric method prefers the derivatizing group to stay with the intact peptide ions and multiple fragments as passive mass tags. This work investigated collisional fragmentation of peptides whose amine groups were derivatized with five linear ω-dimethylamino acids, from 2-(dimethylamino)-acetic acid to 6-(dimethylamino)-hexanoic acid. Tandem mass spectra of the derivatized tryptic peptides revealed different preferential breakdown pathways. Together with energy resolved mass spectrometry, it was found that shutting down the active participation of the terminal dimethylamino group in fragmentation of derivatized peptides is possible. However, it took a separation of five methylene groups between the terminal dimethylamino group and the amide formed upon peptide derivatization. For the first time, the gas-phase fragmentation of peptides derivatized with linear ω-dimethylamino acids of systematically increasing alkyl chain lengths is reported.

  8. Determination of binding capacity and adsorption enthalpy between Human Glutamate Receptor (GluR1) peptide fragments and kynurenic acid by surface plasmon resonance experiments.

    PubMed

    Csapó, E; Majláth, Z; Juhász, Á; Roósz, B; Hetényi, A; Tóth, G K; Tajti, J; Vécsei, L; Dékány, I

    2014-11-01

    The interaction between kynurenic acid (KYNA) and two peptide fragments (ca. 30 residues) of Human Glutamate Receptor 201-300 (GluR1) using surface plasmon resonance (SPR) spectroscopy was investigated. Because of the medical interest in the neuroscience, GluR1 is one of the important subunits of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR). AMPARs are ionotoropic glutamate receptors, which are mediating fast synaptic transmission and are crucial for plasticity in the brain. On the other hand, KYNA has been suggested to have neuroprotective activity and it has been considered for apply in therapy in certain neurobiological disorders. In this article the adsorption of the GluR1201-230 and GluR1231-259 peptides were studied on gold biosensor chip. The peptides were chemically bonded onto the gold surface via thiol group of L-cysteine resulted in the formation of peptide monolayer on the SPR chip surface. Because the GluR1231-259 peptide does not contain L-cysteine the Val256 was replaced by Cys256. The cross sectional area and the surface orientation of the studied peptides were determined by SPR and theoretical calculations (LOMETS) as well. The binding capability of KYNA on the peptide monolayer was studied in the concentration range of 0.1-5.0 mM using 150 mM NaCl ionic strength at pH 7.4 (±0.02) in phosphate buffer solutions. In order to determine the binding enthalpy the experiments were carried out between +10°C and +40°C. The heat of adsorption was calculated by using adsorption isotherms at different surface loading of KYNA on the SPR chip.

  9. Electrospray ionization tandem mass spectrometric study on the effect of N-terminal beta- and gamma-carbo amino acids on fragmentation of GABA-hybrid peptides.

    PubMed

    Ramesh, V; Ramesh, M; Srinivas, R; Sharma, G V M; Jayaprakash, P

    2008-11-01

    The fragmentations of protonated and deprotonated ions of a new class of N-blocked hybrid Boc-carbopeptides containing repeats of gamma-Caa/gammaAbu- and beta-Caa/gammaAbu- (Caa==C-linked carbo gamma(4)-/beta(3)- amino acids derived from D-xylose, gammaAbu = gamma-aminobutyric acid) have been studied using electrospray ionization (ESI) ion-trap tandem mass spectrometry (MS/MS). MS/MS of a pair of these protonated diastereomers produces distinct fragmentation of the Boc group. The formation of [M + H-56](+) corresponding to loss of isobutylene is more pronounced for Boc-NH-(R)-gamma-Caa-gammaAbu-OH (2) whereas it is of low abundance for Boc-NH-(S)-gamma-Caa-gammaAbu--OH (1). Similarly, MS(2) of [M--H](-) of 2 produces an abundant [M--H--C(CH(3))(3)OH--CO(2)](-) ion, which is absent for its diastereomeric isomer 1. From this, it can be suggested that MS/MS of N-blocked Boc-protected carbopeptides may be helpful in distinguishing the stereochemistry of the N-terminus Caa. MS(3) of [M + H-Boc + H](+) ions of peptides with a gamma-amino acid (gamma-Caa/gammaAbu) at the N-terminus produces only abundant y(n) (+) ions. On the other hand, characteristic fragmentations involving the peptide backbone (b(n) (+) and y(n) (+)) and the side chain are seen when beta-Caa is at the N-terminus of the peptides. MS(3) of the [M--H--C(CH(3))(3)OH](-) ion of peptides containing gamma-Caa/gammaAbu at the N-terminus gave y(n) (-) and [M--H--C(CH(3))(3)OH--CO(2)](-) ions, whereas the presence of beta-Caa at the N-terminus yielded predominantly [M--H--C(CH(3))(3)OH--HNCO](-). Thus, on the basis of our previous study and that presented here we propose that the fragmentation of these hybrid carbopeptides is highly influenced by the type of carbo amino acid present at the N-terminus. PMID:18837002

  10. Accurate Peptide Fragment Mass Analysis: Multiplexed Peptide Identification and Quantification

    PubMed Central

    Weisbrod, Chad R.; Eng, Jimmy K.; Hoopmann, Michael R.; Baker, Tahmina; Bruce, James E.

    2012-01-01

    FT All Reaction Monitoring (FT-ARM) is a novel approach for the identification and quantification of peptides that relies upon the selectivity of high mass accuracy data and the specificity of peptide fragmentation patterns. An FT-ARM experiment involves continuous, data-independent, high mass accuracy MS/MS acquisition spanning a defined m/z range. Custom software was developed to search peptides against the multiplexed fragmentation spectra by comparing theoretical or empirical fragment ions against every fragmentation spectrum across the entire acquisition. A dot product score is calculated against each spectrum in order to generate a score chromatogram used for both identification and quantification. Chromatographic elution profile characteristics are not used to cluster precursor peptide signals to their respective fragment ions. FT-ARM identifications are demonstrated to be complementary to conventional data-dependent shotgun analysis, especially in cases where the data-dependent method fails due to fragmenting multiple overlapping precursors. The sensitivity, robustness and specificity of FT-ARM quantification are shown to be analogous to selected reaction monitoring-based peptide quantification with the added benefit of minimal assay development. Thus, FT-ARM is demonstrated to be a novel and complementary data acquisition, identification, and quantification method for the large scale analysis of peptides. PMID:22288382

  11. Pronase E-Based Generation of Fluorescent Peptide Fragments: Tracking Intracellular Peptide Fate in Single Cells.

    PubMed

    Mainz, Emilie R; Dobes, Nicholas C; Allbritton, Nancy L

    2015-08-01

    The ability to track intracellular peptide proteolysis at the single cell level is of growing interest, particularly as short peptide sequences continue to play important roles as biosensors, therapeutics, and endogenous participants in antigen processing and intracellular signaling. We describe a rapid and inexpensive methodology to generate fluorescent peptide fragments from a parent sequence with diverse chemical properties, including aliphatic, nonpolar, basic, acidic, and non-native amino acids. Four peptide sequences with existing biochemical applications were fragmented using incubation with Pronase E and/or formic acid, and in each case a complete set of fluorescent fragments was generated for use as proteolysis standards in chemical cytometry. Fragment formation and identity was monitored with capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) and matrix assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-MS) to confirm the presence of all sequences and yield fragmentation profiles across Pronase E concentrations which can readily be used by others. As a pilot study, Pronase E-generated standards from an Abl kinase sensor and an ovalbumin antigenic peptide were then employed to identify proteolysis products arising from the metabolism of these sequences in single cells. The Abl kinase sensor fragmented at 4.2 ± 4.8 zmol μM(-1) s(-1) and the majority of cells possessed similar fragment identities. In contrast, an ovalbumin epitope peptide was degraded at 8.9 ± 0.1 zmol μM(-1) s(-1), but with differential fragment formation between individual cells. Overall, Pronase E-generated peptide standards were a rapid and efficient method to identify proteolysis products from cells. PMID:26171808

  12. Short communication: Measuring the angiotensin-converting enzyme inhibitory activity of an 8-amino acid (8mer) fragment of the C12 antihypertensive peptide.

    PubMed

    Paul, Moushumi; Phillips, John G; Renye, John A

    2016-05-01

    An 8-AA (8mer) fragment (PFPEVFGK) of a known antihypertensive peptide derived from bovine αS1-casein (C12 antihypertensive peptide) was synthesized by microwave-assisted solid-phase peptide synthesis and purified by reverse phase HPLC. Its ability to inhibit angiotensin-converting enzyme (ACE) was assessed and compared with that of the parent 12mer peptide (FFVAPFPEVFGK) to determine the effect of truncating the sequence on overall hypotensive activity. The activity of the truncated 8mer peptide was found to be almost 1.5 times less active than that of the 12mer, with ACE-inhibiting IC50 (half-maximal inhibitory concentration) values of 108 and 69μM, for the 8mer and 12mer, respectively. Although the 8mer peptide is less active than the original 12mer peptide, its overall activity is comparable to activities reported for other small proteins that elicit physiological responses within humans. These results suggest that microbial degradation of the 12mer peptide would not result in a complete loss of antihypertensive activity if used to supplement fermented foods and that the stable 8mer peptide could have potential as a blood pressure-lowering agent for use in functional foods.

  13. Determination of binding capacity and adsorption enthalpy between Human Glutamate Receptor (GluR1) peptide fragments and kynurenic acid by surface plasmon resonance experiments. Part 2: Interaction of GluR1270-300 with KYNA.

    PubMed

    Csapó, E; Bogár, F; Juhász, Á; Sebők, D; Szolomájer, J; Tóth, G K; Majláth, Z; Vécsei, L; Dékány, I

    2015-09-01

    In the course of our previous work, the interactions of two peptide fragments (GluR1201-230 and GluR1231-259) of human glutamate receptor (GluR1201-300) polypeptide with kynurenic acid (KYNA) were investigated by surface plasmon resonance (SPR) spectroscopy. Besides quantitation of the interactions, the enthalpies of binding of KYNA on certain peptide fragment-modified gold surfaces were also reported. In the present work, a third peptide fragment (GluR1270-300) of the glutamate receptor was synthesized and its interaction with KYNA was investigated by an SPR technique. This 31-membered peptide was chemically bonded onto a gold-coated SPR chip via a cysteine residue. The peptide-functionalized biosensor chip was analyzed by atomic force microscopy (AFM) and theoretical calculations were performed on the structure and dimensions of the peptide on the gold surface. In order to determine the isosteric heat of adsorption of the binding of KYNA on the peptide-functionalized gold thin film, SPR experiments were carried out between +10°C and +40°C. The results on the GluR1270-300-KYNA system were compared with the previously published binding parameters of the interactions of GluR1201-230 and GluR1231-259 with KYNA. The binding abilities of KYNA with all three peptide fragments immobilized on the gold surface were estimated by a molecular docking procedure and the binding free energies of these AMPA receptor subunits with KYNA were determined.

  14. Optimized Fragmentation Regime for Diazirine Photo-Cross-Linked Peptides

    PubMed Central

    2016-01-01

    Cross-linking/mass spectrometry has evolved into a robust technology that reveals structural insights into proteins and protein complexes. We leverage a new tribrid instrument with improved fragmentation capacities in a systematic comparison to identify which fragmentation method would be best for the identification of cross-linked peptides. Specifically, we explored three fragmentation methods and two combinations: collision-induced dissociation (CID), beam-type CID (HCD), electron-transfer dissociation (ETD), ETciD, and EThcD. Trypsin-digested, SDA-cross-linked human serum albumin (HSA) served as a test sample, yielding over all methods and in triplicate analysis in total 2602 matched PSMs and 1390 linked residue pairs at 5% false discovery rate, as confirmed by the crystal structure. HCD wins in number of matched peptide-spectrum-matches (958 PSMs) and identified links (446). CID is most complementary, increasing the number of identified links by 13% (58 links). HCD wins together with EThcD in cross-link site calling precision, with approximately 62% of sites having adjacent backbone cleavages that unambiguously locate the link in both peptides, without assuming any cross-linker preference for amino acids. Overall quality of spectra, as judged by sequence coverage of both peptides, is best for EThcD for the majority of peptides. Sequence coverage might be of particular importance for complex samples, for which we propose a data dependent decision tree, else HCD is the method of choice. The mass spectrometric raw data has been deposited in PRIDE (PXD003737). PMID:27454319

  15. Optimized Fragmentation Regime for Diazirine Photo-Cross-Linked Peptides.

    PubMed

    Giese, Sven H; Belsom, Adam; Rappsilber, Juri

    2016-08-16

    Cross-linking/mass spectrometry has evolved into a robust technology that reveals structural insights into proteins and protein complexes. We leverage a new tribrid instrument with improved fragmentation capacities in a systematic comparison to identify which fragmentation method would be best for the identification of cross-linked peptides. Specifically, we explored three fragmentation methods and two combinations: collision-induced dissociation (CID), beam-type CID (HCD), electron-transfer dissociation (ETD), ETciD, and EThcD. Trypsin-digested, SDA-cross-linked human serum albumin (HSA) served as a test sample, yielding over all methods and in triplicate analysis in total 2602 matched PSMs and 1390 linked residue pairs at 5% false discovery rate, as confirmed by the crystal structure. HCD wins in number of matched peptide-spectrum-matches (958 PSMs) and identified links (446). CID is most complementary, increasing the number of identified links by 13% (58 links). HCD wins together with EThcD in cross-link site calling precision, with approximately 62% of sites having adjacent backbone cleavages that unambiguously locate the link in both peptides, without assuming any cross-linker preference for amino acids. Overall quality of spectra, as judged by sequence coverage of both peptides, is best for EThcD for the majority of peptides. Sequence coverage might be of particular importance for complex samples, for which we propose a data dependent decision tree, else HCD is the method of choice. The mass spectrometric raw data has been deposited in PRIDE (PXD003737). PMID:27454319

  16. Side chain chemistry mediates backbone fragmentation in hydrogen deficient peptide radicals.

    PubMed

    Sun, Qingyu; Nelson, Hosea; Ly, Tony; Stoltz, Brian M; Julian, Ryan R

    2009-02-01

    A crown ether based, photolabile radical precursor which forms noncovalent complexes with peptides has been prepared. The peptide/precursor complexes can be electrosprayed, isolated in an ion trap, and then subjected to laser photolysis and collision induced dissociation to generate hydrogen deficient peptide radicals. It is demonstrated that these peptide radicals behave very differently from the hydrogen rich peptide radicals generated by electron capture methods. In fact, it is shown that side chain chemistry dictates both the occurrence and relative abundance of backbone fragments that are observed. Fragmentation at aromatic residues occurs preferentially over most other amino acids. The origin of this selectivity relates to the mechanism by which backbone dissociation is initiated. The first step is abstraction of a beta-hydrogen from the side chain, followed by beta-elimination to yield primarily a-type fragment ions. Calculations reveal that those side chains which can easily lose a beta-hydrogen correlate well with experimentally favored sites for backbone fragmentation. In addition, radical mediated side chain losses from the parent peptide are frequently observed. Eleven amino acids exhibit unique mass losses from side chains which positively identify that particular amino acid as part of the parent peptide. Therefore, side chain losses allow one to unambiguously narrow the possible sequences for a parent peptide, which when combined with predictable backbone fragmentation should lead to greatly increased confidence in peptide identification.

  17. On the Accuracy and Limits of Peptide Fragmentation Spectrum Prediction

    PubMed Central

    Li, Sujun; Arnold, Randy J.; Tang, Haixu; Radivojac, Predrag

    2011-01-01

    We estimated the reproducibility of tandem mass fragmentation spectra for the widely-used collision-induced dissociation (CID) instruments. Using the Pearson correlation coefficient as a measure of spectral similarity, we found that the within-experiment reproducibility of fragment ion intensities is very high (about 0.85). However, across different experiments and instrument types/setups, the correlation decreases by more than 15% (to about 0.70). We further investigated the accuracy of current predictors of peptide fragmentation spectra and found that they are more accurate than the ad-hoc models generally used by search engines (e.g. SEQUEST) and, surprisingly, approaching the empirical upper limit set by the average across-experiment spectral reproducibility (especially for charge +1 and charge +2 precursor ions). These results provide evidence that, in terms of accuracy of modeling, predicted peptide fragmentation spectra provide a viable alternative to spectral libraries for peptide identification, with a higher coverage of peptides and lower storage requirements. Furthermore, using five data sets of proteome digests by two different proteases, we find that PeptideART (a data-driven machine learning approach) is generally more accurate than MassAnalyzer (an approach based on a kinetic model for peptide fragmentation) in predicting fragmentation spectra, but that both models are significantly more accurate than the ad-hoc models. Availability: PeptideART is freely available at www.informatics.indiana.edu/predrag. PMID:21175207

  18. 213 nm Ultraviolet Photodissociation on Peptide Anions: Radical-Directed Fragmentation Patterns

    NASA Astrophysics Data System (ADS)

    Halim, Mohammad A.; Girod, Marion; MacAleese, Luke; Lemoine, Jérôme; Antoine, Rodolphe; Dugourd, Philippe

    2016-03-01

    Characterization of acidic peptides and proteins is greatly hindered due to lack of suitable analytical techniques. Here we present the implementation of 213 nm ultraviolet photodissociation (UVPD) in high-resolution quadrupole-Orbitrap mass spectrometer in negative polarity for peptide anions. Radical-driven backbone fragmentation provides 22 distinctive fragment ion types, achieving the complete sequence coverage for all reported peptides. Hydrogen-deficient radical anion not only promotes the cleavage of Cα-C bond but also stimulates the breaking of N-Cα and C-N bonds. Radical-directed loss of small molecules and specific side chain of amino acids are detected in these experiments. Radical containing side chain of amino acids (Tyr, Ser, Thr, and Asp) may possibly support the N-Cα backbone fragmentation. Proline comprising peptides exhibit the unusual fragment ions similar to reported earlier. Interestingly, basic amino acids such as Arg and Lys also stimulated the formation of abundant b and y ions of the related peptide anions. Loss of hydrogen atom from the charge-reduced radical anion and fragment ions are rationalized by time-dependent density functional theory (TDDFT) calculation, locating the potential energy surface (PES) of ππ* and repulsive πσ* excited states of a model amide system.

  19. Spatial structure of oligopeptide PAP(248-261), the N-terminal fragment of the HIV enhancer prostatic acid phosphatase peptide PAP(248-286), in aqueous and SDS micelle solutions

    NASA Astrophysics Data System (ADS)

    Blokhin, Dmitriy S.; Filippov, Andrei V.; Antzutkin, Oleg N.; Karataeva, Farida Kh.; Klochkov, Vladimir V.

    2014-07-01

    Prostatic acid phosphatase (PAP) is an enzyme that facilitates infection of cells by HIV. Its peptide fragment PAP(248-286) forms amyloid fibrils known as SEVI, which enhance attachment of the virus by viral adhesion to the host cell prior to receptor-specific binding via reducing the electrostatic repulsion between the membranes of the virus and the target cell. The secondary structure of PAP(248-286) in aqueous and SDS solutions can be divided into an N-terminal disordered region, an α-helical central part and an α/310-helical C-terminal region (Nanga et al., 2009). In this work, we used NMR spectroscopy to study the spatial structure of the isolated N-terminal fragment of PAP(248-286), PAP(248-261) (GIHKQKEKSRLQGG), in aqueous and SDS micelle solutions. Formation of a PAP(248-261)-SDS complex was confirmed by chemical shift alterations in the 1H NMR spectra of the peptide, as well as by the signs and values of Nuclear Overhauser Effect (NOE). In addition, the PAP(248-261) peptide does not form any specified secondary structure in either aqueous or SDS solutions.

  20. Crystallization of the Fab fragments of anti-peptide monoclonal antibodies and a complex with peptide.

    PubMed

    Griest, R E; Jeffrey, P D; Taylor, G L; Rees, A R

    1992-01-01

    The antigen-binding fragments of four monoclonal antibodies that cross-react with both the "loop" peptide of hen egg-white lysozyme (residues 57 to 84) against which they were raised, and with the native protein (HEL) have been crystallized. One of these fragments also crystallizes as a complex with the peptide antigen. PMID:1731084

  1. Chemical macrocyclization of peptides fused to antibody Fc fragments.

    PubMed

    Angelini, Alessandro; Diderich, Philippe; Morales-Sanfrutos, Julia; Thurnheer, Sarah; Hacker, David; Menin, Laure; Heinis, Christian

    2012-09-19

    To extend the plasma half-life of a bicyclic peptide antagonist, we chose to link it to the Fc fragment of the long-lived serum protein IgG1. Instead of chemically conjugating the entire bicyclic peptide, we recombinantly expressed its peptide moiety as a fusion protein to an Fc fragment and subsequently cyclized the peptide by chemically reacting its three cysteine residues with tris-(bromomethyl)benzene. This reaction was efficient and selective, yielding completely modified peptide fusion protein and no side products. After optimization of the linker and the Fc fragment format, the bicyclic peptide was fully functional as an inhibitor (K(i) = 76 nM) and showed an extended terminal half-life of 1.5 days in mice. The unexpectedly clean reaction makes chemical macrocyclization of peptide-Fc fusion proteins an attractive synthetic approach. Its good compatibility with the Fc fragment may lend the bromomethylbenzene-based chemistry also for the generation of antibody-drug conjugates.

  2. Basophile: Accurate Fragment Charge State Prediction Improves Peptide Identification Rates

    DOE PAGES

    Wang, Dong; Dasari, Surendra; Chambers, Matthew C.; Holman, Jerry D.; Chen, Kan; Liebler, Daniel; Orton, Daniel J.; Purvine, Samuel O.; Monroe, Matthew E.; Chung, Chang Y.; et al

    2013-03-07

    In shotgun proteomics, database search algorithms rely on fragmentation models to predict fragment ions that should be observed for a given peptide sequence. The most widely used strategy (Naive model) is oversimplified, cleaving all peptide bonds with equal probability to produce fragments of all charges below that of the precursor ion. More accurate models, based on fragmentation simulation, are too computationally intensive for on-the-fly use in database search algorithms. We have created an ordinal-regression-based model called Basophile that takes fragment size and basic residue distribution into account when determining the charge retention during CID/higher-energy collision induced dissociation (HCD) of chargedmore » peptides. This model improves the accuracy of predictions by reducing the number of unnecessary fragments that are routinely predicted for highly-charged precursors. Basophile increased the identification rates by 26% (on average) over the Naive model, when analyzing triply-charged precursors from ion trap data. Basophile achieves simplicity and speed by solving the prediction problem with an ordinal regression equation, which can be incorporated into any database search software for shotgun proteomic identification.« less

  3. Basophile: Accurate Fragment Charge State Prediction Improves Peptide Identification Rates

    SciTech Connect

    Wang, Dong; Dasari, Surendra; Chambers, Matthew C.; Holman, Jerry D.; Chen, Kan; Liebler, Daniel; Orton, Daniel J.; Purvine, Samuel O.; Monroe, Matthew E.; Chung, Chang Y.; Rose, Kristie L.; Tabb, David L.

    2013-03-07

    In shotgun proteomics, database search algorithms rely on fragmentation models to predict fragment ions that should be observed for a given peptide sequence. The most widely used strategy (Naive model) is oversimplified, cleaving all peptide bonds with equal probability to produce fragments of all charges below that of the precursor ion. More accurate models, based on fragmentation simulation, are too computationally intensive for on-the-fly use in database search algorithms. We have created an ordinal-regression-based model called Basophile that takes fragment size and basic residue distribution into account when determining the charge retention during CID/higher-energy collision induced dissociation (HCD) of charged peptides. This model improves the accuracy of predictions by reducing the number of unnecessary fragments that are routinely predicted for highly-charged precursors. Basophile increased the identification rates by 26% (on average) over the Naive model, when analyzing triply-charged precursors from ion trap data. Basophile achieves simplicity and speed by solving the prediction problem with an ordinal regression equation, which can be incorporated into any database search software for shotgun proteomic identification.

  4. The beneficial effect of BPC 157, a 15 amino acid peptide BPC fragment, on gastric and duodenal lesions induced by restraint stress, cysteamine and 96% ethanol in rats. A comparative study with H2 receptor antagonists, dopamine promotors and gut peptides.

    PubMed

    Sikiric, P; Seiwerth, S; Grabarevic, Z; Petek, M; Rucman, R; Turkovic, B; Rotkvic, I; Jagic, V; Duvnjak, M; Mise, S

    1994-01-01

    The protection of stomach and duodenum in conjecture with anti-inflammatory effect was demonstrated for a novel 15 amino acid peptide, coded BPC 157, a fragment of the recently discovered gastric juice peptide BPC. BPC 157 (i.p./i.g.) was investigated in rats in comparison with several reference standards in three experimental ulcer models (48 h-restraint stress, subcutaneous cysteamine, intragastrical 96% ethanol ulcer tests) (pre-/co-/post-treatment). Only BPC 157 regimens were consistently effective in all of the tested models. On the other hand, bromocriptine, amantadine, famotidine, cimetidine and somatostatin were ineffective (restraint stress). A dose-dependent protection (cysteamine) and/or partial positive effect (related to treatment conditions) (ethanol), was obtained with glucagon, NPY and secretin whereas CCK/26-30/was not effective. Based on Monastral blue studies BPC 157 beneficial effect appears to be related to a strong endothelial protection. PMID:7904712

  5. Peptide Fragmentation by Corona Discharge Induced Electrochemical Ionization

    PubMed Central

    Lloyd, John R.; Hess, Sonja

    2010-01-01

    Fundamental studies have greatly improved our understanding of electrospray, including the underlying electrochemical reactions. Generally regarded as disadvantageous, we have recently shown that corona discharge (CD) can be used as an effective method to create a radical cation species [M]+•, thus optimizing the electrochemical reactions that occur on the surface of the stainless steel (SS) electrospray capillary tip. This technique is known as CD initiated electrochemical ionization (CD-ECI). Here, we report on the fundamental studies using CD-ECI to induce analytically useful in-source fragmentation of a range of molecules that complex transition metals. Compounds that have been selectively fragmented using CD-ECI include enolate forming phenylglycine containing peptides, glycopeptides, nucleosides and phosphopeptides. Collision induced dissociation (CID) or other activation techniques were not necessary for CD-ECI fragmentation. A four step mechanism was proposed: 1. Complexation using either Fe in the SS capillary tip material or Cu(II) as an offline complexation reagent; 2. Electrochemical oxidation of the complexed metal and thus formation of a radical cation (e.g.; Fe - e− → Fe +•); 3. Radical fragmentation of the complexed compound. 4. Electrospray ionization of the fragmented neutrals. Fragmentation patterns resembling b- and y-type ions were observed and allowed the localization of the phosphorylation sites. PMID:20869880

  6. In-Source Fragmentation and the Sources of Partially Tryptic Peptides in Shotgun Proteomics

    SciTech Connect

    Kim, Jong-Seo; Monroe, Matthew E.; Camp, David G.; Smith, Richard D.; Qian, Wei-Jun

    2013-02-01

    Partially tryptic peptides are often identified in shotgun proteomics using trypsin as the proteolytic enzyme; however, it has been controversial regarding the sources of such partially tryptic peptides. Herein we investigate the impact of in-source fragmentation on shotgun proteomics using three biological samples, including a standard protein mixture, a mouse brain tissue homogenate, and a mouse plasma sample. Since the in-source fragments of a peptide retain the same elution time with its parent fully tryptic peptide, the partially tryptic peptides from in-source fragmentation can be distinguished from the other partially tryptic peptides by plotting the observed retention times against the computationally predicted retention times. Most partially tryptic in-source fragmentation artifacts were misaligned from the linear distribution of fully tryptic peptides. The impact of in-source fragmentation on peptide identifications was clearly significant in a less complex sample such as a standard protein digest, where ~60 % of unique peptides were observed as partially tryptic peptides from in-source fragmentation. In mouse brain or mouse plasma samples, in-source fragmentation contributed to 1-3 % of all identified peptides. The other major source of partially tryptic peptides in complex biological samples is presumably proteolytic processing by endogenous proteases in the samples. By filtering out the in-source fragmentation artifacts from the identified partially tryptic or non-tryptic peptides, it is possible to directly survey in-vivo proteolytic processing in biological samples such as blood plasma.

  7. Inhibition of HIV-1 infection by synthetic peptides derived CCR5 fragments

    SciTech Connect

    Imai, Masaki; Baranyi, Lajos; Okada, Noriko; Okada, Hidechika; E-mail: hiokada@med.nagoya-cu.ac.jp

    2007-02-23

    HIV-1 infection requires interaction of viral envelope protein gp160 with CD4 and a chemokine receptor, CCR5 or CXCR4 as entry coreceptor. We designed HIV-inhibitory peptides targeted to CCR5 using a novel computer program (ANTIS), which searched all possible sense-antisense amino acid pairs between proteins. Seven AHBs were found in CCR5 receptor. All AHB peptides were synthesized and tested for their ability to prevent HIV-1 infection to human T cells. A peptide fragment (LC5) which is a part of the CCR5 receptor corresponding to the loop between the fifth and sixth transmembrane regions (amino acids 222-240) proved to inhibit HIV-1{sub IIIB} infection of MT-4 cells. Interaction of these antisense peptides could be involved in sustaining HIV-1 infectivity. LC5 effectively indicated dose-dependent manner, and the suppression was enhanced additively by T20 peptide, which inhibits infection in vitro by disrupting the gp41 conformational changes necessary for membrane fusion. Thus, these results indicate that CCR5-derived AHB peptides could provide a useful tool to define the mechanism(s) of HIV infection, and may provide insight which will contribute to the development of an anti-HIV-1 reagent.

  8. Pro-Cognitive Effects of Non-Peptide Analogues of Soluble Amyloid Peptide Precursor Fragment sAPP.

    PubMed

    Tiunova, A A; Komissarova, N V; Nenaidenko, V G; Makhmutova, A A; Beznosko, B K; Bachurin, S O; Anokhin, K V

    2016-08-01

    We studied pro-cognitive effect of two heterocyclic low-molecular-weight compounds that serve as non-peptide analogues of soluble fragment of amyloid peptide precursor (sAPP). Intracerebroventricular and systemic administration of peptide mimetics P2 and P5 improved weak memory on the model of passive avoidance in chicks and in the object location task in mice. Both compounds were effective if administered close to the moment of training or 4 h after it. The time windows and dose range for the pro-cognitive effects of the mimetics were similar to those observed in previous studies with sAPP peptide fragments. PMID:27590763

  9. Peptide F (pro-enkephalin fragment): radioimmunoassay, and stress-induced changes in adrenal

    SciTech Connect

    Alessi, N.; Taylor, L.; Akil, H.

    1982-10-18

    Utilizing a nine amino-acid (Asp-Glu-Leu-Tyr-Pro-Leu-Glu-Val-Glu) non-enkephalin containing fragment of Peptide F from the pro-enkephalin molecule, a radioimmunoassay was developed. Extraction of bovine, rat, and guinea pig adrenomedullary preparations demonstrated this fragment to be present and apparently partially conserved across species. In rats, acute inescapable foot-shock stress led to a significant decrease of the immunoreactive material in the adrenal medulla. Chronic daily stress for two weeks resulted in an inability of the adrenals to alter F levels upon subsequent stress. The existence of F-like immunoreactivity and its alteration by environmental manipulation, suggest that it may play a unique physiological role.

  10. Proteolytic fragmentation and peptide mapping of human carboxyamidomethylated tracheobronchial mucin

    SciTech Connect

    Rose, M.C.; Kaufman, B.; Martin, B.M.

    1989-05-15

    Human tracheobronchial mucin was isolated from lung mucosal gel by chromatography on Sepharose 4B in the presence of dissociating and reducing agents, and its thiol residues were carboxyamidomethylated with iodo(1(-14)C)acetamide. The 14C-carboxyamido-methylated mucin was purified by chromatography on Sepharose 2B. No low molecular weight components were detected by molecular sieve chromatography or polyacrylamide gel electrophoresis in the presence of dissociating and reducing agents or by analytical density centrifugation in CsCl/guanidinium chloride. After digestion of the purified 14C-mucin with trypsin-L-1-tosylamido-2-phenylethyl chloromethyl ketone, three fractions (TR-1, TR-2, and TR-3) were observed by chromatography on Sepharose 4B. TR-1, a 260-kDa mucin glycopeptide fragment, contained all of the neutral hexose and blood group activity and 20% of the radioactivity in the undigested mucin. TR-1 was refractory to a second incubation with trypsin but could be digested by papain or Pronase to a smaller mucin glycopeptide fraction, as judged by the slight decrease in apparent molecular weight on Sepharose CL-4B. These mucin glycopeptides contained approximately 50% of the radioactivity in the TR-1 fraction, indicating that the glycosylated domains of carboxyamidomethylated tracheobronchial mucin contained thiol residues. The remainder of the radioactivity from papain or Pronase digests of TR-1 eluted, like the TR-3 fractions, in the salt fraction on Sepharose CL-4B. Peptide mapping of the nonglycosylated TR-3 fraction by TLC and high voltage electrophoresis yielded six principal and several less intensely stained ninhydrin reactive components, with the radiolabel concentrated in one of the latter peptides.

  11. Occurrence of C-terminal residue exclusion in peptide fragmentation by ESI and MALDI tandem mass spectrometry.

    PubMed

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

    2012-02-01

    By screening a data set of 392 synthetic peptides MS/MS spectra, we found that a known C-terminal rearrangement was unexpectedly frequently occurring from monoprotonated molecular ions in both ESI and MALDI tandem mass spectrometry upon low and high energy collision activated dissociations with QqTOF and TOF/TOF mass analyzer configuration, respectively. Any residue localized at the C-terminal carboxylic acid end, even a basic one, was lost, provided that a basic amino acid such arginine and to a lesser extent histidine and lysine was present in the sequence leading to a fragment ion, usually depicted as (b(n-1) + H(2)O) ion, corresponding to a shortened non-scrambled peptide chain. Far from being an epiphenomenon, such a residue exclusion from the peptide chain C-terminal extremity gave a fragment ion that was the base peak of the MS/MS spectrum in certain cases. Within the frame of the mobile proton model, the ionizing proton being sequestered onto the basic amino acid side chain, it is known that the charge directed fragmentation mechanism involved the C-terminal carboxylic acid function forming an anhydride intermediate structure. The same mechanism was also demonstrated from cationized peptides. To confirm such assessment, we have prepared some of the peptides that displayed such C-terminal residue exclusion as a C-terminal backbone amide. As expected in this peptide amide series, the production of truncated chains was completely suppressed. Besides, multiply charged molecular ions of all peptides recorded in ESI mass spectrometry did not undergo such fragmentation validating that any mobile ionizing proton will prevent such a competitive C-terminal backbone rearrangement. Among all well-known nondirect sequence fragment ions issued from non specific loss of neutral molecules (mainly H(2)O and NH(3)) and multiple backbone amide ruptures (b-type internal ions), the described C-terminal residue exclusion is highly identifiable giving raise to a single fragment

  12. Occurrence of C-Terminal Residue Exclusion in Peptide Fragmentation by ESI and MALDI Tandem Mass Spectrometry

    NASA Astrophysics Data System (ADS)

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

    2012-02-01

    By screening a data set of 392 synthetic peptides MS/MS spectra, we found that a known C-terminal rearrangement was unexpectedly frequently occurring from monoprotonated molecular ions in both ESI and MALDI tandem mass spectrometry upon low and high energy collision activated dissociations with QqTOF and TOF/TOF mass analyzer configuration, respectively. Any residue localized at the C-terminal carboxylic acid end, even a basic one, was lost, provided that a basic amino acid such arginine and to a lesser extent histidine and lysine was present in the sequence leading to a fragment ion, usually depicted as (bn-1 + H2O) ion, corresponding to a shortened non-scrambled peptide chain. Far from being an epiphenomenon, such a residue exclusion from the peptide chain C-terminal extremity gave a fragment ion that was the base peak of the MS/MS spectrum in certain cases. Within the frame of the mobile proton model, the ionizing proton being sequestered onto the basic amino acid side chain, it is known that the charge directed fragmentation mechanism involved the C-terminal carboxylic acid function forming an anhydride intermediate structure. The same mechanism was also demonstrated from cationized peptides. To confirm such assessment, we have prepared some of the peptides that displayed such C-terminal residue exclusion as a C-terminal backbone amide. As expected in this peptide amide series, the production of truncated chains was completely suppressed. Besides, multiply charged molecular ions of all peptides recorded in ESI mass spectrometry did not undergo such fragmentation validating that any mobile ionizing proton will prevent such a competitive C-terminal backbone rearrangement. Among all well-known nondirect sequence fragment ions issued from non specific loss of neutral molecules (mainly H2O and NH3) and multiple backbone amide ruptures (b-type internal ions), the described C-terminal residue exclusion is highly identifiable giving raise to a single fragment ion in

  13. Conserved Peptide Fragmentation as a Benchmarking Tool for Mass Spectrometers and a Discriminating Feature for Targeted Proteomics*

    PubMed Central

    Toprak, Umut H.; Gillet, Ludovic C.; Maiolica, Alessio; Navarro, Pedro; Leitner, Alexander; Aebersold, Ruedi

    2014-01-01

    Quantifying the similarity of spectra is an important task in various areas of spectroscopy, for example, to identify a compound by comparing sample spectra to those of reference standards. In mass spectrometry based discovery proteomics, spectral comparisons are used to infer the amino acid sequence of peptides. In targeted proteomics by selected reaction monitoring (SRM) or SWATH MS, predetermined sets of fragment ion signals integrated over chromatographic time are used to identify target peptides in complex samples. In both cases, confidence in peptide identification is directly related to the quality of spectral matches. In this study, we used sets of simulated spectra of well-controlled dissimilarity to benchmark different spectral comparison measures and to develop a robust scoring scheme that quantifies the similarity of fragment ion spectra. We applied the normalized spectral contrast angle score to quantify the similarity of spectra to objectively assess fragment ion variability of tandem mass spectrometric datasets, to evaluate portability of peptide fragment ion spectra for targeted mass spectrometry across different types of mass spectrometers and to discriminate target assays from decoys in targeted proteomics. Altogether, this study validates the use of the normalized spectral contrast angle as a sensitive spectral similarity measure for targeted proteomics, and more generally provides a methodology to assess the performance of spectral comparisons and to support the rational selection of the most appropriate similarity measure. The algorithms used in this study are made publicly available as an open source toolset with a graphical user interface. PMID:24623587

  14. 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.

  15. Isolation of cross-linked peptides by diagonal strong cation exchange chromatography for protein complex topology studies by peptide fragment fingerprinting from large sequence databases.

    PubMed

    Buncherd, Hansuk; Roseboom, Winfried; Ghavim, Behrad; Du, Weina; de Koning, Leo J; de Koster, Chris G; de Jong, Luitzen

    2014-06-27

    Knowledge of spatial proximity of amino acid residues obtained by chemical cross-linking and mass spectrometric analysis provides information about protein folding, protein-protein interactions and topology of macromolecular assemblies. We show that the use of bis(succinimidyl)-3-azidomethyl glutarate as a cross-linker provides a solution for two major analytical problems of cross-link mapping by peptide fragment fingerprinting (PFF) from complex sequence databases, i.e., low abundance of protease-generated target peptides and lack of knowledge of the masses of linked peptides. Tris(carboxyethyl)phosphine (TCEP) reduces the azido group in cross-linked peptides to an amine group in competition with cleavage of an amide bond formed in the cross-link reaction. TCEP-induced reaction products were separated by diagonal strong cation exchange (SCX) from unmodified peptides. The relation between the sum of the masses of the cleavage products and the mass of the parent cross-linked peptide enables determination of the masses of candidate linked peptides. By reversed phase LC-MS/MS analysis of secondary SCX fractions, we identified several intraprotein and interprotein cross-links in a HeLa cell nuclear extract, aided by software tools supporting PFF from the entire human sequence database. The data provide new information about interacting protein domains, among others from assemblies involved in splicing.

  16. A peptide hydrogel derived from a fragment of human cardiac troponin C.

    PubMed

    De Leon-Rodriguez, Luis M; Kamalov, Meder; Hemar, Yacine; Mitra, Alok K; Castelletto, Valeria; Hermida-Merino, Daniel; Hamley, Ian W; Brimble, Margaret A

    2016-03-14

    The human cardiac troponin C peptide fragment H-V(9)EQLTEEQKNEFKAAFDIFVLGA(31)-OH, which covers helix-A in the native protein, self-assembles into β-sheet fibrils in solution. These fibrils further entangle to give a hydrogel. This peptide may therefore serve as a template for development of novel biomaterials.

  17. A peptide hydrogel derived from a fragment of human cardiac troponin C.

    PubMed

    De Leon-Rodriguez, Luis M; Kamalov, Meder; Hemar, Yacine; Mitra, Alok K; Castelletto, Valeria; Hermida-Merino, Daniel; Hamley, Ian W; Brimble, Margaret A

    2016-03-14

    The human cardiac troponin C peptide fragment H-V(9)EQLTEEQKNEFKAAFDIFVLGA(31)-OH, which covers helix-A in the native protein, self-assembles into β-sheet fibrils in solution. These fibrils further entangle to give a hydrogel. This peptide may therefore serve as a template for development of novel biomaterials. PMID:26892840

  18. 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.

  19. Fragmentation of singly, doubly, and triply charged hydrogen deficient peptide radical cations in infrared multiphoton dissociation and electron induced dissociation.

