Science.gov

Sample records for plasmamembrane polypeptides final

  1. Preparation of plasma-membrane subfractions from isolated rat hepatocytes.

    PubMed Central

    Wisher, M H; Evans, W H

    1977-01-01

    1. Rat livers were dissociated into their constituent cells by perfusion through the portal vein with a medium containing collagenase, and hepatocytes separated from non-parenchymal cells. 2. It is shown that the procedure described by Wisher & Evans [(1975) Biochem. J. 146, 375-388] for preparation of plasma membranes from liver tissue when applied to isolated hepatocytes also yielded subfractions of similar morphology and marker-enzyme distribution. 3. Thus the distribution of alkaline phosphodiesterase, 5'-nucleotidase and the basal and glucagon-stimulated adenylate cyclase among two 'light' vesicular and one 'heavy' junction-containing plasma-membrane subfractions paralleled that reported for tissue-derived plasma-membrane subfractions. 4. Increased recoveries and specific activities of plasma-membrane marker enzymes were obtained when soya-bean trypsin inhibitor was included in the collagenase-containing perfusion media used to dissociate the liver. 5. Polyacrylamide-gel-electrophoretic analysis of the corresponding plasma-membrane subfractions prepared from liver tissue and isolated hepatocytes were generally similar. 6. The results indicate that the functional polarity of the hepatocyte's plasma membrane is retained after tissue dissociation. The damage occurring to plasma-membrane ectoenzymes by the collagenase-perfusion procedure is discussed. Images PLATE 1 PLATE 2 PLATE 3 PMID:880246

  2. Elastomeric Polypeptides

    PubMed Central

    van Eldijk, Mark B.; McGann, Christopher L.

    2013-01-01

    Elastomeric polypeptides are very interesting biopolymers and are characterized by rubber-like elasticity, large extensibility before rupture, reversible deformation without loss of energy, and high resilience upon stretching. Their useful properties have motivated their use in a wide variety of materials and biological applications. This chapter focuses on elastin and resilin – two elastomeric biopolymers – and the recombinant polypeptides derived from them (elastin-like polypeptides and resilin-like polypeptides). This chapter also discusses the applications of these recombinant polypeptides in the fields of purification, drug delivery, and tissue engineering. PMID:21826606

  3. Antifungal polypeptides

    DOEpatents

    Altier, Daniel J.; Ellanskaya, Irina; Ellanskaya, legal representative, Natalia; Gilliam, Jacob T.; Hunter-Cevera, Jennie; Presnail, James K.; Schepers, Eric; Simmons, Carl R.; Torok, Tamas; Yalpani, Nasser

    2009-09-15

    The invention relates to antifungal compositions and methods for protecting a plant from a fungal pathogen. Compositions including antifungal polypeptides isolated from a fungal fermentation broth are provided.

  4. Antifungal polypeptides

    DOEpatents

    Altier, Daniel J.; Dahlbacka, Glen; Elleskaya, Irina; Ellanskaya, legal representative; Natalia; Herrmann, Rafael; Hunter-Cevera, Jennie; McCutchen, Billy F.; Presnail, James K.; Rice, Janet A.; Schepers, Eric; Simmons, Carl R.; Torok, Tamas; Yalpani, Nasser

    2010-08-10

    Compositions and methods for protecting a plant from a pathogen, particularly a fungal pathogen, are provided. Compositions include novel amino acid sequences, and variants and fragments thereof, for antipathogenic polypeptides that were isolated from microbial fermentation broths. Nucleic acid molecules comprising nucleotide sequences that encode the antipathogenic polypeptides of the invention are also provided. A method for inducing pathogen resistance in a plant using the nucleotide sequences disclosed herein is further provided. The method comprises introducing into a plant an expression cassette comprising a promoter operably linked to a nucleotide sequence that encodes an antipathogenic polypeptide of the invention. Compositions comprising an antipathogenic polypeptide or a transformed microorganism comprising a nucleic acid of the invention in combination with a carrier and methods of using these compositions to protect a plant from a pathogen are further provided. Transformed plants, plant cells, seeds, and microorganisms comprising a nucleotide sequence that encodes an antipathogenic polypeptide of the invention, or variant or fragment thereof, are also disclosed.

  5. Antifungal polypeptides

    DOEpatents

    Altier, Daniel J.; Dahlbacka, Glen; Elleskaya, Irina; Ellanskaya, legal representative, Natalia; Herrmann, Rafael; Hunter-Cevera, Jennie; McCutchen, Billy F.; Presnail, James K.; Rice, Janet A.; Schepers, Eric; Simmons, Carl R.; Torok, Tamas; Yalpani, Nasser

    2011-04-12

    Compositions and methods for protecting a plant from a pathogen, particularly a fungal pathogen, are provided. Compositions include novel amino acid sequences, and variants and fragments thereof, for antipathogenic polypeptides that were isolated from microbial fermentation broths. Nucleic acid molecules comprising nucleotide sequences that encode the antipathogenic polypeptides of the invention are also provided. A method for inducing pathogen resistance in a plant using the nucleotide sequences disclosed herein is further provided. The method comprises introducing into a plant an expression cassette comprising a promoter operably linked to a nucleotide sequence that encodes an antipathogenic polypeptide of the invention. Compositions comprising an antipathogenic polypeptide or a transformed microorganism comprising a nucleic acid of the invention in combination with a carrier and methods of using these compositions to protect a plant from a pathogen are further provided. Transformed plants, plant cells, seeds, and microorganisms comprising a nucleotide sequence that encodes an antipathogenic polypeptide of the invention, or variant or fragment thereof, are also disclosed.