    PubMed

    Kalli, Anastasia; Hess, Sonja

    2012-02-01

    Gas phase fragmentation of hydrogen deficient peptide radical cations continues to be an active area of research. While collision induced dissociation (CID) of singly charged species is widely examined, dissociation channels of singly and multiply charged radical cations in infrared multiphoton dissociation (IRMPD) and electron induced dissociation (EID) have not been, so far, investigated. Here, we report on the gas phase dissociation of singly, doubly and triply charged hydrogen deficient peptide radicals, [M + nH]((n+1)+·) (n=0, 1, 2), in MS(3) IRMPD and EID and compare the observed fragmentation pathways to those obtained in MS(3) CID. Backbone fragmentation in MS(3) IRMPD and EID was highly dependent on the charge state of the radical precursor ions, whereas amino acid side chain cleavages were largely independent of the charge state selected for fragmentation. Cleavages at aromatic amino acids, either through side chain loss or backbone fragmentation, were significantly enhanced over other dissociation channels. For singly charged species, the MS(3) IRMPD and EID spectra were mainly governed by radical-driven dissociation. Fragmentation of doubly and triply charged radical cations proceeded through both radical- and charge-driven processes, resulting in the formation of a wide range of backbone product ions including, a-, b-, c-, y-, x-, and z-type. While similarities existed between MS(3) CID, IRMPD, and EID of the same species, several backbone product ions and side chain losses were unique for each activation method. Furthermore, dominant dissociation pathways in each spectrum were dependent on ion activation method, amino acid composition, and charge state selected for fragmentation.

  20. Conformation-specific spectroscopy of peptide fragment ions in a low-temperature ion trap.

    PubMed

    Wassermann, Tobias N; Boyarkin, Oleg V; Paizs, Béla; Rizzo, Thomas R

    2012-06-01

    We have applied conformer-selective infrared-ultraviolet (IR-UV) double-resonance photofragment spectroscopy at low temperatures in an ion trap mass spectrometer for the spectroscopic characterization of peptide fragment ions. We investigate b- and a-type ions formed by collision-induced dissociation from protonated leucine-enkephalin. The vibrational analysis and assignment are supported by nitrogen-15 isotopic substitution of individual amino acid residues and assisted by density functional theory calculations. Under such conditions, b-type ions of different size are found to appear exclusively as linear oxazolone structures with protonation on the N-terminus, while a rearrangement reaction is confirmed for the a (4) ion in which the side chain of the C-terminal phenylalanine residue is transferred to the N-terminal side of the molecule. The vibrational spectra that we present here provide a particularly stringent test for theoretical approaches.

  1. Conformation-Specific Spectroscopy of Peptide Fragment Ions in a Low-Temperature Ion Trap

    NASA Astrophysics Data System (ADS)

    Wassermann, Tobias N.; Boyarkin, Oleg V.; Paizs, Béla; Rizzo, Thomas R.

    2012-06-01

    We have applied conformer-selective infrared-ultraviolet (IR-UV) double-resonance photofragment spectroscopy at low temperatures in an ion trap mass spectrometer for the spectroscopic characterization of peptide fragment ions. We investigate b- and a-type ions formed by collision-induced dissociation from protonated leucine-enkephalin. The vibrational analysis and assignment are supported by nitrogen-15 isotopic substitution of individual amino acid residues and assisted by density functional theory calculations. Under such conditions, b-type ions of different size are found to appear exclusively as linear oxazolone structures with protonation on the N-terminus, while a rearrangement reaction is confirmed for the a 4 ion in which the side chain of the C-terminal phenylalanine residue is transferred to the N-terminal side of the molecule. The vibrational spectra that we present here provide a particularly stringent test for theoretical approaches.

  2. Solution Structures, Dynamics, and Ice Growth Inhibitory Activity of Peptide Fragments Derived from an Antarctic Yeast Protein

    PubMed Central

    Asmawi, Azren A.; Rahman, Mohd Basyaruddin A.; Murad, Abdul Munir A.; Mahadi, Nor M.; Basri, Mahiran; Rahman, Raja Noor Zaliha A.; Salleh, Abu B.; Chatterjee, Subhrangsu; Tejo, Bimo A.; Bhunia, Anirban

    2012-01-01

    Exotic functions of antifreeze proteins (AFP) and antifreeze glycopeptides (AFGP) have recently been attracted with much interest to develop them as commercial products. AFPs and AFGPs inhibit ice crystal growth by lowering the water freezing point without changing the water melting point. Our group isolated the Antarctic yeast Glaciozyma antarctica that expresses antifreeze protein to assist it in its survival mechanism at sub-zero temperatures. The protein is unique and novel, indicated by its low sequence homology compared to those of other AFPs. We explore the structure-function relationship of G. antarctica AFP using various approaches ranging from protein structure prediction, peptide design and antifreeze activity assays, nuclear magnetic resonance (NMR) studies and molecular dynamics simulation. The predicted secondary structure of G. antarctica AFP shows several α-helices, assumed to be responsible for its antifreeze activity. We designed several peptide fragments derived from the amino acid sequences of α-helical regions of the parent AFP and they also showed substantial antifreeze activities, below that of the original AFP. The relationship between peptide structure and activity was explored by NMR spectroscopy and molecular dynamics simulation. NMR results show that the antifreeze activity of the peptides correlates with their helicity and geometrical straightforwardness. Furthermore, molecular dynamics simulation also suggests that the activity of the designed peptides can be explained in terms of the structural rigidity/flexibility, i.e., the most active peptide demonstrates higher structural stability, lower flexibility than that of the other peptides with lower activities, and of lower rigidity. This report represents the first detailed report of downsizing a yeast AFP into its peptide fragments with measurable antifreeze activities. PMID:23209600

  3. Synthesis and antiviral activity of PB1 component of the influenza A RNA polymerase peptide fragments.

    PubMed

    Matusevich, O V; Egorov, V V; Gluzdikov, I A; Titov, M I; Zarubaev, V V; Shtro, A A; Slita, A V; Dukov, M I; Shurygina, A-P S; Smirnova, T D; Kudryavtsev, I V; Vasin, A V; Kiselev, O I

    2015-01-01

    This study is devoted to the antiviral activity of peptide fragments from the PB1 protein - a component of the influenza A RNA polymerase. The antiviral activity of the peptides synthesized was studied in MDCK cell cultures against the pandemic influenza strain A/California/07/2009 (H1N1) pdm09. We found that peptide fragments 6-13, 6-14, 26-30, 395-400, and 531-540 of the PB1 protein were capable of suppressing viral replication in cell culture. Terminal modifications i.e. N-acetylation and C-amidation increased the antiviral properties of the peptides significantly. Peptide PB1 (6-14) with both termini modified showed maximum antiviral activity, its inhibitory activity manifesting itself during the early stages of viral replication. It was also shown that the fluorescent-labeled analog of this peptide was able to penetrate into the cell. The broad range of virus-inhibiting activity of PB1 (6-14) peptide was confirmed using a panel of influenza A viruses of H1, H3 and H5 subtypes including those resistant to oseltamivir, the leading drug in anti-influenza therapy. Thus, short peptide fragments of the PB1 protein could serve as leads for future development of influenza prevention and/or treatment agents.

  4. Determinants of Curvature-Sensing Behavior for MARCKS-Fragment Peptides.

    PubMed

    de Jesus, Armando J; White, Ormacinda R; Flynn, Aaron D; Yin, Hang

    2016-05-10

    It is increasingly recognized that membrane curvature plays an important role in various cellular activities such as signaling and trafficking, as well as key issues involving health and disease development. Thus, curvature-sensing peptides are essential to the study and detection of highly curved bilayer structures. The effector domain of myristoylated alanine-rich C-kinase substrate (MARCKS-ED) has been demonstrated to have curvature-sensing ability. Research of the MARCKS-ED has further revealed that its Lys and Phe residues play an essential role in how MARCKS-ED detects and binds to curved bilayers. MARCKS-ED has the added property of being a lower-molecular-weight curvature sensor, which offers advantages in production. With that in mind, this work investigates peptide-sequence-related factors that influence curvature sensing and explores whether peptide fragments of even shorter length can function as curvature sensors. Using both experimental and computational methods, we studied the curvature-sensing capabilities of seven fragments of MARCKS-ED. Two of the longer fragments were designed from approximately the two halves of the full-length peptide whereas the five shorter fragments were taken from the central stretch of MARCKS-ED. Fully atomistic molecular dynamics simulations show that the fragments that remain bound to the bilayer exhibit interactions with the bilayer similar to that of the full-length MARCKS-ED peptide. Fluorescence enhancement and anisotropy assays, meanwhile, reveal that five of the MARCKS fragments possess the ability to sense membrane curvature. Based on the sequences of the curvature-sensing fragments, it appears that the ability to sense curvature involves a balance between the numbers of positively charged residues and hydrophobic anchoring residues. Together, these findings help crystallize our understanding of the molecular mechanisms underpinning the curvature-sensing behaviors of peptides, which will prove useful in the

  5. The enantioselective immunoaffinity extraction of an optically active ibuprofen-modified peptide fragment.

    PubMed

    Ikegawa, S; Isriyanthi, N M; Nagata, M; Yahata, K; Ito, H; Mano, N; Goto, J

    2001-09-01

    Acyl glucuronides are known to produce the covalently bound protein adducts which may be the cause of hypersensitivity and toxic responses to acidic drugs. The structural analysis of the drug-protein adducts is therefore needed. From this point of view, we developed an enantioselective immunoaffinity extraction method, which employs an immobilized antibody to specifically isolate peptide fragments that have been modified with optically active ibuprofen. Rabbits were immunized with (S)-ibuprofen coupled to bovine serum albumin through a beta-alanine group. The elicited antibody strongly recognizes the asymmetric center and the isobutylphenyl moiety of (S)-ibuprofen and its conjugates but has a low affinity for their anti podes. A 0.5-mL aliquot of the immunosorbent (11.5 mg of IgG/mL gel) prepared by immobilization of the antibody was capable of retaining up to 1 microg of (S)-ibuprofen. When a mixture of substance P with (R)- and (S)-ibuprofen-modified substance P was loaded on the immunosorbent, the (S)-ibuprofen-modified substance P was selectively retained. The modified peptide was quantitatively recovered by elution with 10 mM ammonium acetate buffer (pH 5.0)/methanol (5:95, v/v). The proposed method would be useful for the structural characterization of optically active ibuprofen-modified human serum albumin.

  6. Spectroscopic Evidence for an Oxazolone Structure in Anionic b-Type Peptide Fragments

    NASA Astrophysics Data System (ADS)

    Grzetic, Josipa; Oomens, Jos

    2012-02-01

    Infrared spectra of anionic b-type fragments generated by collision induced dissociation (CID) from deprotonated peptides are reported. Spectra of the b2 fragments of deprotonated AlaAlaAla and AlaTyrAla have been recorded over the 800-1800 cm-1 spectral range by multiple-photon dissociation (MPD) spectroscopy using an FTICR mass spectrometer in combination with the free electron laser FELIX. Structural characterization of the b-type fragments is accomplished by comparison with density functional theory calculated spectra at the B3LYP/6-31++G(d,p) level for different isomeric structures. Although diketopiperazine structures represent the energetically lowest isomers, the IR spectra suggest an oxazolone structure for the b2 fragments of both peptides. Deprotonation is shown to occur on the oxazolone α-carbon, which leads to a conjugated structure in which the negative charge is practically delocalized over the entire oxazolone ring, providing enhanced gas-phase stability.

  7. Collision-Induced Dissociation Fragmentation Inside Disulfide C-Terminal Loops of Natural Non-Tryptic Peptides

    NASA Astrophysics Data System (ADS)

    Samgina, Tatiana Y.; Vorontsov, Egor A.; Gorshkov, Vladimir A.; Artemenko, Konstantin A.; Zubarev, Roman A.; Ytterberg, Jimmy A.; Lebedev, Albert T.

    2013-07-01

    Collision-induced dissociation (CID) spectra of long non-tryptic peptides are usually quite complicated and rather difficult to interpret. Disulfide bond formed by two cysteine residues at C-terminus of frog skin peptides precludes one to determine sequence inside the forming loop. Thereby, chemical modification of S-S bonds is often used in "bottom up" sequencing approach. However, low-energy CID spectra of natural non-tryptic peptides with C-terminal disulfide cycle demonstrate an unusual fragmentation route, which may be used to elucidate the "hidden" C-terminal sequence. Low charge state protonated molecules experience peptide bond cleavage at the N-terminus of C-terminal cysteine. The forming isomeric acyclic ions serve as precursors for a series of b-type ions revealing sequence inside former disulfide cycle. The reaction is preferable for peptides with basic lysine residues inside the cycle. It may also be activated by acidic protons of Asp and Glu residues neighboring the loop. The observed cleavages may be quite competitive, revealing the sequence inside disulfide cycle, although S-S bond rupture does not occur in this case.

  8. Amyloid-β peptide structure in aqueous solution varies with fragment size

    NASA Astrophysics Data System (ADS)

    Wise-Scira, Olivia; Xu, Liang; Kitahara, Taizo; Perry, George; Coskuner, Orkid

    2011-11-01

    Various fragment sizes of the amyloid-β (Aβ) peptide have been utilized to mimic the properties of the full-length Aβ peptide in solution. Among these smaller fragments, Aβ16 and Aβ28 have been investigated extensively. In this work, we report the structural and thermodynamic properties of the Aβ16, Aβ28, and Aβ42 peptides in an aqueous solution environment. We performed replica exchange molecular dynamics simulations along with thermodynamic calculations for investigating the conformational free energies, secondary and tertiary structures of the Aβ16, Aβ28, and Aβ42 peptides. The results show that the thermodynamic properties vary from each other for these peptides. Furthermore, the secondary structures in the Asp1-Lys16 and Asp1-Lys28 regions of Aβ42 cannot be completely captured by the Aβ16 and Aβ28 fragments. For example, the β-sheet structures in the N-terminal region of Aβ16 and Aβ28 are either not present or the abundance is significantly decreased in Aβ42. The α-helix and β-sheet abundances in Aβ28 and Aβ42 show trends - to some extent - with the potential of mean forces but no such trend could be obtained for Aβ16. Interestingly, Arg5 forms salt bridges with large abundances in all three peptides. The formation of a salt bridge between Asp23-Lys28 is more preferred over the Glu22-Lys28 salt bridge in Aβ28 but this trend is vice versa for Aβ42. This study shows that the Asp1-Lys16 and Asp1-Lys28 regions of the full length Aβ42 peptide cannot be completely mimicked by studying the Aβ16 and Aβ28 peptides.

  9. Fragmentation mechanism of UV-excited peptides in the gas phase

    NASA Astrophysics Data System (ADS)

    Zabuga, Aleksandra V.; Kamrath, Michael Z.; Boyarkin, Oleg V.; Rizzo, Thomas R.

    2014-10-01

    We present evidence that following near-UV excitation, protonated tyrosine- or phenylalanine-containing peptides undergo intersystem crossing to produce a triplet species. This pathway competes with direct dissociation from the excited electronic state and with dissociation from the electronic ground state subsequent to internal conversion. We employ UV-IR double-resonance photofragment spectroscopy to record conformer-specific vibrational spectra of cold peptides pre-excited to their S1 electronic state. The absorption of tunable IR light by these electronically excited peptides leads to a drastic increase in fragmentation, selectively enhancing the loss of neutral phenylalanine or tyrosine side-chain, which are not the lowest dissociation channels in the ground electronic state. The recorded IR spectra evolve upon increasing the time delay between the UV and IR pulses, reflecting the dynamics of the intersystem crossing on a timescale of ˜80 ns and <10 ns for phenylalanine- and tyrosine-containing peptides, respectively. Once in the triplet state, phenylalanine-containing peptides may live for more than 100 ms, unless they absorb IR photons and undergo dissociation by the loss of an aromatic side-chain. We discuss the mechanism of this fragmentation channel and its possible implications for photofragment spectroscopy and peptide photostability.

  10. Fragmentation mechanism of UV-excited peptides in the gas phase

    SciTech Connect

    Zabuga, Aleksandra V. Kamrath, Michael Z.; Boyarkin, Oleg V.; Rizzo, Thomas R.

    2014-10-21

    We present evidence that following near-UV excitation, protonated tyrosine- or phenylalanine–containing peptides undergo intersystem crossing to produce a triplet species. This pathway competes with direct dissociation from the excited electronic state and with dissociation from the electronic ground state subsequent to internal conversion. We employ UV-IR double-resonance photofragment spectroscopy to record conformer-specific vibrational spectra of cold peptides pre-excited to their S{sub 1} electronic state. The absorption of tunable IR light by these electronically excited peptides leads to a drastic increase in fragmentation, selectively enhancing the loss of neutral phenylalanine or tyrosine side-chain, which are not the lowest dissociation channels in the ground electronic state. The recorded IR spectra evolve upon increasing the time delay between the UV and IR pulses, reflecting the dynamics of the intersystem crossing on a timescale of ∼80 ns and <10 ns for phenylalanine- and tyrosine-containing peptides, respectively. Once in the triplet state, phenylalanine-containing peptides may live for more than 100 ms, unless they absorb IR photons and undergo dissociation by the loss of an aromatic side-chain. We discuss the mechanism of this fragmentation channel and its possible implications for photofragment spectroscopy and peptide photostability.

  11. Dualistic nature of adhesive protein function: fibronectin and its biologically active peptide fragments can autoinhibit fibronectin function

    PubMed Central

    1984-01-01

    Fibronectin and certain polypeptide regions of this adhesive glycoprotein mediate cell attachment and spreading on various substrates. We explored the theoretical prediction that this adhesive protein could become a competitive inhibitor of fibronectin-mediated processes if present in solution at appropriately high concentrations. Fibronectin function was inhibited by purified plasma fibronectin at 5- 10 mg/ml, by a 75,000-dalton cell-interaction fragment of the protein at 0.5-1 mg/ml, and even by two synthetic peptides containing a conserved, hydrophilic amino acid sequence at 0.1-0.5 mg/ml. Inhibition of fibronectin-dependent cell spreading was dose dependent, noncytotoxic, and reversible. It was competitive in nature, since increased quantities of substrate-adsorbed fibronectin or longer incubation periods decreased the inhibition. A peptide inhibitory for fibronectin-mediated cell spreading also inhibited fibronectin-mediated attachment of cells to type I collagen, but it did not affect concanavalin A-mediated spreading. These results demonstrate the potential of a cell adhesion molecule and its biologically active peptide fragments to act as competitive inhibitors, and they suggest that fibronectin may act by binding to a saturable cell surface receptor. PMID:6736130

  12. Identification of pro-opiomelanocortin and secretion of its peptide fragments in bovine adrenals

    SciTech Connect

    Tennov, A.V.; Dmitriev, A.D.; Kizim, E.A.; Ustinova, E.E.

    1986-01-01

    This paper describes the results of an investigation to show that biosynthesis of POMC, its proteolytic processing, an secretion of the peptide products of that processing take place in the bovine adrenals. Rabbit antisera against endorphins were obtained and used for radioimmunoassay of peptides. I 125-labeled peptides were obtained by the chloramine method and purified from free I 125 on Sephadex G-10 (0.7 x 5 cm, centrifugation for 10 min at 1500 g). To detect secretion of peptide fragments of POMC in the adrenals experiments were undertaken to determine the beta-endorphin content in perfusates obtained during retrograde perfusion of the bovine adrenals. It was found that immunoreactive compounds, indistinguishable in their immunochemical properties from beta-endorphin, are present in the perfusates, just as in the tissue extracts.

  13. Preparation and analysis of peptide fragments produced by pepsin hydrolysis of human plasma albumin and their relationship to its structure

    PubMed Central

    Franglen, G.; Swaniker, G. R. E.

    1968-01-01

    Human plasma albumin was prepared and subjected to proteolysis by pepsin at pH2·45 at 25° for 10min. with albumin/pepsin ratio 3000:1. Five peptide fragments were detected in the proteolysate by means of zone electrophoresis and gel filtration; these were separated and purified. Molecular weights, amino acid composition and disulphide bond content of the purified fragments were determined. The results show that a high proportion of the polypeptide chain of albumin appears to have a low cystine content, and at low pH values the molecule would be expected to have a considerable degree of freedom in its structure in these regions of the chain. A tripartite model for the structure of plasma albumin is proposed. PMID:4876098

  14. 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.

  15. 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

  16. 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.

  17. [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

  18. 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](+•).

  19. 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

  20. Proton Mobility in b2 Ion Formation and Fragmentation Reactions of Histidine-Containing Peptides

    NASA Astrophysics Data System (ADS)

    Nelson, Carissa R.; Abutokaikah, Maha T.; Harrison, Alex G.; Bythell, Benjamin J.

    2016-03-01

    A detailed energy-resolved study of the fragmentation reactions of protonated histidine-containing peptides and their b2 ions has been undertaken. Density functional theory calculations were utilized to predict how the fragmentation reactions occur so that we might discern why the mass spectra demonstrated particular energy dependencies. We compare our results to the current literature and to synthetic b2 ion standards. We show that the position of the His residue does affect the identity of the subsequent b2 ion (diketopiperazine versus oxazolone versus lactam) and that energy-resolved CID can distinguish these isomeric products based on their fragmentation energetics. The histidine side chain facilitates every major transformation except trans-cis isomerization of the first amide bond, a necessary prerequisite to diketopiperazine b2 ion formation. Despite this lack of catalyzation, trans-cis isomerization is predicted to be facile. Concomitantly, the subsequent amide bond cleavage reaction is rate-limiting.

  1. Human neutrophils are activated by a peptide fragment of Clostridium difficile toxin B presumably via formyl peptide receptor.

    PubMed

    Goy, Sebastian D; Olling, Alexandra; Neumann, Detlef; Pich, Andreas; Gerhard, Ralf

    2015-06-01

    Clostridium difficile may induce antibiotic-associated diarrhoea and, in severe cases, pseudomembranous colitis characterized by tremendous neutrophil infiltration. All symptoms are caused by two exotoxins: TcdA and TcdB. We describe here the activation of isolated human blood neutrophils by TcdB and, moreover, by toxin fragments generated by limited proteolytical digestion. Kinetics and profiles of TcdB-induced rise in intracellular-free Ca(2+) and reactive oxygen species production were similar to that induced by fMLF, which activates the formyl peptide receptor (FPR) recognizing formylated bacterial peptide sequences. Transfection assays with the FPR-1 isoform hFPR26 in HEK293 cells, heterologous desensitization experiments and FPR inhibition via cyclosporine H strongly suggest activation of cells via FPR-1. Domain analyses revealed that the N-terminal glucosyltransferase domain of TcdB is a potent activator of FPR pointing towards an additional mechanism that might contribute to pathogenesis. This pro-inflammatory ligand effect can be triggered even by cleaved and, thus, non-cytotoxic toxin. In summary, we report (i) a ligand effect on neutrophils as completely new molecular mode of action, (ii) pathogenic potential of truncated or proteolytically cleaved 'non-cytotoxic' fragments and (iii) an interaction of the N-terminal glucosyltransferase domain instead of the C-terminal receptor binding domain of TcdB with target cells. PMID:25529763

  2. Formation and Fragmentation of Radical Peptide Anions: Insights from Vacuum Ultra Violet Spectroscopy

    NASA Astrophysics Data System (ADS)

    Brunet, Claire; Antoine, Rodolphe; Dugourd, Philippe; Canon, Francis; Giuliani, Alexandre; Nahon, Laurent

    2012-02-01

    We have studied the photodissociation of gas-phase deprotonated caerulein anions by vacuum ultraviolet (VUV) photons in the 4.5 to 20 eV range, as provided by the DESIRS beamline at the synchrotron radiation facility SOLEIL (France). Caerulein is a sulphated peptide with three aromatic residues and nine amide bonds. Electron loss is found to be the major relaxation channel at every photon energy. However, an increase in the fragmentation efficiency (neutral losses and peptide backbone cleavages) as a function of the energy is also observed. The oxidized ions, generated by electron photodetachment were further isolated and activated by collision (CID) in a MS3 scheme. The branching ratios of the different fragments observed by CID as a function of the initial VUV photon energy are found to be independent of the initial photon energy. Thus, there is no memory effect of the initial excitation energy on the fragmentation channels of the oxidized species on the time scale of our tandem MS experiment. We also report photofragment yields as a function of photon energy for doubly deprotonated caerulein ions, for both closed-shell ([M-2H]2-) non-radical ions and open-shell ([M-3H]2-•) radical ions. These latter ions are generated by electron photodetachment from [M-3H]3- precursor ions. The detachment yield increases monotonically with the energy with the appearance of several absorption bands. Spectra for radical and non-radical ions are quite similar in terms of observed bands; however, the VUV fragmentation yield is enhanced by the presence of a radical in caerulein peptides.

  3. Coordination Environment of Cu(II) Ions Bound to N-Terminal Peptide Fragments of Angiogenin Protein.

    PubMed

    Magrì, Antonio; Munzone, Alessia; Peana, Massimiliano; Medici, Serenella; Zoroddu, Maria Antonietta; Hansson, Orjan; Satriano, Cristina; Rizzarelli, Enrico; La Mendola, Diego

    2016-01-01

    Angiogenin (Ang) is a potent angiogenic factor, strongly overexpressed in patients affected by different types of cancers. The specific Ang cellular receptors have not been identified, but it is known that Ang-actin interaction induces changes both in the cell cytoskeleton and in the extracellular matrix. Most in vitro studies use the recombinant form (r-Ang) instead of the form that is normally present in vivo ("wild-type", wt-Ang). The first residue of r-Ang is a methionine, with a free amino group, whereas wt-Ang has a glutamic acid, whose amino group spontaneously cyclizes in the pyro-glutamate form. The Ang biological activity is influenced by copper ions. To elucidate the role of such a free amino group on the protein-copper binding, we scrutinized the copper(II) complexes with the peptide fragments Ang(1-17) and AcAng(1-17), which encompass the sequence 1-17 of angiogenin (QDNSRYTHFLTQHYDAK-NH₂), with free amino and acetylated N-terminus, respectively. Potentiometric, ultraviolet-visible (UV-vis), nuclear magnetic resonance (NMR) and circular dichroism (CD) studies demonstrate that the two peptides show a different metal coordination environment. Confocal microscopy imaging of neuroblastoma cells with the actin staining supports the spectroscopic results, with the finding of different responses in the cytoskeleton organization upon the interaction, in the presence or not of copper ions, with the free amino and the acetylated N-terminus peptides. PMID:27490533

  4. Coordination Environment of Cu(II) Ions Bound to N-Terminal Peptide Fragments of Angiogenin Protein

    PubMed Central

    Magrì, Antonio; Munzone, Alessia; Peana, Massimiliano; Medici, Serenella; Zoroddu, Maria Antonietta; Hansson, Orjan; Satriano, Cristina; Rizzarelli, Enrico; La Mendola, Diego

    2016-01-01

    Angiogenin (Ang) is a potent angiogenic factor, strongly overexpressed in patients affected by different types of cancers. The specific Ang cellular receptors have not been identified, but it is known that Ang–actin interaction induces changes both in the cell cytoskeleton and in the extracellular matrix. Most in vitro studies use the recombinant form (r-Ang) instead of the form that is normally present in vivo (“wild-type”, wt-Ang). The first residue of r-Ang is a methionine, with a free amino group, whereas wt-Ang has a glutamic acid, whose amino group spontaneously cyclizes in the pyro-glutamate form. The Ang biological activity is influenced by copper ions. To elucidate the role of such a free amino group on the protein–copper binding, we scrutinized the copper(II) complexes with the peptide fragments Ang(1–17) and AcAng(1–17), which encompass the sequence 1–17 of angiogenin (QDNSRYTHFLTQHYDAK-NH2), with free amino and acetylated N-terminus, respectively. Potentiometric, ultraviolet-visible (UV-vis), nuclear magnetic resonance (NMR) and circular dichroism (CD) studies demonstrate that the two peptides show a different metal coordination environment. Confocal microscopy imaging of neuroblastoma cells with the actin staining supports the spectroscopic results, with the finding of different responses in the cytoskeleton organization upon the interaction, in the presence or not of copper ions, with the free amino and the acetylated N-terminus peptides. PMID:27490533

  5. Coordination Environment of Cu(II) Ions Bound to N-Terminal Peptide Fragments of Angiogenin Protein.

    PubMed

    Magrì, Antonio; Munzone, Alessia; Peana, Massimiliano; Medici, Serenella; Zoroddu, Maria Antonietta; Hansson, Orjan; Satriano, Cristina; Rizzarelli, Enrico; La Mendola, Diego

    2016-08-01

    Angiogenin (Ang) is a potent angiogenic factor, strongly overexpressed in patients affected by different types of cancers. The specific Ang cellular receptors have not been identified, but it is known that Ang-actin interaction induces changes both in the cell cytoskeleton and in the extracellular matrix. Most in vitro studies use the recombinant form (r-Ang) instead of the form that is normally present in vivo ("wild-type", wt-Ang). The first residue of r-Ang is a methionine, with a free amino group, whereas wt-Ang has a glutamic acid, whose amino group spontaneously cyclizes in the pyro-glutamate form. The Ang biological activity is influenced by copper ions. To elucidate the role of such a free amino group on the protein-copper binding, we scrutinized the copper(II) complexes with the peptide fragments Ang(1-17) and AcAng(1-17), which encompass the sequence 1-17 of angiogenin (QDNSRYTHFLTQHYDAK-NH₂), with free amino and acetylated N-terminus, respectively. Potentiometric, ultraviolet-visible (UV-vis), nuclear magnetic resonance (NMR) and circular dichroism (CD) studies demonstrate that the two peptides show a different metal coordination environment. Confocal microscopy imaging of neuroblastoma cells with the actin staining supports the spectroscopic results, with the finding of different responses in the cytoskeleton organization upon the interaction, in the presence or not of copper ions, with the free amino and the acetylated N-terminus peptides.

  6. Effect of the basic residue on the energetics, dynamics, and mechanisms of gas-phase fragmentation of protonated peptides.

    PubMed

    Laskin, Julia; Yang, Zhibo; Song, Tao; Lam, Corey; Chu, Ivan K

    2010-11-17

    The effect of the basic residue on the energetics, dynamics, and mechanisms of backbone fragmentation of protonated peptides was investigated. Time-resolved and collision energy-resolved surface-induced dissociation (SID) of singly protonated peptides with the N-terminal arginine residue and their analogues, in which arginine is replaced with less basic lysine and histidine residues, was examined using a specially configured Fourier transform ion cyclotron resonance mass spectrometer (FTICR-MS). SID experiments demonstrated different kinetics of formation of several primary product ions of peptides with and without arginine residue. The energetics and dynamics of these pathways were determined from Rice-Ramsperger-Kassel-Marcus (RRKM) modeling of the experimental data. Comparison between the kinetics and energetics of fragmentation of arginine-containing peptides and the corresponding methyl ester derivatives provides important information on the effect of dissociation pathways involving salt bridge (SB) intermediates on the observed fragmentation behavior. Because pathways involving SB intermediates are characterized by low threshold energies, they efficiently compete with classical oxazolone and imine/enol pathways of arginine-containing peptides on a long time scale of the FTICR instrument. In contrast, fragmentation of histidine- and lysine-containing peptides is largely determined by canonical pathways. Because SB pathways are characterized by negative activation entropies, fragmentation of arginine-containing peptides is kinetically hindered and observed at higher collision energies as compared to their lysine- and histidine-containing analogues.

  7. Peptide linkers lead to modification of liver metabolism and improved tumor targeting of copper-67-labeled antibody fragments.

    PubMed

    Novak-Hofer, I; Zimmermann, K; Schubiger, P A

    2001-12-01

    In order to determine if tumor/nontarget tissue ratios of 67Cu-labeled antibody fragments can be improved, modifying the DO3A copper chelate with tripeptide linkers was investigated. The peptide-linked chelates 1,4,7,10-tetraazacyclodecane-N,N',N",N"'-tetraacetate (DOTA)-triglycyl-L-p-isothiocyanato-phenylalanine (DOTA-R1-NCS), DOTA-glycyl-phenylalanyl-glycyl-L-p-isothiocyanato-phenylalanine (DOTA-R2-NCS), DOTA-glycyl-prolyl-glycyl-L-p-isothiocyanato-phenylalanine (DOTA-R3-NCS) and DOTA-glycyl-L-p-isothiocyanato-phenylalanine (DOTA-R4-NCS) were synthesized and coupled to F(ab')2 fragments of anti-colon carcinoma mAb35. In vitro, the 67Cu-labeled antibody fragments were fully immunoreactive and stable in human serum. In vivo in nude mice bearing human colon carcinoma xenografts the conjugates R1 and R3 showed improved tumor uptake and lower levels of radioactivity in the liver compared with the other conjugates. Biodistributions of the DOTA-R2-F(ab')2 showed at early time points after injection higher levels of radioactivity in the liver, lower levels of activity persisting in the blood and lower accumulation of activity in the tumor. When liver homogenates were analyzed 30 min post injection by SDS-PAGE or FPLC gel chromatography, it was found that radioactivity was released more slowly from the triglycine (R1)-F(ab')2 than from the immunoconjugates with the R2 or the R4 linker. The main radioactive metabolites were protein bands at 66 kD, 31 kD and low molecular weight fragments. The results show that the rate of cleavage of the copper complex from F(ab')2 fragments in vivo can be influenced by the amino acid sequence close to the complex, with significant consequences on biodistributions.

  8. Cytotoxicity and the effect of cationic peptide fragments against cariogenic bacteria under planktonic and biofilm conditions.

    PubMed

    Kreling, Paula Fernanda; Aida, Kelly Limi; Massunari, Loiane; Caiaffa, Karina Sampaio; Percinoto, Célio; Bedran, Telma Blanca Lombardo; Spolidorio, Denise Madalena Palomari; Abuna, Gabriel Flores; Cilli, Eduardo Maffud; Duque, Cristiane

    2016-10-01

    This study evaluated the cytotoxicity and effect of fragments derived from three oral cationic peptides (CP): LL-37, D6-17 and D1-23 against cariogenic bacteria under planktonic and biofilm conditions. For cytotoxicity analysis, two epithelial cell lines were used. The minimum inhibitory concentration and the minimal bactericidal concentration were determined for the CP fragments and the control (chlorhexidine-CHX) against cariogenic bacteria. The fractional inhibitory concentration was obtained for the combinations of CP fragments on Streptococcus mutans. Biofilm assays were conducted with the best antimicrobial CP fragment against S. mutans. The results indicated that D6-17 was not cytotoxic. D1-23, LL-37 and CHX were not cytotoxic in low concentrations. D1-23 presented the best bactericidal activity against S. mutans, S. mitis and S. salivarius. Combinations of CP fragments did not show a synergic effect. D1-23 presented a higher activity against S. mutans biofilm than CHX. It was concluded that D1-23 showed a substantial effect against cariogenic bacteria and low cytotoxicity. PMID:27538256

  9. 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.

  10. 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.

  11. Antimicrobial Peptides: Insights into Membrane Permeabilization, Lipopolysaccharide Fragmentation and Application in Plant Disease Control

    PubMed Central

    Datta, Aritreyee; Ghosh, Anirban; Airoldi, Cristina; Sperandeo, Paola; Mroue, Kamal H.; Jiménez-Barbero, Jesús; Kundu, Pallob; Ramamoorthy, Ayyalusamy; Bhunia, Anirban

    2015-01-01

    The recent increase in multidrug resistance against bacterial infections has become a major concern to human health and global food security. Synthetic antimicrobial peptides (AMPs) have recently received substantial attention as potential alternatives to conventional antibiotics because of their potent broad-spectrum antimicrobial activity. These peptides have also been implicated in plant disease control for replacing conventional treatment methods that are polluting and hazardous to the environment and to human health. Here, we report de novo design and antimicrobial studies of VG16, a 16-residue active fragment of Dengue virus fusion peptide. Our results reveal that VG16KRKP, a non-toxic and non-hemolytic analogue of VG16, shows significant antimicrobial activity against Gram-negative E. coli and plant pathogens X. oryzae and X. campestris, as well as against human fungal pathogens C. albicans and C. grubii. VG16KRKP is also capable of inhibiting bacterial disease progression in plants. The solution-NMR structure of VG16KRKP in lipopolysaccharide features a folded conformation with a centrally located turn-type structure stabilized by aromatic-aromatic packing interactions with extended N- and C-termini. The de novo design of VG16KRKP provides valuable insights into the development of more potent antibacterial and antiendotoxic peptides for the treatment of human and plant infections. PMID:26144972

  12. 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.

  13. Metal ion-catalyzed nucleic acid alkylation and fragmentation.

    PubMed

    Browne, Kenneth A

    2002-07-10

    Nucleic acid microarrays are a growing technology in which high densities of known sequences are attached to a substrate in known locations (addressed). Hybridization of complementary sequences leads to a detectable signal such as an electrical impulse or fluorescence. This combination of sequence addressing, hybridization, and detection increases the efficiency of a variety of genomic disciplines including those that profile genetic expression, search for single nucleotide polymorphisms (SNPs), or diagnose infectious diseases by sequencing portions of microbial or viral genomes. Incorporation of reporter molecules into nucleic acids is essential for the sensitive detection of minute amounts of nucleic acids on most types of microarrays. Furthermore, polynucleic acid size reduction increases hybridization because of increased diffusion rates and decreased competing secondary structure of the target nucleic acids. Typically, these reactions would be performed as two separate processes. An improvement to past techniques, termed labeling-during-cleavage (LDC), is presented in which DNA or RNA is alkylated with fluorescent tags and fragmented in the same reaction mixture. In model studies with 26 nucleotide-long RNA and DNA oligomers using ultraviolet/visible and fluorescence spectroscopies as well as high-pressure liquid chromatography and mass spectrometry, addition of both alkylating agents (5-(bromomethyl)fluorescein, 5- or 6-iodoacetamidofluorescein) and select metal ions (of 21 tested) to nucleic acids in aqueous solutions was critical for significant increases in both labeling and fragmentation, with >or=100-fold increases in alkylation possible relative to metal ion-free reactions. Lanthanide series metal ions, Pb(2+), and Zn(2+) were the most reactive ions in terms of catalyzing alkylation and fragmentation. While oligonucleotides were particularly susceptible to fragmentation at sites containing phosphorothioate moieties, labeling and cleavage reactions

  14. Virtual Screening of Peptide and Peptidomimetic Fragments Targeted to Inhibit Bacterial Dithiol Oxidase DsbA

    PubMed Central

    Stoermer, Martin J.; Tay, Stephanie; McMahon, Róisín M.; Fairlie, David P.; Martin, Jennifer L.

    2015-01-01

    Antibacterial drugs with novel scaffolds and new mechanisms of action are desperately needed to address the growing problem of antibiotic resistance. The periplasmic oxidative folding system in Gram-negative bacteria represents a possible target for anti-virulence antibacterials. By targeting virulence rather than viability, development of resistance and side effects (through killing host native microbiota) might be minimized. Here, we undertook the design of peptidomimetic inhibitors targeting the interaction between the two key enzymes of oxidative folding, DsbA and DsbB, with the ultimate goal of preventing virulence factor assembly. Structures of DsbB - or peptides - complexed with DsbA revealed key interactions with the DsbA active site cysteine, and with a hydrophobic groove adjacent to the active site. The present work aimed to discover peptidomimetics that target the hydrophobic groove to generate non-covalent DsbA inhibitors. The previously reported structure of a Proteus mirabilis DsbA active site cysteine mutant, in a non-covalent complex with the heptapeptide PWATCDS, was used as an in silico template for virtual screening of a peptidomimetic fragment library. The highest scoring fragment compound and nine derivatives were synthesized and evaluated for DsbA binding and inhibition. These experiments discovered peptidomimetic fragments with inhibitory activity at millimolar concentrations. Although only weakly potent relative to larger covalent peptide inhibitors that interact through the active site cysteine, these fragments offer new opportunities as templates to build non-covalent inhibitors. The results suggest that non-covalent peptidomimetics may need to interact with sites beyond the hydrophobic groove in order to produce potent DsbA inhibitors. PMID:26225423

  15. The role of the position of the basic residue in the generation and fragmentation of peptide radical cations

    NASA Astrophysics Data System (ADS)

    Wee, Sheena; O'Hair, Richard A. J.; McFadyen, W. David

    2006-03-01

    Using simple di- and tripeptides GX, GGX, GXG, XG and XGG, the influence of the position of the basic residue, X (X = R, K and H), on the formation of peptide radical cations (M+) from [CuII(tpy)M]2+ complexes (where tpy = 2,2':6',2''-terpyridine) was probed. It was found that M+ is formed with greatest abundance when the basic residue is at the C-terminus. For arginine containing peptides, this may be due to further fragmentation of GRG+, RG+ and RGG+ at the MS2 stage. For lysine and histidine containing peptides, when the basic residue is not located at the C-terminus, competing fragmentation pathways that lead to peptide backbone cleavage are more facile than M+ formation. In order to gain some insights into the binding modes of these peptides to [CuII(tpy)]2+, the formation and fragmentation of copper(II) complexes of tripeptides protected as their carboxy methyl/ethyl esters (M-OR', R' = Me/Et) were also probed. The products of the competing fragmentation pathways of [CuII(tpy)M]2+, as well as the formation and fragmentation of [CuII(tpy)(M-OR')]2+, suggest that the unprotected peptides, M, mainly bind as zwitterions to [CuII(tpy)]2+. The fragmentation reactions of the radical cations (M+) were also studied. Radical driven side chain fragmentation reactions of M+ are dependent on both the position of the residue as well as the identity of other residues present in the peptide radical cations. GR and RG, which undergo rearrangement to form a mixed anhydride in their protonated forms, do not undergo the same rearrangement in their radical cation forms.

  16. Extending the coverage of spectral libraries: a neighbor-based approach to predicting intensities of peptide fragmentation spectra.