  6. Antifungal polypeptides

    DOEpatents

    Altier, Daniel J.; Dahlbacka, Glen; Ellanskaya, legal representative, Natalia; Herrmann, Rafael; Hunter-Cevera, Jennie; McCutchen, Billy F.; Presnail, James K.; Rice, Janet A.; Schepers, Eric; Simmons, Carl R.; Torok, Tamas; Yalpani, Nasser; Ellanskaya, deceased, Irina

    2007-12-11

    Compositions and methods for protecting a plant from a pathogen, particularly a fungal pathogen, are provided. Compositions include novel amino acid sequences, and variants and fragments thereof, for antipathogenic polypeptides that were isolated from microbial fermentation broths. Nucleic acid molecules comprising nucleotide sequences that encode the antipathogenic polypeptides of the invention are also provided. A method for inducing pathogen resistance in a plant using the nucleotide sequences disclosed herein is further provided. The method comprises introducing into a plant an expression cassette comprising a promoter operably linked to a nucleotide sequence that encodes an antipathogenic polypeptide of the invention. Compositions comprising an antipathogenic polypeptide or a transformed microorganism comprising a nucleic acid of the invention in combination with a carrier and methods of using these compositions to protect a plant from a pathogen are further provided. Transformed plants, plant cells, seeds, and microorganisms comprising a nucleotide sequence that encodes an antipathogenic polypeptide of the invention, or variant or fragment thereof, are also disclosed.

  7. Antifungal polypeptides

    DOEpatents

    Altier, Daniel J.; Dahlbacka, Glen; Ellanskaya, Irina; Ellanskaya, legal representative, Natalia; Herrmann, Rafael; Hunter-Cevera, Jennie; McCutchen, Billy F.; Presnail, James K.; Rice, Janet A.; Schepers, Eric; Simmons, Carl R.; Torok, Tamas; Yalpani, Nasser

    2012-04-03

    Compositions and methods for protecting a plant from a pathogen, particularly a fungal pathogen, are provided. Compositions include novel amino acid sequences, and variants and fragments thereof, for antipathogenic polypeptides that were isolated from microbial fermentation broths. Nucleic acid molecules comprising nucleotide sequences that encode the antipathogenic polypeptides of the invention are also provided. A method for inducing pathogen resistance in a plant using the nucleotide sequences disclosed herein is further provided. The method comprises introducing into a plant an expression cassette comprising a promoter operably linked to a nucleotide sequence that encodes an antipathogenic polypeptide of the invention. Compositions comprising an antipathogenic polypeptide or a transformed microorganism comprising a nucleic acid of the invention in combination with a carrier and methods of using these compositions to protect a plant from a pathogen are further provided. Transformed plants, plant cells, seeds, and microorganisms comprising a nucleotide sequence that encodes an antipathogenic polypeptide of the invention, or variant or fragment thereof, are also disclosed.

  8. Combined effects of solvation and aggregation propensity on the final supramolecular structures adopted by hydrophobic, glycine-rich, elastin-like polypeptides.

    PubMed

    Salvi, Anna M; Moscarelli, Pasquale; Bochicchio, Brigida; Lanza, Giuseppe; Castle, James E

    2013-05-01

    Previous work on elastin-like polypeptides (ELPs) made of hydrophobic amino acids of the type XxxGlyGlyZzzGly (Xxx, Zzz = Val, Leu) has consistently shown that differing dominant supramolecular structures were formed when the suspending media were varied: helical, amyloid-like fibers when suspended in water and globules evolving into "string of bead" structures, poly(ValGlyGlyValGly), or cigar-like bundles, poly(ValGlyGlyLeuGly), when suspended in methyl alcohol. Comparative experiments with poly(LeuGlyGlyValGly) have further indicated that the interface energy plays a significant role and that solvation effects act in concomitance with the intrinsic aggregation propensity of the repeat sequence. Continuing our investigation on ELPs using surface (X-ray photoelectron spectroscopy, atomic force microscopy) and bulk (circular dichroism, Fourier transform infrared spectroscopy) techniques for their characterization, here we have compared the effect of suspending solvents (H(2)O, dimethylsulfoxide, ethylene glycol, and MeOH) on poly(ValGlyGlyValGly), the polypeptide most inclined to form long and well-refined helical fibers in water, searching for the signature of intermolecular interactions occurring between the polypeptide chains in the given suspension. The influence of sequence specificities has been studied by comparing poly(ValGlyGlyValGly) and poly(LeuGlyGlyValGly) with a similar degree of polymerization. Deposits on substrates of the polypeptides were characterized taking into account the differing evaporation rate of solvents, and tests on their stability in ultra high vacuum were performed. Finally, combining experimental and computational studies, we have revaluated the three-dimensional modeling previously proposed for the supramolecular assembly in water of poly(ValGlyGlyValGly). The results were discussed and rationalized also in the light of published data. PMID:23426573

  9. In silicio identification of glycosyl-phosphatidylinositol-anchored plasma-membrane and cell wall proteins of Saccharomyces cerevisiae.

    PubMed

    Caro, L H; Tettelin, H; Vossen, J H; Ram, A F; van den Ende, H; Klis, F M

    1997-12-01

    Use of the Von Heijne algorithm allowed the identification of 686 open reading frames (ORFs) in the genome of Saccharomyces cerevisiae that encode proteins with a potential N-terminal signal sequence for entering the secretory pathway. On further analysis, 51 of these proteins contain a potential glycosyl-phosphatidylinositol (GPI)-attachment signal. Seven additional ORFs were found to belong to this group. Upon examination of the possible GPI-attachment sites, it was found that in yeast the most probable amino acids for GPI-attachment as asparagine and glycine. In yeast, GPI-proteins are found at the cell surface, either attached to the plasma-membrane or as an intrinsic part of the cell wall. It was noted that plasma-membrane GPI-proteins possess a dibasic residue motif just before their predicted GPI-attachment site. Based on this, and on homologies between proteins, families of plasma-membrane and cell wall proteins were assigned, revealing 20 potential plasma-membrane and 38 potential cell wall proteins. For members of three plasma-membrane protein families, a function has been described. On the other hand, most of the cell wall proteins seem to be structural components of the wall, responsive to different growth conditions. The GPI-attachment site of yeast slightly differs from mammalian cells. This might be of use in the development of anti-fungal drugs.

  10. Targeted polypeptide degradation

    DOEpatents

    Church, George M.; Janse, Daniel M.

    2008-05-13

    This invention pertains to compositions, methods, cells and organisms useful for selectively localizing polypeptides to the proteasome for degradation. Therapeutic methods and pharmaceutical compositions for treating disorders associated with the expression and/or activity of a polypeptide by targeting these polypeptides for degradation, as well as methods for targeting therapeutic polypeptides for degradation and/or activating therapeutic polypeptides by degradation are provided. The invention provides methods for identifying compounds that mediate proteasome localization and/or polypeptide degradation. The invention also provides research tools for the study of protein function.

  11. Large plasma-membrane depolarization precedes rapid blue-light-induced growth inhibition in cucumber

    NASA Technical Reports Server (NTRS)

    Spalding, E. P.; Cosgrove, D. J.

    1989-01-01

    Blue-light (BL)-induced suppression of elongation of etiolated Cucumis sativus L. hypocotyls began after a 30-s lag time, which was halved by increasing the fluence rate from 10 to 100 micromoles m-2 s-1. Prior to the growth suppression, the plasma-membrane of the irradiated cells depolarized by as much as 100 mV, then returned within 2-3 min to near its initial value. The potential difference measured with surface electrodes changed with an identical time course but opposite polarity. The lag time for the change in surface potential showed an inverse dependence on fluence rate, similar to the lag for the growth inhibition. Green light and red light caused neither the electrical response nor the rapid inhibition of growth. The depolarization by BL did not propagate to nonirradiated regions and exhibited a refractory period of about 10 min following a BL pulse. Fluence-response relationships for the electrical and growth responses provide correlational evidence that the plasma-membrane depolarization reflects an event in the transduction chain of this light-growth response.

  12. Organic Anion Transporting Polypeptides

    PubMed Central

    Stieger, Bruno; Hagenbuch, Bruno

    2013-01-01

    Organic anion transporting polypeptides or OATPs are central transporters in the disposition of drugs and other xenobiotics. In addition, they mediate transport of a wide variety of endogenous substrates. The critical role of OATPs in drug disposition has spurred research both in academia and in the pharmaceutical industry. Translational aspects with clinical questions are the focus in academia, while the pharmaceutical industry tries to define and understand the role these transporters play in pharmacotherapy. The present overview summarizes our knowledge on the interaction of food constituents with OATPs, and on the OATP transport mechanisms. Further, it gives an update on the available information on the structure-function relationship of the OATPs, and finally, covers the transcriptional and posttranscriptional regulation of OATPs. PMID:24745984

  13. SLC41A2 encodes a plasma-membrane Mg2+ transporter

    PubMed Central

    Sahni, Jaya; Nelson, Bruce; Scharenberg, Andrew M.

    2006-01-01

    The TRPM7 (transient receptor potential melastatin 7) ion channel has been implicated in the uptake of Mg2+ into vertebrate cells, as elimination of TRPM7 expression through gene targeting in DT40 B-lymphocytes renders them unable to grow in the absence of supplemental Mg2+. However, a residual capacity of TRPM7-deficient cells to accumulate Mg2+ and proliferate when provided with supplemental Mg2+ suggests the existence of Mg2+ uptake mechanism(s) other than TRPM7. Evaluation of the expression of several members of the SLC41 (solute carrier family 41) family, which exhibit homology with the MgtE class of prokaryotic putative bivalent-cation transporters, demonstrated that one, SLC41A2 (solute carrier family 41 member 2), is expressed in both wild-type and TRPM7-deficient DT40 cells. Characterization of heterologously expressed SLC41A2 protein indicated that it is a plasma-membrane protein with an N-terminus-outside/C-terminus-inside 11-TM (transmembrane)-span topology, consistent with its functioning as a trans-plasma-membrane transporter. In contrast with a previous report of ion-channel activity associated with SLC41A2 expression in oocytes, investigation of whole cell currents in SLC41A2-expressing DT40 cells revealed no novel currents of any type associated with SLC41A2 expression. However, expression of SLC41A2 in TRPM7-deficient cells under the control of a doxycycline-inducible promoter was able to conditionally enhance their net uptake of 26Mg2+ and conditionally and dose-dependently provide them with the capacity to grow in the absence of supplemental Mg2+, observations strongly supporting a model whereby SLC41A2 directly mediates trans-plasma-membrane Mg2+ transport. Overall, our results suggest that SLC41A2 functions as a plasma-membrane Mg2+ transporter in vertebrate cells. PMID:16984228

  14. Agouti polypeptide compositions

    DOEpatents

    Woychik, Richard P.; Bultman, Scott J.; Michaud, Edward J.

    2001-10-30

    Disclosed are methods and compositions comprising novel agouti polypeptides and the polynucleotides which encode them. Also disclosed are DNA segments encoding these proteins derived from human and murine cell lines, and the use of these polynucleotides and polypeptides in a variety of diagnostic and therapeutic applications. Methods, compositions, kits, and devices are also provided for identifying compounds which are inhibitors of agouti activity, and for altering fatty acid synthetase activity and intracellular calcium levels in transformed cells.

  15. Cell wall constrains lateral diffusion of plant plasma-membrane proteins

    PubMed Central

    Martinière, Alexandre; Lavagi, Irene; Nageswaran, Gayathri; Rolfe, Daniel J.; Maneta-Peyret, Lilly; Luu, Doan-Trung; Botchway, Stanley W.; Webb, Stephen E. D.; Mongrand, Sebastien; Maurel, Christophe; Martin-Fernandez, Marisa L.; Kleine-Vehn, Jürgen; Friml, Jirí; Moreau, Patrick; Runions, John