    PubMed

    Ji, Chao; Arnold, Randy J; Sokoloski, Kevin J; Hardy, Richard W; Tang, Haixu; Radivojac, Predrag

    2013-03-01

    Searching spectral libraries in MS/MS is an important new approach to improving the quality of peptide and protein identification. The idea relies on the observation that ion intensities in an MS/MS spectrum of a given peptide are generally reproducible across experiments, and thus, matching between spectra from an experiment and the spectra of previously identified peptides stored in a spectral library can lead to better peptide identification compared to the traditional database search. However, the use of libraries is greatly limited by their coverage of peptide sequences: even for well-studied organisms a large fraction of peptides have not been previously identified. To address this issue, we propose to expand spectral libraries by predicting the MS/MS spectra of peptides based on the spectra of peptides with similar sequences. We first demonstrate that the intensity patterns of dominant fragment ions between similar peptides tend to be similar. In accordance with this observation, we develop a neighbor-based approach that first selects peptides that are likely to have spectra similar to the target peptide and then combines their spectra using a weighted K-nearest neighbor method to accurately predict fragment ion intensities corresponding to the target peptide. This approach has the potential to predict spectra for every peptide in the proteome. When rigorous quality criteria are applied, we estimate that the method increases the coverage of spectral libraries available from the National Institute of Standards and Technology by 20-60%, although the values vary with peptide length and charge state. We find that the overall best search performance is achieved when spectral libraries are supplemented by the high quality predicted spectra.

  17. Structural analysis of a functional DIAP1 fragment bound to grim and hid peptides.

    PubMed

    Wu, J W; Cocina, A E; Chai, J; Hay, B A; Shi, Y

    2001-07-01

    The inhibitor of apoptosis protein DIAP1 suppresses apoptosis in Drosophila, with the second BIR domain (BIR2) playing an important role. Three proteins, Hid, Grim, and Reaper, promote apoptosis, in part by binding to DIAP1 through their conserved N-terminal sequences. The crystal structures of DIAP1-BIR2 by itself and in complex with the N-terminal peptides from Hid and Grim reveal that these peptides bind a surface groove on DIAP1, with the first four amino acids mimicking the binding of the Smac tetrapeptide to XIAP. The next 3 residues also contribute to binding through hydrophobic interactions. Interestingly, peptide binding induces the formation of an additional alpha helix in DIAP1. Our study reveals the structural conservation and diversity necessary for the binding of IAPs by the Drosophila Hid/Grim/Reaper and the mammalian Smac proteins.

  18. Structural and Thermodynamic Properties of Amyloid-β Peptides: Impact of Fragment Size

    NASA Astrophysics Data System (ADS)

    Kitahara, T.; Wise-Scira, O.; Coskuner, O.

    2010-10-01

    Alzheimer's disease is a progressive neurodegenerative disease whose physiological characteristics include the accumulation of amyloid-containing deposits in the brain and consequent synapse and neuron loss. Unfortunately, most widely used drugs for the treatment can palliate the outer symptoms but cannot cure the disease itself. Hence, developing a new drug that can cure it. Most recently, the ``early aggregation and monomer'' hypothesis has become popular and a few drugs have been developed based on this hypothesis. Detailed understanding of the amyloid-β peptide structure can better help us to determine more effective treatment strategies; indeed, the structure of Amyloid has been studied extensively employing experimental and theoretical tools. Nevertheless, those studies have employed different fragment sizes of Amyloid and characterized its conformational nature in different media. Thus, the structural properties might be different from each other and provide a reason for the existing debates in the literature. Here, we performed all-atom MD simulations and present the structural and thermodynamic properties of Aβ1-16, Aβ1-28, and Aβ1-42 in the gas phase and in aqueous solution. Our studies show that the overall structures, secondary structures, and the calculated thermodynamic properties change with increasing peptide size. In addition, we find that the structural properties of those peptides are different from each other in the gas phase and in aqueous solution.

  19. Structure and further fragmentation of significant [a3 + Na - H]+ ions from sodium-cationized peptides.

    PubMed

    Wang, Huixin; Wang, Bing; Wei, Zhonglin; Zhang, Hao; Guo, Xinhua

    2015-01-01

    A good understanding of gas-phase fragmentation chemistry of peptides is important for accurate protein identification. Additional product ions obtained by sodiated peptides can provide useful sequence information supplementary to protonated peptides and improve protein identification. In this work, we first demonstrate that the sodiated a3 ions are abundant in the tandem mass spectra of sodium-cationized peptides although observations of a3 ions have rarely been reported in protonated peptides. Quantum chemical calculations combined with tandem mass spectrometry are used to investigate this phenomenon by using a model tetrapeptide GGAG. Our results reveal that the most stable [a3 + Na - H](+) ion is present as a bidentate linear structure in which the sodium cation coordinates to the two backbone carbonyl oxygen atoms. Due to structural inflexibility, further fragmentation of the [a3 + Na - H](+) ion needs to overcome several relatively high energetic barriers to form [b2 + Na - H](+) ion with a diketopiperazine structure. As a result, low abundance of [b2 + Na - H](+) ion is detected at relatively high collision energy. In addition, our computational data also indicate that the common oxazolone pathway to generate [b2 + Na - H](+) from the [a3 + Na - H](+) ion is unlikely. The present work provides a mechanistic insight into how a sodium ion affects the fragmentation behaviors of peptides.

  20. Mechanisms of peptide fragmentation from time- and energy-resolved surface-induced dissociation studies: Dissociation of angiotensin analogs

    NASA Astrophysics Data System (ADS)

    Laskin, Julia; Bailey, Thomas H.; Futrell, Jean H.

    2006-03-01

    Energetics and mechanism of dissociation of singly protonated angiotensin III (RVYIHPF) and its analogs RVYIFPF, RVYIYPF, RVYIHAF and RVYIHDF was studied using surface-induced dissociation (SID) in a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) specially configured for studying ion activation by collisions with surfaces. The energetics and dynamics of peptide fragmentation were deduced by modeling the time- and energy-resolved survival curves for each precursor ion using an RRKM-based approach developed in our laboratory. Fragmentation mechanisms were inferred from comparison of time- and energy-resolved fragmentation efficiency curves (TFECs) of different fragment ions followed by RRKM modeling of dissociation of angiotensin III into six major families of fragment ions. Detailed modeling demonstrated that dissociation of these peptides is dominated by loss of ammonia from the precursor ion and characterized by a high-energy barrier of 1.6 eV. Loss of NH3 and subsequent rearrangement of the MH+-NH3 ion results in proton mobilization and release of ca. 30 kcal/mol into internal excitation of the MH+-NH3 ion. The resulting highly excited ion accesses a variety of non-specific dissociation pathways with very high rate constants. Fast fragmentation of excited MH+-NH3 ion forms a variety of abundant bn-NH3 and an-NH3 fragment ions. Abundant XH and HX internal fragments are also formed, reflecting the stability of histidine-containing diketopiperazine structures.

  1. 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

  2. Fragmentation of peptide radical cations containing a tyrosine or tryptophan residue: structural features that favor formation of [x(n-1) + H]˙⁺ and [z(n-1) + H]˙⁺ ions.

    PubMed

    Mädler, Stefanie; Lau, Justin Kai-Chi; Williams, Declan; Wang, Yating; Saminathan, Irine S; Zhao, Junfang; Siu, K W Michael; Hopkinson, Alan C

    2014-06-12

    Peptide radical cations A(n)Y(•+) (where n = 3, 4, or 5) and A5W(•+) have been generated by collision-induced dissociation (CID) of [Cu(II)(tpy)(peptide)](•2+) complexes. Apart from the charge-driven fragmentation at the N-Cα bond of the hetero residue producing either [c + 2H](+) or [z - H](•+) ions and radical-driven fragmentation at the Cα-C bond to give a(+) ions, unusual product ions [x + H](•+) and [z + H](•+) are abundant in the CID spectra of the peptides with the hetero residue in the second or third position of the chain. The formation of these ions requires that both the charge and radical be located on the peptide backbone. Energy-resolved spectra established that the [z + H](•+) ion can be produced either directly from the peptide radical cation or via the fragment ion [x + H](•+). Additionally, backbone dissociation by loss of the C-terminal amino acid giving [b(n-1) - H](•+) increases in abundance with the length of the peptides. Mechanisms by which peptide radical cations dissociate have been modeled using density functional theory (B3LYP/6-31++G** level) on tetrapeptides AYAG(•+), AAYG(•+), and AWAG(•+).

  3. Correlations Between Amino Acids at Different Sites in Local Sequences of Protein Fragments with Given Structural Patterns

    NASA Astrophysics Data System (ADS)

    Lu, Wen; Liu, Hai-yan

    2007-02-01

    Ample evidence suggests that the local structures of peptide fragments in native proteins are to some extent encoded by their local sequences. Detecting such local correlations is important but it is still an open question what would be the most appropriate method. This is partly because conventional sequence analyses treat amino acid preferences at each site of a protein sequence independently, while it is often the inter-site interactions that bring about local sequence-structure correlations. Here a new scheme is introduced to capture the correlation between amino acid preferences at different sites for different local structure types. A library of nine-residue fragments is constructed, and the fragments are divided into clusters based on their local structures. For each local structure cluster or type, chi-square tests are used to identify correlated preferences of amino acid combinations at pairs of sites. A score function is constructed including both the single site amino acid preferences and the dual-site amino acid combination preferences, which can be used to identify whether a sequence fragment would have a strong tendency to form a particular local structure in native proteins. The results show that, given a local structure pattern, dual-site amino acid combinations contain different information from single site amino acid preferences. Representative examples show that many of the statistically identified correlations agree with previously-proposed heuristic rules about local sequence-structure correlations, or are consistent with physical-chemical interactions required to stabilize particular local structures. Results also show that such dual-site correlations in the score function significantly improves the Z-score matching a sequence fragment to its native local structure relative to non-native local structures, and certain local structure types are highly predictable from the local sequence alone if inter-site correlations are considered.

  4. 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

  5. Gas-phase peptide fragmentation: how understanding the fundamentals provides a springboard to developing new chemistry and novel proteomic tools.

    PubMed

    Barlow, Christopher K; O'Hair, Richard A J

    2008-10-01

    This tutorial provides an overview of the evolution of some of the key concepts in the gas-phase fragmentation of different classes of peptide ions under various conditions [e.g. collision-induced dissociation (CID) and electron transfer dissociation (ETD)], and then demonstrates how these concepts can be used to develop new methods. For example, an understanding of the role of the mobile proton and neighboring group interactions in the fragmentation reactions of protonated peptides has led to the design of the 'SELECT' method. For ETD, a model based on the Landau-Zener theory reveals the role of both thermodynamic and geometric effects in the electron transfer from polyatomic reagent anions to multiply protonated peptides, and this predictive model has facilitated the design of a new strategy to form ETD reagent anions from precursors generated via ESI. Finally, two promising, emerging areas of gas-phase ion chemistry of peptides are also described: (1) the design of new gas-phase radical chemistry to probe peptide structure, and (2) selective cleavage of disulfide bonds of peptides in the gas phase via various physicochemical approaches.

  6. Peptide fragments of the dihydropyridine receptor can modulate cardiac ryanodine receptor channel activity and sarcoplasmic reticulum Ca2+ release.

    PubMed Central

    Dulhunty, Angela F; Curtis, Suzanne M; Cengia, Louise; Sakowska, Magdalena; Casarotto, Marco G

    2004-01-01

    We show that peptide fragments of the dihydropyridine receptor II-III loop alter cardiac RyR (ryanodine receptor) channel activity in a cytoplasmic Ca2+-dependent manner. The peptides were AC (Thr-793-Ala-812 of the cardiac dihydropyridine receptor), AS (Thr-671-Leu-690 of the skeletal dihydropyridine receptor), and a modified AS peptide [AS(D-R18)], with an extended helical structure. The peptides added to the cytoplasmic side of channels in lipid bilayers at > or = 10 nM activated channels when the cytoplasmic [Ca2+] was 100 nM, but either inhibited or did not affect channel activity when the cytoplasmic [Ca2+] was 10 or 100 microM. Both activation and inhibition were independent of bilayer potential. Activation by AS, but not by AC or AS(D-R18), was reduced at peptide concentrations >1 mM in a voltage-dependent manner (at +40 mV). In control experiments, channels were not activated by the scrambled AS sequence (ASS) or skeletal II-III loop peptide (NB). Resting Ca2+ release from cardiac sarcoplasmic reticulum was not altered by peptide AC, but Ca2+-induced Ca2+ release was depressed. Resting and Ca2+-induced Ca2+ release were enhanced by both the native and modified AS peptides. NMR revealed (i) that the structure of peptide AS(D-R18) is not influenced by [Ca2+] and (ii) that peptide AC adopts a helical structure, particularly in the region containing positively charged residues. This is the first report of specific functional interactions between dihydropyridine receptor A region peptides and cardiac RyR ion channels in lipid bilayers. PMID:14678014

  7. 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

  8. Conformational Dynamics of Two Natively Unfolded Fragment Peptides: Comparison of the AMBER and CHARMM Force Fields.

    PubMed

    Chen, Wei; Shi, Chuanyin; MacKerell, Alexander D; Shen, Jana

    2015-06-25

    Physics-based force fields are the backbone of molecular dynamics simulations. In recent years, significant progress has been made in the assessment and improvement of commonly used force fields for describing conformational dynamics of folded proteins. However, the accuracy for the unfolded states remains unclear. The latter is however important for detailed studies of protein folding pathways, conformational transitions involving unfolded states, and dynamics of intrinsically disordered proteins. In this work, we compare the three commonly used force fields, AMBER ff99SB-ILDN, CHARMM22/CMAP, and CHARMM36, for modeling the natively unfolded fragment peptides, NTL9(1-22) and NTL9(6-17), using explicit-solvent replica-exchange molecular dynamics simulations. All three simulations show that NTL9(6-17) is completely unstructured, while NTL9(1-22) transiently samples various β-hairpin states, reminiscent of the first β-hairpin in the structure of the intact NTL9 protein. The radius of gyration of the two peptides is force field independent but likely underestimated due to the current deficiency of additive force fields. Compared to the CHARMM force fields, ff99SB-ILDN gives slightly higher β-sheet propensity and more native-like residual structures for NTL9(1-22), which may be attributed to its known β preference. Surprisingly, only two sequence-local pairs of charged residues make appreciable ionic contacts in the simulations of NTL9(1-22), which are sampled slightly more by the CHARMM force fields. Taken together, these data suggest that the current CHARMM and AMBER force fields are globally in agreement in modeling the unfolded states corresponding to β-sheet in the folded structure, while differing in details such as the native-likeness of the residual structures and interactions. PMID:26020564

  9. Conformational Dynamics of Two Natively Unfolded Fragment Peptides: Comparison of the AMBER and CHARMM Force Fields.

    PubMed

    Chen, Wei; Shi, Chuanyin; MacKerell, Alexander D; Shen, Jana

    2015-06-25

    Physics-based force fields are the backbone of molecular dynamics simulations. In recent years, significant progress has been made in the assessment and improvement of commonly used force fields for describing conformational dynamics of folded proteins. However, the accuracy for the unfolded states remains unclear. The latter is however important for detailed studies of protein folding pathways, conformational transitions involving unfolded states, and dynamics of intrinsically disordered proteins. In this work, we compare the three commonly used force fields, AMBER ff99SB-ILDN, CHARMM22/CMAP, and CHARMM36, for modeling the natively unfolded fragment peptides, NTL9(1-22) and NTL9(6-17), using explicit-solvent replica-exchange molecular dynamics simulations. All three simulations show that NTL9(6-17) is completely unstructured, while NTL9(1-22) transiently samples various β-hairpin states, reminiscent of the first β-hairpin in the structure of the intact NTL9 protein. The radius of gyration of the two peptides is force field independent but likely underestimated due to the current deficiency of additive force fields. Compared to the CHARMM force fields, ff99SB-ILDN gives slightly higher β-sheet propensity and more native-like residual structures for NTL9(1-22), which may be attributed to its known β preference. Surprisingly, only two sequence-local pairs of charged residues make appreciable ionic contacts in the simulations of NTL9(1-22), which are sampled slightly more by the CHARMM force fields. Taken together, these data suggest that the current CHARMM and AMBER force fields are globally in agreement in modeling the unfolded states corresponding to β-sheet in the folded structure, while differing in details such as the native-likeness of the residual structures and interactions.

  10. 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

  11. Tandem Mass Spectrometric Characterization of Thiol Peptides Modified by the Chemoselective Cationic Sulfhydryl Reagent (4-Iodobutyl)Triphenylphosphonium—. Effects of a Cationic Thiol Derivatization on Peptide Fragmentation

    NASA Astrophysics Data System (ADS)

    Wang, Jing; Zhang, Jie; Arbogast, Brian; Maier, Claudia S.

    2011-10-01

    Fixed charge chemical modifications on peptides and proteins can impact fragmentation behaviors in tandem mass spectrometry (MS/MS). In this study, we employed a thiol-specific cationic alkylation reagent, (4-iodobutyl)triphenylphosphonium (IBTP), to selectively modify cysteine thiol groups in mitochondrial proteome samples. Tandem mass spectrometric characteristics of butyltriphenylphosphonium (BTP)-modified peptides were evaluated by comparison to their carbamidomethylated (CAM) analogues using a quadrupole time-of-flight (Q-TOF) instrument under low energy collision-induced dissociation (CID) conditions. Introduction of the fixed charge modification resulted in the observation of peptide and fragment (bn and yn) ions with higher charge states than those observed for CAM-modified analogues. The charged BTP moiety had a significant effect on the neighboring amide bond fragmentation products. A decrease in relative abundances of the product ions at the corresponding cleavage sites was observed compared with those from the CAM-modified derivatives. This effect was particularly noticeable when an Xxx-Pro bond was in the vicinity of a BTP group. We hypothesized that the presence of a phosphonium moiety will reduce the tendency for protonation of the proximal amide bonds in the peptide backbone. Indeed, calculations indicated that proton affinities of backbone amide bonds close to the modified cysteine residues were generally 20-50 kcal/mol lower for BTP-modified peptides than for the unmodified or CAM-modified analogues with the sequence motif -Ala-Cys-Alan-Ala2-, -Ala-Cys-Alan-Pro-Ala-, and -Ala-Pro-Alan-Cys-Ala-, n = 0-3.

  12. Investigation of cis/trans proline isomerism in a multiply occurring peptide fragment from human salivary proline-rich glycoprotein.

    PubMed

    Loomis, R E; Gonzalez, M; Loomis, P M

    1991-11-01

    The solution-state conformations of eight proline-containing peptide fragments found in human salivary proline-rich glycoprotein (PRG) were investigated in 2 x distilled water (treated with metal ion chelating resin) using 13C-nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy. The peptide sequences and acronyms were as follows: PRG9-2 = NH2-G(1)-P(2)-CONH2, PRG9-3 = NH2-G(1)P(2)-P(3)-CONH2, PRG9-4 = NH2-G(1)-P(2)-P(3)-P(4)-CONH2, PRG9-5 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-CONH2, PRG9-6 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-CONH2, PRG9-7 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-G(7)-CONH2, PRG9-8 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-G(7)-K(8)-CONH2 and PRG9-9 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-G(7)-K(8)-P(9)-CONH2. Sequence-specific resonance assignments from the 13C-NMR spectra indicated that the trans proline isomer dominated the conformations of the peptides. CD results clearly showed the presence of the poly-L-proline II helix as the major conformation in PRG9-3----PRG9-5, supplemented by beta- and/or gamma-turns in PRG9-6----PRG9-9. These data suggest that in "metal free" water, native PRG could contain several small poly-L-proline II helices along with beta- and/or gamma-turns. Since proline is the major amino acid present in native PRG, these localized conformations may contribute to PRG's global conformation and act as a primary force in determining its biological activities.

  13. Fragment-based solid-phase assembly of oligonucleotide conjugates with peptide and polyethylene glycol ligands.

    PubMed

    Dirin, Mehrdad; Urban, Ernst; Noe, Christian R; Winkler, Johannes

    2016-10-01

    Ligand conjugation to oligonucleotides is an attractive strategy for enhancing the therapeutic potential of antisense and siRNA agents by inferring properties such as improved cellular uptake or better pharmacokinetic properties. Disulfide linkages enable dissociation of ligands and oligonucleotides in reducing environments found in endosomal compartments after cellular uptake. Solution-phase fragment coupling procedures for producing oligonucleotide conjugates are often tedious, produce moderate yields and reaction byproducts are frequently difficult to remove. We have developed an improved method for solid-phase coupling of ligands to oligonucleotides via disulfides directly after solid-phase synthesis. A 2'-thiol introduced using a modified nucleotide building block was orthogonally deprotected on the controlled pore glass solid support with N-butylphosphine. Oligolysine peptides and a short monodisperse ethylene glycol chain were successfully coupled to the deprotected thiol. Cleavage from the resin and full removal of oligonucleotide protection groups were achieved using methanolic ammonia. After standard desalting, and without further purification, homogenous conjugates were obtained as demonstrated by HPLC, gel electrophoresis, and mass spectrometry. The attachment of both amphiphilic and cationic ligands proves the versatility of the conjugation procedure. An antisense oligonucleotide conjugate with hexalysine showed pronounced gene silencing in a cell culture tumor model in the absence of a transfection reagent and the corresponding ethylene glycol conjugate resulted in down regulation of the target gene to nearly 50% after naked application. PMID:27236069

  14. 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

  15. The synthesis of new amphiphilic p-tert-butylthiacalix[4]arenes containing peptide fragments and their interaction with DNA.

    PubMed

    Padnya, Pavel L; Andreyko, Elena A; Mostovaya, Olga A; Rizvanov, Ildar Kh; Stoikov, Ivan I

    2015-06-01

    New water-soluble p-tert-butylthiacalix[4]arenes containing peptide and quaternary ammonium fragments in cone and 1,3-alternate conformations were synthesized and characterized. The interaction of the macrocycles with DNA was studied by UV-spectroscopy, DLS and TEM. It was shown that the interaction of the self-associates based on p-tert-butylthiacalix[4]arenes tetrasubstituted at the lower rim with glycine and quaternary ammonium fragments in cone and 1,3-alternate conformations with DNA led to the formation of particles of about 99-192 nm in size.

  16. 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

  17. Diagnostic and prognostic values of B-type natriuretic peptides (BNP) and N-terminal fragment brain natriuretic peptides (NT-pro-BNP).

    PubMed

    Maries, Lorena; Manitiu, Ioan

    2013-08-01

    B-type natriuretic peptide (BNP) is a member of a fournatriuretic peptide family that shares a common 17-peptide ring structure. The N-terminal fragment (NT-pro-BNP) is biologically inert, but both are secreted in the plasma in equimolar quantities and both have been evaluated for use in the management of congestive heart failure. BNP and NT-pro-BNP are frequently used in the diagnosis of congestive heart failure and distinguishing between patients with dyspnoea of cardiac or pulmonary origin. Values of NT-pro-BNP are affected by age or the presence of one or several co-morbidities such as chronic renal failure, type 2 diabetes, and acute coronary syndrome. 'Normal' values of these peptides also vary depending on the type of test used. The performance characteristics of these tests vary depending on the patients on whom they are used and the manufacturer. For this reason, the determination of reference values for this peptide represents such a challenge.

  18. Antimicrobial activity of pleurocidin is retained in Plc-2, a C-terminal 12-amino acid fragment.

    PubMed

    Souza, Andre L A; Díaz-Dellavalle, Paola; Cabrera, Andrea; Larrañaga, Patricia; Dalla-Rizza, Marco; De-Simone, Salvatore G

    2013-07-01

    An analysis of a series of five peptides composed of various portions of the pleurocidin (Plc) sequence identified a l2-amino acid fragment from the C-terminus of Plc, designated Plc-2, as the smallest fragment that retained a antimicrobial activity comparable to that of the parent compound. MIC tests in vitro with low-ionic-strength medium showed that Plc-2 has potent activity against Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus but not against Enterococcus faecalis. The antifungal activity of the synthetic peptides against phytopathogenic fungi, such as Fusarium oxysporum, Colletotrichum sp., Aspergillus niger and Alternaria sp., also identified Plc-2 as a biologically active peptide. Microscopy studies of fluorescently stained fungi treated with Plc-2 demonstrated that cytoplasmic and nuclear membranes were compromised in all strains of phytopathogenic fungi tested. Together, these results identify Plc-2 as a potential antimicrobial agent with similar properties to its parent compound, pleurocidin. In addition, it demonstrated that the KHVGKAALTHYL residues are critical for the antimicrobial activity described for pleurocidin.

  19. Highly Amyloidogenic Two-chain Peptide Fragments Are Released upon Partial Digestion of Insulin with Pepsin*♦

    PubMed Central

    Piejko, Marcin; Dec, Robert; Babenko, Viktoria; Hoang, Agnieszka; Szewczyk, Monika; Mak, Paweł; Dzwolak, Wojciech

    2015-01-01

    Proteases play a well recognized role in the emergence of highly aggregation-prone protein fragments in vivo, whereas in vitro limited proteolysis is often employed to probe different phases of amyloidogenic pathways. Here, we show that addition of moderate amounts of pepsin to acidified bovine insulin at close to physiological temperature results in an abrupt self-assembly of amyloid-like fibrils from partially digested insulin fragments. Biochemical analysis of the pepsin-induced fibrils implicates peptide fragments (named H) consisting of the 13 or 15 N-terminal residues of the A-chain and 11 or 13 N-terminal residues of the B-chain linked by the disulfide bond between Cys-7A–Cys-7B as the main constituents. There are up to eight pepsin-cleavage sites remaining within the double chain peptide, which become protected upon fast fibrillation unless concentration of the enzyme is increased resulting in complete digestion of insulin. Controlled re-association of H-peptides leads to “explosive” fibrillation only under nonreducing conditions implying the key role of the disulfide bond in their amyloidogenicity. Such re-assembled amyloid is similar in terms of morphology and infrared features to typical bovine insulin fibrils, although it lacks the ability to seed the intact protein. PMID:25586185

  20. Photocross-Linked Peptide-Protein Complexes Analysis: A Comparative Study of CID and ETD Fragmentation Modes

    NASA Astrophysics Data System (ADS)

    Clavier, Séverine; Bolbach, Gérard; Sachon, Emmanuelle

    2015-06-01

    Protein-protein interactions are among the keys to organizing cellular processes in space and time. One of the only direct ways to identify such interactions in their cellular environment is to covalently bond the interacting partners to fix the interaction. Photocross-linking in living cells is thus a very promising technique. The feasibility of in cellulo photocross-linking reactions has been shown and mass spectrometry is a tool of choice to analyze photocross-linked proteins. However, the interpretation of the MS and MS/MS spectra of photocross-linked peptides remains one of the most important bottlenecks of the method and still limits its potential for large-scale applications (interactomics). Fundamental studies are still necessary to understand and characterize the fragmentation behavior of photocross-linked peptides. Here, we report the successful identification of the interaction sites in a well-characterized model of in vitro interaction between a protein and a peptide. We describe in detail the fragmentation pattern of these photocross-linked species in order to identify trends that could be generalized. In particular, we compare CID and ETD fragmentation modes (and HCD in a lesser extent), demonstrating the complementarity of both methods and the advantage of ETD for the analysis of photocross-linked species. The information should help further development of dedicated software to properly score MS/MS spectra of photocross-linked species.

  1. Changes in the molecular ion yield and fragmentation of peptides under various primary ions in ToF-SIMS and matrix-enhanced ToF-SIMS.

    PubMed

    Körsgen, Martin; Tyler, Bonnie J; Pelster, Andreas; Lipinsky, Dieter; Dreisewerd, Klaus; Arlinghaus, Heinrich F

    2016-06-01

    Time of flight secondary ion mass spectrometry (ToF-SIMS) is a powerful technique for the nanoanalysis of biological samples, but improvements in sensitivity are needed in order to detect large biomolecules, such as peptides, on the individual cell level at physiological concentrations. Two promising options to improve the sensitivity of SIMS to large peptides are the use of cluster primary ions to increase desorption of intact molecules or the use of matrix-assisted laser desorption/ionization (MALDI) matrices to increase the ionization probability. In this paper, the authors have combined these two approaches in order to improve understanding of the interaction between ionization and fragmentation processes. The peptides bradykinin and melittin were prepared as neat monolayers on silicon, in a Dextran-40 matrix and in two common MALDI matrices, 2,5-dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxy cinnamic acid (HCCA). ToF-SIMS spectra of these samples were collected using a range of small Bi cluster primary ions and large Ar cluster primary ions. The trends observed in the molecular ion yield and the [M+H](+)/C4H8N(+) ratio with primary ion cluster size were sample system dependent. The molecular ion yield of the bradykinin was maximized by using 30 keV Bi3 (+) primary ions in a DHB matrix but in the HCCA matrix, the maximum molecular ion yield was obtained by using 30 keV Bi7 (+) primary ions. In contrast, the molecular ion yield for melittin in both matrices was greatest using 20 keV Ar2000 (+) primary ions. Improvements in the molecular ion yield were only loosely correlated with a decrease in small fragment ions. The data indicate a complex interplay between desorption processes and ion formation processes which mean that the optimal analytical conditions depend on both the target analyte and the matrix.

  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. Peptide docking of HIV-1 p24 with single chain fragment variable (scFv) by CDOCKER algorithm

    NASA Astrophysics Data System (ADS)

    Karim, Hana Atiqah Abdul; Tayapiwatana, Chatchai; Nimmanpipug, Piyarat; Zain, Sharifuddin M.; Rahman, Noorsaadah Abdul; Lee, Vannajan Sanghiran

    2014-10-01

    In search for the important residues that might have involve in the binding interaction between the p24 caspid protein of HIV-1 fragment (MET68 - PRO90) with the single chain fragment variable (scFv) of FAB23.5, modern computational chemistry approach has been conducted and applied. The p24 fragment was initially taken out from the 1AFV protein molecule consisting of both light (VL) and heavy (VH) chains of FAB23.5 as well as the HIV-1 caspid protein. From there, the p24 (antigen) fragment was made to dock back into the protein pocket receptor (antibody) by using the CDOCKER algorithm to conduct the molecular docking process. The score calculated from the CDOCKER gave 15 possible docked poses with various docked ligand's positions, the interaction energy as well as the binding energy. The best docked pose that imitates the original antigen's position was determined and further processed to the In Situ minimization to obtain the residues interaction energy as well as to observe the hydrogen bonds interaction in the protein-peptide complex. Based on the results demonstrated, the specific residues in the complex that have shown immense lower interaction energies in the 5Å vicinity region from the peptide are from the heavy chain (VH:TYR105) and light chain (VL: ASN31, TYR32, and GLU97). Those residues play vital roles in the binding mechanism of Antibody-Antigen (Ab-Ag) complex of p24 with FAB23.5.

  4. N-Terminal Peptide Sequence Repetition Influences the Kinetics of Backbone Fragmentation: A Manifestation of the Jahn-Teller Effect?

    NASA Astrophysics Data System (ADS)

    Good, David M.; Yang, Hongqian; Zubarev, Roman A.

    2013-11-01

    Analysis of large (>10,000 entries) databases consisting of high-resolution tandem mass spectra of peptide dications revealed with high statistical significance ( P < 1ṡ10-3) that peptides with non-identical first two N-terminal amino acids undergo cleavages of the second peptide bond at higher rates than repetitive sequences composed of the same amino acids (i.e., in general AB- and BA- bonds cleave more often than AA- and BB- bonds). This effect seems to depend upon the collisional energy, being stronger at lower energies. The phenomenon is likely to indicate the presence of the diketopiperazine structure for at least some b2 + ions. When consisting of two identical amino acids, these species should form through intermediates that have a symmetric geometry and, thus, must be subject to the Jahn-Teller effect that reduces the stability of such systems.

  5. Identifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in protein sequence databases.

    PubMed

    Henzel, W J; Billeci, T M; Stults, J T; Wong, S C; Grimley, C; Watanabe, C

    1993-06-01

    A rapid method for the identification of known proteins separated by two-dimensional gel electrophoresis is described in which molecular masses of peptide fragments are used to search a protein sequence database. The peptides are generated by in situ reduction, alkylation, and tryptic digestion of proteins electroblotted from two-dimensional gels. Masses are determined at the subpicomole level by matrix-assisted laser desorption/ionization mass spectrometry of the unfractionated digest. A computer program has been developed that searches the protein sequence database for multiple peptides of individual proteins that match the measured masses. To ensure that the most recent database updates are included, a theoretical digest of the entire database is generated each time the program is executed. This method facilitates simultaneous processing of a large number of two-dimensional gel spots. The method was applied to a two-dimensional gel of a crude Escherichia coli extract that was electroblotted onto poly(vinylidene difluoride) membrane. Ten randomly chosen spots were analyzed. With as few as three peptide masses, each protein was uniquely identified from over 91,000 protein sequences. All identifications were verified by concurrent N-terminal sequencing of identical spots from a second blot. One of the spots contained an N-terminally blocked protein that required enzymatic cleavage, peptide separation, and Edman degradation for confirmation of its identity.

  6. IR action spectroscopy shows competitive oxazolone and diketopiperazine formation in peptides depends on peptide length and identity of terminal residue in the departing fragment.

    PubMed

    Morrison, L J; Chamot-Rooke, J; Wysocki, V H

    2014-05-01

    The interplay between the entropically and enthalpically favored products of peptide fragmentation is probed using a combined experimental and theoretical approach. These b2 ion products can take either an oxazolone or diketopiperazine structure. Cleavage after the second amide bond is often a favorable process because the products are small ring structures that are particularly stable. These structures are structurally characterized by action IRMPD spectroscopy and semi-quantified using gas-phase hydrogen-deuterium exchange. The formation of the oxazolone and diketopiperazine has been thought to be largely governed by the identity of the first two residues at the N-terminus of the peptide. We show here that the length of the precursor peptide and identity of the third residue play a significant role in the formation of the diketopiperazine structure in peptides containing an N-terminal asparagine residue. This is additionally the first instance showing an N-terminal residue with an amide side chain can promote formation of the diketopiperazine b2 ion structure.

  7. 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.

  8. The Effect of the Secondary Structure on Dissociation of Peptide Radical Cations: Fragmentation of Angiotensin III and Its Analogues

    SciTech Connect

    Yang, Zhibo; Lam, Corey; Chu, Ivan K.; Laskin, Julia

    2008-09-28

    Fragmentation of protonated RVYIHPF and RVYIHPF-OMe and the corresponding radical cations was studied using time- and collision energy-resolved surface-induced dissociation (SID) in a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) specially equipped to perform SID experiments. Peptide radical cations were produced by gas-phase fragmentation of CoIII(salen)-peptide complexes. Both the energetics and mechanisms of dissociation of even-electron and odd-electron angiotensin III ions are quite different. Protonated molecules are much more stable towards fragmentation than the corresponding radical cations. RRKM modeling of the experimental data suggests that this stability is largely attributed to differences in threshold energies for dissociation while activation entropies are very similar. Detailed analysis of the experimental data obtained for radical cations demonstrated the presence of two distinct structures separated by a high free-energy barrier. The two families of structures were ascribed to the canonical and zwitterionic forms of the radical cations produced in our experiments.

  9. A comparison of the effects of amide and acid groups at the C-terminus on the collision-induced dissociation of deprotonated peptides.

    PubMed

    Bokatzian-Johnson, Samantha S; Stover, Michele L; Dixon, David A; Cassady, Carolyn J

    2012-09-01

    The dissociative behavior of peptide amides and free acids was explored using low-energy collision-induced dissociation and high level computational theory. Both positive and negative ion modes were utilized, but the most profound differences were observed for the deprotonated species. Deprotonated peptide amides produce a characteristic c(m-2)(-) product ion (where m is the number of residues in the peptide) that is either absent or in low abundance in the analogous peptide acid spectrum. Peptide acids show an enhanced formation of c(m-3)(-); however, this is not generally as pronounced as c(m-2)(-) production from amides. The most notable occurrence of an amide-specific product ion is for laminin amide (YIGSR-NH(2)) and this case was investigated using several modified peptides. Mechanisms involving 6- and 9-membered ring formation were proposed, and their energetic properties were investigated using G3(MP2) molecular orbital theory calculations. For example, with C-terminal deprotonation of pentaglycine amide, formation of c(m-2)(-) and a 6-membered ring diketopiperazine neutral requires >31.6 kcal/mol, which is 26.1 kcal/mol less than the analogous process involving the peptide acid. The end group specific fragmentation of peptide amides in the negative ion mode may be useful for identifying such groups in proteomic applications.

  10. A Comparison of the Effects of Amide and Acid Groups at the C-Terminus on the Collision-Induced Dissociation of Deprotonated Peptides

    NASA Astrophysics Data System (ADS)

    Bokatzian-Johnson, Samantha S.; Stover, Michele L.; Dixon, David A.; Cassady, Carolyn J.

    2012-09-01

    The dissociative behavior of peptide amides and free acids was explored using low-energy collision-induced dissociation and high level computational theory. Both positive and negative ion modes were utilized, but the most profound differences were observed for the deprotonated species. Deprotonated peptide amides produce a characteristic cm-2 - product ion (where m is the number of residues in the peptide) that is either absent or in low abundance in the analogous peptide acid spectrum. Peptide acids show an enhanced formation of cm-3 -; however, this is not generally as pronounced as cm-2 - production from amides. The most notable occurrence of an amide-specific product ion is for laminin amide (YIGSR-NH2) and this case was investigated using several modified peptides. Mechanisms involving 6- and 9-membered ring formation were proposed, and their energetic properties were investigated using G3(MP2) molecular orbital theory calculations. For example, with C-terminal deprotonation of pentaglycine amide, formation of cm-2 - and a 6-membered ring diketopiperazine neutral requires >31.6 kcal/mol, which is 26.1 kcal/mol less than the analogous process involving the peptide acid. The end group specific fragmentation of peptide amides in the negative ion mode may be useful for identifying such groups in proteomic applications.

  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. 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.

  13. The molecular mechanism of fullerene-inhibited aggregation of Alzheimer's β-amyloid peptide fragment.

    PubMed

    Xie, Luogang; Luo, Yin; Lin, Dongdong; Xi, Wenhui; Yang, Xinju; Wei, Guanghong

    2014-08-21

    Amyloid deposits are implicated in the pathogenesis of many neurodegenerative diseases such as Alzheimer's disease (AD). The inhibition of β-sheet formation has been considered as the primary therapeutic strategy for AD. Increasing data show that nanoparticles can retard or promote the fibrillation of amyloid-β (Aβ) peptides depending on the physicochemical properties of nanoparticles, however, the underlying molecular mechanism remains elusive. In this study, our replica exchange molecular dynamics (REMD) simulations show that fullerene nanoparticle - C60 (with a fullerene :  peptide molar ratio greater than 1 : 8) can dramatically prevent β-sheet formation of Aβ(16-22) peptides. Atomic force microscopy (AFM) experiments further confirm the inhibitory effect of C60 on Aβ(16-22) fibrillation, in support of our REMD simulations. An important finding from our REMD simulations is that fullerene C180, albeit with the same number of carbon atoms as three C60 molecules (3C60) and smaller surface area than 3C60, displays an unexpected stronger inhibitory effect on the β-sheet formation of Aβ(16-22) peptides. A detailed analysis of the fullerene-peptide interaction reveals that the stronger inhibition of β-sheet formation by C180 results from the strong hydrophobic and aromatic-stacking interactions of the fullerene hexagonal rings with the Phe rings relative to the pentagonal rings. The strong interactions between the fullerene nanoparticles and Aβ(16-22) peptides significantly weaken the peptide-peptide interaction that is important for β-sheet formation, thus retarding Aβ(16-22) fibrillation. Overall, our studies reveal the significant role of fullerene hexagonal rings in the inhibition of Aβ(16-22) fibrillation and provide novel insight into the development of drug candidates against Alzheimer's disease.

  14. Fragmentation of amino acids induced by collisions with low-energy highly charged ions

    NASA Astrophysics Data System (ADS)

    Piekarski, D. G.; Maclot, S.; Domaracka, A.; Adoui, L.; Alcamí, M.; Rousseau, P.; Díaz-Tendero, S.; Huber, B. A.; Martín, F.

    2014-04-01

    Fragmentation of amino acids NH2-(CH2)n-COOH (n=1 glycine; n=2 β-alanine and n=3 γ-aminobutyric acid GABA) following collisions with slow highly charged ions has been studied in the gas phase by a combined experimental and theoretical approach. In the experiments, a multi-coincidence detection method was used to deduce the charge state of the molecules before fragmentation. Quantum chemistry calculations have been carried out in the basis of the density functional theory and ab initio molecular dynamics. The combination of both methodologies is essential to unambiguously unravel the different fragmentation pathways.

  15. Identification of non-peptidic cysteine reactive fragments as inhibitors of cysteine protease rhodesain.

    PubMed

    McShan, Danielle; Kathman, Stefan; Lowe, Brittiney; Xu, Ziyang; Zhan, Jennifer; Statsyuk, Alexander; Ogungbe, Ifedayo Victor

    2015-10-15

    Rhodesain, the major cathepsin L-like cysteine protease in the protozoan Trypanosoma brucei rhodesiense, the causative agent of African sleeping sickness, is a well-validated drug target. In this work, we used a fragment-based approach to identify inhibitors of this cysteine protease, and identified inhibitors of T. brucei. To discover inhibitors active against rhodesain and T. brucei, we screened a library of covalent fragments against rhodesain and conducted preliminary SAR studies. We envision that in vitro enzymatic assays will further expand the use of the covalent tethering method, a simple fragment-based drug discovery technique to discover covalent drug leads.

  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. The recombinant C-terminal fragment of tetanus toxin protects against cholinotoxicity by intraseptal injection of β-amyloid peptide (25-35) in rats.

    PubMed

    Patricio-Martínez, A; Mendieta, L; Martínez, I; Aguilera, J; Limón, I D

    2016-02-19

    The recombinant C-terminal domain of tetanus toxin (Hc-TeTx) is a new non-toxic peptide of the tetanus toxin that exerts a protective action against glutamate excitotoxicity in motoneurons. Moreover, its efficacy as a neuroprotective agent has been demonstrated in several animal models of neurodegeneration. The eleven amino acids in the β amyloid peptide (Aβ25-35) mimic the toxic effects of the full β amyloid peptide (Aβ1-42), causing the impairment of the cholinergic system in the medial septum (MS) which, in turn, alters the septo-hippocampal pathway and leads to learning and memory impairments. The aim of this study was to examine the neuroprotective effects of the Hc-TeTx fragment against cholinotoxicity. The Hc-TeTx fragment (100 ng) was injected into the rats intercranially, with the Aβ(25-35) (2 μg) then injected into their MS. The animals were tested for spatial learning and memory in the eight-arm radial maze. The brains were removed to assess cholinergic markers, such as choline acetyltransferase (ChAT) and acetylcholinesterase (AChE), and to explore neurodegeneration in the MS and hippocampus, using amino-cupric silver and H&E staining. Finally, capase-3, a marker of apoptosis, was examined in the MS. Our results clearly demonstrate that the application of Hc-TeTx prevents the loss of cholinergic markers (ChAT and AChE), the activation of capase-3, and neurodegeneration in the MS and the CA1 and CA3 subfields of the hippocampus. All these improvements were reflected in spatial learning and memory performance, and were significantly higher compared with animals treated with Aβ(25-35). Interestingly, the single administration of Hc-TeTx into the MS modified the ChAT and AChE expression that affect cognitive processes, without inducing neurodegeneration or an increase in capase-3 expression in the MS and hippocampus. In summary, our findings suggest that the recombinant Hc-TeTx fragment offers effective protection for the septo-hippocampal pathway

  19. [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.

  20. 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.

  1. 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.

  2. Fragmentation Mechanisms of Oxidized Peptides Elucidated by SID, RRKM Modeling and Molecular Dynamics

    SciTech Connect

    Spraggins, Jeffrey M.; Lloyd, Julie A.; Johnston, Murray V.; Laskin, Julia; Ridge, Douglas P.

    2009-09-08

    The gas phase fragmentation reactions of singly charged angiotensin II (AngII, DRVYIHPF) and the ozonolysis products AngII+O (DRVY*IHPF), AngII+3O (DRVYIH*PF), and AngII+4O (DRVY*IH*PF) were studied using SID FT-ICR mass spectrometry and molecular dynamics. Oxidation of Tyr (AngII+O) leads to an additional low-energy charge-remote selective fragmentation channel resulting in the b4 fragment ion. Modification of His leads to a series of new selective dissociation channels. For AngII+3O and AngII+4O, the formation of [MH+3O]+-45 and [MH+3O]+-71 are driven by charge-remote processes while it is suggested that the b5 and [MH+3O]+-88 fragments are a result of charge-directed reactions. Energy-resolved SID experiments and RRKM modeling were able to provide insight into the energetics of the lowest energy fragmentation channel for each of the parent ions. Destabilization of the ozonolysis products was found to be strictly due to entropic effects. Mechanistic details for each of the new dissociation pathways were determined by relating the SID FT-ICR MS results to parent ion conformations samples using molecular dynamics.

  3. The molecular mechanism of fullerene-inhibited aggregation of Alzheimer's β-amyloid peptide fragment

    NASA Astrophysics Data System (ADS)

    Xie, Luogang; Luo, Yin; Lin, Dongdong; Xi, Wenhui; Yang, Xinju; Wei, Guanghong

    2014-07-01

    Amyloid deposits are implicated in the pathogenesis of many neurodegenerative diseases such as Alzheimer's disease (AD). The inhibition of β-sheet formation has been considered as the primary therapeutic strategy for AD. Increasing data show that nanoparticles can retard or promote the fibrillation of amyloid-β (Aβ) peptides depending on the physicochemical properties of nanoparticles, however, the underlying molecular mechanism remains elusive. In this study, our replica exchange molecular dynamics (REMD) simulations show that fullerene nanoparticle - C60 (with a fullerene : peptide molar ratio greater than 1 : 8) can dramatically prevent β-sheet formation of Aβ(16-22) peptides. Atomic force microscopy (AFM) experiments further confirm the inhibitory effect of C60 on Aβ(16-22) fibrillation, in support of our REMD simulations. An important finding from our REMD simulations is that fullerene C180, albeit with the same number of carbon atoms as three C60 molecules (3C60) and smaller surface area than 3C60, displays an unexpected stronger inhibitory effect on the β-sheet formation of Aβ(16-22) peptides. A detailed analysis of the fullerene-peptide interaction reveals that the stronger inhibition of β-sheet formation by C180 results from the strong hydrophobic and aromatic-stacking interactions of the fullerene hexagonal rings with the Phe rings relative to the pentagonal rings. The strong interactions between the fullerene nanoparticles and Aβ(16-22) peptides significantly weaken the peptide-peptide interaction that is important for β-sheet formation, thus retarding Aβ(16-22) fibrillation. Overall, our studies reveal the significant role of fullerene hexagonal rings in the inhibition of Aβ(16-22) fibrillation and provide novel insight into the development of drug candidates against Alzheimer's disease.Amyloid deposits are implicated in the pathogenesis of many neurodegenerative diseases such as Alzheimer's disease (AD). The inhibition of

  4. Synthesis of peptide fragments of the hemagglutinin of influenza virus A/Aichi/2/68 (H/sub 3/N/sub 2/)

    SciTech Connect

    Samukov, V.V.; Kalashnikov, V.V.; Ofitserov, V.I.; Shval'e, A.F.

    1986-06-01

    Peptides corresponding to sections 122-133, 136-147, and 154-164 of the heavy chain of the hemagglutinin of influenza virus A/Aichi/2/68 (H/sub 3/N/sub 2/) have been synthesized. The synthesis was performed by the stepwise growth of the peptide chain by means of activated esters of Boc-amino acids and the condensation of peptide blocks by the carbodiimide method in solution. The colored 2-(4-(phenylazo)benylsulfonyl)ethyl group, which can be removed by bases, was used for the protection of the C-terminal carboxy groups of the peptides and peptide blocks, facilitating the purification of the peptides synthesized. After the elimination of the N- and C-terminal carboxy groups of the peptides and peptide blocks, facilitating the purification of the peptides synthesized. After the elimination of the N- and C-terminal protective groups, the peptides were deblocked with 1 M trifluoromethanesulfonic acid in trifluoroacetic acid containing 10% of thioanisole and were purified by prepatative reversed-phase chromatography. Conjugates of the peptides synthesized with bovine serum albumin have been obtained.

  5. Zinc(II) interactions with brain-derived neurotrophic factor N-terminal peptide fragments: inorganic features and biological perspectives.

    PubMed

    Travaglia, Alessio; La Mendola, Diego; Magrì, Antonio; Pietropaolo, Adriana; Nicoletti, Vincenzo G; Grasso, Giuseppe; Malgieri, Gaetano; Fattorusso, Roberto; Isernia, Carla; Rizzarelli, Enrico

    2013-10-01

    Brain-derived neurotrophic factor (BDNF) is a neurotrophin essential for neuronal differentiation, growth, and survival; it is involved in memory formation and higher cognitive functions. The N-terminal domain of BDNF is crucial for the binding selectivity and activation of its specific TrkB receptor. Zn(2+) ion binding may influence BDNF activity. Zn(2+) complexes with the peptide fragment BDNF(1-12) encompassing the sequence 1-12 of the N-terminal domain of BDNF were studied by means of potentiometry, electrospray mass spectrometry, NMR, and density functional theory (DFT) approaches. The predominant Zn(2+) complex species, at physiological pH, is [ZnL] in which the metal ion is bound to an amino, an imidazole, and two water molecules (NH2, N(Im), and 2O(water)) in a tetrahedral environment. DFT-based geometry optimization of the zinc coordination environment showed a hydrogen bond between the carboxylate and a water molecule bound to zinc in [ZnL]. The coordination features of the acetylated form [AcBDNF(1-12)] and of a single mutated peptide [BDNF(1-12)D3N] were also characterized, highlighting the role of the imidazole side chain as the first anchoring site and ruling out the direct involvement of the aspartate residue in the metal binding. Zn(2+) addition to the cell culture medium induces an increase in the proliferative activity of the BDNF(1-12) peptide and of the whole protein on the SHSY5Y neuroblastoma cell line. The effect of Zn(2+) is opposite to that previously observed for Cu(2+) addition, which determines a decrease in the proliferative activity for both peptide and protein, suggesting that these metals might discriminate and modulate differently the activity of BDNF.

  6. Identification and Investigation of Novel Binding Fragments in the Fatty Acid Binding Protein 6 (FABP6).

    PubMed

    Hendrick, Alan G; Müller, Ilka; Willems, Henriëtte; Leonard, Philip M; Irving, Steve; Davenport, Richard; Ito, Takashi; Reeves, Jenny; Wright, Susanne; Allen, Vivienne; Wilkinson, Stephen; Heffron, Helen; Bazin, Richard; Turney, Jennifer; Mitchell, Philip J

    2016-09-01

    Fatty acid binding protein 6 (FABP6) is a potential drug discovery target, which, if inhibited, may have a therapeutic benefit for the treatment of diabetes. Currently, there are no published inhibitors of FABP6, and with the target believed to be amenable to fragment-based drug discovery, a structurally enabled program was initiated. This program successfully identified fragment hits using the surface plasmon resonance (SPR) platform. Several hits were validated with SAR and were found to be displaced by the natural ligand taurocholate. We report the first crystal structure of human FABP6 in the unbound form, in complex with cholate, and with one of the key fragments. PMID:27500412

  7. Epitope mapping of antigenic MUC1 peptides to breast cancer antibody fragment B27.29: a heteronuclear NMR study.

    PubMed

    Grinstead, Jeffrey S; Schuman, Jason T; Campbell, A Patricia

    2003-12-01

    MUC1 mucin is a breast cancer-associated transmembrane glycoprotein, of which the extracellular domain is formed by the repeating 20-amino acid sequence GVTSAPDTRPAPGSTAPPAH. In neoplastic breast tissue, the highly immunogenic sequence PDTRPAP (in bold above) is exposed. Antibodies raised directly against MUC1-expressing tumors offer unique access to this neoplastic state, as they represent immunologically relevant "reverse templates" of the tumor-associated mucin. In a previous study [Grinstead, J. S., et al. (2002) Biochemistry 41, 9946-9961], (1)H NMR methods were used to correlate the effects of cryptic glycosylation outside of the PDTRPAP core epitope sequence on the recognition and binding of Mab B27.29, a monoclonal antibody raised against breast tumor cells. In the study presented here, isotope-edited NMR methods, including (15)N and (13)C relaxation measurements, were used to probe the recognition and binding of the PDTRPAP epitope sequence to Fab B27.29. Two peptides were studied: a one-repeat MUC1 16mer peptide of the sequence GVTSAPDTRPAPGSTA and a two-repeat MUC1 40mer peptide of the sequence (VTSAPDTRPAPGSTAPPAHG)(2). (15)N and (13)C NMR relaxation parameters were measured for both peptides free in solution and bound to Fab B27.29. The (13)C(alpha) T(1) values best represent changes in the local correlation time of the peptide epitope upon binding antibody, and demonstrate that the PDTRPAP sequence is immobilized in the antibody-combining site. This result is also reflected in the appearance of the (15)N- and (13)C-edited HSQC spectra, where line broadening of the same peptide epitope resonances is observed. The PDTRPAP peptide epitope expands upon the peptide epitope identified previously in our group as PDTRP by homonuclear NMR experiments [Grinstead, J. S., et al. (2002) Biochemistry 41, 9946-9961], and illustrates the usefulness of the heteronuclear NMR experiments. The implications of these results are discussed within the context of MUC1 breast

  8. Structural Studies of Copper(I) Complexes of Amyloid-Beta Peptide Fragments: Formation of Two-Coordinate Bis(Histidine) Complexes

    SciTech Connect

    Himes, R.A.; Park, G.Young.; Siluvai, G.Sutha.; Blackburn, N.J.; Karlin, K.D.

    2009-05-18

    The beta bind: Copper(I) binds to amyloid {beta}-peptide fragments (see structure) as a stable bis(histidine), two-coordinate, near-linear complex, even in the presence of potential additional ligands. As has been proposed or assumed in other studies, the copper(I)-peptide complexes react with dioxygen to form the reactive oxygen species H{sub 2}O{sub 2}, without the need for a third histidine ligand to promote the chemistry.

  9. A crystallographic fragment screen identifies cinnamic acid derivatives as starting points for potent Pim-1 inhibitors.

    PubMed

    Schulz, Michèle N; Fanghänel, Jörg; Schäfer, Martina; Badock, Volker; Briem, Hans; Boemer, Ulf; Nguyen, Duy; Husemann, Manfred; Hillig, Roman C

    2011-03-01

    A crystallographic fragment screen was carried out to identify starting points for the development of inhibitors of protein kinase Pim-1, a potential target for tumour therapy. All fragment hits identified via soaking in this study turned out to bind to the unusually hydrophobic pocket at the hinge region. The most potent fragments, two cinnamic acid derivatives (with a best IC(50) of 130 µM), additionally form a well defined hydrogen bond. The balance between hydrophobic and polar interactions makes these molecules good starting points for further optimization. Pim-2 inhibitors from a recently reported high-throughput screening campaign also feature a cinnamic acid moiety. Two of these Pim-2 inhibitors were synthesized, their potencies against Pim-1 were determined and their cocrystal structures were elucidated in order to determine to what degree the binding modes identified by fragment screening are conserved in optimized inhibitors. The structures show that the cinnamic acid moieties indeed adopt the same binding mode. Fragment screening thus correctly identified binding modes which are maintained when fragments are grown into larger and higher affinity inhibitors. The high-throughput screening-derived compound (E)-3-{3-[6-(4-aminocyclohexylamino)-pyrazin-2-yl]phenyl}acrylic acid (compound 1) is the most potent inhibitor of the cinnamic acid series for which the three-dimensional binding mode is known (IC(50) = 17 nM, K(d) = 28 nM). The structure reveals the molecular basis for the large gain in potency between the initial fragment hit and this optimized inhibitor.

  10. 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

  11. 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

  12. 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.

  13. Conformations of Prolyl-Peptide Bonds in the Bradykinin 1-5 Fragment in Solution and in the Gas Phase.

    PubMed

    Voronina, Liudmila; Masson, Antoine; Kamrath, Michael; Schubert, Franziska; Clemmer, David; Baldauf, Carsten; Rizzo, Thomas

    2016-07-27

    The dynamic nature of intrinsically disordered peptides makes them a challenge to characterize by solution-phase techniques. In order to gain insight into the relation between the disordered state and the environment, we explore the conformational space of the N-terminal 1-5 fragment of bradykinin (BK[1-5](2+)) in the gas phase by combining drift tube ion mobility, cold-ion spectroscopy, and first-principles simulations. The ion-mobility distribution of BK[1-5](2+) consists of two well-separated peaks. We demonstrate that the conformations within the peak with larger cross-section are kinetically trapped, while the more compact peak contains low-energy structures. This is a result of cis-trans isomerization of the two prolyl-peptide bonds in BK[1-5](2+). Density-functional theory calculations reveal that the compact structures have two very different geometries with cis-trans and trans-cis backbone conformations. Using the experimental CCSs to guide the conformational search, we find that the kinetically trapped species have a trans-trans configuration. This is consistent with NMR measurements performed in a solution, which show that 82% of the molecules adopt a trans-trans configuration and behave as a random coil. PMID:27366919

  14. 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

  15. EIMS Fragmentation Pathways and MRM Quantification of 7α/β-Hydroxy-Dehydroabietic Acid TMS Derivatives

    NASA Astrophysics Data System (ADS)

    Rontani, Jean-François; Aubert, Claude; Belt, Simon T.

    2015-09-01

    EI mass fragmentation pathways of TMS derivatives οf 7α/β-hydroxy-dehydroabietic acids resulting from NaBH4-reduction of oxidation products of dehydroabietic acid (a component of conifers) were investigated and deduced by a combination of (1) low energy CID-GC-MS/MS, (2) deuterium labeling, (3) different derivatization methods, and (4) GC-QTOF accurate mass measurements. Having identified the main fragmentation pathways, the TMS-derivatized 7α/β-hydroxy-dehydroabietic acids could be quantified in multiple reaction monitoring (MRM) mode in sea ice and sediment samples collected from the Arctic. These newly characterized transformation products of dehydroabietic acid constitute potential tracers of biotic and abiotic degradation of terrestrial higher plants in the environment.

  16. EIMS Fragmentation Pathways and MRM Quantification of 7α/β-Hydroxy-Dehydroabietic Acid TMS Derivatives.

    PubMed

    Rontani, Jean-François; Aubert, Claude; Belt, Simon T

    2015-09-01

    EI mass fragmentation pathways of TMS derivatives οf 7α/β-hydroxy-dehydroabietic acids resulting from NaBH(4)-reduction of oxidation products of dehydroabietic acid (a component of conifers) were investigated and deduced by a combination of (1) low energy CID-GC-MS/MS, (2) deuterium labeling, (3) different derivatization methods, and (4) GC-QTOF accurate mass measurements. Having identified the main fragmentation pathways, the TMS-derivatized 7α/β-hydroxy-dehydroabietic acids could be quantified in multiple reaction monitoring (MRM) mode in sea ice and sediment samples collected from the Arctic. These newly characterized transformation products of dehydroabietic acid constitute potential tracers of biotic and abiotic degradation of terrestrial higher plants in the environment.

  17. Molecular resolution and fragmentation of fulvic acid by electrospray ionization/multistage tandem mass spectrometry

    USGS Publications Warehouse

    Leenheer, J.A.; Rostad, C.E.; Gates, Paul M.; Furlong, E.T.; Ferrer, I.

    2001-01-01

    Molecular weight distributions of fulvic acid from the Suwannee River, Georgia, were investigated by electrospray ionization/quadrupole mass spectrometry (ESI/QMS), and fragmentation pathways of specific fulvic acid masses were investigated by electrospray ionization/ion trap multistage tandem mass spectrometry (ESI/MST/MS). ESI/QMS studies of the free acid form of low molecular weight poly(carboxylic acid) standards in 75% methanol/25% water mobile phase found that negative ion detection gave the optimum generation of parent ions that can be used for molecular weight determinations. However, experiments with poly(acrylic acid) mixtures and specific high molecular weight standards found multiply charged negative ions that gave a low bias to molecular mass distributions. The number of negative charges on a molecule is dependent on the distance between charges. ESI/MST/MS of model compounds found characteristic water loss from alcohol dehydration and anhydride formation, as well as CO2 loss from decarboxylation, and CO loss from ester structures. Application of these fragmentation pathways to specific masses of fulvic acid isolated and fragmented by ESI/MST/MS is indicative of specific structures that can serve as a basis for future structural confirmation after these hypothesized structures are synthesized.

  18. Imidate-Based Cross-Linkers for Structural Proteomics: Increased Charge of Protein and Peptide Ions and CID and ECD Fragmentation Studies

    NASA Astrophysics Data System (ADS)

    Koolen, Hector H. F.; Gomes, Alexandre F.; Schwab, Nicolas V.; Eberlin, Marcos N.; Gozzo, Fabio C.

    2014-07-01

    Chemical cross-linking is an attractive low-resolution technique for structural studies of protein complexes. Distance constraints obtained from cross-linked peptides identified by mass spectrometry (MS) are used to construct and validate protein models. Amidinating cross-linkers such as diethyl suberthioimidate (DEST) have been used successfully in chemical cross-linking experiments. In this work, the application of a commercial diimidate cross-linking reagent, dimethyl suberimidate (DMS), was evaluated with model peptides and proteins. The peptides were designed with acetylated N-termini followed by random sequences containing two Lys residues separated by an Arg residue. After cross-linking reactions, intra- and intermolecular cross-linked species were submitted to CID and ECD dissociations to study their fragmentation features in the gas phase. Fragmentation of intramolecular peptides by collision induced dissociation (CID) demonstrates a unique two-step fragmentation pathway involving formation of a ketimine as intermediate. Electron capture and electron transfer dissociation (ECD and ETD) experiments demonstrated that the cyclic moiety is not dissociated. Intermolecular species demonstrated previously described fragmentation behavior in both CID and ECD experiments. The charge state distributions (CSD) obtained after reaction with DMS were compared with those obtained with disuccinimidyl suberate (DSS). CSDs for peptides and proteins were increased after their reaction with DMS, owing to the higher basicity of DMS modified species. These features were also observed in LC-MS experiments with bovine carbonic anhydrase II (BCA) after cross-linking with DMS and tryptic proteolysis. Cross-linked peptides derived from this protein were identified at high confidence and those species were in agreement with the crystal structure of BCA.

  19. 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

  20. 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.

  1. Analysis of isotopic labeling in peptide fragments by tandem mass spectrometry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The cellular phenotype is the consequence of dynamic metabolic events that occur in a spacially dependent fashion. This spatial and temporal complexity presents challenges for investigating primary metabolism and improved methods to probe biochemical events such as amino acid biosynthesis may be nee...

  2. Selective detection of carbohydrates and their peptide conjugates by ESI-MS using synthetic quaternary ammonium salt derivatives of phenylboronic acids.

    PubMed

    Kijewska, Monika; Kuc, Adam; Kluczyk, Alicja; Waliczek, Mateusz; Man-Kupisinska, Aleksandra; Lukasiewicz, Jolanta; Stefanowicz, Piotr; Szewczuk, Zbigniew

    2014-06-01

    We present new tags based on the derivatives of phenylboronic acid and apply them for the selective detection of sugars and peptide-sugar conjugates in mass spectrometry. We investigated the binding of phenylboronic acid and its quaternary ammonium salt (QAS) derivatives to carbohydrates and peptide-derived Amadori products by HR-MS and MS/MS experiments. The formation of complexes between sugar or sugar-peptide conjugates and synthetic tags was confirmed on the basis of the unique isotopic distribution resulting from the presence of boron atom. Moreover, incorporation of a quaternary ammonium salt dramatically improved the efficiency of ionization in mass spectrometry. It was found that the formation of a complex with phenylboronic acid stabilizes the sugar moiety in glycated peptides, resulting in simplification of the fragmentation pattern of peptide-derived Amadori products. The obtained results suggest that derivatization of phenylboronic acid as QAS is a promising method for sensitive ESI-MS detection of carbohydrates and their conjugates formed by non-enzymatic glycation or glycosylation.

  3. Correlation of Multiple Peptide Mass Spectra for Phosphoprotein Identification

    Technology Transfer Automated Retrieval System (TEKTRAN)

    When collision induced dissociation is used to fragment phosphorylated peptides during tandem mass spectrometry (MS2), an ion exhibiting the neutral loss of phosphoric acid can be the major product. The neutral loss ion can then be fragmented during MS3 for additional resolution of the peptide sequ...

  4. 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.

  5. 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

  6. 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.

  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. Diagnosing the Protonation Site of b 2 Peptide Fragment Ions using IRMPD in the X-H (X = O, N, and C) Stretching Region

    NASA Astrophysics Data System (ADS)

    Sinha, Rajeev K.; Erlekam, Undine; Bythell, Benjamin J.; Paizs, Béla; Maître, Philippe

    2011-09-01

    Charge-directed fragmentation has been shown to be the prevalent dissociation step for protonated peptides under the low-energy activation (eV) regime. Thus, the determination of the ion structure and, in particular, the characterization of the protonation site(s) of peptides and their fragments is a key approach to substantiate and refine peptide fragmentation mechanisms. Here we report on the characterization of the protonation site of oxazolone b 2 ions formed in collision-induced dissociation (CID) of the doubly protonated tryptic model-peptide YIGSR. In support of earlier work, here we provide complementary IR spectra in the 2800-3800 cm-1 range acquired on a table-top laser system. Combining this tunable laser with a high power CO2 laser to improve spectroscopic sensitivity, well resolved bands are observed, with an excellent correspondence to the IR absorption bands of the ring-protonated oxazolone isomer as predicted by quantum chemical calculations. In particular, it is shown that a band at 3445 cm-1, corresponding to the asymmetric N-H stretch of the (nonprotonated) N-terminal NH2 group, is a distinct vibrational signature of the ring-protonated oxazolone structure.

  10. Studies on adenosine triphosphate transphosphorylases. XVIII. Synthesis and preparation of peptides and peptide fragments of rabbit muscle ATP-AMP transphosphorylase (adenylate kinase) and their nucleotide-binding properties.

    PubMed

    Kuby, S A; Hamada, M; Johnson, M S; Russell, G A; Manship, M; Palmieri, R H; Fleming, G; Bredt, D S; Mildvan, A S

    1989-08-01

    Two peptide fragments, derived from the head and tail of rabbit muscle myokinase, were found to possess remarkable and specific ligand-binding properties (Hamada et al., 1979). By initiating systematic syntheses and measurements of equilibrium substrate-binding properties of these two sets of peptides, or portions thereof, which encompass the binding sites for (a) the magnesium complexes of the nucleotide substrates (MgATP2- and MgADP-) and (b) the uncomplexed nucleotide substrates (ADP3- and AMP2-) of rabbit muscle myokinase, some of the requirements for binding of the substrates to ATP-AMP transphosphorylase are being deduced and chemically outlined. One requirement for tight nucleotide binding appears to be a minimum peptide length of 15-25 residues. In addition, Lys-172 and/or Lys-194 may be involved in the binding of epsilon AMP. The syntheses are described as a set of peptides corresponding to residues 31-45, 20-45, 5-45, and 1-45, and a set of peptides corresponding to residues 178-192, 178-194, and 172-194 of rabbit muscle adenylate kinase. The ligand-binding properties of the first set of synthetic peptides to the fluorescent ligands: epsilon MgATP/epsilon ATP and epsilon MgADP/epsilon ADP are quantitatively presented in terms of their intrinsic dissociation constants (K'd) and values of N (maximal number of moles bound per mole of peptide); and compared with the peptide fragment MT-I (1-44) obtained from rabbit muscle myokinase (Kuby et al., 1984) and with the native enzyme (Hamada et al., 1979). In addition, the values of N and K'd are given for the second set of synthetic peptides to the fluorescent ligands epsilon AMP and epsilon ADP as well as for the peptide fragments MT-XII(172-194) and CB-VI(126-194) (Kuby et al., 1984) and, in turn, compared with the native enzyme. A few miscellaneous dissociation constants which had been derived kinetically are also given for comparison (e.g., the Ki for epsilon AMP and the value of KMg epsilon ATP obtained for

  11. 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.

  12. 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.

  13. 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

  14. Anti-inflammatory effect of a human prothrombin fragment-2-derived peptide, NSA9, in EOC2 microglia

    SciTech Connect

    Kim, Ji Yeon; Kim, Tae Hyong; Kim, Soung Soo

    2008-04-11

    Pro-inflammatory mediators, such as nitric oxide (NO), prostaglandin E{sub 2} (PGE{sub 2}), and several cytokines (tumor necrosis factor (TNF)-{alpha}, interleukin (IL)-1{beta}, and IL-6) are responsible for central nervous system (CNS) injuries that include ischemia, Alzheimer's disease, and neural death. Inhibition of these pro-inflammatory mediators would be an effective therapy to reduce the progression of neurodegenerative diseases. In this study, we examined the anti-inflammatory effects of a human prothrombin fragment-2-derived peptide, NSA9 (NSAVQLVEN), on the production of pro-inflammatory mediators in lipopolysaccharide (LPS)-activated brain microglia. NSA9 significantly inhibited the release of NO, PGE{sub 2}, and pro-inflammatory cytokines in a dose-dependent manner. Furthermore, NSA9 reduced the expression of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 mRNA and protein, which control the production of NO and PGE{sub 2}, respectively. Moreover, NSA9 suppressed the LPS-induced nuclear translocation and activation of nuclear factor-{kappa}B (NF-{kappa}B). These results suggest that NSA9 strongly inhibits the pro-inflammatory responses of microglia through the modulation of NF-{kappa}B activity.

  15. Fragmentation of doubly-protonated peptide ion populations labeled by H/D exchange with CD3OD

    NASA Astrophysics Data System (ADS)

    Herrmann, Kristin A.; Kuppannan, Krishna; Wysocki, Vicki H.

    2006-03-01

    Doubly-protonated bradykinin (RPPGFSPFR) and an angiotensin III analogue (RVYIFPF) were subjected to hydrogen/deuterium (H/D) exchange with CD3OD in a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. A bimodal distribution of deuterium incorporation was present for bradykinin after H/D exchange for 90 s at a CD3OD pressure of 4 × 10-7 Torr, indicating the existence of at least two distinct populations. Bradykinin ion populations corresponding to 0-2 and 5-11 deuteriums (i.e., D0, D1, D2, D5, D6, D7, D8, D9, D10, and D11) were each monoisotopically selected and fragmented via sustained off-resonance irradiation (SORI) collision-induced dissociation (CID). The D0-D2 ion populations, which correspond to the slower exchanging population, consistently require lower SORI amplitude to achieve a similar precursor ion survival yield as the faster-reacting (D5-D11) populations. These results demonstrate that conformation/protonation motif has an effect on fragmentation efficiency for bradykinin. Also, the partitioning of the deuterium atoms into fragment ions suggests that the C-terminal arginine residue exchanges more rapidly than the N-terminal arginine. Total deuterium incorporation in the b1/y8 and b2/y7 ion pairs matches very closely the theoretical values for all ion populations studied, indicating that the ions of a complementary pair are likely formed during the same fragmentation event, or that no scrambling occurs upon SORI. Deuterium incorporation into the y1/a8 pseudo-ion pair does not closely match the expected theoretical values. The other peptide, doubly-protonated RVYIFPF, has a trimodal distribution of deuterium incorporation upon H/D exchange with CD3OD at a pressure of 1 × 10-7 Torr for 600 s, indicating at least three distinct ion populations. After 90 s of H/D exchange where at least two distinct populations are detected, the D0-D7 ion populations were monoisotopically selected and fragmented via SORI-CID over a range of SORI

  16. 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

  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. Total synthesis of microcin B17 via a fragment condensation approach.

    PubMed

    Thompson, Robert E; Jolliffe, Katrina A; Payne, Richard J

    2011-02-18

    The total synthesis of the 43 amino acid antibacterial peptide Microcin B17 (MccB17) is described. The natural product was synthesized via a convergent approach from a heterocycle-derived peptide and peptide thioester fragments prepared via Fmoc-strategy solid phase peptide synthesis (SPPS). Final assembly was achieved in an efficient manner using two Ag(I)-assisted peptide ligation reactions to afford MccB17 in excellent overall yield.

  19. 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

  20. 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.

  1. 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

  2. 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

  3. 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.

  4. 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

  5. Identification of a novel angiotensin-I-converting enzyme inhibitory peptide corresponding to a tryptic fragment of bovine beta-lactoglobulin.

    PubMed

    Mullally, M M; Meisel, H; FitzGerald, R J

    1997-02-01

    The angiotensin-I-converting enzyme (ACE) inhibitory activity of a tryptic digest of bovine beta-lactoglobulin (beta-lg) was investigated. Intact beta-lg essentially did not inhibit ACE while the tryptic digest gave an 84.3% inhibition of ACE. Peptide material eluting between 20 and 25% acetonitrile during C18 solid-phase extraction of the beta-lg tryptic digest inhibited ACE by 93.6%. This solid-phase extraction fraction was shown by mass spectroscopy to contain beta-lg f(142-148). This peptide had an ACE IC50 value of 42.6 micromol/l. The peptide was resistant to further digestion with pepsin and was hydrolysed to a very low extent with chymotrypsin. The contribution of specific amino acid residues within the peptide to ACE inhibitory activity and the potential application of this peptide as a nutraceutical is discussed.

  6. [An improved method of preparing protein and peptide probes in mass spectrometry with ionization of division fragments by californium-252 (TOF-PDMS)].

    PubMed

    Chivanov, V D; Zubarev, R A; Aksenov, S A; Bordunova, O G; Eremenko, V I; Kabanets, V M; Tatarinova, V I; Mishnev, A K; Kuraev, V V; Knysh, A N; Eremenko, I A

    1996-08-01

    The addition of organic acids (picric, oxalic, citric, or tartaric) to peptide and protein samples was found to significantly increase the yield of their quasi-molecular ions (QMI) in time-of-flight 252Cf plasma desorption mass spectrometry. The yield of the ions depended on the pKa of the acid added.

  7. 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.

  8. 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.

  9. Peptide mapping and amino acid sequencing of two catechol 1,2-dioxygenases (CD I1 and CD I2) from Acinetobacter lwoffii K24.

    PubMed

    Kim, S I; Ha, K S

    1997-10-31

    The partial amino acid sequences of two catechol 1,2-dioxygenases (CD I1 and CD I2) from Acinetobacter lwoffii K24 have been determined by analysis of peptides after cleavages with endopeptidase Lys-C, endopeptidase Glu-C, trypsin, and chemicals (cyanogen bromide and BNPS-skatole). They include 248 amino acid sequences (4 fragments) of CD I1 and 211 amino acid sequences (5 fragments) of CD I2. Two enzymes have more than 50% sequence homology with type I catechol 1,2-dioxygenases and less than 30% sequence homology with type II catechol 1,2-dioxygenases. Two enzymes have similar hydropathy profiles in the N-terminal region, suggesting that they have similar secondary structures. PMID:9387151

  10. The human antimicrobial peptide LL-37 and its fragments possess both antimicrobial and antibiofilm activities against multidrug-resistant Acinetobacter baumannii.

    PubMed

    Feng, Xiaorong; Sambanthamoorthy, Karthik; Palys, Thomas; Paranavitana, Chrysanthi

    2013-11-01

    Acinetobacter baumannii infections are difficult to treat due to multidrug resistance. Biofilm formation by A. baumannii is an additional factor in its ability to resist antimicrobial therapy. The antibacterial and antibiofilm activities of the human antimicrobial peptide LL-37 and its fragments KS-30, KR-20 and KR-12 against clinical isolates of multidrug-resistant (MDR) A. baumannii were evaluated. The minimal inhibitory concentration (MIC) of LL-37 against MDR A. baumannii isolates ranged from 16 to 32 μg/mL. The MIC of KS-30 fragment varied from 8.0 to 16 μg/mL and the KR-20 fragment MIC ranged from 16 to 64 μg/mL. LL-37 and KS-30 fragment exhibited 100% bactericidal activity against five A. baumannii strains, including four MDR clinical isolates, within 30 min at concentrations of 0.25-1 μg/mL. By 0.5h, the fragments KR-20 and KR-12 eliminated all tested strains at 8 and 64 μg/mL respectively. LL-37 and its fragments displayed anti-adherence activities between 32-128 μg/mL. A minimum biofilm eradication concentration (MBEC) biofilm assay demonstrated that LL-37 inhibited and dispersed A. baumannii biofilms at 32 μg/mL respectively. Truncated fragments of LL-37 inhibited biofilms at concentrations of 64-128 μg/mL. KS-30, the truncated variant of LL-37, effectively dispersed biofilms at 64 μg/mL. At 24h, no detectable toxicity was observed at the efficacious doses when cytotoxicity assays were performed. Thus, LL-37, KS-30 and KR-20 exhibit significant antimicrobial activity against MDR A. baumannii. The prevention of biofilm formation in vitro by LL-37, KS-30 and KR-20 adds significance to their efficacy. These peptides can be potential therapeutics against MDR A. baumannii infections.

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. Studies of peptide a- and b-type fragment ions using stable isotope labeling and integrated ion mobility/tandem mass spectrometry.

    PubMed

    Riba Garcia, Isabel; Giles, Kevin; Bateman, Robert H; Gaskell, Simon J

    2008-12-01

    The structures of peptide a- and b-type fragment ions were studied using synthetic peptides including a set of isomeric peptides, differing in the sequence location of an alanine residue labeled with (15)N and uniformly with (13)C. The pattern of isotope labeling of second-generation fragment ions derived via a(n) and b(n) ions (where n = 4 or 5) suggested that these intermediates existed in part as macrocyclic structures, where alternative sites of ring opening gave rise to different linear forms whose simple cleavage might give rise to the observed final products. Similar conclusions were derived from combined ion mobility/tandem MS analyses where different fragmentation patterns were observed for isomeric a- or b-type ions that display different ion mobilities. These analyses were facilitated by a new approach to the processing of ion mobility/tandem MS data, from which distinct and separate product ion spectra are derived from ions that are incompletely separated by ion mobility. Finally, an example is provided of evidence for a macrocyclic structure for b(n) ions where n = 8 or 9.

  18. Fragmentation processes of hydrogen-deficient peptide radicals in matrix-assisted laser desorption/ionization in-source decay mass spectrometry.

    PubMed

    Asakawa, Daiki; Takayama, Mitsuo

    2012-04-01

    The mechanism of in-source decay (ISD) in matrix-assisted laser desorption/ionization (MALDI) has been described. The MALDI-ISD with an oxidizing matrix is initiated by hydrogen abstraction from peptides to matrix molecules, leading to hydrogen-deficient peptide radicals. Subsequently, the C(α)-C and C(α)-H bonds are cleaved, forming the a•/x fragment pair and [M-2H], respectively. Those reactions competitively occur during MALDI-ISD processes. Our results suggest that the C(α)-H bond cleavage to form [M-2H] was induced by collisions between hydrogen-deficient peptide radicals and matrix molecules in the MALDI plume. In contrast, the C(α)-C bond cleavages occur via a unimolecular dissociation process and independently of the collision rate in the MALDI plume. The formation mechanism of the a-, b-, and d-series fragments are also described. We report 2,5-bis(2-hydroxyethoxy)-7,7,8,8-tetracyanoquinodimethane (bisHE-TCNQ), being known as an organic semiconductor and an electron acceptor, as a novel suitable matrix for the MALDI-ISD of peptides via hydrogen abstraction.

  19. 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

  20. The hinge region fragment of immunoglobulin G improves immunogenicity of recombinant gonadotrophin-releasing hormone conjugated to the T-helper epitope in designing peptide vaccines

    PubMed Central

    Xu, Jinshu; Wu, Jie; Wang, Xuejun; Zhang, Yin; Li, Wenjia; Zhu, Zheng; Zhu, Dongya; Hu, Zhuoyi; Roque, Rouel S; Liu, Jingjing

    2009-01-01

    In our previous study, the hinge fragment (225–232/225′–232′) of human immunoglobulin G1 (IgG1) was used as a space peptide linker for synthesizing the GnRH3–hinge–MVP chimeric peptide, whereby three repeated gonadotrophin-releasing hormone (GnRH) units and a T-cell epitope from measles virus fusion protein (MVP) were amide-bond-linked at the N and C terminus, respectively, to the hinge peptide for producing anti-GnRH antibody responses. To investigate whether or not the hinge region fragment can improve the immunogenicity of GnRH, we further synthesized and purified GnRH3–hinge–MVP, GnRH3–hinge and GnRH3–MVP using recombinant DNA technology. Under high pH conditions, GnRH3–hinge–MVP was capable of forming double-chain structures. Immunization of male mice with the immunogens of GnRH3–hinge–MVP resulted in the generation of high-titre antibodies specific for GnRH. The synthetic GnRH3–hinge and GnRH3–MVP induced a lower titre of anti-GnRH antibody than GnRH3–hinge–MVP. This was followed by a decrease in serum testosterone levels, which resulted in a low level of expression of the relaxin-like factor gene in the testis. Our data suggest that peptide and T-cell epitopes oriented at the N-terminus or C-terminus of hinge peptides simplify the antigenic peptide conjugates and may be considered as potential synthetic immunogens. PMID:19740311

  1. Mitochondrial DNA Fragmentation to Monitor Processing Parameters in High Acid, Plant-Derived Foods.

    PubMed

    Caldwell, Jane M; Pérez-Díaz, Ilenys M; Harris, Keith; Hassan, Hosni M; Simunovic, Josip; Sandeep, K P

    2015-12-01

    Mitochondrial DNA (mtDNA) fragmentation was assessed in acidified foods. Using quantitative polymerase chain reaction, Ct values measured from fresh, fermented, pasteurized, and stored cucumber mtDNA were determined to be significantly different (P > 0.05) based on processing and shelf-life. This indicated that the combination of lower temperature thermal processes (hot-fill at 75 °C for 15 min) and acidified conditions (pH = 3.8) was sufficient to cause mtDNA fragmentation. In studies modeling high acid juices, pasteurization (96 °C, 0 to 24 min) of tomato serum produced Ct values which had high correlation to time-temperature treatment. Primers producing longer amplicons (approximately 1 kb) targeting the same mitochondrial gene gave greater sensitivity in correlating time-temperature treatments to Ct values. Lab-scale pasteurization studies using Ct values derived from the longer amplicon differentiated between heat treatments of tomato serum (95 °C for <2 min). MtDNA fragmentation was shown to be a potential new tool to characterize low temperature (<100 °C) high acid processes (pH < 4.6), nonthermal processes such as vegetable fermentation and holding times of acidified, plant-derived products. PMID:26556214

  2. Mitochondrial DNA Fragmentation to Monitor Processing Parameters in High Acid, Plant-Derived Foods.

    PubMed

    Caldwell, Jane M; Pérez-Díaz, Ilenys M; Harris, Keith; Hassan, Hosni M; Simunovic, Josip; Sandeep, K P

    2015-12-01

    Mitochondrial DNA (mtDNA) fragmentation was assessed in acidified foods. Using quantitative polymerase chain reaction, Ct values measured from fresh, fermented, pasteurized, and stored cucumber mtDNA were determined to be significantly different (P > 0.05) based on processing and shelf-life. This indicated that the combination of lower temperature thermal processes (hot-fill at 75 °C for 15 min) and acidified conditions (pH = 3.8) was sufficient to cause mtDNA fragmentation. In studies modeling high acid juices, pasteurization (96 °C, 0 to 24 min) of tomato serum produced Ct values which had high correlation to time-temperature treatment. Primers producing longer amplicons (approximately 1 kb) targeting the same mitochondrial gene gave greater sensitivity in correlating time-temperature treatments to Ct values. Lab-scale pasteurization studies using Ct values derived from the longer amplicon differentiated between heat treatments of tomato serum (95 °C for <2 min). MtDNA fragmentation was shown to be a potential new tool to characterize low temperature (<100 °C) high acid processes (pH < 4.6), nonthermal processes such as vegetable fermentation and holding times of acidified, plant-derived products.