    2012-01-01

    A cell membrane can be considered a liquid-phase plane in which lipids and proteins theoretically are free to diffuse. Numerous reports, however, describe retarded diffusion of membrane proteins in animal cells. This anomalous diffusion results from a combination of structuring factors including protein–protein interactions, cytoskeleton corralling, and lipid organization into microdomains. In plant cells, plasma-membrane (PM) proteins have been described as relatively immobile, but the control mechanisms that structure the PM have not been studied. Here, we use fluorescence recovery after photobleaching to estimate mobility of a set of minimal PM proteins. These proteins consist only of a PM-anchoring domain fused to a fluorescent protein, but their mobilities remained limited, as is the case for many full-length proteins. Neither the cytoskeleton nor membrane microdomain structure was involved in constraining the diffusion of these proteins. The cell wall, however, was shown to have a crucial role in immobilizing PM proteins. In addition, by single-molecule fluorescence imaging we confirmed that the pattern of cellulose deposition in the cell wall affects the trajectory and speed of PM protein diffusion. Regulation of PM protein dynamics by the plant cell wall can be interpreted as a mechanism for regulating protein interactions in processes such as trafficking and signal transduction. PMID:22689944

  16. Arrays of plasma-membrane "rosettes" involved in cellulose microfibril formation of Spirogyra.

    PubMed

    Herth, W

    1983-11-01

    The cell-wall structure and plasma-membrane particle arrangement during cell wall formation of the filamentous chlorophycean alga Spirogyra sp. was investigated with the freeze-fracture technique. The cell wall consists of a thick outer slime layer and a multilayered inner wall with ribbon-like microfibrils. This inner wall shows three differing orientations of microfibrils: random orientation on its outside, followed by axial bundles of parallel microfibrils, and several internal layers of bands of mostly five to six parallel associated microfibrils with transverse to oblique orientation. The extraplasmatic fracture face of the plasma membrane shows microfibril imprints, relatively few particles, and "terminal complexes" arranged in a hexagonal package at the end of the imprint of a microfibril band. The plasmatic fracture face of the plasma membrane is rich in particles. In places, it reveals hexagonal arrays of "rosettes". These rosettes are best demonstrable with the double-replica technique. These findings on rosette arrays of the zygnematacean alga Spirogyra are compared in detail with the published data on the desmidiacean algae Micrasterias and Closterium.

  17. Mechanism of blue-light-induced plasma-membrane depolarization in etiolated cucumber hypocotyls

    NASA Technical Reports Server (NTRS)

    Spalding, E. P.; Cosgrove, D. J.

    1992-01-01

    A large, transient depolarization of the plasma membrane precedes the rapid blue-light (BL)-induced growth suppression in etiolated seedlings of Cucumis sativus L. The mechanism of this voltage transient was investigated by applying inhibitors of ion channels and the plasma-membrane H(+)-ATPase, by manipulating extracellular ion concentrations, and by measuring cell input resistance and ATP levels. The depolarizing phase was not affected by Ca(2+)-channel blockers (verapamil, La3+) or by reducing extracellular free Ca2+ by treatment with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). However, these treatments did reduce the rate of repolarization, indicating an inward movement of Ca2+ is involved. No effects of the K(+)-channel blocker tetraethylammonium (TEA+) were detected. Vanadate and KCN, used to inhibit the H(+)-ATPase, reduced or completely inhibited the BL-induced depolarization. Levels of ATP increased by 11-26% after 1-2 min of BL. Input resistance of trichrome cells, measured with double-barreled microelectrodes, remained constant during the onset of the depolarization but decreased as the membrane voltage became more positive than -90 mV. The results indicate that the depolarization mechanism initially involves inactivation of the H(+)-ATPase with subsequent transient activation of one or more types of ion channels.

  18. ATP-dependent bile-salt transport in canalicular rat liver plasma-membrane vesicles.

    PubMed Central

    Stieger, B; O'Neill, B; Meier, P J

    1992-01-01

    The present study identifies and characterizes a novel ATP-dependent bile-salt transport system in isolated canalicular rat liver plasma-membrane (cLPM) vesicles. ATP (1-5 mM) stimulated taurocholate uptake into cLPM vesicles between 6- and 8-fold above equilibrium uptake values (overshoot) and above values for incubations in the absence of ATP. The ATP-dependent portion of taurocholate uptake was 2-fold higher in the presence of equilibrated KNO3 as compared with potassium gluconate, indicating that the stimulatory effect of ATP was not due to the generation of an intravesicular positive membrane potential. Saturation kinetics revealed a very high affinity (Km approximately 2.1 microM) of the system for taurocholate. The system could only minimally be stimulated by nucleotides other than ATP. Furthermore, it was preferentially inhibited by conjugated univalent bile salts. Further strong inhibitory effects were observed with valinomycin, oligomycin, 4,4'-di-isothiocyano-2,2'-stilbene disulphonate, sulphobromophthalein, leukotriene C4 and N-ethylmaleimide, whereas nigericin, vanadate, GSH, GSSG and daunomycin exerted only weak inhibitory effects or none at all. These results indicate the presence of a high-affinity primary ATP-dependent bile-salt transport system in cLPM vesicles. This transport system might be regulated in vivo by the number of carriers present at the perspective transport site(s), which, in addition to the canalicular membrane, might also include pericanalicular membrane vesicles. PMID:1599411

  19. Mechanism of glucose and maltose transport in plasma-membrane vesicles from the yeast Candida utilis.

    PubMed Central

    van den Broek, P J; van Gompel, A E; Luttik, M A; Pronk, J T; van Leeuwen, C C

    1997-01-01

    Transport of glucose and maltose was studied in plasma-membrane vesicles from Candida utilis. The yeast was grown on a mixture of glucose and maltose in aerobic carbon-limited continuous cultures which enabled transport to be studied for both sugars with the same vesicles. Vesicles were prepared by fusion of isolated plasma membranes with proteoliposomes containing bovine heart cytochrome c oxidase as a proton-motive-force-generating system. Addition of reduced cytochrome c generated a proton-motive force, consisting of a membrane potential, negative inside, and a pH gradient, alkaline inside. Energization led to accumulation of glucose and maltose in these vesicles, reaching accumulation ratios of about 40-50. Accumulation also occurred in the presence of valinomycin or nigericin, but was prevented by a combination of the two ionophores or by uncoupler, showing that glucose and maltose transport are dependent on the proton-motive force. Comparison of sugar accumulation with quantitative data on the proton-motive force indicated a 1:1 H+/sugar stoichiometry for both transport systems. Efflux of accumulated glucose was observed on dissipation of the proton-motive force. Exchange and counterflow experiments confirmed the reversible character of the H+-glucose symporter. In contrast, uncoupler or a mixture of valinomycin plus nigericin induced only a slow efflux of accumulated maltose. Moreover under counterflow conditions, the expected transient accumulation was small. Thus the H+-maltose symporter has some characteristics of a carrier that is not readily reversible. It is concluded that in C. utilis the transport systems for glucose and maltose are both driven by the proton-motive force, but the mechanisms are different. PMID:9020885