  3. 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

  4. Synthetic peptides.

    PubMed

    Francis, M J

    1996-01-01

    Efforts to produce more stable and defined vaccines have concentrated on studying, in detail, the immune response to many infectious diseases in order to identify the antigenic sites on the pathogens that are involved in stimulating protective immumty. Armed with this knowledge, it is possible to mimic such sites by producing short chains of amino acids (peptides) and to use these as the basis for novel vaccines. The earliest documented work on peptide immunization is actually for a plant virus, tobacco mosaic virus. In 1963, Anderer (1) demonstrated that rabbit antibodies to an isolated hexapeptide fragment from the virus-coat protein coupled to bovine serum albumm would neutralize the infectious vn-us in culture. Two years later, he used a synthetically produced copy of the same peptide to confirm this observation. This was pioneering work, and it was over 10 years before the next example of a peptide that elicited antivirus antibody appeared following work by Sela and his colleagues (2) on a virus, MS2 bacteriophage, which infects bacteria. The emergence of more accessible techniques for sequencing proteins in 1977, coupled with the ability to synthesize readily peptides already developed in 1963, heralded a decade of intensive research into experimental peptide vaccines. The first demonstration that peptides could elicit protective immunity in vivo, in addition to neutralizing activity in vitro, was obtained using a peptide from the VP1 coat protein of foot-and-mouth disease virus (FMDV) in 1982, with the guinea pig as a laboratory animal model (3, 4). PMID:21359696

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. Fragmentation of D- and L-enantiomers of amino acids through interaction with 3He2+ ions

    NASA Astrophysics Data System (ADS)

    Smirnov, O. V.; Basalaev, A. A.; Boitsov, V. M.; Vyaz'min, S. Yu.; Orbeli, A. L.; Dubina, M. V.

    2014-11-01

    The relative cross section of processes attendant on the capture of an electron by 12-keV 3He2+ ions are measured by time-of-flight mass spectrometry for leucine (C6H13NO2), methionine (C5H11NO2S), and glutmic acid (C5H9NO4) molecules. No differences between the formation relative cross sections of different fragment ions for the D- and L-enantiomeric forms of the amino acids are revealed. The spectrum of glutamic acid fragments taken at temperatures above 110°C is explained by decomposition of the acid with the formation of pyroglutamic acid (C5H7NO3) and water. The results are compared with published data on fragmentation of the same molecules via electron-impact ionization.

  13. 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.

  14. 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

  15. 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.

  16. Adolescent Femoral Chondral Fragment Fixation With Poly-L-Lactic Acid Chondral Darts.

    PubMed

    Morris, John K; Weber, Alexander E; Morris, Mark S

    2016-01-01

    Large chondral injuries without attached bone are uncommon. This report describes a 14-year-old boy who had a unique stress reaction between the bone and the overlying cartilage, predominantly of the anterior lateral femoral condyle, during a week-long basketball camp, resulting in complete displacement of a 2.5 × 2.5-cm full-thickness articular cartilage lesion. There was a 6-day interval from the time of the injury to the first office appointment. Scheduling of magnetic resonance imaging and insurance approval took another week, and then surgery scheduling, including insurance approval and arranging for surgical supplies, took another week. Three weeks after the initial injury, the patient underwent diagnostic arthroscopy and open arthrotomy, and the cartilage-free fragment was returned to the donor site and fixed with poly-L-lactic acid chondral darts. Considerable delamination of the shoulders of the defect was noted on preoperative magnetic resonance imaging and at the time of surgery, suggesting an unusual prodromal stress reaction. Although there was no underlying subchondral bone on the free cartilage fragment, the injury healed. The patient had return of full knee range of motion and strength. Magnetic resonance imaging performed 3 months postoperatively showed healed cartilage. At 1 year of clinical follow-up, the patient had no clinical sequelae from the initial injury and had returned to competitive basketball. Prompt recognition of this injury pattern and subsequent surgical repair are necessary because the window of opportunity closes as fibrous healing occurs and the cartilage fragment deforms. The poly-L-lactic acid chondral dart system was instrumental to the success of this case. PMID:26840696

  17. Adolescent Femoral Chondral Fragment Fixation With Poly-L-Lactic Acid Chondral Darts.

    PubMed

    Morris, John K; Weber, Alexander E; Morris, Mark S

    2016-01-01

    Large chondral injuries without attached bone are uncommon. This report describes a 14-year-old boy who had a unique stress reaction between the bone and the overlying cartilage, predominantly of the anterior lateral femoral condyle, during a week-long basketball camp, resulting in complete displacement of a 2.5 × 2.5-cm full-thickness articular cartilage lesion. There was a 6-day interval from the time of the injury to the first office appointment. Scheduling of magnetic resonance imaging and insurance approval took another week, and then surgery scheduling, including insurance approval and arranging for surgical supplies, took another week. Three weeks after the initial injury, the patient underwent diagnostic arthroscopy and open arthrotomy, and the cartilage-free fragment was returned to the donor site and fixed with poly-L-lactic acid chondral darts. Considerable delamination of the shoulders of the defect was noted on preoperative magnetic resonance imaging and at the time of surgery, suggesting an unusual prodromal stress reaction. Although there was no underlying subchondral bone on the free cartilage fragment, the injury healed. The patient had return of full knee range of motion and strength. Magnetic resonance imaging performed 3 months postoperatively showed healed cartilage. At 1 year of clinical follow-up, the patient had no clinical sequelae from the initial injury and had returned to competitive basketball. Prompt recognition of this injury pattern and subsequent surgical repair are necessary because the window of opportunity closes as fibrous healing occurs and the cartilage fragment deforms. The poly-L-lactic acid chondral dart system was instrumental to the success of this case.

  18. 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.

  19. Expression and characterization of an enantioselective antigen-binding fragment directed against α-amino acids

    PubMed Central

    Eleniste, Pierre P.; Hofstetter, Heike; Hofstetter, Oliver

    2013-01-01

    This work describes the design and expression of a stereoselective Fab that possesses binding properties comparable to those displayed by the parent monoclonal antibody. Utilizing mRNA from hybridoma clones that secrete a stereoselective anti-L-amino acid antibody, a corresponding biotechnologically produced Fab was generated. For that, appropriate primers were designed based on extensive literature and databank searches. Using these primers in PCR resulted in successful amplification of the VH, VL, CL and CH1 gene fragments. Overlap PCR was utilized to combine the VH and CH1 sequences and the VL and CL sequences, respectively, to obtain the genes encoding the HC and LC fragments. These sequences were separately cloned into the pEXP5-CT/TOPO expression vector and used for transfection of BL21(DE3) cells. Separate expression of the two chains, followed by assembly in a refolding buffer, yielded an Fab that was demonstrated to bind to L-amino acids but not to recognize the corresponding D-enantiomers. PMID:23827208

  20. 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

  1. Angiotensin I-converting enzyme inhibitor peptides derived from the endostatin-containing NC1 fragment of human collagen XVIII.

    PubMed

    Farias, Shirley L; Sabatini, Regiane A; Sampaio, Tatiana C; Hirata, Izaura Y; Cezari, Maria Helena S; Juliano, Maria A; Sturrock, Edward D; Carmona, Adriana K; Juliano, Luiz

    2006-05-01

    Extracellular matrix and soluble plasma proteins generate peptides that regulate biological activities such as cell growth, differentiation and migration. Bradykinin, a peptide released from kininogen by kallikreins, stimulates vasodilatation and endothelial cell proliferation. Various classes of substances can potentiate these biological actions of bradykinin. Among them, the best studied are bradykinin potentiating peptides (BPPs) derived from snake venom, which can also strongly inhibit angiotensin I-converting enzyme (ACE) activity. We identified and synthesized sequences resembling BPPs in the vicinity of potential proteolytic cleavage sites in the collagen XVIII molecule, close to endostatin. These peptides were screened as inhibitors of human recombinant wild-type ACE containing two intact functional domains; two full-length ACE mutants containing only a functional C- or N-domain catalytic site; and human testicular ACE, a natural form of the enzyme that only contains the C-domain. The BPP-like peptides inhibited ACE in the micromolar range and interacted preferentially with the C-domain. The proteolytic activity involved in the release of BPP-like peptides was studied in human serum and human umbilical-vein endothelial cells. The presence of enzymes able to release these peptides in blood led us to speculate on a physiological mechanism for the control of ACE activities.

  2. Fragment-based discovery of a new family of non-peptidic small-molecule cyclophilin inhibitors with potent antiviral activities

    PubMed Central

    Ahmed-Belkacem, Abdelhakim; Colliandre, Lionel; Ahnou, Nazim; Nevers, Quentin; Gelin, Muriel; Bessin, Yannick; Brillet, Rozenn; Cala, Olivier; Douguet, Dominique; Bourguet, William; Krimm, Isabelle; Pawlotsky, Jean-Michel; Guichou, Jean- François

    2016-01-01

    Cyclophilins are peptidyl-prolyl cis/trans isomerases (PPIase) that catalyse the interconversion of the peptide bond at proline residues. Several cyclophilins play a pivotal role in the life cycle of a number of viruses. The existing cyclophilin inhibitors, all derived from cyclosporine A or sanglifehrin A, have disadvantages, including their size, potential for side effects unrelated to cyclophilin inhibition and drug–drug interactions, unclear antiviral spectrum and manufacturing issues. Here we use a fragment-based drug discovery approach using nucleic magnetic resonance, X-ray crystallography and structure-based compound optimization to generate a new family of non-peptidic, small-molecule cyclophilin inhibitors with potent in vitro PPIase inhibitory activity and antiviral activity against hepatitis C virus, human immunodeficiency virus and coronaviruses. This family of compounds has the potential for broad-spectrum, high-barrier-to-resistance treatment of viral infections. PMID:27652979

  3. 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.

  4. 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.

  5. 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.

  6. Crystallization and preliminary X-ray diffraction analysis of the Fab fragment of WO2, an antibody specific for the Aβ peptides associated with Alzheimer’s disease

    SciTech Connect

    Wun, Kwok S.; Miles, Luke A.; Crespi, Gabriela A. N.; Wycherley, Kaye; Ascher, David B.; Barnham, Kevin J.; Cappai, Roberto; Beyreuther, Konrad; Masters, Colin L.; Parker, Michael W.; McKinstry, William J.

    2008-05-01

    Crystallization and X-ray diffraction data collection of the Fab fragment of the monoclonal antibody WO2 in the absence or presence of amyloid β peptides associated with Alzheimer’s disease are reported. The murine monoclonal antibody WO2 specifically binds the N-terminal region of the amyloid β peptide (Aβ) associated with Alzheimer’s disease. This region of Aβ has been shown to be the immunodominant B-cell epitope of the peptide and hence is considered to be a basis for the development of immunotherapeutic strategies against this prevalent cause of dementia. Structural studies have been undertaken in order to characterize the molecular basis for antibody recognition of this important epitope. Here, details of the crystallization and X-ray analysis of the Fab fragment of the unliganded WO2 antibody in two crystal forms and of the complexes that it forms with the truncated Aβ peptides Aβ{sub 1–16} and Aβ{sub 1–28} are presented. These crystals were all obtained using the hanging-drop vapour-diffusion method at 295 K. Crystals of WO2 Fab were grown in polyethylene glycol solutions containing ZnSO{sub 4}; they belonged to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1} and diffracted to 1.6 Å resolution. The complexes of WO2 Fab with either Aβ{sub 1–@}@{sub 16} or Aβ{sub 1–28} were cocrystallized from polyethylene glycol solutions. These two complex crystals grew in the same space group, P2{sub 1}2{sub 1}2{sub 1}, and diffracted to 1.6 Å resolution. A second crystal form of WO2 Fab was grown in the presence of the sparingly soluble Aβ{sub 1–42} in PEG 550 MME. This second form belonged to space group P2{sub 1} and diffracted to 1.9 Å resolution.

  7. 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

  8. Formation and Fragmentation of Unsaturated Fatty Acid [M - 2H + Na]- Ions: Stabilized Carbanions for Charge-Directed Fragmentation

    NASA Astrophysics Data System (ADS)

    Thomas, Michael C.; Kirk, Benjamin B.; Altvater, Jens; Blanksby, Stephen J.; Nette, Geoffrey W.

    2013-12-01

    Fatty acids are long-chain carboxylic acids that readily produce [M - H]- ions upon negative ion electrospray ionization (ESI) and cationic complexes with alkali, alkaline earth, and transition metals in positive ion ESI. In contrast, only one anionic monomeric fatty acid-metal ion complex has been reported in the literature, namely [M - 2H + FeIICl]-. In this manuscript, we present two methods to form anionic unsaturated fatty acid-sodium ion complexes (i.e., [M - 2H + Na]-). We find that these ions may be generated efficiently by two distinct methods: (1) negative ion ESI of a methanolic solution containing the fatty acid and sodium fluoride forming an [M - H + NaF]- ion. Subsequent collision-induced dissociation (CID) results in the desired [M - 2H + Na]- ion via the neutral loss of HF. (2) Direct formation of the [M - 2H + Na]- ion by negative ion ESI of a methanolic solution containing the fatty acid and sodium hydroxide or bicarbonate. In addition to deprotonation of the carboxylic acid moiety, formation of [M - 2H + Na]- ions requires the removal of a proton from the fatty acid acyl chain. We propose that this deprotonation occurs at the bis-allylic position(s) of polyunsaturated fatty acids resulting in the formation of a resonance-stabilized carbanion. This proposal is supported by ab initio calculations, which reveal that removal of a proton from the bis-allylic position, followed by neutral loss of HX (where X = F- and -OH), is the lowest energy dissociation pathway.

  9. Formation and fragmentation of unsaturated fatty acid [M - 2H + Na]- ions: stabilized carbanions for charge-directed fragmentation.

    PubMed

    Thomas, Michael C; Kirk, Benjamin B; Altvater, Jens; Blanksby, Stephen J; Nette, Geoffrey W

    2014-02-01

    Fatty acids are long-chain carboxylic acids that readily produce [M - H](-) ions upon negative ion electrospray ionization (ESI) and cationic complexes with alkali, alkaline earth, and transition metals in positive ion ESI. In contrast, only one anionic monomeric fatty acid-metal ion complex has been reported in the literature, namely [M - 2H  +  Fe(II)Cl](-). In this manuscript, we present two methods to form anionic unsaturated fatty acid-sodium ion complexes (i.e., [M - 2H  +  Na](-)). We find that these ions may be generated efficiently by two distinct methods: (1) negative ion ESI of a methanolic solution containing the fatty acid and sodium fluoride forming an [M - H  +  NaF](-) ion. Subsequent collision-induced dissociation (CID) results in the desired [M - 2H  +  Na](-) ion via the neutral loss of HF. (2) Direct formation of the [M - 2H  +  Na](-) ion by negative ion ESI of a methanolic solution containing the fatty acid and sodium hydroxide or bicarbonate. In addition to deprotonation of the carboxylic acid moiety, formation of [M - 2H  +  Na](-) ions requires the removal of a proton from the fatty acid acyl chain. We propose that this deprotonation occurs at the bis-allylic position(s) of polyunsaturated fatty acids resulting in the formation of a resonance-stabilized carbanion. This proposal is supported by ab initio calculations, which reveal that removal of a proton from the bis-allylic position, followed by neutral loss of HX (where X = F(-) and (-)OH), is the lowest energy dissociation pathway. PMID:24338213

  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. Formation and Fragmentation of Protonated Molecules after Ionization of Amino Acid and Lactic Acid Clusters by Collision with Ions in the Gas Phase.

    PubMed

    Poully, Jean-Christophe; Vizcaino, Violaine; Schwob, Lucas; Delaunay, Rudy; Kocisek, Jaroslav; Eden, Samuel; Chesnel, Jean-Yves; Méry, Alain; Rangama, Jimmy; Adoui, Lamri; Huber, Bernd

    2015-08-01

    Collisions between O(3+) ions and neutral clusters of amino acids (alanine, valine and glycine) as well as lactic acid are performed in the gas phase, in order to investigate the effect of ionizing radiation on these biologically relevant molecular systems. All monomers and dimers are found to be predominantly protonated, and ab initio quantum-chemical calculations on model systems indicate that for amino acids, this is due to proton transfer within the clusters after ionization. For lactic acid, which has a lower proton affinity than amino acids, a significant non-negligible amount of the radical cation monomer is observed. New fragment-ion channels observed from clusters, as opposed to isolated molecules, are assigned to the statistical dissociation of protonated molecules formed upon ionization of the clusters. These new dissociation channels exhibit strong delayed fragmentation on the microsecond time scale, especially after multiple ionization.

  12. 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

  13. 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 ...

  14. 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

  15. 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.

  16. 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)

  17. Effect of D-amino acids at Asp{sup 23} and Ser{sup 26} residues on the conformational preference of A{beta}{sub 20-29} peptides

    SciTech Connect

    Shanmugam, Ganesh; Polavarapu, Prasad L. . E-mail: Prasad.L.Polavarapu@Vanderbilt.edu; Hallgas, Balazs; Majer, Zsuzsa

    2005-09-30

    The effects of D-amino acids at Asp{sup 23} and Ser{sup 26} residues on the conformational preference of {beta}-amyloid (A{beta}) peptide fragment (A{beta}{sub 20-29}) have been studied using different spectroscopic techniques, namely vibrational circular dichroism (VCD), vibrational absorption, and electronic circular dichroism. To study the structure of the A{beta}{sub 20-29}, [D-Asp{sup 23}]A{beta}{sub 20-29}, and [D-Ser{sup 26}]A{beta}{sub 20-29} peptides under different conditions, the spectra were measured in 10 mM acetate buffer (pH 3) and in 2,2,2-trifluoroethanol (TFE). The spectroscopic results indicated that at pH 3, A{beta}{sub 20-29} peptide takes random coil with {beta}-turn structure, while [D-Ser{sup 26}]A{beta}{sub 20-29} peptide adopts significant amount of polyproline II (PPII) type structure along with {beta}-turn contribution and D-Asp-substituted peptide ([D-Asp{sup 23}]A{beta}{sub 20-29}) adopts predominantly PPII type structure. The increased propensity for PPII conformation upon D-amino acid substitution, in acidic medium, has important biological implications. In TFE, A{beta}{sub 20-29}, [D-Asp{sup 23}]A{beta}{sub 20-29}, and [D-Ser{sup 26}]A{beta}{sub 20-29} peptides adopt 3{sub 10}-helix, {alpha}-helix, and random coil with some {beta}-turn structures, respectively. The VCD data obtained for the A{beta} peptide films suggested that the secondary structures for the peptide films are not the same as those for corresponding solution and are also different among the A{beta} peptides studied here. This observation suggests that dehydration can have a significant influence on the structural preferences of these peptides.

  18. Fragmentation and dimerization of aliphatic amino acid films induced by vacuum ultraviolet irradiation

    NASA Astrophysics Data System (ADS)

    Tanaka, Masahito; Kaneko, Fusae; Koketsu, Toshiyuki; Nakagawa, Kazumichi; Yamada, Toru

    2008-10-01

    The chemical reaction of aliphatic amino acid, such as alanine (Ala) and leucine (Leu), in the solid phase induced by vacuum ultraviolet (VUV) irradiation was studied by high-performance liquid chromatography technique and mass spectroscopic method. Quantum efficiencies of dimerization of Ala in the solid phase obviously showed irradiated VUV wavelength dependence. The values of quantum efficiencies of formation of Ala dimer were determined to be 5.7×10-5, 1.3×10-3, and 2.4×10-4 for 208, 183, and 87 nm irradiation, respectively. VUV-induced fragment desorption from Ala and Leu films has also been examined by mass spectroscopic method. Observed mass spectra clearly indicated that both the deamination and decarboxylation reactions were common in both Ala and Leu films, and the dissociation of side chain occurred only in Leu film.

  19. Concise synthesis of the A/BCD-ring fragment of gambieric acid A

    PubMed Central

    Fuwa, Haruhiko; Fukazawa, Ryo; Sasaki, Makoto

    2014-01-01

    Gambieric acid A (GAA) and its congeners belong to the family of marine polycyclic ether natural products. Their highly complex molecular architecture and unique biological activities have been of intense interest within the synthetic community. We have previously reported the first total synthesis, stereochemical reassignment, and preliminary structure–activity relationships of GAA. Here we disclose a concise synthesis of the A/BCD-ring fragment of GAA. The synthesis started from our previously reported synthetic intermediate that represents the A/B-ring. The C-ring was synthesized via an oxiranyl anion coupling and a 6-endo cyclization, and the D-ring was forged by means of an oxidative lactonization and subsequent palladium-catalyzed functionalization of the lactone ring. In this manner, the number of linear synthetic steps required for the construction of the C- and D-rings was reduced from 22 to 11. PMID:25629027

  20. Moving Away from the Reference Genome: Evaluating a Peptide Sequencing Tagging Approach for Single Amino Acid Polymorphism Identifications in the Genus Populus

    SciTech Connect

    Abraham, Paul E; Adams, Rachel M; Tuskan, Gerald A; Hettich, Robert {Bob} L

    2013-01-01

    The genetic diversity across natural populations of the model organism, Populus, is extensive, containing a single nucleotide polymorphism roughly every 200 base pairs. When deviations from the reference genome occur in coding regions, they can impact protein sequences. Rather than relying on a static reference database to profile protein expression, we employed a peptide sequence tagging (PST) approach capable of decoding the plasticity of the Populus proteome. Using shotgun proteomics data from two genotypes of P. trichocarpa, a tag-based approach enabled the detection of 6,653 unexpected sequence variants. Through manual validation, our study investigated how the most abundant chemical modification (methionine oxidation) could masquerade as a sequence variant (AlaSer) when few site-determining ions existed. In fact, precise localization of an oxidation site for peptides with more than one potential placement was indeterminate for 70% of the MS/MS spectra. We demonstrate that additional fragment ions made available by high energy collisional dissociation enhances the robustness of the peptide sequence tagging approach (81% of oxidation events could be exclusively localized to a methionine). We are confident that augmenting fragmentation processes for a PST approach will further improve the identification of single amino acid polymorphism in Populus and potentially other species as well.

  1. 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.

  2. Vaccination of non-obese diabetic mice with a fragment of peptide P277 attenuates insulin-dependent diabetes mellitus.

    PubMed

    Ma, Yan-Jun; Lu, Yong; Hou, Jing; Dong, Yuan-Kai; Du, Ming-Zhu; Xing, Yun; Ge, Chi-Yu; Xu, Mao-Lei; Jin, Liang; Cao, Rong-Yue; Li, Tai-Ming; Wu, Jie; Liu, Jing-Jing

    2011-09-01

    P277 is a peptide derived from the HSP60 regions, have potent immunological effect on insulin-dependent diabetes mellitus (IDDM) and its phase III clinical trials are currently under investigation. However, we recently discovered a positive correlation between anti-P277 autoantibodies and the presence of endothelial cells damage in inducing vascular leak syndrome. Therefore, the aim of our study was to demonstrate the critical peptide epitope of P277 to IDDM and to highlight the effects of this peptide therapy on inflammation of the islets. Groups of 4-week old female non-obese diabetic (NOD) mice were immunized one time every three weeks for three times with a residue of P277, showing a significant effect of down-regulating immunity to P277 protein and preventing the development of IDDM. Immunologic results including the suppression of T-cell proliferation, the increase of interleukin-10 (IL-10) production and reduction of interferon-γ (IFN-γ) production caused immune tolerance to P277. Hence, a functional role of the key epitope in P277 peptide capable of preventing IDDM is suggested, which could be modified to develop a novel safe and effective peptide vaccine against IDDM by reconstructing P277 in the further studies.

  3. Neuropeptides: metabolism to bioactive fragments and the pharmacology of their receptors.

    PubMed

    Hallberg, Mathias

    2015-05-01

    The proteolytic processing of neuropeptides has an important regulatory function and the peptide fragments resulting from the enzymatic degradation often exert essential physiological roles. The proteolytic processing generates, not only biologically inactive fragments, but also bioactive fragments that modulate or even counteract the response of their parent peptides. Frequently, these peptide fragments interact with receptors that are not recognized by the parent peptides. This review discusses tachykinins, opioid peptides, angiotensins, bradykinins, and neuropeptide Y that are present in the central nervous system and their processing to bioactive degradation products. These well-known neuropeptide systems have been selected since they provide illustrative examples that proteolytic degradation of parent peptides can lead to bioactive metabolites with different biological activities as compared to their parent peptides. For example, substance P, dynorphin A, angiotensin I and II, bradykinin, and neuropeptide Y are all degraded to bioactive fragments with pharmacological profiles that differ considerably from those of the parent peptides. The review discusses a selection of the large number of drug-like molecules that act as agonists or antagonists at receptors of neuropeptides. It focuses in particular on the efforts to identify selective drug-like agonists and antagonists mimicking the effects of the endogenous peptide fragments formed. As exemplified in this review, many common neuropeptides are degraded to a variety of smaller fragments but many of the fragments generated have not yet been examined in detail with regard to their potential biological activities. Since these bioactive fragments contain a small number of amino acid residues, they provide an ideal starting point for the development of drug-like substances with ability to mimic the effects of the degradation products. Thus, these substances could provide a rich source of new pharmaceuticals

  4. 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.

  5. The ability of apolipoprotein E fragments to promote intraneuronal accumulation of amyloid beta peptide 42 is both isoform and size-specific.

    PubMed

    Dafnis, Ioannis; Argyri, Letta; Sagnou, Marina; Tzinia, Athina; Tsilibary, Effie C; Stratikos, Efstratios; Chroni, Angeliki

    2016-01-01

    The apolipoprotein (apo) E4 isoform is the strongest risk factor for late-onset Alzheimer's disease (AD). ApoE4 is more susceptible to proteolysis than apoE2 and apoE3 isoforms and carboxyl-terminal truncated apoE4 forms have been found in AD patients' brain. We have previously shown that a specific apoE4 fragment, apoE4-165, promotes amyloid-peptide beta 42 (Aβ42) accumulation in human neuroblastoma SK-N-SH cells and increased intracellular reactive oxygen species formation, two events considered to occur early in AD pathogenesis. Here, we show that these effects are allele-dependent and absolutely require the apoE4 background. Furthermore, the exact length of the fragment is critical since longer or shorter length carboxyl-terminal truncated apoE4 forms do not elicit the same effects. Structural and thermodynamic analyses showed that apoE4-165 has a compact structure, in contrast to other carboxyl-terminal truncated apoE4 forms that are instead destabilized. Compared however to other allelic backgrounds, apoE4-165 is structurally distinct and less thermodynamically stable suggesting that the combination of a well-folded structure with structural plasticity is a unique characteristic of this fragment. Overall, our findings suggest that the ability of apoE fragments to promote Aβ42 intraneuronal accumulation is specific for both the apoE4 isoform and the particular structural and thermodynamic properties of the fragment. PMID:27476701

  6. The ability of apolipoprotein E fragments to promote intraneuronal accumulation of amyloid beta peptide 42 is both isoform and size-specific

    PubMed Central

    Dafnis, Ioannis; Argyri, Letta; Sagnou, Marina; Tzinia, Athina; Tsilibary, Effie C.; Stratikos, Efstratios; Chroni, Angeliki

    2016-01-01

    The apolipoprotein (apo) E4 isoform is the strongest risk factor for late-onset Alzheimer’s disease (AD). ApoE4 is more susceptible to proteolysis than apoE2 and apoE3 isoforms and carboxyl-terminal truncated apoE4 forms have been found in AD patients’ brain. We have previously shown that a specific apoE4 fragment, apoE4-165, promotes amyloid-peptide beta 42 (Aβ42) accumulation in human neuroblastoma SK-N-SH cells and increased intracellular reactive oxygen species formation, two events considered to occur early in AD pathogenesis. Here, we show that these effects are allele-dependent and absolutely require the apoE4 background. Furthermore, the exact length of the fragment is critical since longer or shorter length carboxyl-terminal truncated apoE4 forms do not elicit the same effects. Structural and thermodynamic analyses showed that apoE4-165 has a compact structure, in contrast to other carboxyl-terminal truncated apoE4 forms that are instead destabilized. Compared however to other allelic backgrounds, apoE4-165 is structurally distinct and less thermodynamically stable suggesting that the combination of a well-folded structure with structural plasticity is a unique characteristic of this fragment. Overall, our findings suggest that the ability of apoE fragments to promote Aβ42 intraneuronal accumulation is specific for both the apoE4 isoform and the particular structural and thermodynamic properties of the fragment. PMID:27476701

  7. The ability of apolipoprotein E fragments to promote intraneuronal accumulation of amyloid beta peptide 42 is both isoform and size-specific.

    PubMed

    Dafnis, Ioannis; Argyri, Letta; Sagnou, Marina; Tzinia, Athina; Tsilibary, Effie C; Stratikos, Efstratios; Chroni, Angeliki

    2016-08-01

    The apolipoprotein (apo) E4 isoform is the strongest risk factor for late-onset Alzheimer's disease (AD). ApoE4 is more susceptible to proteolysis than apoE2 and apoE3 isoforms and carboxyl-terminal truncated apoE4 forms have been found in AD patients' brain. We have previously shown that a specific apoE4 fragment, apoE4-165, promotes amyloid-peptide beta 42 (Aβ42) accumulation in human neuroblastoma SK-N-SH cells and increased intracellular reactive oxygen species formation, two events considered to occur early in AD pathogenesis. Here, we show that these effects are allele-dependent and absolutely require the apoE4 background. Furthermore, the exact length of the fragment is critical since longer or shorter length carboxyl-terminal truncated apoE4 forms do not elicit the same effects. Structural and thermodynamic analyses showed that apoE4-165 has a compact structure, in contrast to other carboxyl-terminal truncated apoE4 forms that are instead destabilized. Compared however to other allelic backgrounds, apoE4-165 is structurally distinct and less thermodynamically stable suggesting that the combination of a well-folded structure with structural plasticity is a unique characteristic of this fragment. Overall, our findings suggest that the ability of apoE fragments to promote Aβ42 intraneuronal accumulation is specific for both the apoE4 isoform and the particular structural and thermodynamic properties of the fragment.

  8. 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.

  9. 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.

  10. Key Role for the 12-Hydroxy Group in the Negative Ion Fragmentation of Unconjugated C24 Bile Acids.

    PubMed

    Lan, Ke; Su, Mingming; Xie, Guoxiang; Ferslew, Brian C; Brouwer, Kim L R; Rajani, Cynthia; Liu, Changxiao; Jia, Wei

    2016-07-19

    Host-gut microbial interactions contribute to human health and disease states and an important manifestation resulting from this cometabolism is a vast diversity of bile acids (BAs). There is increasing interest in using BAs as biomarkers to assess the health status of individuals and, therefore, an increased need for their accurate separation and identification. In this study, the negative ion fragmentation behaviors of C24 BAs were investigated by UPLC-ESI-QTOF-MS. The step-by-step fragmentation analysis revealed a distinct fragmentation mechanism for the unconjugated BAs containing a 12-hydroxyl group. The unconjugated BAs lacking 12-hydroxylation fragmented via dehydration and dehydrogenation. In contrast, the 12-hydroxylated ones, such as deoxycholic acid (DCA) and cholic acid (CA), employed dissociation routes including dehydration, loss of carbon monoxide or carbon dioxide, and dehydrogenation. All fragmentations of the 12-hydroxylated unconjugated BAs, characterized by means of stable isotope labeled standards, were associated with the rotation of the carboxylate side chain and the subsequent rearrangements accompanied by proton transfer between 12-hydroxyl and 24-carboxyl groups. Compared to DCA, CA underwent further cleavages of the steroid skeleton. Accordingly, the effects of stereochemistry on the fragmentation pattern of CA were investigated using its stereoisomers. Based on the knowledge gained from the fragmentation analysis, a novel BA, 3β,7β,12α-trihydroxy-5β-cholanic acid, was identified in the postprandial urine samples of patients with nonalcoholic steatohepatitis. The analyses used in this study may contribute to a better understanding of the chemical diversity of BAs and the molecular basis of human liver diseases that involve BA synthesis, transport, and metabolism. PMID:27322813

  11. 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.

  12. 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

  13. Monitoring β-arrestin recruitment via β-lactamase enzyme fragment complementation: purification of peptide E as a low-affinity ligand for mammalian bombesin receptors.

    PubMed

    Ikeda, Yuichi; Kumagai, Hidetoshi; Okazaki, Hiroaki; Fujishiro, Mitsuhiro; Motozawa, Yoshihiro; Nomura, Seitaro; Takeda, Norifumi; Toko, Haruhiro; Takimoto, Eiki; Akazawa, Hiroshi; Morita, Hiroyuki; Suzuki, Jun-ichi; Yamazaki, Tsutomu; Komuro, Issei; Yanagisawa, Masashi

    2015-01-01

    Identification of cognate ligands for G protein-coupled receptors (GPCRs) provides a starting point for understanding novel regulatory mechanisms. Although GPCR ligands have typically been evaluated through the activation of heterotrimeric G proteins, recent studies have shown that GPCRs signal not only through G proteins but also through β-arrestins. As such, monitoring β-arrestin signaling instead of G protein signaling will increase the likelihood of identifying currently unknown ligands, including β-arrestin-biased agonists. Here, we developed a cell-based assay for monitoring ligand-dependent GPCR-β-arrestin interaction via β-lactamase enzyme fragment complementation. Inter alia, β-lactamase is a superior reporter enzyme because of its cell-permeable fluorescent substrate. This substrate makes the assay non-destructive and compatible with fluorescence-activated cell sorting (FACS). In a reporter cell, complementary fragments of β-lactamase (α and ω) were fused to β-arrestin 2 and GPCR, respectively. Ligand stimulation initiated the interaction of these chimeric proteins (β-arrestin-α and GPCR-ω), and this inducible interaction was measured through reconstituted β-lactamase activity. Utilizing this system, we screened various mammalian tissue extracts for agonistic activities on human bombesin receptor subtype 3 (hBRS3). We purified peptide E as a low-affinity ligand for hBRS3, which was also found to be an agonist for the other two mammalian bombesin receptors such as gastrin-releasing peptide receptor (GRPR) and neuromedin B receptor (NMBR). Successful purification of peptide E has validated the robustness of this assay. We conclude that our newly developed system will facilitate the discovery of GPCR ligands.

  14. R vs. S fluoroproline ring substitution: trans/cis effects on the formation of b2 ions in gas-phase peptide fragmentation.

    PubMed

    Bernier, Matthew C; Chamot-Rooke, Julia; Wysocki, Vicki H

    2016-01-21

    The b2 structures of model systems Xxx-Flp-Ala (Flp = 4R-fluoroproline) and Xxx-flp-Ala (flp = 4S-fluoroproline) (where Xxx is Val or Tyr) were studied by action IRMPD spectroscopy. Proline ring substitutions influence the trans/cis isomerization of the precursor ion, resulting in different b2 fragment ion structures by collision induced dissociation. Vibrational spectra of the b2 ions of Val-Flp and Val-flp exhibit highly intense bands at ~1970 cm(-1), revealing that the dominant ion in each case is an oxazolone. The major difference between the spectra of b2 ions for R vs. S fluoroproline is a collection of peaks at 1690 and 1750 cm(-1), characteristic of a diketopiperazine structure, which were only present in the 4S-fluoroproline (flp) cases. This suggests only one b2 ion structure (oxazolone) is being formed for Flp-containing peptides, whereas flp-containing peptides produce a mixture of a dominant oxazolone with a lower population of diketopiperazine. In solution, Flp is known to possess a higher trans percentage in the N-terminally adjacent peptide bond, with flp inducing a greater proportion of the cis conformation. The diketopiperazine formation observed here correlates directly with the Ktrans/cis trend previously shown in solution, highlighting that the trans/cis isomerization likelihood for proline residues modified in the 4(th) position is retained in the gas-phase.

  15. Can collision-induced negative-ion fragmentations of [M-H](-) anions be used to identify phosphorylation sites in peptides?

    PubMed

    Tran, T T Nha; Wang, Tianfang; Hack, Sandra; Hoffmann, Peter; Bowie, John H

    2011-12-15

    A joint experimental and theoretical investigation of the fragmentation behaviour of energised [M-H](-) anions from selected phosphorylated peptides has confirmed some of the most complex rearrangement processes yet to be reported for peptide negative ions. In particular: pSer and pThr (like pTyr) may transfer phosphate groups to C-terminal carboxyl anions and to the carboxyl anion side chains of Asp and Glu, and characteristic nucleophilic/cleavage reactions accompany or follow these rearrangements. pTyr may transfer phosphate to the side chains of Ser and Thr. The reverse reaction, namely transfer of a phosphate group from pSer or pThr to Tyr, is energetically unfavourable in comparison. pSer can transfer phosphate to a non-phosphorylated Ser. The non-rearranged [M-H](-) species yields more abundant product anions than its rearranged counterpart. If a peptide containing any or all of Ser, Thr and Tyr is not completely phosphorylated, negative-ion cleavages can determine the number of phosphated residues, and normally the positions of Ser, Thr and Tyr, but not which specific residues are phosphorylated. This is in accord with comments made earlier by Lehmann and coworkers.

  16. R vs. S fluoroproline ring substitution: trans/cis effects on the formation of b2 ions in gas-phase peptide fragmentation.

    PubMed

    Bernier, Matthew C; Chamot-Rooke, Julia; Wysocki, Vicki H

    2016-01-21

    The b2 structures of model systems Xxx-Flp-Ala (Flp = 4R-fluoroproline) and Xxx-flp-Ala (flp = 4S-fluoroproline) (where Xxx is Val or Tyr) were studied by action IRMPD spectroscopy. Proline ring substitutions influence the trans/cis isomerization of the precursor ion, resulting in different b2 fragment ion structures by collision induced dissociation. Vibrational spectra of the b2 ions of Val-Flp and Val-flp exhibit highly intense bands at ~1970 cm(-1), revealing that the dominant ion in each case is an oxazolone. The major difference between the spectra of b2 ions for R vs. S fluoroproline is a collection of peaks at 1690 and 1750 cm(-1), characteristic of a diketopiperazine structure, which were only present in the 4S-fluoroproline (flp) cases. This suggests only one b2 ion structure (oxazolone) is being formed for Flp-containing peptides, whereas flp-containing peptides produce a mixture of a dominant oxazolone with a lower population of diketopiperazine. In solution, Flp is known to possess a higher trans percentage in the N-terminally adjacent peptide bond, with flp inducing a greater proportion of the cis conformation. The diketopiperazine formation observed here correlates directly with the Ktrans/cis trend previously shown in solution, highlighting that the trans/cis isomerization likelihood for proline residues modified in the 4(th) position is retained in the gas-phase. PMID:26690386

  17. 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.

  18. A tandem mass spectrometric study of bile acids: interpretation of fragmentation pathways and differentiation of steroid isomers.

    PubMed

    Qiao, Xue; Ye, Min; Liu, Chun-fang; Yang, Wen-zhi; Miao, Wen-juan; Dong, Jing; Guo, De-an

    2012-02-01

    Bile acids are steroids with a pentanoic acid substituent at C-17. They are the terminal products of cholesterol excretion, and play critical physiological roles in human and animals. Bile acids are easy to detect but difficult to identify by using mass spectrometry due to their poly-ring structure and various hydroxylation patterns. In this study, fragmentation pathways of 18 free and conjugated bile acids were interpreted by using tandem mass spectrometry. The analyses were conducted on ion trap and triple quadrupole mass spectrometers. Upon collision-induced dissociation, the conjugated bile acids could cleave into glycine or taurine related fragments, together with the steroid skeleton. Fragmentations of free bile acids were further elucidated, especially by atmospheric pressure chemical ionization mass spectrometry in positive ion mode. Aside from universally observed neutral losses, eliminations occurred on bile acid carbon rings were proposed for the first time. Moreover, four isomeric 5β-cholanic acid hydroxyl derivatives (3α,6α-, 3α,7β-, 3α,7α-, and 3α,12α-) were differentiated using electrospray ionization in negative ion mode: 3α,7β-OH substituent inclined to eliminate H(2)O and CH(2)O(2) groups; 3α,6α-OH substituent preferred neutral loss of two H(2)O molecules; 3α,12α-OH substituent apt to lose the carboxyl in the form of CO(2) molecule; and 3α,7α-OH substituent exhibited no further fragmentation after dehydration. This study provided specific interpretation for mass spectra of bile acids. The results could contribute to bile acid analyses, especially in clinical assays and metabonomic studies.

  19. 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.

  20. Influence of turn (or fold) and local charge in fragmentation of the peptide analogue molecule CH3CO-Gly-NH2 following single-photon VUV (118.22 nm) ionization.

    PubMed

    Bhattacharya, Atanu; Bernstein, Elliot R

    2011-10-01

    The radical cationic reactivity of the peptide analogue molecule CH(3)CO-Gly-NH(2) is addressed both experimentally and theoretically. The radical cation intermediate of CH(3)CO-Gly-NH(2) is created by single-photon ionization of this molecule at 118.22 nm (~10.5 eV). The two most stable conformers (C(7) and C(5)) of this molecule exhibit different folds along the backbone: the C(7) conformer has a γ-turn structure, and the C(5) conformer has a β-strand structure. The experimental results show that the radical cation intermediate of CH(3)CO-Gly-NH(2) dissociates and generates a fragment-ion signal at 73 amu that is observed through TOFMS. Theoretical results show how the fragment-ion signal at 73 amu is generated by only one conformer of CH(3)CO-Gly-NH(2) (C(7)) and how local charge and specific hydrogen bonding in the molecule influence fragmentation of the radical cation intermediate of CH(3)CO-Gly-NH(2). The specific fold of the molecule controls fragmentation of this reactive radical cation intermediate. Whereas the radical cation of the C(7) conformer dissociates through a hydrogen-transfer mechanism followed by HNCO elimination, the radical cation of the C(5) conformer does not dissociate at all. CASSCF calculations show that positive charge in the radical cationic C(7) conformer is localized at the NH(2)CO moiety of the molecular ion. This site-specific localization of the positive charge enhances the acidity of the terminal NH(2) group, facilitating hydrogen transfer from the NH(2) to the COCH(3) end of the molecular ion. Positive charge in the C(5) conformer of the CH(3)CO-Gly-NH(2) radical cation is, however, localized at the COCH(3) end of the molecular ion, and this conformer does not have enough energy to surmount the energy barrier to dissociation on the ion potential energy surface. CASSCF results show that conformation-specific localization of charge in the CH(3)CO-Gly-NH(2) molecular ion occurs as a result of the different hydrogen

  1. 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

  2. Radical generating coordination complexes as tools for rapid and effective fragmentation and fluorescent labeling of nucleic acids for microchip hybridization.

    SciTech Connect

    Kelly, J. J.; Chernov, B. N.; Mirzabekov, A. D.; Bavykin, S. G.; Biochip Technology Center; Northwestern Univ.; Engelhardt Inst. of Molecular Biology

    2002-01-01

    DNA microchip technology is a rapid, high-throughput method for nucleic acid hybridization reactions. This technology requires random fragmentation and fluorescent labeling of target nucleic acids prior to hybridization. Radical-generating coordination complexes, such as 1,10-phenanthroline-Cu(II) (OP-Cu) and Fe(II)-EDTA (Fe-EDTA), have been commonly used as sequence nonspecific 'chemical nucleases' to introduce single-strand breaks in nucleic acids. Here we describe a new method based on these radical-generating complexes for random fragmentation and labeling of both single- and double-stranded forms of RNA and DNA. Nucleic acids labeled with the OP-Cu and the Fe-EDTA protocols revealed high hybridization specificity in hybridization with DNA microchips containing oligonucleotide probes selected for identification of 16S rRNA sequences of the Bacillus group microorganisms.We also demonstrated cDNA- and cRNA-labeling and fragmentation with this method. Both the OP-Cu and Fe-EDTA fragmentation and labeling procedures are quick and inexpensive compared to other commonly used methods. A column-based version of the described method does not require centrifugation and therefore is promising for the automation of sample preparations in DNA microchip technology as well as in other nucleic acid hybridization studies.

  3. 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.

  4. 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

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. Amino acid sequence of the 203-residue fragment of the heavy chain of chicken gizzard myosin containing the SH1-type cysteine residue.

    PubMed

    Onishi, H; Maita, T; Miyanishi, T; Watanabe, S; Matsuda, G

    1986-12-01

    A fluorescent fragment of Mr = 23,800 was obtained by the papain digestion of N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylene diamine (abbreviated as IAEDANS)-modified chicken gizzard myosin. The fragment was isolated by gel filtration on a Sephadex G-100 column in the presence of 5 M guanidine-HCl followed by anion exchange chromatography on a QAE Sephadex A-50 column. This fragment contained 203 amino acid residues which could be assigned as a COOH-terminal part of the S-1 heavy chain based on the homology with the known sequence of rabbit skeletal myosin fragment. The amino acid sequence was K-G-M-F-R-T-V- G-Q-L-Y-K-E-Q-L-T-K-L-M-T-T-L-R-N-T-N-P-N-F-V-R-C-I-I-P-N-H-E-K-R-A- G-K-L-D-A-H-L-V-L-E-Q-L-R-C-N-G-V-L-E-G-I-R-I-C-R-Q-G-F-P-N-R-I-V-F-Q- E-F-R-Q-R-Y-E-I-L-A-A-N-A-I-P-K-G-F-M-D-G-K-Q-A-C-I-L-M -I-K-A-L-E-L- D-P-N-L-Y-R-I-G-Q-S-K-I-F-F-R-T-G-V-L-A-H-L-E-E-E-R-D-L-K- I-T-D-V-I-I-A- F-Q-A-Q-C-R-G-Y-L-A-R-K-A-F-A-K-R-Q-Q-Q-L-T-A-M-K-V-I-Q-R-N-C-A -A-Y-L-K-L-R-N-W-Q-W-W-R-L-F-T-K-V-K-P-L-L-Q-V-T-R. The cysteine residue which was modified with IAEDANS was of the SH1 type (Cys-65). Pro-197 was suggested to be the NH2-terminal boundary of the alpha-helical coiled-coil rod sequence of gizzard myosin, based on the homology with the nematode sequence reported by MacLachlan and Karn (Proc. Natl. Acad. Sci. U.S. 80, 4253-4257 (1983)). Three different COOH-terminal peptides (Val-Lys-Pro-Leu-Leu-Gln-Val-Thr-Arg, Val-Lys-Pro-Leu-Leu-Gln, and Val-Lys-Pro-Leu-Leu) were isolated from the tryptic digest of this fragment.(ABSTRACT TRUNCATED AT 400 WORDS)

  12. 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.

  13. Interpretation of sucrose gradient sedimentation pattern of deoxyribonucleic acid fragments resulting from random breaks.

    PubMed

    Litwin, S; Shahn, E; Kozinski, A W

    1969-07-01

    Mass distribution in a sucrose gradient of deoxyribonucleic acid (DNA) fragments arising as a result of random breaks is predicted by analytical means from which computer evaluations are plotted. The analytical results are compared with the results of verifying experiments: (i) a Monte Carlo computer experiment in which simulated molecules of DNA were individuals of unit length subjected to random "breaks" applied by a random number generator, and (ii) an in vitro experiment in which molecules of T4 DNA, highly labeled with (32)P, were stored in liquid nitrogen for variable periods of time during which a precisely known number of (32)P atoms decayed, causing single-stranded breaks. The distribution of sizes of the resulting fragments was measured in an alkaline sucrose gradient. The profiles obtained in this fashion were compared with the mathematical predictions. Both experiments agree with the analytical approach and thus permit the use of the graphs obtained from the latter as a means of determining the average number of random breaks in DNA from distributions obtained experimentally in a sucrose gradient. An example of the application of this procedure to a previously unresolved problem is provided in the case of DNA from ultraviolet-irradiated phage which undergoes a dose-dependent intracellular breakdown. The relationship between the number of lethal hits and the number of single-stranded breaks was not previously established. A comparison of the calculated number of nicks per strand of DNA with the known dose in phage-lethal hits reveals a relationship closely approximating one lethal hit to one single-stranded break. PMID:5804949

  14. 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.

  15. Effect of Basic Residue on the Kinetics of Peptide Fragmentation Examined Using Surface-Induced Dissociation Combined with Resonant Ejection

    SciTech Connect

    Laskin, Julia

    2015-11-30

    In this work, resonant ejection coupled with surface-induced dissociation (SID) in a Fourier transform ion cyclotron resonance mass spectrometer is used to examine fragmentation kinetics of two singly protonated hexapeptides, RYGGFL and KYGGFL, containing the basic arginine residue and less basic lysine residue at the N-terminus. The kinetics of individual reaction channels at different collision energies are probed by applying a short ejection pulse (1 ms) in resonance with the cyclotron frequency of a selected fragment ion and varying the delay time between ion-surface collision and resonant ejection while keeping total reaction delay time constant. Rice-Ramsperger-Kassel-Marcus (RRKM) modeling of the experimental data provides accurate threshold energies and activation entropies of individual reaction channels. Substitution of arginine with less basic lysine has a pronounced effect on the observed fragmentation kinetics of several pathways, including the b2 ion formation, but has little or no effect on formation of the b5+H2O fragment ion. The combination of resonant ejection SID, time- and collision energy-resolved SID, and RRKM modeling of both types of experimental data provides a detailed mechanistic understanding of the primary dissociation pathways of complex gaseous ions.

  16. 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).

  17. 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

  18. 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.

  19. Construction, expression, and characterization of a single-chain variable fragment antibody against 2,4-dichlorophenoxyacetic acid in the hemolymph of silkworm larvae.

    PubMed

    Sakamoto, Seiichi; Pongkitwitoon, Benyakan; Nakamura, Seiko; Sasaki-Tabata, Kaori; Tanizaki, Yusuke; Maenaka, Katsumi; Tanaka, Hiroyuki; Morimoto, Satoshi

    2011-07-01

    A single-chain variable fragment antibody against herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D-scFv) has been successfully expressed in the hemolymph of silkworm larvae using a rapid Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system. Variable heavy- and light-chain domains were cloned directly from the cDNA of the hybridoma cell line 2C4 and assembled together with flexible peptide linker (Gly(4)Ser)(3) between two domains. The yield of functional 2,4-D-scFv after purification was 640 μg per 30 ml of hemolymph, which is equivalent to 21.3 mg per liter of hemolymph. The characterization of 2,4-D-scFv using an indirect competitive enzyme-linked immunosorbent assay (icELISA) revealed that it has wide cross-reactivities against 2,4,5-trichlorophenoxyacetic acid (65.5%), 2,4-dichlorophenol (47.9%), and 2,4-dichlorobenzoic acid (26.0%), making it possible to apply 2,4-D-scFv to icELISA for detecting/determining 2,4-D and its metabolites. Judging from its cost and time requirements and its ease of handling, this BmNPV bacmid DNA expression system is more useful for expressing functional scFv than bacterial systems, which frequently require costly and time-consuming refolding.

  20. 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.

  1. NMR studies of the MgATP binding site of adenylate kinase and of a 45-residue peptide fragment of the enzyme.

    PubMed

    Fry, D C; Kuby, S A; Mildvan, A S

    1985-08-13

    Proton NMR was used to study the interaction of beta,gamma-bidentate Cr3+ATP and MgATP with rabbit muscle adenylate kinase, which has 194 amino acids, and with a synthetic peptide consisting of residues 1-45 of the enzyme, which has previously been shown to bind MgepsilonATP [Hamada, M., Palmieri, R. H., Russell, G. A., & Kuby, S. A. (1979) Arch. Biochem. Biophys. 195, 155-177]. The peptide is globular and binds Cr3+ATP competitively with MgATP with a dissociation constant, KD(Cr3+ATP) = 35 microM, comparable to that of the complete enzyme [KI(Cr3+ATP) = 12 microM]. Time-dependent nuclear Overhauser effects (NOE's) were used to measure interproton distances on enzyme- and peptide-bound MgATP. The correlation time was measured directly for peptide-bound MgATP by studying the frequency dependence of the NOE's at 250 and 500 MHz. The H2' to H1' distance so obtained (3.07 A) was within the range established by X-ray and model-building studies of nucleotides (2.9 +/- 0.2 A). Interproton distances yielded conformations of enzyme- and peptide-bound MgATP with indistinguishable anti-glycosyl torsional angles (chi = 63 +/- 12 degrees) and 3'-endo/O1'-endo ribose puckers (sigma = 96 +/- 12 degrees). Enzyme- and peptide-bound MgATP molecules exhibited different C4'-C5' torsional angles (gamma) of 170 degrees and 50 degrees, respectively. Ten intermolecular NOE's from protons of the enzyme and four such NOE's from protons of the peptide to protons of bound MgATP were detected, which indicated proximity of the adenine ribose moiety to the same residues on both the enzyme and the peptide. Paramagnetic effects of beta,gamma-bidentate Cr3+ATP on the longitudinal relaxation rates of protons of the peptide provided a set of distances to the side chains of five residues, which allowed the location of the bound Cr3+ atom to be uniquely defined. Distances from enzyme-bound Cr3+ATP to the side chains of three residues of the protein agreed with those measured for the peptide. The mutual

  2. Enzymatic fragmentation of the antimicrobial peptides casocidin and isracidin by Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus.

    PubMed

    Somkuti, George A; Paul, Moushumi

    2010-06-01

    The cumulative effect of peptidase and protease activities associated with cells of Streptococcus thermophilus (ST) and Lactobacillus delbrueckii subsp. bulgaricus (LB) was evaluated on the milk protein-based antimicrobial peptides casocidin and isracidin. Reaction mixtures of casocidin or isracidin and nonproliferating mid-log cells of these essential yogurt starter cultures were individually incubated for up to 4 h at pH 4.5 and 7.0, and samples removed at various time points were analyzed by reverse phase-high performance liquid chromatography (RP-HPLC) and MALDI-TOF/TOF-MS. Both casocidin and isracidin remained largely unchanged following exposure to cell suspensions of ST or LB strains at pH 4.5. Casocidin was extensively degraded by both ST and LB strains at pH 7.0, whereas isracidin remained largely intact after incubation for 4 h with ST strains but was degraded by exposure to LB strains. The results showed the feasibility of using the bovine casein-based peptides casocidin and isracidin as food grade antimicrobial supplements to impart fermented dairy foods additional protection against bacterial contamination. The structural integrity and efficacy of these biodefensive peptides may be preserved by timing their addition near the end of the fermentation of yogurt-like dairy foods (at or below pH 4.5), when conditions for bacterial proteolytic activity become unfavorable.

  3. 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

  4. 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.

  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. Association of restriction fragment length polymorphism at the atrial natriuretic peptide gene locus with aldosterone responsiveness to angiotensin in aldosterone-producing adenoma.

    PubMed

    Tunny, T J; Jonsson, J R; Klemm, S A; Ballantine, D M; Stowasser, M; Gordon, R D

    1994-11-15

    Primary aldosteronism is an important, potentially curable, form of hypertension. We examined the possible association between restriction fragment length polymorphisms in the atrial natriuretic peptide (ANP) gene and responsiveness of aldosterone to angiotensin II in 59 patients with primary aldosteronism due to aldosterone-producing adenoma (APA). Significant differences in the allelic frequencies of the BglI, TaqI and XhoI polymorphic sites at the ANP gene locus (chromosome 1; 1p36) between angiotensin II-unresponsive and angiotensin II-responsive tumors were observed. Variation in the ANP gene between the two groups may result in altered expression of ANP within the adrenal gland, and may contribute to the biochemical regulation of aldosterone production of these two subgroups of patients with APA.

  8. 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)

  9. 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.

  10. 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.

  11. 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.

  12. Anti-angiogenic peptides for cancer therapeutics.

    PubMed

    Rosca, Elena V; Koskimaki, Jacob E; Rivera, Corban G; Pandey, Niranjan B; Tamiz, Amir P; Popel, Aleksander S

    2011-08-01

    Peptides have emerged as important therapeutics that are being rigorously tested in angiogenesis-dependent diseases due to their low toxicity and high specificity. Since the discovery of endogenous proteins and protein fragments that inhibit microvessel formation (thrombospondin, endostatin) several peptides have shown promise in pre-clinical and clinical studies for cancer. Peptides have been derived from thrombospondin, collagens, chemokines, coagulation cascade proteins, growth factors, and other classes of proteins and target different receptors. Here we survey recent developments for anti-angiogenic peptides with length not exceeding 50 amino acid residues that have shown activity in pre-clinical models of cancer or have been tested in clinical trials; some of the peptides have been modified and optimized, e.g., through L-to-D and non-natural amino acid substitutions. We highlight technological advances in peptide discovery and optimization including computational and bioinformatics tools and novel experimental techniques.

  13. 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.

  14. 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

  15. 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.

  16. A fragment of the LG3 peptide of endorepellin is present in the urine of physically active mining workers: a potential marker of physical activity.

    PubMed

    Parker, Tony J; Sampson, Dayle L; Broszczak, Daniel; Chng, Yee L; Carter, Shea L; Leavesley, David I; Parker, Anthony W; Upton, Zee

    2012-01-01

    Biomarker analysis has been implemented in sports research in an attempt to monitor the effects of exertion and fatigue in athletes. This study proposed that while such biomarkers may be useful for monitoring injury risk in workers, proteomic approaches might also be utilised to identify novel exertion or injury markers. We found that urinary urea and cortisol levels were significantly elevated in mining workers following a 12 hour overnight shift. These levels failed to return to baseline over 24 h in the more active maintenance crew compared to truck drivers (operators) suggesting a lack of recovery between shifts. Use of a SELDI-TOF MS approach to detect novel exertion or injury markers revealed a spectral feature which was associated with workers in both work categories who were engaged in higher levels of physical activity. This feature was identified as the LG3 peptide, a C-terminal fragment of the anti-angiogenic/anti-tumourigenic protein endorepellin. This finding suggests that urinary LG3 peptide may be a biomarker of physical activity. It is also possible that the activity mediated release of LG3/endorepellin into the circulation may represent a biological mechanism for the known inverse association between physical activity and cancer risk/survival.

  17. 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

  18. Crystallization and preliminary X-ray diffraction analysis of the Fab fragment of WO2, an antibody specific for the A[beta] peptides associated with Alzheimer's disease

    SciTech Connect

    Wun, Kwok S.; Miles, Luke A.; Crespi, Gabriela A.N.; Wycherley, Kaye; Ascher, David B.; Barnham, Kevin J.; Cappai, Roberto; Beyreuther, Konrad; Masters, Colin L.; Parker, Michael W.; McKinstry, William J.

    2008-05-28

    The murine monoclonal antibody WO2 specifically binds the N-terminal region of the amyloid {beta} peptide (A{beta}) associated with Alzheimer's disease. This region of A{beta} has been shown to be the immunodominant B-cell epitope of the peptide and hence is considered to be a basis for the development of immunotherapeutic strategies against this prevalent cause of dementia. Structural studies have been undertaken in order to characterize the molecular basis for antibody recognition of this important epitope. Here, details of the crystallization and X-ray analysis of the Fab fragment of the unliganded WO2 antibody in two crystal forms and of the complexes that it forms with the truncated Az{beta} peptides A{beta}{sub 1-16} and A{beta}{sub 1-28} are presented. These crystals were all obtained using the hanging-drop vapour-diffusion method at 295 K. Crystals of WO2 Fab were grown in polyethylene glycol solutions containing ZnSO{sub 4}; they belonged to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1} and diffracted to 1.6 {angstrom} resolution. The complexes of WO2 Fab with either A{beta}{sub 1-16} or A{beta}{sub 1-28} were cocrystallized from polyethylene glycol solutions. These two complex crystals grew in the same space group, P2{sub 1}2{sub 1}2{sub 1}, and diffracted to 1.6 {angstrom} resolution. A second crystal form of WO2 Fab was grown in the presence of the sparingly soluble A{beta}{sub 1-42} in PEG 550 MME. This second form belonged to space group P2{sub 1} and diffracted to 1.9 {angstrom} resolution.

  19. Specific cleavage at peptide backbone Cα-C and CO-N bonds during matrix-assisted laser desorption/ionization in-source decay mass spectrometry with 5-nitrosalicylic acid as the matrix.

    PubMed

    Asakawa, Daiki; Takayama, Mitsuo

    2011-09-15

    The use of 5-nitrosalicylic acid (5-NSA) as a matrix for in-source decay (ISD) of peptides during matrix-assisted laser desorption/ionization (MALDI) is described herein. Mechanistically, the decay process is initiated by a hydrogen abstraction from a peptide backbone amide nitrogen by 5-NSA. Hydrogen abstraction results in formation of an oxidized peptide containing a radical amide nitrogen. Subsequently, the C(α)-C bond N-terminal to the peptide bond is cleaved to form an a·/x fragment pair. The C(α)-C bonds C-terminal to Gly residues were less susceptible to cleavage than were those of other residues. C(α)-C bonds N-terminal to Pro and Sar residues were not cleaved by the aforementioned mechanism; instead, after hydrogen abstraction from a Pro or Sar C(α)-H bond, the peptide bond N-terminal to the Pro was cleaved yielding b- and y-series ions. We also show that fragments produced by MALDI 5-NSA-induced ISD were formed independently of the ionization process.

  20. Peptide Fragments of Odin-Sam1: Conformational Analysis and Interaction Studies with EphA2-Sam.

    PubMed

    Mercurio, Flavia A; Di Natale, Concetta; Pirone, Luciano; Scognamiglio, Pasqualina L; Marasco, Daniela; Pedone, Emilia M; Saviano, Michele; Leone, Marilisa

    2015-07-27

    Odin is a protein belonging to the ANKS family, and has two tandem Sam domains. The first, Odin-Sam1, binds to the Sam domain of the EphA2 receptor (EphA2-Sam); this interaction could be crucial for the regulation of receptor endocytosis and might have an impact on cancer. Odin-Sam1 associates with EphA2-Sam by adopting a "mid-loop/end-helix" model. In this study three peptide sequences, encompassing the mid-loop interacting portion of Odin-Sam1 and its C-terminal α5 helix, were designed. Their conformational properties were analyzed by CD and NMR. In addition, their abilities to interact with EphA2-Sam were investigated by SPR studies. The peptides adopt a predominantly disordered state in aqueous buffer, but a higher helical content is evident in the presence of the cosolvent trifluoroethanol. Dissociation constants towards EphA2-Sam were in the high micromolar range. The structural findings suggest further routes for the design of potential anti-cancer therapeutics as inhibitors of EphA2-Sam heterotypic interactions.

  1. Peptide Fragments of Odin-Sam1: Conformational Analysis and Interaction Studies with EphA2-Sam.

    PubMed

    Mercurio, Flavia A; Di Natale, Concetta; Pirone, Luciano; Scognamiglio, Pasqualina L; Marasco, Daniela; Pedone, Emilia M; Saviano, Michele; Leone, Marilisa

    2015-07-27

    Odin is a protein belonging to the ANKS family, and has two tandem Sam domains. The first, Odin-Sam1, binds to the Sam domain of the EphA2 receptor (EphA2-Sam); this interaction could be crucial for the regulation of receptor endocytosis and might have an impact on cancer. Odin-Sam1 associates with EphA2-Sam by adopting a "mid-loop/end-helix" model. In this study three peptide sequences, encompassing the mid-loop interacting portion of Odin-Sam1 and its C-terminal α5 helix, were designed. Their conformational properties were analyzed by CD and NMR. In addition, their abilities to interact with EphA2-Sam were investigated by SPR studies. The peptides adopt a predominantly disordered state in aqueous buffer, but a higher helical content is evident in the presence of the cosolvent trifluoroethanol. Dissociation constants towards EphA2-Sam were in the high micromolar range. The structural findings suggest further routes for the design of potential anti-cancer therapeutics as inhibitors of EphA2-Sam heterotypic interactions. PMID:26120079

  2. Glycoprotein identification and localization of O-glycosylation sites by mass spectrometric analysis of deglycosylated/alkylaminylated peptide fragments.

    PubMed

    Hanisch, F G; Jovanovic, M; Peter-Katalinic, J

    2001-03-01

    In-gel digestion of densely O-glycosylated proteins, an essential step in proteome analysis, is often hampered by steric hindrance of the proteases. To overcome this technical problem a simple and convenient method has been developed, which combines several advantages: (1) Approximately 70% of the oligosaccharides are cleaved without significant protein hydrolysis at the optimal reaction conditions of 70% ethylamine, and quantitative cleavage is achieved with 40% methylamine, at 50 degrees C. (2) To the unsaturated derivatives of Ser and Thr the alkylamine is added as a label of previous O-glycosylation sites. (3) The alkylaminylated protein is effectively cleaved by proteolysis. (4) The modified peptides are identified by MALDI mass spectrometry under consideration of incremental mass increases. (5) The alkylamine label is stable under MALDI post-source-decay analysis as well as in collision-induced dissociation experiments allowing sequencing and peptide localization of O-glycosylation sites. Applicability of the method is evaluated with a series of synthetic glycopeptides, the densely O-glycosylated human glycophorin A, and with the mucin MUC1 from human milk fat globule membranes.

  3. Structures of endothiapepsin-fragment complexes from crystallographic fragment screening using a novel, diverse and affordable 96-compound fragment library.

    PubMed

    Huschmann, Franziska U; Linnik, Janina; Sparta, Karine; Ühlein, Monika; Wang, Xiaojie; Metz, Alexander; Schiebel, Johannes; Heine, Andreas; Klebe, Gerhard; Weiss, Manfred S; Mueller, Uwe

    2016-05-01

    Crystallographic screening of the binding of small organic compounds (termed fragments) to proteins is increasingly important for medicinal chemistry-oriented drug discovery. To enable such experiments in a widespread manner, an affordable 96-compound library has been assembled for fragment screening in both academia and industry. The library is selected from already existing protein-ligand structures and is characterized by a broad ligand diversity, including buffer ingredients, carbohydrates, nucleotides, amino acids, peptide-like fragments and various drug-like organic compounds. When applied to the model protease endothiapepsin in a crystallographic screening experiment, a hit rate of nearly 10% was obtained. In comparison to other fragment libraries and considering that no pre-screening was performed, this hit rate is remarkably high. This demonstrates the general suitability of the selected compounds for an initial fragment-screening campaign. The library composition, experimental considerations and time requirements for a complete crystallographic fragment-screening campaign are discussed as well as the nine fully refined obtained endothiapepsin-fragment structures. While most of the fragments bind close to the catalytic centre of endothiapepsin in poses that have been observed previously, two fragments address new sites on the protein surface. ITC measurements show that the fragments bind to endothiapepsin with millimolar affinity. PMID:27139825

  4. 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

  5. Function of streptokinase fragments in plasminogen activation.

    PubMed Central

    Shi, G Y; Chang, B I; Chen, S M; Wu, D H; Wu, H L

    1994-01-01

    Several peptide fragments of streptokinase (SK) were prepared by incubating SK with immobilized human plasmin (hPlm) and purified by h.p.l.c. with a reverse-phase phenyl column. The N-terminal sequences, amino acid compositions and molecular masses of these peptide fragments were determined. The SK peptide fragment of 36 kDa consisting of Ser60-Lys387 (SK-p), was the only peptide fragment that could be tightly bound to immobilized hPlm. Another three large SK peptide fragments, SK-m, SK-n and SK-o, with molecular masses of 7 kDa, 18 kDa and 30 kDa, and consisting of Ile1-Lys59, Glu148-Lys333, Ser60-Lys333 respectively, were also obtained from the supernatant of the reaction mixture. The purified SK-p had high affinity with hPlm and could activate human plasminogen (hPlg) with a kPlg one-sixth that of the native SK. SK-o had low affinity with hPlm and could also activate hPlg, although the catalytic constant was less than 1% of the native SK. SK-n, as well as SK-m, which is the N-terminal 59 amino acid peptide of the native SK, had no activator activity. However, SK-m could enhance the activator activity of both SK-o and SK-p and increase their second-order rate constants by two- and six-fold respectively. It was concluded from these studies that (1) SK-o, the Ser60-Lys333 peptide of SK, was essential for minimal SK activator activity, (2) the C-terminal peptide of SK-p, Ala334-Lys387, was essential for high affinity with hPlm, and (3) the N-terminal 59-amino-acid peptide was important in maintaining the proper conformation of SK to have its full activator activity. Images Figure 1 Figure 2 PMID:7998939

  6. The fragmentations of [M-H]- anions derived from underivatised peptides. The side-chain loss of H2S from Cys. A joint experimental and theoretical study.

    PubMed

    Bilusich, Daniel; Brinkworth, Craig S; McAnoy, Andrew M; Bowie, John H

    2003-01-01

    Loss of H2S is the characteristic Cys side-chain fragmentation of the [M-H]- anions of Cys-containing peptides. A combination of experiment and theory suggests that this reaction is initiated from the Cys enolate anion as follows: RNH-(-)C(CH2SH)CONHR' Ø [RNHC(=CH2)CONHR' (HS-)] Ø [RNHC(=CH2)CO-HNR'-H]-+H2S. This process is facile. Calculations at the HF/6-31G(d)//AM1 level of theory indicate that the initial anion needs only > or =20.1 kcal mol(-1) of excess energy to effect loss of H2S. Loss of CH2S is a minor process, RNHCH(CH2SH)CON(-)-R' Ø RNHCH(CH2S-)CONHR' Ø RNH -CHCONHR+CH2S, requiring an excess energy of > or =50.2 kcal mol(-1). When Cys occupies the C-terminal end of a peptide, the major fragmentation from the [M-H]- species involves loss of (H2S+CO2). A deuterium-labelling study suggests that this could either be a charge-remote reaction (a process which occurs remote from and uninfluenced by the charged centre in the molecule), or an anionic reaction initiated from the C-terminal CO2- group. These processes have barriers requiring the starting material to have an excess energy of > or =79.6 (charge-remote) or > or =67.1 (anion-directed) kcal mol(-1), respectively, at the HF/6-31G(d)//AM1 level of theory. The corresponding losses of CH2O and H2O from the [M-H]- anions of Ser-containing peptides require > or =35.6 and > or =44.4 kcal mol(-1) of excess energy (calculated at the AM1 level of theory), explaining why loss of CH2O is the characteristic side-chain loss of Ser in the negative ion mode. PMID:14608618

  7. 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.

  8. Amino-terminal fragment of C-type natriuretic peptide precursor and C-type natriuretic peptide do not correlate in patients with Chagas disease: role for neutral endopeptidase.

    PubMed

    Wang, Yong; Moreira, Maria da Consolação V; Heringer-Walther, Silvia; Schultheiss, Heinz-Peter; Siems, Wolf-Eberhard; Wessel, Niels; Walther, Thomas

    2010-01-01

    Atrial and B-type natriuretic peptides (ANP and BNP), but not C-type natriuretic peptide (CNP), have been identified to be diagnostic and prognostic markers in Chagas disease (CD). Although ANP and BNP excessively rise in patients with CD, increase in CNP is just minor. Our study aimed to investigate the mechanisms leading to CNP insensitivity to heart failure (HF) stimuli. Amino-terminal fragment of CNP precursor (NT-proCNP) and activity of neutral endopeptidase (NEP) were quantified to monitor CNP generation and degradation, respectively. Blood samples were collected from patients with CD and control healthy subjects. NT-proCNP concentrations were significantly lower in patients with CD without systolic dysfunction compared with healthy subjects. Despite a trend toward increase with rising heart failure clinical severity, it was significantly correlated with left ventricular ejection fraction and other echocardiographic parameters. As shown for CNP before, NT-proCNP could not predict mortality and heart transplant. Importantly, it had no statistical correlation with CNP. Additionally, NEP activity was significantly increased in New York Heart Association III and IV patients with HF but was positively correlated with CNP concentration. Our data demonstrates that generation of CNP is not enhanced under HF condition like CD. Thus, CNP rise by severe HF is caused by its less degradation that is independent of NEP activity.

  9. 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.

  10. 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.

  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. A 193-amino acid fragment of the SARS coronavirus S protein efficiently binds angiotensin-converting enzyme 2.

    PubMed

    Wong, Swee Kee; Li, Wenhui; Moore, Michael J; Choe, Hyeryun; Farzan, Michael

    2004-01-30

    The coronavirus spike (S) protein mediates infection of receptor-expressing host cells and is a critical target for antiviral neutralizing antibodies. Angiotensin-converting enzyme 2 (ACE2) is a functional receptor for the coronavirus (severe acute respiratory syndrome (SARS)-CoV) that causes SARS. Here we demonstrate that a 193-amino acid fragment of the S protein (residues 318-510) bound ACE2 more efficiently than did the full S1 domain (residues 12-672). Smaller S protein fragments, expressing residues 327-510 or 318-490, did not detectably bind ACE2. A point mutation at aspartic acid 454 abolished association of the full S1 domain and of the 193-residue fragment with ACE2. The 193-residue fragment blocked S protein-mediated infection with an IC(50) of less than 10 nm, whereas the IC(50) of the S1 domain was approximately 50 nm. These data identify an independently folded receptor-binding domain of the SARS-CoV S protein.

  13. 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.

  14. [Synthesis and immunogenic properties of peptides--fragments of the immunodominant regions of the VP1 protein of the Asia-1 type of foot- and-mouth disease virus].

    PubMed

    Petrov, V N; Rybakov, S S; Petrova, O N; Chepurkin, A V; Gulenkin, V M

    1998-12-01

    Potential immunodominant epitopes were predicted on the basis of a theoretical analysis of the antigenic structure of the VP1 protein of the type Asia-1 foot-and-mouth disease virus. Peptides corresponding to the 140-153, 136-153, 132-153, 143-157, 137-157, and 193-208 fragments of the VP1 protein sequence were synthesized by the solid phase method, and the immunogenic properties of the peptides were studied on guinea pigs. The shortest peptide exhibiting the protective effect was found to correspond to the, 140-153 fragment of the VP1 sequence. The Plm-(Gly)3-(140-153)-(Gly)2-Lys(Plm)-Leu and [Ac-(140-153)-(Gly)3]8-(Lys)7-Gly synthetic constructions in combination with adjuvants provided up to 80% protection of immunized animals against infection with the foot-and-mouth disease virus.

  15. Influence of the solvent on the self-assembly of a modified amyloid beta peptide fragment. II. NMR and computer simulation investigation.

    PubMed

    Hamley, I W; Nutt, D R; Brown, G D; Miravet, J F; Escuder, B; Rodríguez-Llansola, F

    2010-01-21

    The conformation of a model peptide AAKLVFF based on a fragment of the amyloid beta peptide Abeta16-20, KLVFF, is investigated in methanol and water via solution NMR experiments and molecular dynamics computer simulations. In previous work, we have shown that AAKLVFF forms peptide nanotubes in methanol and twisted fibrils in water. Chemical shift measurements were used to investigate the solubility of the peptide as a function of concentration in methanol and water. This enabled the determination of critical aggregation concentrations. The solubility was lower in water. In dilute solution, diffusion coefficients revealed the presence of intermediate aggregates in concentrated solution, coexisting with NMR-silent larger aggregates, presumed to be beta-sheets. In water, diffusion coefficients did not change appreciably with concentration, indicating the presence mainly of monomers, coexisting with larger aggregates in more concentrated solution. Concentration-dependent chemical shift measurements indicated a folded conformation for the monomers/intermediate aggregates in dilute methanol, with unfolding at higher concentration. In water, an antiparallel arrangement of strands was indicated by certain ROESY peak correlations. The temperature-dependent solubility of AAKLVFF in methanol was well described by a van't Hoff analysis, providing a solubilization enthalpy and entropy. This pointed to the importance of solvophobic interactions in the self-assembly process. Molecular dynamics simulations constrained by NOE values from NMR suggested disordered reverse turn structures for the monomer, with an antiparallel twisted conformation for dimers. To model the beta-sheet structures formed at higher concentration, possible model arrangements of strands into beta-sheets with parallel and antiparallel configurations and different stacking sequences were used as the basis for MD simulations; two particular arrangements of antiparallel beta-sheets were found to be stable, one

  16. 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

  17. 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

  18. 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.

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. Competitive fragmentation pathways of acetic acid dimer explored by synchrotron VUV photoionization mass spectrometry and electronic structure calculations

    SciTech Connect

    Guan Jiwen; Hu Yongjun; Zou Hao; Cao Lanlan; Liu Fuyi; Shan Xiaobin; Sheng Liusi

    2012-09-28

    In present study, photoionization and dissociation of acetic acid dimers have been studied with the synchrotron vacuum ultraviolet photoionization mass spectrometry and theoretical calculations. Besides the intense signal corresponding to protonated cluster ions (CH{sub 3}COOH){sub n}{center_dot}H{sup +}, the feature related to the fragment ions (CH{sub 3}COOH)H{sup +}{center_dot}COO (105 amu) via {beta}-carbon-carbon bond cleavage is observed. By scanning photoionization efficiency spectra, appearance energies of the fragments (CH{sub 3}COOH){center_dot}H{sup +} and (CH{sub 3}COOH)H{sup +}{center_dot}COO are obtained. With the aid of theoretical calculations, seven fragmentation channels of acetic acid dimer cations were discussed, where five cation isomers of acetic acid dimer are involved. While four of them are found to generate the protonated species, only one of them can dissociate into a C-C bond cleavage product (CH{sub 3}COOH)H{sup +}{center_dot}COO. After surmounting the methyl hydrogen-transfer barrier 10.84 {+-} 0.05 eV, the opening of dissociative channel to produce ions (CH{sub 3}COOH){sup +} becomes the most competitive path. When photon energy increases to 12.4 eV, we also found dimer cations can be fragmented and generate new cations (CH{sub 3}COOH){center_dot}CH{sub 3}CO{sup +}. Kinetics, thermodynamics, and entropy factors for these competitive dissociation pathways are discussed. The present report provides a clear picture of the photoionization and dissociation processes of the acetic acid dimer in the range of the photon energy 9-15 eV.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. Requirement of novel amino acid fragments of orphan nuclear receptor TR3/Nur77 for its functions in angiogenesis

    PubMed Central

    Grant, Marianne A.; Peng, Jin; Ye, Taiyang; Zhao, Dezheng; Zeng, Huiyan

    2015-01-01

    Pathological angiogenesis is a hallmark of many diseases. We demonstrated that TR3/Nur77 is an excellent target for pro-angiogenesis and anti-angiogenesis therapies. Here, we report that TR3 transcriptionally regulates endothelial cell migration, permeability and the formation of actin stress fibers that is independent of RhoA GTPase. 1) Amino acid residues 344-GRR-346 and de-phosphorylation of amino acid residue serine 351 in the DNA binding domain, and 2) phosphorylation of amino acid residues in the 41-61 amino acid fragment of the transactivation domain, of TR3 are required for its induction of the formation of actin stress fibers, cell proliferation, migration and permeability. The 41-61 amino acid fragment contains one of the three potential protein interaction motifs in the transactivation domain of TR3, predicted by computational modeling and analysis. These studies further our understanding of the molecular mechanism, by which TR3 regulates angiogenesis, identify novel therapeutic targeted sites of TR3, and set the foundation for the development of high-throughput screening assays to identify compounds targeting TR3/Nur77 for pro-angiogenesis and anti-angiogenesis therapies. PMID:26155943

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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

  17. 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.

  18. 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

  19. 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

  20. 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

  1. 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.

  2. 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

  3. 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

  4. Relationship between pregnancy, embryo development, and sperm deoxyribonucleic acid fragmentation dynamics.

    PubMed

    Wdowiak, Artur; Bojar, Iwona

    2016-09-01

    The way the dynamics of DNA fragmentation affects the growth of embryos in real time, and effectiveness of infertility treatment using the ICSI procedure were determined in 148 couples treated with the ICSI technique. The percentage of sperm with fragmented DNA (known as the DNA fragmentation index [DFI]) in semen samples was determined at 3, 6 and 12 h. Embryo culture was assessed continuously during 12 h of observation monitoring. Statistically significant difference was found in DFI at 12 h and outcome of treatment. For the remaining time intervals, no statistically significant differences were noted. An analysis of relationship between the DFI dynamics over time at individual measurements and achievement of pregnancy, confirmed a statistically significant relationship between the rate measured at 6-12 h of observations of DFI changes (DFI 12 h%/h), and achieving pregnancy. Correlation was observed between DFI (during 0, 3, 6 and 12 h), the growth rate in DFI, and time of embryo development. A statistically significant relationship was found between the rate from the start to the end of observations of the DFI, and outcome of treatment. Intensity level regarding fragmentation of sperm DNA and its growth rate affected the time of embryo development in the ICSI procedure. The most significant prognostic factor for achieving pregnancy was intensification of sperm DNA fragmentation after 12 h. PMID:27579009

  5. Internal energy distribution of the NCO fragment from near-threshold photolysis of isocyanic acid, HNCO

    SciTech Connect

    Brown, S.S.; Berghout, H.L.; Crim, F.F.

    1996-05-09

    We report the first measurement of the vibrational- and rotational-state distributions in the NCO fragment resulting from photolysis of HNCO. Recent studies have drawn conclusions about the photochemistry of HNCO and the vibrational distribution in the NCO fragment from observations of kinetic energy distribution of the H atom produced in this photolysis; however, there has been no direct observation of the NCO fragment itself. We use laser-induced fluorescence to detect the nascent NCO photoproducts and spectral simulations to extract vibrational-state populations. The rotational distributions, where we can measure them, show little excitation, and the vibrational energy preferentially appears in the bending mode. The vibrational-state distribution results directly from the excited-state geometry of the HNCO parent, in which the NCO group is bent. The dissociation proceeds from this bent NCO group to a linear NCO fragment, strongly exciting the bending mode. We find about 65% of the total energy in relative translation of the fragments, while 30% goes into vibration and 5% into rotation of NCO. 49 refs., 7 figs., 2 tabs.

  6. Low Mass MS/MS Fragments of Protonated Amino Acids Used for Distinction of Their 13C- Isotopomers in Metabolic Studies

    NASA Astrophysics Data System (ADS)

    Ma, Xin; Dagan, Shai; Somogyi, Árpád; Wysocki, Vicki H.; Scaraffia, Patricia Y.

    2013-04-01

    Glu, Gln, Pro, and Ala are the main amino acids involved in ammonia detoxification in mosquitoes. In order to develop a tandem mass spectrometry method (MS2) to monitor each carbon of the above isotopically-labeled 13C-amino acids for metabolic studies, the compositions and origins of atoms in fragments of the protonated amino acid should be first elucidated. Thus, various electrospray (ESI)-based MS2 tools were employed to study the fragmentation of these unlabeled and isotopically-labeled amino acids and better understand their dissociation pathways. A broad range of fragments, including previously-undescribed low m/z fragments was revealed. The formulae of the fragments (from m/z 130 down to m/z 27) were confirmed by their accurate masses. The structures and conformations of the larger fragments of Glu were also explored by ion mobility mass spectrometry (IM-MS) and gas-phase hydrogen/deuterium exchange (HDX) experiments. It was found that some low m/z fragments ( m/z 27-30) are common to Glu, Gln, Pro, and Ala. The origins of carbons in these small fragments are discussed and additional collision induced dissociation (CID) MS2 fragmentation pathways are proposed for them. It was also found that small fragments (≤ m/z 84) of protonated, methylated Glu, and methylated Gln are the same as those of the underivatized Glu and Gln. Taken together, the new approach of utilizing low m/z fragments can be applied to distinguish, identify, and quantify 13C-amino acids labeled at various positions, either in the backbone or side chain.