  20. Resynthesis of sphingomyelin from plasma-membrane phosphatidylcholine in BHK cells treated with Staphylococcus aureus sphingomyelinase.

    PubMed Central

    Allan, D; Quinn, P

    1988-01-01

    About 60-65% of the total sphingomyelin in intact BHK cells is in a readily accessible pool which is rapidly degraded by Staphylococcus aureus sphingomyelinase. No more sphingomyelin is broken down in cells which have been fixed with glutaraldehyde or lysed with streptolysin O, suggesting that all the sphingomyelin which is available to the enzyme is on the cell surface. The inaccessible pool of sphingomyelin does not equilibrate with the plasma-membrane pool, even after prolonged incubation. Experiments using [3H]-choline show that much more phosphocholine is released from the intact cells treated with sphingomyelinase than can be accounted for by breakdown of the original cell-surface pool of sphingomyelin; the excess appears to be a consequence of the breakdown of sphingomyelin newly resynthesized at the expense of a pool of phosphatidylcholine which represents about 8% of total cell phosphatidylcholine and may reside in the plasma membrane. This would be consistent with resynthesis of cell-surface sphingomyelin by the phosphatidylcholine: ceramide phosphocholinetransferase pathway, which has previously been shown to be localized in the plasma membrane. However, in [3H]palmitate-labelled cells there appeared to be no accumulation of the diacylglycerol expected to be produced by this reaction, and no enhanced synthesis of phosphatidate or phosphatidylinositol; instead there was an increased synthesis of triacylglycerol. A similar increase in labelling of triacylglycerol was seen in enzyme-treated cells where the sphingomyelinase was subsequently removed, allowing resynthesis of sphingomyelin which occurred at a rate of about 25% of total sphingomyelin/h. Treatment of BHK cells with sphingomyelinase caused no change in the rates of fluid-phase endocytosis or exocytosis as measured with [3H]inulin. PMID:2848498

  1. The relationship of detergent-solubilization plasma-membrane components of rabbit alveolar macrophages to an isolated inhibitor of phagocytosis.

    PubMed Central

    Pratt, R S; Cook, G M

    1979-01-01

    1. A plasma-membrane fraction prepared from rabbit alveolar macrophages by hyposmotic borate lysis is described. 2. Rabbit lung lavages, containing a glycoprotein inhibitor of phagocytosis, may be fractionated by preparative isoelectric focusing in the presence of Triton X-100. 3. Chemical analysis indicates that the glycoproteins of the lung lavage contain sialic acid, fucose, mannose, galactose, hexosamine and appreciable quantities of glucose. 4. The relationship of macrophage membrane glycoproteins, solubilized with Triton X-100 in the presence of borate, to the lung lavage glycoproteins is demonstrated immunoelectrophoretically. Images PLATE 1 Fig. 1. Fig. 2. PMID:486083

  2. Histones and basic polypeptides activate Ca2+/cation influx in various cell types.

    PubMed Central

    Gamberucci, A; Fulceri, R; Marcolongo, P; Pralong, W F; Benedetti, A

    1998-01-01

    Histone H2A (1-10 microg/ml) added to Ehrlich ascite cell suspensions promoted: (i) Ca2+ influx, but no apparent intracellular Ca2+ mobilization; (ii) plasma-membrane depolarization and Na+ influx in Ca2+-free medium, which were recovered by Ca2+ readmission; (iii) influx of other cations such as Ba2+, Mn2+, choline+ and N-methyl-d-glucamine+, but not of propidium+, ethidium bromide and Trypan Blue. H2A-induced Ca2+ influx and cell depolarization were: (i) blocked by La3+ and Gd3+, but not by various inhibitors of receptor-activated Ca2+-influx pathways/channels; (ii) mimicked by various basic polypeptides, with Mr>4000; (iii) prevented or reversed by polyanions such as polyglutamate or heparin; (iv) present in other cell types, such as Jurkat, PC12 and Friend erythroleukaemia cells, but virtually absent from rat hepatocytes and thymocytes. We conclude that cationic proteins/polypeptides, by interacting in a cell-specific manner with the cell surface, can activate in those cells putative non-selective Ca2+ channels and membrane depolarization. PMID:9531506

  3. Plasma-membrane H(+)-ATPase gene expression is regulated by NaCl in cells of the halophyte Atriplex nummularia L.

    PubMed

    Niu, X; Zhu, J K; Narasimhan, M L; Bressan, R A; Hasegawa, P M

    1993-01-01

    An Atriplex nummularia L. cDNA probe encoding the partial sequence of an isoform of the plasma-membrane H(+)-ATPase was isolated, and used to characterize the NaCl regulation of mRNA accumulation in cultured cells of this halophyte. The peptide (477 amino acids) translated from the open reading frame has the highest sequence homology to the Nicotiana plumbaginifolia plasma-membrane H(+)-ATPase isoform pma4 (greater than 80% identity) and detected a transcript of approximately 3.7 kb on Northern blots of both total and poly(A)+ RNA. The mRNA levels were comparable in unadapted cells, adapted cells (cells adapted to and growing in 342 mM NaCl) and deadapted cells (cells previously adapted to 342 mM NaCl that are now growing without salt). Increased mRNA abundance was detected in deadapted cells within 24 h after exposure to NaCl but not in unadapted cells with similar salt treatments. The NaCl up-regulation of message abundance in deadapted cells was subject to developmental control. Analogous to those reported for glycophytes, the plasma-membrane H(+)-ATPase are encoded by a multigene family in the halophyte. PMID:7763822

  4. Methods for producing secreted polypeptides

    DOEpatents

    Maiyuran, Suchindra; Fidantsef, Ana; Brody, Howard

    2008-07-01

    The present invention relates to methods for producing a polypeptide, comprising: (a) cultivating a fungal host cell in a medium conducive for the production of the polypeptide, wherein the fungal host cell comprises a nucleic acid construct comprising a first nucleotide sequence encoding a signal peptide operably linked to a second nucleotide sequence encoding the polypeptide, wherein the first nucleotide sequence is foreign to the second nucleotide sequence and the 3' end of the first nucleotide sequence is immediately upstream of the initiator codon of the second nucleotide sequence. The present invention also relates to the isolated signal peptide sequences and to constructs, vectors, and fungal host cells comprising the signal peptide sequences operably linked to nucleotide sequences encoding polypeptides.

  5. Hydrogenase polypeptide and methods of use

    DOEpatents

    Adams, Michael W.W.; Hopkins, Robert C.; Jenney, JR, Francis E.; Sun, Junsong

    2016-02-02

    Provided herein are polypeptides having hydrogenase activity. The polypeptide may be multimeric, and may have hydrogenase activity of at least 0.05 micromoles H.sub.2 produced min.sup.-1 mg protein.sup.-1. Also provided herein are polynucleotides encoding the polypeptides, genetically modified microbes that include polynucleotides encoding one or more subunits of the multimeric polypeptide, and methods for making and using the polypeptides.

  6. Plasma-membrane hyperpolarization diminishes the cation efflux via Nha1 antiporter and Ena ATPase under potassium-limiting conditions.

    PubMed

    Zahrádka, Jaromír; Sychrová, Hana

    2012-06-01

    Saccharomyces cerevisiae extrudes K(+) cations even when potassium is only present in scarce amounts in the environment. Lost potassium is taken up by the Trk1 and Trk2 uptake systems. If the Trk transporters are absent or nonfunctional, the efflux of potassium is significantly diminished. A series of experiments with strains lacking various combinations of potassium efflux and uptake systems revealed that all three potassium-exporting systems the Nha1 antiporter, Ena ATPase and Tok1 channel contribute to potassium homeostasis and are active upon potassium limitation in wild-type cells. In trk1Δ trk2Δ mutants, the potassium efflux via potassium exporters Nha1 and Ena1 is diminished and can be restored either by the expression of TRK1 or deletion of TOK1. In both cases, the relative hyperpolarization of trk1Δ trk2Δ cells is decreased. Thus, it is the plasma-membrane potential which serves as the common mechanism regulating the activity of K(+) exporting systems. There is a continuous uptake and efflux of potassium in yeast cells to regulate their membrane potential and thereby other physiological parameters, and the cells are able to quickly and efficiently compensate for a malfunction of potassium transport in one direction by diminishing the transport in the other direction.

  7. Machupo virus polypeptides: identification by immunoprecipitation.

    PubMed

    Lukashevich, I S; Lemeshko, N N

    1985-01-01

    The most abundant protein in purified Machupo virions (Corvallo strain) labelled with 14C-Protein hydrolysate is a 64 K polypeptide which is associated with virion RNAs. Another structural polypeptide, 37 K, solubilized by nonionic detergent seems to be a major surface glycoprotein. In addition to this, a 78 K polypeptide and a minor 50 K polypeptide have been detected. In Machupo virus infected cells three virus-specific polypeptides similar in size to those described for structural polypeptides were immunoprecipitated with anti-Machupo virus serum. The most abundant virus-specific polypeptide was nonglycosylated (64 K, NP), and the others were glycosylated polypeptides (78 K and 37 K). The synthesis of NP and 78 K polypeptides was recognized at the beginning of a log phase of virus replication. Pulse-chase experiments as well as experiments with an arginine analogue, canavanine (to block proteolytic processing) suggest that 78 K is a precursor for structural glycoproteins of Machupo virions.

  8. Biological Synthesis of Circular Polypeptides*

    PubMed Central

    Aboye, Teshome L.; Camarero, Julio A.

    2012-01-01

    Here, we review the use of different biochemical approaches for biological synthesis of circular or backbone-cyclized proteins and peptides. These methods allow the production of circular polypeptides either in vitro or in vivo using standard recombinant DNA expression techniques. Protein circularization can significantly impact protein engineering and research in protein folding. Basic polymer theory predicts that circularization should lead to a net thermodynamic stabilization of a folded protein by reducing the entropy associated with the unfolded state. Protein cyclization also provides a valuable tool for exploring the effects of topology on protein folding kinetics. Furthermore, the biological production of cyclic polypeptides makes possible the production of cyclic polypeptide libraries. The generation of such libraries, which was previously restricted to the domain of synthetic chemists, now offers biologists access to highly diverse and stable molecular libraries for probing protein structure and function. PMID:22707722