  7. 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

  8. 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

  9. Bioactive Molecules Released in Food by Lactic Acid Bacteria: Encrypted Peptides and Biogenic Amines

    PubMed Central

    Pessione, Enrica; Cirrincione, Simona

    2016-01-01

    Lactic acid bacteria (LAB) can produce a huge amount of bioactive compounds. Since their elective habitat is food, especially dairy but also vegetal food, it is frequent to find bioactive molecules in fermented products. Sometimes these compounds can have adverse effects on human health such as biogenic amines (tyramine and histamine), causing allergies, hypertensive crises, and headache. However, some LAB products also display benefits for the consumers. In the present review article, the main nitrogen compounds produced by LAB are considered. Besides biogenic amines derived from the amino acids tyrosine, histidine, phenylalanine, lysine, ornithine, and glutamate by decarboxylation, interesting peptides can be decrypted by the proteolytic activity of LAB. LAB proteolytic system is very efficient in releasing encrypted molecules from several proteins present in different food matrices. Alpha and beta-caseins, albumin and globulin from milk and dairy products, rubisco from spinach, beta-conglycinin from soy and gluten from cereals constitute a good source of important bioactive compounds. These encrypted peptides are able to control nutrition (mineral absorption and oxidative stress protection), metabolism (blood glucose and cholesterol lowering) cardiovascular function (antithrombotic and hypotensive action), infection (microbial inhibition and immunomodulation) and gut-brain axis (opioids and anti-opioids controlling mood and food intake). Very recent results underline the role of food-encrypted peptides in protein folding (chaperone-like molecules) as well as in cell cycle and apoptosis control, suggesting new and positive aspects of fermented food, still unexplored. In this context, the detailed (transcriptomic, proteomic, and metabolomic) characterization of LAB of food interest (as starters, biocontrol agents, nutraceuticals, and probiotics) can supply a solid evidence-based science to support beneficial effects and it is a promising approach as well to obtain

  10. Bioactive Molecules Released in Food by Lactic Acid Bacteria: Encrypted Peptides and Biogenic Amines.

    PubMed

    Pessione, Enrica; Cirrincione, Simona

    2016-01-01

    Lactic acid bacteria (LAB) can produce a huge amount of bioactive compounds. Since their elective habitat is food, especially dairy but also vegetal food, it is frequent to find bioactive molecules in fermented products. Sometimes these compounds can have adverse effects on human health such as biogenic amines (tyramine and histamine), causing allergies, hypertensive crises, and headache. However, some LAB products also display benefits for the consumers. In the present review article, the main nitrogen compounds produced by LAB are considered. Besides biogenic amines derived from the amino acids tyrosine, histidine, phenylalanine, lysine, ornithine, and glutamate by decarboxylation, interesting peptides can be decrypted by the proteolytic activity of LAB. LAB proteolytic system is very efficient in releasing encrypted molecules from several proteins present in different food matrices. Alpha and beta-caseins, albumin and globulin from milk and dairy products, rubisco from spinach, beta-conglycinin from soy and gluten from cereals constitute a good source of important bioactive compounds. These encrypted peptides are able to control nutrition (mineral absorption and oxidative stress protection), metabolism (blood glucose and cholesterol lowering) cardiovascular function (antithrombotic and hypotensive action), infection (microbial inhibition and immunomodulation) and gut-brain axis (opioids and anti-opioids controlling mood and food intake). Very recent results underline the role of food-encrypted peptides in protein folding (chaperone-like molecules) as well as in cell cycle and apoptosis control, suggesting new and positive aspects of fermented food, still unexplored. In this context, the detailed (transcriptomic, proteomic, and metabolomic) characterization of LAB of food interest (as starters, biocontrol agents, nutraceuticals, and probiotics) can supply a solid evidence-based science to support beneficial effects and it is a promising approach as well to obtain

  11. 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.

  12. Gliadin fragments and a specific gliadin 33-mer peptide close KATP channels and induce insulin secretion in INS-1E cells and rat islets of langerhans.

    PubMed

    Dall, Morten; Calloe, Kirstine; Haupt-Jorgensen, Martin; Larsen, Jesper; Schmitt, Nicole; Josefsen, Knud; Buschard, Karsten

    2013-01-01

    In non-obese diabetic (NOD) mice, diabetes incidence is reduced by a gluten-free diet. Gluten peptides, such as the compound gliadin, can cross the intestinal barrier and may directly affect pancreatic beta cells. We investigated the effects of enzymatically-digested gliadin in NOD mice, INS-1E cells and rat islets. Six injections of gliadin digest in 6-week-old NOD mice did not affect diabetes development, but increased weight gain (20% increase by day 100). In INS-1E cells, incubation with gliadin digest induced a dose-dependent increase in insulin secretion, up to 2.5-fold after 24 hours. A similar effect was observed in isolated rat islets (1.6-fold increase). In INS-1E cells, diazoxide reduced the stimulatory effect of gliadin digest. Additionally, gliadin digest was shown to decrease current through KATP-channels. A specific gliadin 33-mer had a similar effect, both on current and insulin secretion. Finally, INS-1E incubation with gliadin digest potentiated palmitate-induced insulin secretion by 13% compared to controls. Our data suggest that gliadin fragments may contribute to the beta-cell hyperactivity observed prior to the development of type 1 diabetes. PMID:23785500

  13. 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

  14. 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.

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. 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

  20. Identification of purple acid phosphatase inhibitors by fragment-based screening: promising new leads for osteoporosis therapeutics.

    PubMed

    Feder, Daniel; Hussein, Waleed M; Clayton, Daniel J; Kan, Meng-Wei; Schenk, Gerhard; McGeary, Ross P; Guddat, Luke W

    2012-11-01

    Purple acid phosphatases are metalloenzymes found in animals, plants and fungi. They possess a binuclear metal centre to catalyse the hydrolysis of phosphate esters and anhydrides under acidic conditions. In humans, elevated purple acid phosphatases levels in sera are correlated with the progression of osteoporosis and metabolic bone malignancies, making this enzyme a target for the development of new chemotherapeutics to treat bone-related illnesses. To date, little progress has been achieved towards the design of specific and potent inhibitors of this enzyme that have drug-like properties. Here, we have undertaken a fragment-based screening approach using a 500-compound library identifying three inhibitors of purple acid phosphatases with K(i) values in the 30-60 μm range. Ligand efficiency values are 0.39-0.44 kcal/mol per heavy atom. X-ray crystal structures of these compounds in complex with a plant purple acid phosphatases (2.3-2.7 Å resolution) have been determined and show that all bind in the active site within contact of the binuclear centre. For one of these compounds, the phenyl ring is positioned within 3.5 Å of the binuclear centre. Docking simulations indicate that the three compounds fit into the active site of human purple acid phosphatases. These studies open the way to the design of more potent and selective inhibitors of purple acid phosphatases that can be tested as anti-osteoporotic drug leads.

  1. Synthetic Peptides as Protein Mimics

    PubMed Central

    Groß, Andrea; Hashimoto, Chie; Sticht, Heinrich; Eichler, Jutta

    2016-01-01

    The design and generation of molecules capable of mimicking the binding and/or functional sites of proteins represents a promising strategy for the exploration and modulation of protein function through controlled interference with the underlying molecular interactions. Synthetic peptides have proven an excellent type of molecule for the mimicry of protein sites because such peptides can be generated as exact copies of protein fragments, as well as in diverse chemical modifications, which includes the incorporation of a large range of non-proteinogenic amino acids as well as the modification of the peptide backbone. Apart from extending the chemical and structural diversity presented by peptides, such modifications also increase the proteolytic stability of the molecules, enhancing their utility for biological applications. This article reviews recent advances by this and other laboratories in the use of synthetic protein mimics to modulate protein function, as well as to provide building blocks for synthetic biology. PMID:26835447

  2. Hyperpurification of paired helical filaments reveals elevations in hydroxyproline content and a core structure related peptide fragment.

    PubMed

    Vogelsang, G D; Zemlan, F P; Dean, G E

    1989-01-01

    , rather than being non-covalently bound proteins or co-purifying contaminants. The present study extends previously published purification procedures, resulting in an isolated stable PHF core structure which is indistinguishable from previously published descriptions of PHF. The amino acid composition (Bidlingmeyer et al., 1984) of the PHF core structure appears to include hydroxyproline, an amino acid not commonly found in cytosolic proteins. We also demonstrate that some monoclonal antibodies previously raised against semi-purified PHF recognize determinants which are not related to the PHF core structure but rather recognize PHF-associated proteins or contaminants of the purification process.

  3. 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.

  4. 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

  5. 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.

  6. 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

  7. Maize EMBRYO SAC family peptides interact differentially with pollen tubes and fungal cells

    PubMed Central

    Woriedh, Mayada; Merkl, Rainer; Dresselhaus, Thomas

    2015-01-01

    EMBRYO SAC1-4 (ES1-4) peptides belong to the defensin subgroup of cysteine-rich peptides known to mediate pollen tube burst in Zea mays (maize). ES1-4 are reported here to also be capable of inhibiting germination and growth of the maize fungal pathogens Fusarium graminearum and Ustilago maydis at higher concentrations. Dividing the peptides into smaller pieces showed that a 15-amino-acid peptide located in a highly variable loop region lacking similarity to other defensins or defensin-like peptides binds to maize pollen tube surfaces, causing swelling prior to burst. This peptide fragment and a second conserved neighbouring fragment showed suppression of fungal germination and growth. The two peptides caused swelling of fungal cells, production of reactive oxygen species, and finally the formation of big vacuoles prior to burst at high peptide concentration. Furthermore, peptide fragments were found to bind differently to fungal cells. In necrotrophic F. graminearum, a peptide fragment named ES-d bound only at cell surfaces whereas the peptide ES-c bound at cell surfaces and also accumulated inside cells. Conversely, in biotrophic U. maydis, both peptide fragments accumulated inside cells, but, if applied at higher concentration, ES-c but not ES-d accumulated mainly in vacuoles. Mapping of peptide interaction sites identified amino acids differing in pollen tube burst and fungal response reactions. In summary, these findings indicate that residues targeting pollen tube burst in maize are specific to the ES family, while residues targeting fungal growth are conserved within defensins and defensin-like peptides. PMID:26071527

  8. 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

  9. 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

  10. Microscopic factors that control beta-sheet registry in amyloid fibrils formed by fragment 11-25 of amyloid beta peptide: insights from computer simulations.

    PubMed

    Negureanu, Lacramioara; Baumketner, Andrij

    2009-06-26

    Short fragments of amyloidogenic proteins are widely used as model systems in studies of amyloid formation. Fragment 11-25 of the amyloid beta protein involved in Alzheimer's disease (Abeta11-25) was recently shown to form amyloid fibrils composed of anti-parallel beta-sheets. Interestingly, fibrils grown under neutral and acidic conditions were seen to possess different registries of their inter-beta-strand hydrogen bonds. In an effort to explain the microscopic origin of this pH dependence, we studied Abeta11-25 fibrils using methods of theoretical modeling. Several structural models were built for fibrils at low and neutral pH levels and these were examined in short molecular dynamics simulations in explicit water. The models that displayed the lowest free energy, as estimated using an implicit solvent model, were selected as representative of the true fibrillar structure. It was shown that the registry of these models agrees well with the experimental results. At neutral pH, the main contribution to the free energy difference between the two registries comes from the electrostatic interactions. The charge group of the carboxy terminus makes a large contribution to these interactions and thus appears to have a critical role in determining the registry.

  11. 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

  12. 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.

  13. Lewis acid fragmentation of a lithium aryloxide cage: generation of new heterometallic aluminium-lithium species.

    PubMed

    Muñoz, Ma Teresa; Urbaneja, Carmen; Temprado, Manuel; Mosquera, Marta E G; Cuenca, Tomás

    2011-11-14

    Heterometallic aluminium-lithium species were prepared by the fragmentation reaction of the hexametallic cage compound [Li{2,6-(MeO)(2)C(6)H(3)O}](6) (1) with alkyl aluminium derivatives. Depending on the aluminium precursor, the species formed present different nuclearities in the solid state as shown by single crystal X-ray analysis. Spectroscopic and computational studies have been performed to study the nuclearity of the synthesized compounds in solution.

  14. 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

  15. 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

  16. 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

  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. 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.

  19. Conformational analysis of amyloid precursor protein fragment containing amino acids 667-676, and the effect of D-Asp and iso-Asp substitution at Asp₆₇₂ residue.

    PubMed

    Shanmugam, Ganesh; Polavarapu, Prasad L; Láng, Emma; Majer, Zsuzsa

    2012-03-01

    Amyloid precursor protein (APP) fragment containing amino acids 667-676, (APP₆₆₇₋₆₇₆), is a substrate for β-secretase which is responsible for generating amyloid β peptides. Conformational analysis of APP₆₆₇₋₆₇₆ peptide [Ac-Ser-Glu-Val-Lys-Met-Asp-Ala-Glu-Phe-Arg-NH₂] and the effect of substitution of Asp₆₇₂ with D-Asp and iso-L-Asp, studied for the first time, demonstrate that the peptide backbone of APP₆₆₇₋₆₇₆ is flexible and adopts different conformations in different solvent environments (water, trifluoroethanol and dimethylsulfoxide). A major conformational difference was observed in trifluoroethanol solvent when Asp₆₇₂ is substituted with D-Asp and iso-Asp. These conformational changes involved in APP₆₆₇₋₆₇₆ may assist in understanding the interactions between β-secretase and APP₆₆₇₋₆₇₆, with relevance to Alzheimer's disease.

  20. 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.

  1. 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.

  2. 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

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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

  8. 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

  9. Does the marine biotoxin okadaic acid cause DNA fragmentation in the blue mussel and the pacific oyster?

    PubMed

    McCarthy, Moira; O'Halloran, John; O'Brien, Nora M; van Pelt, Frank F N A M

    2014-10-01

    Two bivalve species of global economic importance: the blue mussel, Mytilus edulis and the pacific oyster, Crassostrea gigas were exposed in vivo, to the diarrhoetic shellfish toxin okadaic acid (OA), and impacts on DNA fragmentation were measured. Shellfish were exposed using two different regimes, the first was a single (24 h) exposure of 2.5 nM OA (∼0.1 μg/shellfish) and algal feed at the beginning of the trial (T0), after which shellfish were only fed algae. The second was daily exposure of shellfish to two different concentrations of OA mixed with the algal feed over 7 days; 1.2 nM OA (∼0.05 μg OA/shellfish/day) and 50 nM OA (∼2 μg OA/shellfish/day). Haemolymph and hepatopancreas cells were extracted following 1, 3 and 7 days exposure. Cell viability was measured using the trypan blue exclusion assay and remained above 85% for both cell types. DNA fragmentation was examined using the single-cell gel electrophoresis (comet) assay. A significant increase in DNA fragmentation was observed in the two cell types from both species relative to the controls. This increase was greater in the pacific oyster at the higher toxin concentration. However, there was no difference in the proportion of damage measured between the two cell types, and a classic dose response was not observed, increasing toxin concentration did not correspond to increased DNA fragmentation. PMID:25440785

  10. Differentiating chondroitin sulfate glycosaminoglycans using collision-induced dissociation; uronic acid cross-ring diagnostic fragments in a single stage of tandem mass spectrometry.

    PubMed

    Kailemia, Muchena J; Patel, Anish B; Johnson, Dane T; Li, Lingyun; Linhardt, Robert J; Amster, I Jonathan

    2015-01-01

    The stereochemistry of the hexuronic acid residues of the structure of glycosaminoglycans (GAGs) is a key feature that affects their interactions with proteins and other biological functions. Electron based tandem mass spectrometry methods, in particular electron detachment dissociation (EDD), have been able to distinguish glucuronic acid (GlcA) from iduronic acid (IdoA) residues in some heparan sulfate tetrasaccharides by producing epimer-specific fragments. Similarly, the relative abundance of glycosidic fragment ions produced by collision-induced dissociation (CID) or EDD has been shown to correlate with the type of hexuronic acid present in chondroitin sulfate GAGs. The present work examines the effect of charge state and degree of sodium cationization on the CID fragmentation products that can be used to distinguish GlcA and IdoA containing chondroitin sulfate A and dermatan sulfate chains. The cross-ring fragments (2,4)A(n) and (0,2)X(n) formed within the hexuronic acid residues are highly preferential for chains containing GlcA, distinguishing it from IdoA. The diagnostic capability of the fragments requires the selection of a molecular ion and fragment ions with specific ionization characteristics, namely charge state and number of ionizable protons. The ions with the appropriate characteristics display diagnostic properties for all the chondroitin sulfate and dermatan sulfate chains (degree of polymerization of 4-10) studied.

  11. Differentiation of mixed lactic acid bacteria communities in beverage fermentations using targeted terminal restriction fragment length polymorphism.

    PubMed

    Bokulich, Nicholas A; Mills, David A

    2012-08-01

    Lactic acid bacteria (LAB) are an important group of bacteria in beer and wine fermentations both as beneficial organisms and as spoilage agents. However, sensitive, rapid, culture-independent methods for identification and community analyses of LAB in mixed-culture fermentations are limited. We developed a terminal restriction fragment length polymorphism (TRFLP)-based assay for the detection and identification of lactic acid bacteria and Bacilli during wine, beer, and food fermentations. This technique can sensitively discriminate most species of Lactobacillales, and most genera of Bacillales, in mixed culture, as indicated by both bioinformatic predictions and empirical observations. This method was tested on a range of beer and wine fermentations containing mixed LAB communities, demonstrating the efficacy of this technique for discriminating LAB in mixed culture. PMID:22475950

  12. 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

    The origins of our nearly ten-year research program of chemical and biological investigations into peptides based on homologated proteinogenic amino acids are described. The road from the biopolymer poly[ethyl (R)-3-hydroxybutanoate] to the beta-peptides was primarily a step from organic synthesis methodology (the preparation of enantiomerically pure compounds (EPCs)) to supramolecular chemistry (higher-order structures maintained through non-covalent interactions). The performing of biochemical and biological tests on the beta- and gamma-peptides, which differ from natural peptides/proteins by a single or two additional CH(2) groups per amino acid, then led into bioorganic chemistry and medicinal chemistry. The individual chapters of this review article begin with descriptions of work on beta-amino acids, beta-peptides, and polymers (Nylon-3) that dates back to the 1960s, even to the times of Emil Fischer, but did not yield insights into structures or biological properties. The numerous, often highly physiologically active, or even toxic, natural products containing beta- and gamma-amino acid moieties are then presented. Chapters on the preparation of homologated amino acids with proteinogenic side chains, their coupling to provide the corresponding peptides, both in solution (including thioligation) and on the solid phase, their isolation by preparative HPLC, and their characterization by mass spectrometry (HR-MS and MS sequencing) follow. After that, their structures, predominantly determined by NMR spectroscopy in methanolic solution, are described: helices, pleated sheets, and turns, together with stack-, crankshaft-, paddlewheel-, and staircase-like patterns. The presence of the additional C--C bonds in the backbones of the new peptides did not give rise to a chaotic increase in their secondary structures as many protein specialists might have expected: while there are indeed more structure types than are observed in the alpha-peptide realm - three different

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. 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.

  18. 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

  19. 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

  20. 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

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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

  6. 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

  7. 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.

  8. 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.

  9. Evaluation of an Antimicrobial L-Amino Acid Oxidase and Peptide Derivatives from Bothropoides mattogrosensis Pitviper Venom

    PubMed Central

    Gomes, Diego G.; Porto, William F.; Batista, Carla L.; Ramos, Carmel S.; Holanda, Hortência H. S.; Dias, Simoni C.; Franco, Octavio L.; Moreno, Susana E.

    2012-01-01

    Healthcare-associated infections (HAIs) are causes of mortality and morbidity worldwide. The prevalence of bacterial resistance to common antibiotics has increased in recent years, highlighting the need to develop novel alternatives for controlling these pathogens. Pitviper venoms are composed of a multifaceted mixture of peptides, proteins and inorganic components. L-amino oxidase (LAO) is a multifunctional enzyme that is able to develop different activities including antibacterial activity. In this study a novel LAO from Bothrops mattogrosensis (BmLAO) was isolated and biochemically characterized. Partial enzyme sequence showed full identity to Bothrops pauloensis LAO. Moreover, LAO here isolated showed remarkable antibacterial activity against Gram-positive and -negative bacteria, clearly suggesting a secondary protective function. Otherwise, no cytotoxic activities against macrophages and erythrocytes were observed. Finally, some LAO fragments (BmLAO-f1, BmLAO-f2 and BmLAO-f3) were synthesized and further evaluated, also showing enhanced antimicrobial activity. Peptide fragments, which are the key residues involved in antimicrobial activity, were also structurally studied by using theoretical models. The fragments reported here may be promising candidates in the rational design of new antibiotics that could be used to control resistant microorganisms. PMID:22438972

  10. A bottom-up approach to build the hyperpolarizability of peptides and proteins from their amino acids.

    PubMed

    Duboisset, Julien; Deniset-Besseau, Ariane; Benichou, Emmanuel; Russier-Antoine, Isabelle; Lascoux, Noelle; Jonin, Christian; Hache, François; Schanne-Klein, Marie-Claire; Brevet, Pierre-François

    2013-08-29

    We experimentally demonstrate that some peptides and proteins lend themselves to an elementary analysis where their first hyperpolarizability can be decomposed into the coherent superposition of the first hyperpolarizability of their elementary units. We then show that those elementary units can be associated with the amino acids themselves in the case of nonaromatic amino acids and nonresonant second harmonic generation. As a case study, this work investigates the experimentally determined first hyperpolarizability of rat tail Type I collagen and compares it to that of the shorter peptide [(PPG)10]3, where P and G are the one-letter code for Proline and Glycine, respectively, and that of the triamino acid peptides PPG and GGG. An absolute value of (0.16 ± 0.01) × 10(-30) esu for the first hyperpolarizability of nonaromatic amino acids is then obtained by using the newly defined 0.087 × 10(-30) esu reference value for water. By using a collagen like model, the microscopic hyperpolarizability along the peptide bond can be evaluated at (0.7 ± 0.1) × 10(-30) esu. PMID:23879840

  11. 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...

  12. 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.

  13. PFR peptide, one of the antimicrobial peptides identified from the derivatives of lactoferrin, induces necrosis in leukemia cells

    PubMed Central

    Lu, Yan; Zhang, Teng-Fei; Shi, Yue; Zhou, Han-Wei; Chen, Qi; Wei, Bu-Yun; Wang, Xi; Yang, Tian-Xin; Chinn, Y. Eugene; Kang, Jian; Fu, Cai-Yun

    2016-01-01

    LF11-322 (PFWRIRIRR-NH2) (PFR peptide), a nine amino acid-residue peptide fragment derived from human lactoferricin, possesses potent cytotoxicity against bacteria. We report here the discovery and characterization of its antitumor activity in leukemia cells. PFR peptide inhibited the proliferation of MEL and HL-60 leukemia cells by inducing cell death in the absence of the classical features of apoptosis, including chromatin condensation, Annexin V staining, Caspase activation and increase of abundance of pro-apoptotic proteins. Instead, necrotic cell death as evidenced by increasing intracellular PI staining and LDH release, inducing membrane disruption and up-regulating intracellular calcium level, was observed following PFR peptide treatment. In addition to necrotic cell death, PFR peptide also induced G0/G1 cell cycle arrest. Moreover, PFR peptide exhibited favorable antitumor activity and tolerability in vivo. These findings thus provide a new clue of antimicrobial peptides as a potential novel therapy for leukemia. PMID:26860588

  14. Scandium triflate-catalyzed nucleophilic additions to indolylmethyl Meldrum's acid derivatives via a gramine-type fragmentation: synthesis of substituted indolemethanes.

    PubMed

    Armstrong, Erin L; Grover, Huck K; Kerr, Michael A

    2013-10-18

    Treatment of indolylmethyl Meldrum's acids with catalytic scandium triflate and a variety of nucleophiles results in the nucleophilic displacement of the Meldrum's acid moiety via a gramine-type fragmentation. The reaction is useful for the generation of heterocyclic compounds of significant molecular complexity.

  15. Reversible phospholipid nanogels for deoxyribonucleic acid fragment size determinations up to 1500 base pairs and integrated sample stacking.

    PubMed

    Durney, Brandon C; Bachert, Beth A; Sloane, Hillary S; Lukomski, Slawomir; Landers, James P; Holland, Lisa A

    2015-06-23

    Phospholipid additives are a cost-effective medium to separate deoxyribonucleic acid (DNA) fragments and possess a thermally-responsive viscosity. This provides a mechanism to easily create and replace a highly viscous nanogel in a narrow bore capillary with only a 10°C change in temperature. Preparations composed of dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) self-assemble, forming structures such as nanodisks and wormlike micelles. Factors that influence the morphology of a particular DMPC-DHPC preparation include the concentration of lipid in solution, the temperature, and the ratio of DMPC and DHPC. It has previously been established that an aqueous solution containing 10% phospholipid with a ratio of [DMPC]/[DHPC]=2.5 separates DNA fragments with nearly single base resolution for DNA fragments up to 500 base pairs in length, but beyond this size the resolution decreases dramatically. A new DMPC-DHPC medium is developed to effectively separate and size DNA fragments up to 1500 base pairs by decreasing the total lipid concentration to 2.5%. A 2.5% phospholipid nanogel generates a resolution of 1% of the DNA fragment size up to 1500 base pairs. This increase in the upper size limit is accomplished using commercially available phospholipids at an even lower material cost than is achieved with the 10% preparation. The separation additive is used to evaluate size markers ranging between 200 and 1500 base pairs in order to distinguish invasive strains of Streptococcus pyogenes and Aspergillus species by harnessing differences in gene sequences of collagen-like proteins in these organisms. For the first time, a reversible stacking gel is integrated in a capillary sieving separation by utilizing the thermally-responsive viscosity of these self-assembled phospholipid preparations. A discontinuous matrix is created that is composed of a cartridge of highly viscous phospholipid assimilated into a separation matrix

  16. 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.

  17. Host-Pathogen interactions. 25. Endopolygalacturonic acid lyase from Erwinia carotovora elicits phytoalexin accumulation by releasing plant cell wall fragments

    SciTech Connect

    Davis, K.R.; Lyon, G.D.; Darvill, A.G.; Albersheim, P.

    1984-01-01

    Heat-labile elicitors of phytoalexin accumulation in soybeans (Glycine max L. Merr. cv Wayne) were detected in culture filtrates of Erwinia carotovora grown on a defined medium containing citrus pectin as the sole carbon source. The heat-labile elicitors were highly purified by cation-exchange chromatography on a CM-Sephadex (C-50) column, followed by agarose-affinity chromatography on a Bio-Gel A-0.5m gel filtration column. The heat-labile elicitor activity co-purified with two ..cap alpha..-1,4-endopolygalacturonic acid lyases (EC 4 x 2 x 2 x 2). Endopolygalacturonic acid lyase activity appeared to be necessary for elicitor activity because heat-inactivated enzyme preparations did not elicit phytoalexins. The purified endopolygalacturonic acid lyases elicited pterocarpan phytoalexins at microbial-inhibitory concentrations in the soybean-cotyledon bioassay when applied at a concentration of 55 nanograms per milliliter (1 x 10/sup -9/ molar). One of these lyases released heat-stable elicitors from soybean cell walls, citrus pectin, and sodium polypectate. The heat-stable elicitor-active material solubilized from soybean cell walls by the lyase was composed of at least 90% (w/v) uronosyl residues. These results demonstrate that endopolygalacturonic acid lyase elicits phytoalexin accumulation by releasing fragments from pectic polysaccharides in plant cell walls.

  18. 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

  19. On the specificity of lipid hydroperoxide fragmentation by fatty acid hydroperoxide lyase from Arabidopsis thaliana.

    PubMed

    Kandzia, Romy; Stumpe, Michael; Berndt, Ekkehardt; Szalata, Marlena; Matsui, Kenji; Feussner, Ivo

    2003-07-01

    Fatty acid hydroperoxide lyase (HPL) is a membrane associated P450 enzyme that cleaves fatty acid hydroperoxides into aldehydes and omega-oxo fatty acids. One of the major products of this reaction is (3Z)-hexenal. It is a constituent of many fresh smelling fruit aromas. For its biotechnological production and because of the lack of structural data on the HPL enzyme family, we investigated the mechanistic reasons for the substrate specificity of HPL by using various structural analogues of HPL substrates. To approach this 13-HPL from Arabidopsis thaliana was cloned and expressed in E. coli utilising a His-Tag expression vector. The fusion protein was purified by affinity chromatography from the E. coli membrane fractions and its pH optimum was detected to be pH 7.2. Then, HPL activity against the respective (9S)- and (13S)-hydroperoxides derived either from linoleic, alpha-linolenic or gamma-linolenic acid, respectively, as well as that against the corresponding methyl esters was analysed. Highest enzyme activity was observed with the (13S)-hydroperoxide of alpha-linolenic acid (13alpha-HPOT) followed by that with its methyl ester. Most interestingly, when the hydroperoxy isomers of gamma-linolenic acid were tested as substrates, 9gamma-HPOT and not 13gamma-HPOT was found to be a better substrate of the enzyme. Taken together from these studies on the substrate specificity it is concluded that At13HPL may not recognise the absolute position of the hydroperoxy group within the substrate, but shows highest activities against substrates with a (1Z4S,5E,7Z)-4-hydroperoxy-1,5,7-triene motif. Thus, At13HPL may not only be used for the production of C6-derived volatiles, but depending on the substrate may be further used for the production of Cg-derived volatiles as well. PMID:12940547

  20. 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.

  1. 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.

  2. 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.

  3. 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

  4. 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.

  5. 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).

  6. 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

  7. 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.

  8. 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

  9. Neuropeptides: metabolism to bioactive fragments and the pharmacology of their receptors.

    PubMed

    Hallberg, Mathias

    2015-05-01

    The proteolytic processing of neuropeptides has an important regulatory function and the peptide fragments resulting from the enzymatic degradation often exert essential physiological roles. The proteolytic processing generates, not only biologically inactive fragments, but also bioactive fragments that modulate or even counteract the response of their parent peptides. Frequently, these peptide fragments interact with receptors that are not recognized by the parent peptides. This review discusses tachykinins, opioid peptides, angiotensins, bradykinins, and neuropeptide Y that are present in the central nervous system and their processing to bioactive degradation products. These well-known neuropeptide systems have been selected since they provide illustrative examples that proteolytic degradation of parent peptides can lead to bioactive metabolites with different biological activities as compared to their parent peptides. For example, substance P, dynorphin A, angiotensin I and II, bradykinin, and neuropeptide Y are all degraded to bioactive fragments with pharmacological profiles that differ considerably from those of the parent peptides. The review discusses a selection of the large number of drug-like molecules that act as agonists or antagonists at receptors of neuropeptides. It focuses in particular on the efforts to identify selective drug-like agonists and antagonists mimicking the effects of the endogenous peptide fragments formed. As exemplified in this review, many common neuropeptides are degraded to a variety of smaller fragments but many of the fragments generated have not yet been examined in detail with regard to their potential biological activities. Since these bioactive fragments contain a small number of amino acid residues, they provide an ideal starting point for the development of drug-like substances with ability to mimic the effects of the degradation products. Thus, these substances could provide a rich source of new pharmaceuticals

  10. 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

  11. Okadaic acid induces DNA fragmentation via caspase-3-dependent and caspase-3-independent pathways in Chinese hamster ovary (CHO)-K1 cells.

    PubMed

    Kitazumi, Ikuko; Maseki, Yoko; Nomura, Yoshiko; Shimanuki, Akiko; Sugita, Yumi; Tsukahara, Masayoshi

    2010-01-01

    DNA fragmentation is a hallmark of apoptosis that occurs in a variety of cell types; however, it remains unclear whether caspase-3 is required for its induction. To investigate this, we produced caspase-3 knockout Chinese hamster ovary (CHO)-K1 cells and examined the effects of gene knockout and treatment with caspase-3 inhibitors. Okadaic acid (OA) is a potent inhibitor of the serine/threonine protein phosphatases (PPs) PP1 and PP2A, which induce apoptotic cellular reactions. Treatment of caspase-3(-/-) cells with OA induced DNA fragmentation, indicating that caspase-3 is not an essential requirement. However, in the presence of benzyloxycarbonyl-Asp-Glu-Val-Asp (OMe) fluoromethylketone (z-DEVD-fmk), DNA fragmentation occurred in CHO-K1 cells but not in caspase-3(-/-) cells, suggesting that caspase-3 is involved in OA-induced DNA fragmentation that does not utilize DEVDase activity. In the absence of caspase-3, DEVDase activity may play an important role. In addition, OA-induced DNA fragmentation was reduced but not blocked in CHO-K1 cells, suggesting that caspase-3 is involved in caspase-independent OA-induced DNA fragmentation. Furthermore, OA-induced cleavage of caspase-3 and DNA fragmentation were blocked by pretreatment with the wide-ranging serine protease inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride. These results suggest that serine proteases regulate DNA fragmentation upstream of caspase-3.

  12. 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

  13. 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

  14. 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

  15. 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.

  16. Utility of point-of-care testing of natriuretic peptides (brain natriuretic peptide and n-terminal pro-brain natriuretic peptide) in the emergency department.

    PubMed

    Nayer, Jamshed; Aggarwal, Praveen; Galwankar, Sagar

    2014-07-01

    Rapid and accurate diagnosis of a patient with an acute disease is a challenge for emergency physicians. Natriuretic peptides have emerged as important tools for diagnosis, risk stratification and therapeutic decision making for some categories of emergency patients. Brain natriuretic peptide (BNP) is a member of a four natriuretic peptides family that shares a common 17-peptide ring structure. Atrial natriuretic peptide, C-natriuretic peptide (CNP), and D-type natriuretic peptide are the other natriuretic peptide, which share the same common 17-peptide ring structure. The N-terminal fragment of pro-BNP, N-terminal pro-brain natriuretic peptide (NT-proBNP) consists of 76 amino acids, which is biologically inert, while the active component BNP contains 32 amino acids. BNP and NT-proBNP are secreted in the plasma in equimolar quantities and are frequently used in the diagnosis of congestive heart failure, and distinguishing between patients with dyspnea of cardiac or pulmonary origin. Both natriuretic peptides have also been evaluated for use in the assessment and management of several other conditions including sepsis, cirrhosis of liver and renal failure. However, one should remember that the values of natriuretic peptides are affected by age and weight of the patients, and presence of several comorbidities such as chronic renal failure, type 2 diabetes mellitus, anemia, pulmonary embolism, and acute coronary syndrome. Values of these peptides also vary depending on the type of test used. The performance characteristics of these natriuretic peptides vary depending on the patients on whom they are used. Therefore determination of reference values for these peptides represents a challenge.

  17. Selective Deletion of the Internal Lysine Residue from the Peptide Sequence by Collisional Activation

    NASA Astrophysics Data System (ADS)

    Banerjee, Shibdas; Mazumdar, Shyamalava

    2012-11-01

    The gas-phase peptide ion fragmentation chemistry is always the center of attraction in proteomics to analyze the amino acid sequence of peptides and proteins. In this work, we describe the formation of an anomalous fragment ion, which corresponds to the selective deletion of the internal lysine residue from a series of lysine containing peptides upon collisional activation in the ion trap. We detected several water-loss fragment ions and the maximum number of water molecules lost from a particular fragment ion was equal to the number of lysine residues in that fragment. As a consequence of this water-loss phenomenon, internal lysine residues were found to be deleted from the peptide ion. The N,N-dimethylation of all the amine functional groups of the peptide stopped the internal lysine deletion reaction, but selective N-terminal α-amino acetylation had no effect on this process indicating involvement of the side chains of the lysine residues. The detailed mechanism of the lysine deletion was investigated by multistage CID of the modified and unmodified peptides, by isotope labeling and by energy resolved CID studies. The results suggest that the lysine deletion might occur through a unimolecular multistep mechanism involving a seven-membered cyclic imine intermediate formed by the loss of water from a lysine residue in the protonated peptide. This intermediate subsequently undergoes degradation reaction to deplete the interior imine ring from the peptide backbone leading to the deletion of an internal lysine residue.

  18. 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.

  19. 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

  20. 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.

  1. 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

  2. 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.

  3. Effects of an acidic fibroblast growth factor fragment analog on learning and memory and on medial septum cholinergic neurons in senescence-accelerated mice.

    PubMed

    Sasaki, K; Tooyama, I; Li, A J; Oomura, Y; Kimura, H

    1999-01-01

    We examined the effects of repeated subcutaneous injections of an acidic fibroblast growth factor fragment analog, [Ala16] acidic fibroblast growth factor (1-29), on learning and memory and on the choline acetyltransferase immunoreactivity of forebrain neurons in senescence-accelerated mice. One group of accelerated senescence-prone mice (accelerated senescence-prone-8) received [Ala16] acidic fibroblast growth factor (1-29), whereas the other group of accelerated senescence-prone-8 mice and a group of accelerated senescence-resistant mice (control) received vehicle solution. Injections began at three weeks after birth and were given weekly for 10 months. In a passive avoidance test, the mean retention latency at three, six and nine months of age was significantly longer in controls (vehicle-treated accelerated senescence-resistant-1) and acidic fibroblast growth factor fragment-treated accelerated senescence-prone-8 than in vehicle-treated accelerated senescence-prone-8 mice, and the latency in acidic fibroblast growth factor fragment-treated accelerated senescence-prone-8 mice was significantly shorter than that in controls only at nine months of age. In the Morris water maze task, the mean latency to climb onto the platform was significantly longer in acidic fibroblast growth factor fragment- and vehicle-treated accelerated senescence-prone-8 mice than in controls. However, the mean latency in the third and fourth trial blocks was significantly shorter for acidic fibroblast growth factor fragment-treated accelerated senescence-prone-8 than for vehicle-treated accelerated senescence-prone-8 mice. In the probe trials, controls and acidic fibroblast growth factor fragment-treated accelerated senescence-prone-8 mice spent significantly more time in the quadrant in which the platform had previously been located than in the other three quadrants. In acidic fibroblast growth factor fragment-treated accelerated senescence-prone-8 mice, the density of medial septum

  4. 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

  5. Peptide identification

    DOEpatents

    Jarman, Kristin H [Richland, WA; Cannon, William R [Richland, WA; Jarman, Kenneth D [Richland, WA; Heredia-Langner, Alejandro [Richland, WA

    2011-07-12

    Peptides are identified from a list of candidates using collision-induced dissociation tandem mass spectrometry data. A probabilistic model for the occurrence of spectral peaks corresponding to frequently observed partial peptide fragment ions is applied. As part of the identification procedure, a probability score is produced that indicates the likelihood of any given candidate being the correct match. The statistical significance of the score is known without necessarily having reference to the actual identity of the peptide. In one form of the invention, a genetic algorithm is applied to candidate peptides using an objective function that takes into account the number of shifted peaks appearing in the candidate spectrum relative to the test spectrum.

  6. Interpretation of tandem mass spectra obtained from cyclic nonribosomal peptides.

    PubMed

    Liu, Wei-Ting; Ng, Julio; Meluzzi, Dario; Bandeira, Nuno; Gutierrez, Marcelino; Simmons, Thomas L; Schultz, Andrew W; Linington, Roger G; Moore, Bradley S; Gerwick, William H; Pevzner, Pavel A; Dorrestein, Pieter C

    2009-06-01

    Natural and non-natural cyclic peptides are a crucial component in drug discovery programs because of their considerable pharmaceutical properties. Cyclosporin, microcystins, and nodularins are all notable pharmacologically important cyclic peptides. Because these biologically active peptides are often biosynthesized nonribosomally, they often contain nonstandard amino acids, thus increasing the complexity of the resulting tandem mass spectrometry data. In addition, because of the cyclic nature, the fragmentation patterns of many of these peptides showed much higher complexity when compared to related counterparts. Therefore, at the present time it is still difficult to annotate cyclic peptides MS/MS spectra. In this current work, an annotation program was developed for the annotation and characterization of tandem mass spectra obtained from cyclic peptides. This program, which we call MS-CPA is available as a web tool (http://lol.ucsd.edu/ms-cpa_v1/Input.py). Using this program, we have successfully annotated the sequence of representative cyclic peptides, such as seglitide, tyrothricin, desmethoxymajusculamide C, dudawalamide A, and cyclomarins, in a rapid manner and also were able to provide the first-pass structure evidence of a newly discovered natural product based on predicted sequence. This compound is not available in sufficient quantities for structural elucidation by other means such as NMR. In addition to the development of this cyclic annotation program, it was observed that some cyclic peptides fragmented in unexpected ways resulting in the scrambling of sequences. In summary, MS-CPA not only provides a platform for rapid confirmation and annotation of tandem mass spectrometry data obtained with cyclic peptides but also enables quantitative analysis of the ion intensities. This program facilitates cyclic peptide analysis, sequencing, and also acts as a useful tool to investigate the uncommon fragmentation phenomena of cyclic peptides and aids the

  7. 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

  8. Effects of amyloid β-peptide fragment 31-35 on the BK channel-mediated K⁺ current and intracellular free Ca²⁺ concentration of hippocampal CA1 neurons.