  9. Distinct plasma-membrane PtdIns(4)P and PtdIns(4,5)P2 dynamics in secretagogue-stimulated beta-cells.

    PubMed

    Wuttke, Anne; Sågetorp, Jenny; Tengholm, Anders

    2010-05-01

    Phosphoinositides regulate numerous processes in various subcellular compartments. Whereas many stimuli trigger changes in the plasma-membrane PtdIns(4,5)P(2) concentration, little is known about its precursor, PtdIns(4)P, in particular whether there are stimulus-induced alterations independent of those of PtdIns(4,5)P(2). We investigated plasma-membrane PtdIns(4)P and PtdIns(4,5)P(2) dynamics in insulin-secreting MIN6 cells using fluorescent translocation biosensors and total internal reflection microscopy. Loss of PtdIns(4,5)P(2) induced by phospholipase C (PLC)-activating receptor agonists or stimulatory glucose concentrations was paralleled by increased PtdIns(4)P levels. In addition, glucose-stimulated cells regularly showed anti-synchronous oscillations of the two lipids. Whereas glucose-induced PtdIns(4)P elevation required voltage-gated Ca(2+) entry and was mimicked by membrane-depolarizing stimuli, the receptor-induced response was Ca(2+) independent, but sensitive to protein kinase C (PKC) inhibition and mimicked by phorbol ester stimulation. We conclude that glucose and PLC-activating receptor stimuli trigger Ca(2+)- and PKC-dependent changes in the plasma-membrane PtdIns(4)P concentration that are independent of the effects on PtdIns(4,5)P(2). These findings indicate that enhanced formation of PtdIns(4)P, apart from ensuring efficient replenishment of the PtdIns(4,5)P(2) pool, might serve an independent signalling function by regulating the association of PtdIns(4)P-binding proteins with the plasma membrane.

  10. Splice-Site A Choice Targets Plasma-Membrane Ca2+-ATPase Isoform 2 to Hair Bundles

    PubMed Central

    Hill, Jennifer K.; Williams, Diane E.; LeMasurier, Meredith; Dumont, Rachel A.; Strehler, Emanuel E.; Gillespie, Peter G.

    2007-01-01

    Localization of mechanotransduction in sensory hair cells to hair bundles requires selective targeting of essential proteins to specific locations. Isoform 2 of the plasma-membrane Ca2+-ATPase (PMCA2), required for hearing and balance, is found exclusively in hair bundles. We determined the contribution of splicing at the two major splicing sites (A and C) to hair-cell targeting of PMCA2. When PMCA2 isoforms were immunoprecipitated from purified hair bundles of rat utricle, 2w was the only site A variant detected; moreover, immunocytochemistry for 2w in rat vestibular and cochlear tissues indicated that this splice form was located solely in bundles. To demonstrate the necessity of the 2w sequence, we transfected hair cells with PMCA2 containing different variants at splice sites A and C. Although native hair bundles exclusively use the 2a form at splice-site C, epitope-tagged PMCA2w/a and PMCA2w/b were both concentrated in bundles, indicating that site C is not involved in bundle targeting. In contrast, PMCA2z/a was excluded from bundles and was instead targeted to the basolateral plasma membrane. Bundle-specific targeting of PMCA2w/a tagged with green fluorescent protein (GFP) was diminished, suggesting that GFP interfered with splice-site A. Together, these data demonstrate that PMCA2w/a is the hair-bundle isoform of PMCA in rat hair cells and that 2w targets PMCA2 to bundles. The 2w sequence is thus the first targeting signal identified for a hair-bundle membrane protein; moreover, the striking distribution of inner-ear PMCA isoforms dictated by selective targeting suggests a critical functional role for segregated pathways of Ca2+ transport. PMID:16763025

  11. Polypeptide cartography of Spiroplasma taiwanense.

    PubMed

    Humphery-Smith, I; Guyonnet, F; Chastel, C

    1994-01-01

    Spiroplasma taiwanense is the first member of the Class Mollicutes to be subjected to polypeptide cartography using computerized image analysis. The small genome size characteristic of this group was shown to code for low numbers of polypeptides when compared to other bacterial species. Silver-stained two-dimensional electrophoresis gels, following separation by either isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (ISO-DALT) or nonequilibrium pH gradient electrophoresis (NEPHGE), were used to create databases from 10 and 6 gels, respectively, for each technique and produced, respectively, 263 and 287 replicated spots. Polypeptides were mapped with respect to molecular mass and glyceraldehyde-3-phosphate dehydrogenase carbamylation standards. Of interest was the unexpectedly high percentage (50.2%) of the total normalised optical intensity associated with all 263 spots detected by ISO-DALT electrophoresis, having been contributed by just 29 dominant protein spots. These 29 polypeptides are to be given priority in microsequencing and microanalysis aimed at their identification.

  12. Methods for using polypeptides having cellobiohydrolase activity

    DOEpatents

    Morant, Marc D; Harris, Paul

    2016-08-23

    The present invention relates to isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  13. Physical association between a novel plasma-membrane structure and centrosome orients cell division.

    PubMed

    Negishi, Takefumi; Miyazaki, Naoyuki; Murata, Kazuyoshi; Yasuo, Hitoyoshi; Ueno, Naoto

    2016-01-01

    In the last mitotic division of the epidermal lineage in the ascidian embryo, the cells divide stereotypically along the anterior-posterior axis. During interphase, we found that a unique membrane structure invaginates from the posterior to the centre of the cell, in a microtubule-dependent manner. The invagination projects toward centrioles on the apical side of the nucleus and associates with one of them. Further, a cilium forms on the posterior side of the cell and its basal body remains associated with the invagination. A laser ablation experiment suggests that the invagination is under tensile force and promotes the posterior positioning of the centrosome. Finally, we showed that the orientation of the invaginations is coupled with the polarized dynamics of centrosome movements and the orientation of cell division. Based on these findings, we propose a model whereby this novel membrane structure orchestrates centrosome positioning and thus the orientation of cell division axis. PMID:27502556

  14. Physical association between a novel plasma-membrane structure and centrosome orients cell division.

    PubMed

    Negishi, Takefumi; Miyazaki, Naoyuki; Murata, Kazuyoshi; Yasuo, Hitoyoshi; Ueno, Naoto

    2016-08-09

    In the last mitotic division of the epidermal lineage in the ascidian embryo, the cells divide stereotypically along the anterior-posterior axis. During interphase, we found that a unique membrane structure invaginates from the posterior to the centre of the cell, in a microtubule-dependent manner. The invagination projects toward centrioles on the apical side of the nucleus and associates with one of them. Further, a cilium forms on the posterior side of the cell and its basal body remains associated with the invagination. A laser ablation experiment suggests that the invagination is under tensile force and promotes the posterior positioning of the centrosome. Finally, we showed that the orientation of the invaginations is coupled with the polarized dynamics of centrosome movements and the orientation of cell division. Based on these findings, we propose a model whereby this novel membrane structure orchestrates centrosome positioning and thus the orientation of cell division axis.