    PubMed

    Zhang, Yu; Shi, Zhi-Gang; Wang, Zhi-Hua; Li, Jian-Guo; Chen, Jin-Yuan; Zhang, Ce

    2014-05-01

    The present study characterizes the effects of Aβ31-35, a short active fragment of amyloid β-peptide (Aβ), upon the BK channel-mediated K⁺ current and intracellular free Ca²⁺ concentration ([Ca²⁺]i) of freshly dissociated pyramidal cells from rat CA1 hippocampus by using whole-cell patch-clamp recording and single cell Ca²⁺ imaging techniques. The results show that: (1) in the presence of voltage- and ATP-gated K⁺ channel blockers application of 5.0 μM Aβ31-35 significantly diminished transient outward K⁺ current amplitudes at clamped voltages between 0 and 45mV; (2) under the same conditions [Ca²⁺]i was minimally affected by 5.0 μM but significantly increased by 12.5 μM and 25 μM Aβ31-35; and (3) when 25 μM of a larger fragment of the amyloid β-peptide, Aβ25-35, was applied, the results were similar to those obtained with the same concentration of Aβ31-35. These results indicate that Aβ31-35 is likely to be the shortest active fragment of the full Aβ sequence, and can be as effectively as the full-length Aβ peptide in suppressing BK-channel mediated K⁺ currents and significantly elevating [Ca²⁺]i in hippocampal CA1 neurons.

  9. Self-Assembly of a 9-Residue Amyloid-Forming Peptide Fragment of SARS Corona Virus E-protein: Mechanism of Self Aggregation and Amyloid-Inhibition of hIAPP

    PubMed Central

    Bhat, Jyotsna; Bera, Supriyo; Midya, Anupam; Fierke, Carol A.; Ramamoorthy, Ayyalusamy; Bhunia, Anirban

    2016-01-01

    Molecular self-assembly, a phenomenon widely observed in nature, has been exploited through organic molecules, proteins, DNA and peptides to study complex biological systems. These self-assembly systems may also be used in understanding the molecular and structural biology which can inspire the design and synthesis of increasingly complex biomaterials. Specifically, use of these building blocks to investigate protein folding and misfolding has been of particular value since it can provide tremendous insights into peptide aggregation related to a variety of protein misfolding diseases, or amyloid diseases (e.g. Alzheimer’s disease, Parkinson’s disease, type-II diabetes). Herein, the self-assembly of TK9, a 9 residue peptide of the extra membrane C-terminal tail of the SARS Corona virus envelope, and its variants were characterized through biophysical, spectroscopic and simulated studies, and it was confirmed that the structure of these peptides influence their aggregation propensity, hence, mimicking amyloid proteins. TK9, which forms a beta-sheet rich fibril, contains a key sequence motif that may be critical for beta-sheet formation, thus making it an interesting system to study amyloid fibrillation. TK9 aggregates were further examined through simulations to evaluate the possible intra- and inter peptide interactions at the molecular level. These self-assembly peptides can also serve as amyloid inhibitors through hydrophobic and electrophilic recognition interactions. Our results show that TK9 inhibits the fibrillation of hIAPP, a 37 amino acid peptide implicated in the pathology of type-II diabetes. Thus, biophysical and NMR experimental results have revealed a molecular level understanding of peptide folding events, as well as the inhibition of amyloid-protein aggregation are reported. PMID:25785896

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. 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

  15. 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

  16. 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.

  17. 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.

  18. 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

  19. 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.

  20. 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

  1. 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

  2. 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.

  3. Diagnosis of bacterial vaginosis by a new multiplex peptide nucleic acid fluorescence in situ hybridization method

    PubMed Central

    Machado, António; Castro, Joana; Cereija, Tatiana; Almeida, Carina

    2015-01-01

    Bacterial vaginosis (BV) is one of most common vaginal infections. However, its diagnosis by classical methods reveals low specificity. Our goal was to evaluate the accuracy diagnosis of 150 vaginal samples with research gold standard methods and our Peptide Nucleic Acid (PNA) probes by Fluorescence in situ Hybridization (FISH) methodology. Also, we described the first PNA-FISH methodology for BV diagnosis, which provides results in approximately 3 h. The results showed a sensitivity of 84.6% (95% confidence interval (CI), from 64.3 to 95.0%) and a specificity of 97.6% (95% CI [92.6–99.4%]), demonstrating the higher specificity of the PNA-FISH method and showing false positive results in BV diagnosis commonly obtained by the classical methods. This methodology combines the specificity of PNA probes for Lactobacillus species and G. vaginalis visualization and the calculation of the microscopic field by Nugent score, allowing a trustful evaluation of the bacteria present in vaginal microflora and avoiding the occurrence of misleading diagnostics. Therefore, the PNA-FISH methodology represents a valuable alternative for BV diagnosis. PMID:25737820

  4. Isodesmic reaction for accurate theoretical pKa calculations of amino acids and peptides.

    PubMed

    Sastre, S; Casasnovas, R; Muñoz, F; Frau, J

    2016-04-28

    Theoretical and quantitative prediction of pKa values at low computational cost is a current challenge in computational chemistry. We report that the isodesmic reaction scheme provides semi-quantitative predictions (i.e. mean absolute errors of 0.5-1.0 pKa unit) for the pKa1 (α-carboxyl), pKa2 (α-amino) and pKa3 (sidechain groups) of a broad set of amino acids and peptides. This method fills the gaps of thermodynamic cycles for the computational pKa calculation of molecules that are unstable in the gas phase or undergo proton transfer reactions or large conformational changes from solution to the gas phase. We also report the key criteria to choose a reference species to make accurate predictions. This method is computationally inexpensive and makes use of standard density functional theory (DFT) and continuum solvent models. It is also conceptually simple and easy to use for researchers not specialized in theoretical chemistry methods. PMID:27052591

  5. 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.

  6. New formulation of vasoactive intestinal peptide using liposomes in hyaluronic acid gel for uveitis.

    PubMed

    Lajavardi, Laure; Camelo, Serge; Agnely, Florence; Luo, Wei; Goldenberg, Brigitte; Naud, Marie-Christine; Behar-Cohen, Francine; de Kozak, Yvonne; Bochot, Amélie

    2009-10-01

    We evaluated the benefits of a novel formulation of vasoactive intestinal peptide (VIP) based on the incorporation of VIP-loaded rhodamine-conjugated liposomes (VIP-Rh-Lip) within hyaluronic acid (HA) gel (Gel-VIP-Rh-Lip) for the treatment of endotoxin-induced uveitis (EIU) in comparison with VIP-Rh-Lip alone. In vitro release study and rheological analysis showed that interactions between HA chains and liposomes resulted in increased viscosity and reinforced elasticity of the gel. In vivo a single intravitreal injection of Gel-VIP-Rh-Lip was performed in rats 7 days prior to uveitis induction by subcutaneous lipopolysaccharide injection. The maximal ocular inflammation occurs within 16-24 h in controls (VIP-Rh-Lip, unloaded-Rh-Lip). Whereas intraocular injection of VIP-Rh-Lip had no effect on EIU severity compared with controls, Gel-VIP-Rh-Lip reduced significantly the clinical score and number of inflammatory cells infiltrating the eye. The fate of liposomes, VIP and HA in the eyes, regional and inguinal lymph nodes and spleen was analyzed by immunostaining and fluorescence microscopy. Retention of liposomes by HA gel was observed in vitro and in vivo. Inflammation severity seemed to impact on system stability resulting in the delayed release of VIP. Thus, HA gel containing VIP-Rh-Lip is an efficient strategy to obtain a sustained delivery of VIP in ocular and lymph node tissues.

  7. 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

  8. Peptide nucleic acid probe detection of mutations in Mycobacterium tuberculosis genes associated with drug resistance.

    PubMed

    Bockstahler, L E; Li, Z; Nguyen, N Y; Van Houten, K A; Brennan, M J; Langone, J J; Morris, S L

    2002-03-01

    The emergence of drug-resistant strains of Mycobacterium tuberculosis is a serious public health problem. Many of the specific gene mutations that cause drug resistance in M. tuberculosis are point mutations. We are developing a PCR-peptide nucleic acid (PNA)-based ELISA as a diagnostic method to recognize point mutations in genes associated with isoniazid and rifampin resistance in M. tuberculosis. Specific point mutation-containing sequences and wild-type sequences of cloned mycobacterial genes were PCR-amplified, denatured, and hybridized with PNA probes bound to microplate wells. Using 15-base PNA probes, we established the hybridization temperatures (50 degrees C-55 degrees C) and other experimental conditions suitable for detecting clinically relevant point mutations in the katG and rpoB genes. Hybridization of PCR-amplified sequences that contained these point mutations with complementary mutation-specific PNAs resulted in significant increases in ELISA response compared with hybridization using wild-type-specific PNAs. Conversely, PCR-amplified wild-type sequences hybridized much more efficiently with wild-type PNAs than with the mutation-specific PNAs. Using the M. tuberculosis cloned genes and PCR-PNA-ELISA format developed here, M. tuberculosis sequences containing point mutations associated with drug resistance can be identified in less than 24 h. PMID:11926172

  9. Boswellic acids target the human immune system-modulating antimicrobial peptide LL-37.

    PubMed

    Henkel, Arne; Tausch, Lars; Pillong, Max; Jauch, Johann; Karas, Michael; Schneider, Gisbert; Werz, Oliver

    2015-12-01

    The antimicrobial peptide LL-37 is the sole member of the human cathelicidin family with immune system-modulating properties and roles in autoimmune disease development. Small molecules able to interact with LL-37 and to modulate its functions have not been described yet. Boswellic acids (BAs) are pentacyclic triterpene acids that are bioactive principles of frankincense extracts used as anti-inflammatory remedies. Although various anti-inflammatory modes of action have been proposed for BAs, the pharmacological profile of these compounds is still incompletely understood. Here, we describe the identification of human LL-37 as functional target of BAs. In unbiased target fishing experiments using immobilized BAs as bait and human neutrophils as target source, LL-37 was identified as binding partner assisted by MALDI-TOF mass spectrometry. Thermal stability experiments using circular dichroism spectroscopy confirm direct interaction between BAs and LL-37. Of interest, this binding of BAs resulted in an inhibition of the functionality of LL-37. Thus, the LPS-neutralizing properties of isolated LL-37 were inhibited by 3-O-acetyl-β-BA (Aβ-BA) and 3-O-acetyl-11-keto-β-BA (AKβ-BA) in a cell-free limulus amoebocyte lysate assay with EC50=0.2 and 0.8 μM, respectively. Also, LL-37 activity was inhibited by these BAs in LL-37-enriched supernatants of stimulated neutrophils or human plasma derived from stimulated human whole blood. Together, we reveal BAs as inhibitors of LL-37, which might be a relevant mechanism underlying the anti-inflammatory properties of BAs and suggests BAs as suitable chemical tools or potential agents for intervention with LL-37 and related disorders. PMID:26361729

  10. Strand displacement and duplex invasion into double-stranded DNA by pyrrolidinyl peptide nucleic acids.

    PubMed

    Bohländer, Peggy R; Vilaivan, Tirayut; Wagenknecht, Hans-Achim

    2015-09-21

    The so-called acpcPNA system bears a peptide backbone consisting of 4'-substituted proline units with (2'R,4'R) configuration in an alternating combination with (2S)-amino-cyclopentane-(1S)-carboxylic acids. acpcPNA forms exceptionally stable hybrids with complementary DNA. We demonstrate herein (i) strand displacements by single-stranded DNA from acpcPNA-DNA hybrids, and by acpcPNA strands from DNA duplexes, and (ii) strand invasions by acpcPNA into double-stranded DNA. These processes were studied in vitro using synthetic oligonucleotides and by means of our concept of wavelength-shifting fluorescent nucleic acid probes, including fluorescence lifetime measurements that allow quantifying energy transfer efficiencies. The strand displacements of preannealed 14mer acpcPNA-7mer DNA hybrids consecutively by 10mer and 14mer DNA strands occur with rather slow kinetics but yield high fluorescence color ratios (blue : yellow or blue : red), fluorescence intensity enhancements, and energy transfer efficiencies. Furthermore, 14mer acpcPNA strands are able to invade into 30mer double-stranded DNA, remarkably with quantitative efficiency in all studied cases. These processes can also be quantified by means of fluorescence. This remarkable behavior corroborates the extraordinary versatile properties of acpcPNA. In contrast to conventional PNA systems which require 3 or more equivalents PNA, only 1.5 equivalents acpcPNA are sufficient to get efficient double duplex invasion. Invasions also take place even in the presence of 250 mM NaCl which represents an ionic strength nearly twice as high as the physiological ion concentration. These remarkable results corroborate the extraordinary properties of acpcPNA, and thus acpcPNA represents an eligible tool for biological analytics and antigene applications.

  11. Radical-generating coordination complexes as tools for rapid and effective fragmentation and fluorescent labeling of nucleic acids for microchip hybridization.

    SciTech Connect

    Kelly, J. J.; Chernov, B. K.; Tovstanovsky, I.; Mirzabekov, A. D.; Bavykin, S. G.; Biochip Technology Center; Northwestern Univ.; Engelhardt Inst. of Molecular Biology

    2002-12-15

    DNA microchip technology is a rapid, high-throughput method for nucleic acid hybridization reactions. This technology requires random fragmentation and fluorescent labeling of target nucleic acids prior to hybridization. Radical-generating coordination complexes, such as 1,10-phenanthroline-Cu(II) (OP-Cu) and Fe(II)-EDTA (Fe-EDTA), have been commonly used as sequence nonspecific 'chemical nucleases' to introduce single-strand breaks in nucleic acids. Here we describe a new method based on these radical-generating complexes for random fragmentation and labeling of both single- and double-stranded forms of RNA and DNA. Nucleic acids labeled with the OP-Cu and the Fe-EDTA protocols revealed high hybridization specificity in hybridization with DNA microchips containing oligonucleotide probes selected for identification of 16S rRNA sequences of the Bacillus group microorganisms.We also demonstrated cDNA- and cRNA-labeling and fragmentation with this method. Both the OP-Cu and Fe-EDTA fragmentation and labeling procedures are quick and inexpensive compared to other commonly used methods. A column-based version of the described method does not require centrifugation and therefore is promising for the automation of sample preparations in DNA microchip technology as well as in other nucleic acid hybridization studies.

  12. BIOSYNTHESIS OF MYCOBACILLIN, A NEW ANTIFUNGAL PEPTIDE I.

    PubMed Central

    Banerjee, Arun B.; Bose, S. K.

    1964-01-01

    Banerjee, Arun B. (University of Calcutta, Calcutta, India), and S. K. Bose. Biosynthesis of mycobacillin, a new antifungal peptide. I. Role of nucleic acid. J. Bacteriol. 87:1397–1401. 1964.—The biosynthesis of mycobacillin, a cyclic polypeptide antifungal antibiotic, was studied in relation to the effect of chloramphenicol, 6-azathymine, and 5-bromouracil on the process. It was found that chloramphenicol inhibits both mycobacillin synthesis and growth, whereas nucleic acid base analogues inhibit only growth and nucleic acid synthesis but not mycobacillin formation. A change in the concentration of labeled aspartic acid in the general metabolic pool led to a corresponding change in the specific activity of aspartic acid isolated from different peptide fragments of the mycobacillin molecule, suggesting that mycobacillin synthesis occurs by way of linear addition of amino acid to the peptide chain. PMID:14188719

  13. Lactic acid bacterial population dynamics during fermentation and storage of Thai fermented sausage according to restriction fragment length polymorphism analysis.

    PubMed

    Wanangkarn, Amornrat; Liu, Deng-Cheng; Swetwiwathana, Adisorn; Jindaprasert, Aphacha; Phraephaisarn, Chirapiphat; Chumnqoen, Wanwisa; Tan, Fa-Jui

    2014-09-01

    This study applied restriction fragment length polymorphism (RFLP) analysis to identify the lactic acid bacteria (LAB) isolated from "mum" Thai fermented sausages during fermentation and storage. A total of 630 lactic acid bacteria were isolated from the sausages prepared using 2 methods. In Method 1, after stuffing, the sausages were stored at 30 °C for 14 days. In Method 2, after stuffing and storage at 30 °C for 3 days, the sausages were vacuum-packed and stored at 4 °C until Day 28. The sausages were sampled on Days 0, 3, 14, and 28 for analyses. The 16S rDNA was amplified and digested using restriction enzymes. Of the restriction enzymes evaluated, Dde I displayed the highest discrimination capacity. The LAB were classified and 7 species were identified For Methods 1 and 2, during fermentation, the Lactobacillus sakei and Lactobacillus plantarum species were dominant. For Method 2, the proportion of Leuconostoc mesenteroides markedly increased during storage, until L. sakei and Ln. mesenteroides represented the dominant species. The identification of LAB in the sausage samples could facilitate the selection of appropriate microorganisms for candidate starter cultures for future controlled mum production.

  14. Synthesis of a chiral amino acid with bicyclo[1.1.1]pentane moiety and its incorporation into linear and cyclic antimicrobial peptides.

    PubMed

    Pritz, Stephan; Pätzel, Michael; Szeimies, Günter; Dathe, Margitta; Bienert, Michael

    2007-06-01

    The synthesis of the lipophilic chiral amino acid 1 bearing the bicyclo[1.1.1]pentane moiety is described. Linear and cyclic hexapeptides of the type Arg-Arg-Xaa-Yaa-Arg-Phe containing 1 instead of one or two tryptophan residues are prepared by solid phase peptide synthesis and the antimicrobial and hemolytic activity of the peptides obtained are discussed.

  15. Folic Acid Inhibits Amyloid β-Peptide Production through Modulating DNA Methyltransferase Activity in N2a-APP Cells.

    PubMed

    Li, Wen; Jiang, Mingyue; Zhao, Shijing; Liu, Huan; Zhang, Xumei; Wilson, John X; Huang, Guowei

    2015-10-20

    Alzheimer's disease (AD) is a common neurodegenerative disease resulting in progressive dementia, and is a principal cause of dementia among older adults. Folate acts through one-carbon metabolism to support the methylation of multiple substrates. We hypothesized that folic acid supplementation modulates DNA methyltransferase (DNMT) activity and may alter amyloid β-peptide (Aβ) production in AD. Mouse Neuro-2a cells expressing human APP695 were incubated with folic acid (2.8-40 μmol/L), and with or without zebularine (the DNMT inhibitor). DNMT activity, cell viability, Aβ and DNMTs expression were then examined. The results showed that folic acid stimulated DNMT gene and protein expression, and DNMT activity. Furthermore, folic acid decreased Aβ protein production, whereas inhibition of DNMT activity by zebularine increased Aβ production. The results indicate that folic acid induces methylation potential-dependent DNMT enzymes, thereby attenuating Aβ production.

  16. Radiation chemistry of amino acids, peptides and proteins in relation to the radiation sterilization of high-protein foods

    SciTech Connect

    Garrison, W. M.

    1981-12-01

    An important source of information on the question of whether or not toxic or other deleterious substances are formed in the radiation sterilization of foods is the chemical study of reaction products and reaction mechanisms in the radiolysis of individual food components. The present evaluation of the radiation chemistry of amino acids, peptides, and proteins outlines the various radiation-induced processes which lead to amino acid degradation and to the synthesis of amino acid derivatives of higher molecular weight. Among the latter are the ..cap alpha..,..cap alpha..'-diamino dicarboxylic acids which are formed as major products in the radiolysis of peptides both in aqueous solution and in the solid state. The ..cap alpha..,..cap alpha..'-diamino acids are of particular interest as irradiation products because they represent a class of compounds not normally encountered in plant and animal protein sources. Such compounds have, however, been isolated from certain types of bacteria and bacterial products. All of the available data strongly suggest that the ..cap alpha..,..cap alpha..'-diamino acids are produced in significant yield in the radiation sterilization of high protein foods. The importance of initiating extensive chemical and biological studies of these and of other high molecular weight products in irradiated food is emphasized.

  17. Tri-peptide reference structures for the calculation of relative solvent accessible surface area in protein amino acid residues.

    PubMed

    Topham, Christopher M; Smith, Jeremy C

    2015-02-01

    Relative amino acid residue solvent accessibility values allow the quantitative comparison of atomic solvent-accessible surface areas in different residue types and physical environments in proteins and in protein structural alignments. Geometry-optimised tri-peptide structures in extended solvent-exposed reference conformations have been obtained for 43 amino acid residue types at a high level of quantum chemical theory. Significant increases in side-chain solvent accessibility, offset by reductions in main-chain atom solvent exposure, were observed for standard residue types in partially geometry-optimised structures when compared to non-minimised models built from identical sets of proper dihedral angles abstracted from the literature. Optimisation of proper dihedral angles led most notably to marked increases of up to 54% in proline main-chain atom solvent accessibility compared to literature values. Similar effects were observed for fully-optimised tri-peptides in implicit solvent. The relief of internal strain energy was associated with systematic variation in N, C(α) and C(β) atom solvent accessibility across all standard residue types. The results underline the importance of optimisation of 'hard' degrees of freedom (bond lengths and valence bond angles) and improper dihedral angle values from force field or other context-independent reference values, and impact on the use of standardised fixed internal co-ordinate geometry in sampling approaches to the determination of absolute values of protein amino acid residue solvent accessibility. Quantum chemical methods provide a useful and accurate alternative to molecular mechanics methods to perform energy minimisation of peptides containing non-standard (chemically modified) amino acid residues frequently present in experimental protein structure data sets, for which force field parameters may not be available. Reference tri-peptide atomic co-ordinate sets including hydrogen atoms are made freely available

  18. Measurement and modeling of acid dissociation constants of tri-peptides containing Glu, Gly, and His using potentiometry and generalized multiplicative analysis of variance.

    PubMed

    Khoury, Rima Raffoul; Sutton, Gordon J; Hibbert, D Brynn; Ebrahimi, Diako

    2013-02-28

    We report pK(a) values with measurement uncertainties for all labile protons of the 27 tri-peptides prepared from the amino acids glutamic acid (E), glycine (G) and histidine (H). Each tri-peptide (GGG, GGE, GGH, …, HHH) was subjected to alkali titration and pK(a) values were calculated from triplicate potentiometric titrations data using HyperQuad 2008 software. A generalized multiplicative analysis of variance (GEMANOVA) of pK(a) values for the most acidic proton gave the optimum model having two terms, an interaction between the end amino acids plus an isolated main effect of the central amino acid.

  19. Measurement and modeling of acid dissociation constants of tri-peptides containing Glu, Gly, and His using potentiometry and generalized multiplicative analysis of variance.

    PubMed

    Khoury, Rima Raffoul; Sutton, Gordon J; Hibbert, D Brynn; Ebrahimi, Diako

    2013-02-28

    We report pK(a) values with measurement uncertainties for all labile protons of the 27 tri-peptides prepared from the amino acids glutamic acid (E), glycine (G) and histidine (H). Each tri-peptide (GGG, GGE, GGH, …, HHH) was subjected to alkali titration and pK(a) values were calculated from triplicate potentiometric titrations data using HyperQuad 2008 software. A generalized multiplicative analysis of variance (GEMANOVA) of pK(a) values for the most acidic proton gave the optimum model having two terms, an interaction between the end amino acids plus an isolated main effect of the central amino acid. PMID:23247603

  20. Enrichment of ACE inhibitory peptides in navy bean (Phaseolus vulgaris) using lactic acid bacteria.

    PubMed

    Rui, Xin; Wen, Delan; Li, Wei; Chen, Xiaohong; Jiang, Mei; Dong, Mingsheng

    2015-02-01

    The present study was conducted to explore a novel strategy to enhance angiotensin I-converting enzyme (ACE) inhibitory activities of navy bean by preparation of navy bean milk (NBM) which was then subjected to fermentation of four lactic acid bacteria (LAB) strains, namely, Lactobacillus bulgaricus, Lactobacillus helveticus MB2-1, Lactobacillus plantarum B1-6, and Lactobacillus plantarum 70810. With the exception of L. helveticus MB2-1, the other three selected strains had good growth performances in NBM with viable counts increased to log 8.30-8.39 cfu ml(-1) during 6 h of fermentation, and thus were selected for the following investigations. Protein contents of NBM significantly reduced when treated with L. bulgaricus and L. plantarum B1-6, and the electrophoresis patterns showed the preferable proteins for LAB strains to hydrolyze were α- and β-type phaseolins, whereas γ-type phaseolin was resistant to hydrolysis. RP-HPLC analysis demonstrated all fermented NBM had higher intensities of peaks with retention times between 2.5 and 3.5 min indicative of formation of small peptides. All fermented NBM showed higher ACE inhibitory activity compared to the unfermented ones, for which 2 h, 3 h, and 5 h were found to be the optimum fermentation periods for respectively L. plantarum 70810, L. plantarum B1-6 and L. bulgaricus, with IC50 values of 109 ± 5.1, 108 ± 1.1, and 101 ± 2.2 μg protein ml(-1). The subsequent in vitro gastrointestinal simulation afforded all fermented extracts reduced IC50 values and the extracts fermented by L. plantarum B1-6 exerted the lowest IC50 value of 21 ± 2.1 μg protein ml(-1). The research has broadened our knowledge bases on the effect of LAB fermentation on the degradation of navy bean proteins and the capacity to release ACE inhibitory peptides. The approach was promising to obtain probiotic products with potential to serve as functional ingredients targeting hypertension.

  1. Differential 14N/15N-Labeling of Peptides Using N-Terminal Charge Derivatization with a High-Proton Affinity for Straightforward de novo Peptide Sequencing

    PubMed Central

    Nihashi, Yoichiro; Miyashita, Masahiro; Awane, Hiroyuki; Miyagawa, Hisashi

    2013-01-01

    While de novo peptide sequencing is essential in many situations, it remains a difficult task. This is because peptide fragmentation results in complicated and often incomplete product ion spectra. In a previous study, we demonstrated that N-terminal charge derivatization with 4-amidinobenzoic acid (Aba) resulted in improved peptide fragmentation under low-energy CID conditions. However, even with this derivatization, some ambiguity exists, due to difficulties in discriminating between N- and C-terminal fragments. In this study, to specifically identify b-ions from complex product ion spectra, the differential 14N/15N-labeling of peptides was performed using Aba derivatization. 15N-Labeled Aba was synthesized in the form of a succinimide ester. Peptides were derivatized individually with 14N-Aba or 15N-Aba and analyzed by ESI-MS/MS using a linear ion trap-Orbitrap hybrid FTMS system. The N-terminal fragments (i.e., b-ions) were then identified based on m/z differences arising from isotope labeling. By comparing the spectra between 14N- and 15N-Aba derivatized peptides, b-ions could be successfully identified based on the m/z shifts, which provided reliable sequencing results for all of the peptides examined in this study. The method developed in this study allows the easy and reliable de novo sequencing of peptides, which is useful in peptidomics and proteomics studies. PMID:24860714

  2. Vibrational spectroscopy and DFT calculations of the di-amino acid peptide L-aspartyl-L-glutamic acid in the zwitterionic state.

    PubMed

    Kausar, Nighat; Dines, Trevor J; Chowdhry, Babur Z; Alexander, Bruce D

    2009-08-14

    Solid state IR and Raman as well as aqueous solution state Raman spectra are reported for the linear di-amino acid peptide L-aspartyl-L-glutamic acid (L-Asp-L-Glu); the solution state Raman spectrum has also been obtained for the N,O-deuterated derivative. SCF-DFT calculations at the B3-LYP/cc-pVDZ level established that the structure and vibrational spectra of L-Asp-L-Glu can be interpreted using a model of the peptide with ten hydrogen-bonded water molecules, in conjunction with the conductor-like polarizable continuum solvation method. The DFT calculations resulted in the computation of a stable zwitterionic structure, which displays trans-amide conformation. The vibrational spectra were computed at the optimised molecular geometry, enabling normal coordinate analysis, which yielded satisfactory agreement with the experimental IR and Raman data. Computed potential energy distributions of the normal modes provided detailed vibrational assignments.

  3. 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

  4. 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

  5. Human immunodeficiency virus trans-activator of transcription peptide detection via ribonucleic acid aptamer on aminated diamond biosensor

    NASA Astrophysics Data System (ADS)

    Rahim Ruslinda, A.; Wang, Xianfen; Ishii, Yoko; Ishiyama, Yuichiro; Tanabe, Kyosuke; Kawarada, Hiroshi

    2011-09-01

    The potential of ribonucleic acid (RNA) as both informational and ligand binding molecule have opened a scenario in the development of biosensors. An aminated diamond-based RNA aptasensor is presented for human immunodeficiency virus (HIV) trans-activator of transcription (Tat) peptide protein detection that not only gives a labeled or label-free detection method but also provides a reusable platform for a simple, sensitive, and selective detection of proteins. The immobilized procedure was based on the binding interaction between positively charged amine terminated diamond and the RNA aptamer probe molecules with the negatively charged surface carboxylic compound linker molecule such as terephthalic acid.

  6. Self-assembly of a nine-residue amyloid-forming peptide fragment of SARS corona virus E-protein: mechanism of self aggregation and amyloid-inhibition of hIAPP.

    PubMed

    Ghosh, Anirban; Pithadia, Amit S; Bhat, Jyotsna; Bera, Supriyo; Midya, Anupam; Fierke, Carol A; Ramamoorthy, Ayyalusamy; Bhunia, Anirban

    2015-04-01

    Molecular self-assembly, a phenomenon widely observed in nature, has been exploited through organic molecules, proteins, DNA, and peptides to study complex biological systems. These self-assembly systems may also be used in understanding the molecular and structural biology which can inspire the design and synthesis of increasingly complex biomaterials. Specifically, use of these building blocks to investigate protein folding and misfolding has been of particular value since it can provide tremendous insights into peptide aggregation related to a variety of protein misfolding diseases, or amyloid diseases (e.g., Alzheimer's disease, Parkinson's disease, type-II diabetes). Herein, the self-assembly of TK9, a nine-residue peptide of the extra membrane C-terminal tail of the SARS corona virus envelope, and its variants were characterized through biophysical, spectroscopic, and simulated studies, and it was confirmed that the structure of these peptides influences their aggregation propensity, hence, mimicking amyloid proteins. TK9, which forms a beta-sheet rich fibril, contains a key sequence motif that may be critical for beta-sheet formation, thus making it an interesting system to study amyloid fibrillation. TK9 aggregates were further examined through simulations to evaluate the possible intra- and interpeptide interactions at the molecular level. These self-assembly peptides can also serve as amyloid inhibitors through hydrophobic and electrophilic recognition interactions. Our results show that TK9 inhibits the fibrillation of hIAPP, a 37 amino acid peptide implicated in the pathology of type-II diabetes. Thus, biophysical and NMR experimental results have revealed a molecular level understanding of peptide folding events, as well as the inhibition of amyloid-protein aggregation are reported.

  7. Engineered Adhesion Peptides for Improved Silicon Adsorption.

    PubMed

    Ramakrishnan, Sathish Kumar; Jebors, Said; Martin, Marta; Cloitre, Thierry; Agarwal, Vivechana; Mehdi, Ahmad; Martinez, Jean; Subra, Gilles; Gergely, Csilla

    2015-11-01

    Engineering peptides that present selective recognition and high affinity for a material is a major challenge for assembly-driven elaboration of complex systems with wide applications in the field of biomaterials, hard-tissue regeneration, and functional materials for therapeutics. Peptide-material interactions are of vital importance in natural processes but less exploited for the design of novel systems for practical applications because of our poor understanding of mechanisms underlying these interactions. Here, we present an approach based on the synthesis of several truncated peptides issued from a silicon-specific peptide recovered via phage display technology. We use the photonic response provided by porous silicon microcavities to evaluate the binding efficiency of 14 different peptide derivatives. We identify and engineer a short peptide sequence (SLVSHMQT), revealing the highest affinity for p(+)-Si. The molecular recognition behavior of the obtained peptide fragment can be revealed through mutations allowing identification of the preferential affinity of certain amino acids toward silicon. These results constitute an advance in both the engineering of peptides that reveal recognition properties for silicon and the understanding of biomolecule-material interactions.

  8. Evolutionary importance of the intramolecular pathways of hydrolysis of phosphate ester mixed anhydrides with amino acids and peptides.

    PubMed

    Liu, Ziwei; Beaufils, Damien; Rossi, Jean-Christophe; Pascal, Robert

    2014-12-11

    Aminoacyl adenylates (aa-AMPs) constitute essential intermediates of protein biosynthesis. Their polymerization in aqueous solution has often been claimed as a potential route to abiotic peptides in spite of a highly efficient CO2-promoted pathway of hydrolysis. Here we investigate the efficiency and relevance of this frequently overlooked pathway from model amino acid phosphate mixed anhydrides including aa-AMPs. Its predominance was demonstrated at CO2 concentrations matching that of physiological fluids or that of the present-day ocean, making a direct polymerization pathway unlikely. By contrast, the occurrence of the CO2-promoted pathway was observed to increase the efficiency of peptide bond formation owing to the high reactivity of the N-carboxyanhydride (NCA) intermediate. Even considering CO2 concentrations in early Earth liquid environments equivalent to present levels, mixed anhydrides would have polymerized predominantly through NCAs. The issue of a potential involvement of NCAs as biochemical metabolites could even be raised. The formation of peptide-phosphate mixed anhydrides from 5(4H)-oxazolones (transiently formed through prebiotically relevant peptide activation pathways) was also observed as well as the occurrence of the reverse cyclization process in the reactions of these mixed anhydrides. These processes constitute the core of a reaction network that could potentially have evolved towards the emergence of translation.

  9. Evolutionary Importance of the Intramolecular Pathways of Hydrolysis of Phosphate Ester Mixed Anhydrides with Amino Acids and Peptides

    NASA Astrophysics Data System (ADS)

    Liu, Ziwei; Beaufils, Damien; Rossi, Jean-Christophe; Pascal, Robert

    2014-12-01

    Aminoacyl adenylates (aa-AMPs) constitute essential intermediates of protein biosynthesis. Their polymerization in aqueous solution has often been claimed as a potential route to abiotic peptides in spite of a highly efficient CO2-promoted pathway of hydrolysis. Here we investigate the efficiency and relevance of this frequently overlooked pathway from model amino acid phosphate mixed anhydrides including aa-AMPs. Its predominance was demonstrated at CO2 concentrations matching that of physiological fluids or that of the present-day ocean, making a direct polymerization pathway unlikely. By contrast, the occurrence of the CO2-promoted pathway was observed to increase the efficiency of peptide bond formation owing to the high reactivity of the N-carboxyanhydride (NCA) intermediate. Even considering CO2 concentrations in early Earth liquid environments equivalent to present levels, mixed anhydrides would have polymerized predominantly through NCAs. The issue of a potential involvement of NCAs as biochemical metabolites could even be raised. The formation of peptide-phosphate mixed anhydrides from 5(4H)-oxazolones (transiently formed through prebiotically relevant peptide activation pathways) was also observed as well as the occurrence of the reverse cyclization process in the reactions of these mixed anhydrides. These processes constitute the core of a reaction network that could potentially have evolved towards the emergence of translation.

  10. Proline-glutamic acid-proline-lysine peptide set as a specific antigen for the serological diagnosis of strangles.

    PubMed

    Hobo, S; Niwa, H; Anzai, T

    2006-11-01

    The reactivity of synthesised peptide sets for the M-like proteins SeM and SzPSe with sera from horses infected with Streptococcus equi or Streptococcus zooepidemicus, or control horses, was investigated by an ELISA. Seventeen horses were infected experimentally with S equi or S zooepidemicus, convalescent sera were obtained from 25 horses and control sera were obtained from 1945 horses. The serum antibody responses of individual horses to the peptide sets were highly variable. Some of the peptide sets for SeM reacted strongly with the sera from the horses infected experimentally with S equi, but also reacted with sera from some of the horses infected experimentally with S zooepidemicus. However, the proline-glutamic acid-proline-lysine (PEPK) repeats peptide set, synthesised from the PEPK repeats areas of SzPSe, reacted most strongly with the sera from the horses infected experimentally with S equi and the horses convalescing from strangles, and reacted only minimally with the sera from the horses infected experimentally with S zooepidemicus and the control horses.

  11. Targeting acidity in diseased tissues: mechanism and applications of the membrane-inserting peptide, pHLIP

    PubMed Central

    Deacon, John; Engelman, Donald M.; Barrera, Francisco N.

    2014-01-01

    pHLIPs are a family of soluble ~36 amino acid peptides, which bind to membrane surfaces. If the environment is acidic, a pHLIP folds and inserts across the membrane to form a stable transmembrane helix, thus preferentially locating itself in acidic tissues. Since tumors and other disease tissues are acidic, pHLIPs’ low-pH targeting behavior leads to applications as carriers for diagnostic and surgical imaging agents. The energy of membrane insertion can also be used to promote the insertion of modestly polar, normally cell-impermeable cargos across the cell membrane into the cytosol of targeted cells, leading to applications in tumor-targeted delivery of therapeutic molecules. We review the biochemical and biophysical basis of pHLIPs’ unique properties, diagnostic and therapeutic applications, and the principles upon which translational applications are being developed. PMID:25444855

  12. Ribosome-mediated incorporation of a non-standard amino acid into a peptide through expansion of the genetic code.

    PubMed

    Bain, J D; Switzer, C; Chamberlin, A R; Benner, S A

    1992-04-01

    One serious limitation facing protein engineers is the availability of only 20 'proteinogenic' amino acids encoded by natural messenger RNA. The lack of structural diversity among these amino acids restricts the mechanistic and structural issues that can be addressed by site-directed mutagenesis. Here we describe a new technology for incorporating non-standard amino acids into polypeptides by ribosome-based translation. In this technology, the genetic code is expanded through the creation of a 65th codon-anticodon pair from unnatural nucleoside bases having non-standard hydrogen-bonding patterns. This new codon-anticodon pair efficiently supports translation in vitro to yield peptides containing a non-standard amino acid. The versatility of the ribosome as a synthetic tool offers new possibilities for protein engineering, and compares favourably with another recently described approach in which the genetic code is simply rearranged to recruit stop codons to play a coding role. PMID:1560827

  13. Immobilization-free electrochemical DNA detection with anthraquinone-labeled pyrrolidinyl peptide nucleic acid probe.

    PubMed

    Kongpeth, Jutatip; Jampasa, Sakda; Chaumpluk, Piyasak; Chailapakul, Orawon; Vilaivan, Tirayut

    2016-01-01

    Electrochemical detection provides a simple, rapid, sensitive and inexpensive method for DNA detection. In traditional electrochemical DNA biosensors, the probe is immobilized onto the electrode. Hybridization with the DNA target causes a change in electrochemical signal, either from the intrinsic signal of the probe/target or through a label or a redox indicator. The major drawback of this approach is the requirement for probe immobilization in a controlled fashion. In this research, we take the advantage of different electrostatic properties between PNA and DNA to develop an immobilization-free approach for highly sequence-specific electrochemical DNA sensing on a screen-printed carbon electrode (SPCE) using a square-wave voltammetric (SWV) technique. Anthraquinone-labeled pyrrolidinyl peptide nucleic acid (AQ-PNA) was employed as a probe together with an SPCE that was modified with a positively-charged polymer (poly quaternized-(dimethylamino-ethyl)methacrylate, PQDMAEMA). The electrostatic attraction between the negatively-charged PNA-DNA duplex and the positively-charged modified SPCE attributes to the higher signal of PNA-DNA duplex than that of the electrostatically neutral PNA probe, resulting in a signal change. The calibration curve of this proposed method exhibited a linear range between 0.35 and 50 nM of DNA target with a limit of detection of 0.13 nM (3SD(blank)/Slope). The sub-nanomolar detection limit together with a small sample volume required (20 μL) allowed detection of <10 fmol (<1 ng) of DNA. With the high specificity of the pyrrolidinyl PNA probe used, excellent discrimination between complementary and various single-mismatched DNA targets was obtained. An application of this new platform for a sensitive and specific detection of isothermally-amplified shrimp's white spot syndrome virus (WSSV) DNA was successfully demonstrated. PMID:26695270

  14. 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

  15. Immobilization-free electrochemical DNA detection with anthraquinone-labeled pyrrolidinyl peptide nucleic acid probe.

    PubMed

    Kongpeth, Jutatip; Jampasa, Sakda; Chaumpluk, Piyasak; Chailapakul, Orawon; Vilaivan, Tirayut

    2016-01-01

    Electrochemical detection provides a simple, rapid, sensitive and inexpensive method for DNA detection. In traditional electrochemical DNA biosensors, the probe is immobilized onto the electrode. Hybridization with the DNA target causes a change in electrochemical signal, either from the intrinsic signal of the probe/target or through a label or a redox indicator. The major drawback of this approach is the requirement for probe immobilization in a controlled fashion. In this research, we take the advantage of different electrostatic properties between PNA and DNA to develop an immobilization-free approach for highly sequence-specific electrochemical DNA sensing on a screen-printed carbon electrode (SPCE) using a square-wave voltammetric (SWV) technique. Anthraquinone-labeled pyrrolidinyl peptide nucleic acid (AQ-PNA) was employed as a probe together with an SPCE that was modified with a positively-charged polymer (poly quaternized-(dimethylamino-ethyl)methacrylate, PQDMAEMA). The electrostatic attraction between the negatively-charged PNA-DNA duplex and the positively-charged modified SPCE attributes to the higher signal of PNA-DNA duplex than that of the electrostatically neutral PNA probe, resulting in a signal change. The calibration curve of this proposed method exhibited a linear range between 0.35 and 50 nM of DNA target with a limit of detection of 0.13 nM (3SD(blank)/Slope). The sub-nanomolar detection limit together with a small sample volume required (20 μL) allowed detection of <10 fmol (<1 ng) of DNA. With the high specificity of the pyrrolidinyl PNA probe used, excellent discrimination between complementary and various single-mismatched DNA targets was obtained. An application of this new platform for a sensitive and specific detection of isothermally-amplified shrimp's white spot syndrome virus (WSSV) DNA was successfully demonstrated.

  16. 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 o