  15. Nano polypeptide particles reinforced polymer composite fibers.

    PubMed

    Li, Jiashen; Li, Yi; Zhang, Jing; Li, Gang; Liu, Xuan; Li, Zhi; Liu, Xuqing; Han, Yanxia; Zhao, Zheng

    2015-02-25

    Because of the intensified competition of land resources for growing food and natural textile fibers, there is an urgent need to reuse and recycle the consumed/wasted natural fibers as regenerated green materials. Although polypeptide was extracted from wool by alkaline hydrolysis, the size of the polypeptide fragments could be reduced to nanoscale. The wool polypeptide particles were fragile and could be crushed down to nano size again and dispersed evenly among polymer matrix under melt extrusion condition. The nano polypeptide particles could reinforce antiultraviolet capability, moisture regain, and mechanical properties of the polymer-polypeptide composite fibers.

  16. Elastomeric polypeptide-based biomaterials

    PubMed Central

    Li, Linqing; Charati, Manoj B.; Kiick, Kristi L.

    2011-01-01

    Elastomeric proteins are characterized by their large extensibility before rupture, reversible deformation without loss of energy, and high resilience upon stretching. Motivated by their unique mechanical properties, there has been tremendous research in understanding and manipulating elastomeric polypeptides, with most work conducted on the elastins but more recent work on an expanded set of polypeptide elastomers. Facilitated by biosynthetic strategies, it has been possible to manipulate the physical properties, conformation, and mechanical properties of these materials. Detailed understanding of the roles and organization of the natural structural proteins has permitted the design of elastomeric materials with engineered properties, and has thus expanded the scope of applications from elucidation of the mechanisms of elasticity to the development of advanced drug delivery systems and tissue engineering substrates. PMID:21637725

  17. Phycobilisome structure of porphyridium cruentum: polypeptide composition

    SciTech Connect

    Redlinger, T.; Gantt, E.

    1981-01-01

    Purified phycobilisomes of porphyridium cruentum were solubilized in sodium dodecyl sulfate and resolved by sodium dodecyl sulfate-acrylamide gel electrophoresis into nine colored and nine colorless polypeptides. The colored polypeptides accounted for about 84% of the total stainable protein, and the colorless polypeptides accounted for the remaining 16%. Five of the colored polypeptides ranging in molecular weight from 13,300 to 19,500 were identified as the ..cap alpha.. and ..beta.. subunits of allophycocyanin, R-phycocyanin, and phycoerythrin. Three others (29,000-30,500) were orange and are probably related to the ..gamma.. subunit of phycoerythrin. Sequential dissociation of phycobilisomes, and analysis of the polypeptides in each fraction, revealed the association of a 32,500 molecular weight colorless polypeptide with a phycoerythrin fraction. The remaining eight colorless polypeptides were in the core fraction of the phycobilisome, which also was enriched in allophycocyanin.

  18. Physical association between a novel plasma-membrane structure and centrosome orients cell division

    PubMed Central

    Negishi, Takefumi; Miyazaki, Naoyuki; Murata, Kazuyoshi; Yasuo, Hitoyoshi; Ueno, Naoto

    2016-01-01

    In the last mitotic division of the epidermal lineage in the ascidian embryo, the cells divide stereotypically along the anterior-posterior axis. During interphase, we found that a unique membrane structure invaginates from the posterior to the centre of the cell, in a microtubule-dependent manner. The invagination projects toward centrioles on the apical side of the nucleus and associates with one of them. Further, a cilium forms on the posterior side of the cell and its basal body remains associated with the invagination. A laser ablation experiment suggests that the invagination is under tensile force and promotes the posterior positioning of the centrosome. Finally, we showed that the orientation of the invaginations is coupled with the polarized dynamics of centrosome movements and the orientation of cell division. Based on these findings, we propose a model whereby this novel membrane structure orchestrates centrosome positioning and thus the orientation of cell division axis. DOI: http://dx.doi.org/10.7554/eLife.16550.001 PMID:27502556

  19. Electrospun Synthetic Polypeptide Nanofibrous Biomaterials

    NASA Astrophysics Data System (ADS)

    Khadka, Dhan; Haynie, Donald

    2011-03-01

    Water-insoluble nanofiber mats of synthetic polypeptides of defined composition have been prepared from fibers electrospun from aqueous solution in the absence of organic co-solvents. 20-50 kDa poly(L-glutamate, L-tyrosine) 4:1 (PLGY) but not 15-50 kDa or 50-100 kDa poly(L-glutamate) was spinnable at 20-55% (w/v) polymer in water. Applied voltage and needle-collector distance were crucial for spinnability. Attractive fibers were obtained at 50% polymer. Fiber diameter and mat morphology have been characterized by electron microscopy. Exposure of spun fiber mats to 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), which reacts with carboxylate, decreased fiber solubility. Fluorescein-conjugated poly(L-lysine) (FITC-PLL) but not the fluorophore alone was able bind PLGY fiber mats electrostatically, judging by fluorescence microscopy. Key advances of this work are the avoidance of an animal source of peptides and of an inorganic co-solvent to achieve polypeptide spinnability. Polypeptide fiber mats are a promising type of nano-structured biomaterial for applications in biomedicine and biotechnology.

  20. Polypeptide having swollenin activity and uses thereof

    DOEpatents

    Schoonneveld-Bergmans, Margot Elizabeth Francoise; Heijne, Wilbert Herman Marie; Vlasie, Monica D; Damveld, Robbertus Antonius

    2015-11-04

    The invention relates to a polypeptide comprising the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 73% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 73% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional polypeptide and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.

  1. Polypeptide having cellobiohydrolase activity and uses thereof

    DOEpatents

    Sagt, Cornelis Maria Jacobus; Schooneveld-Bergmans, Margot Elisabeth Francoise; Roubos, Johannes Andries; Los, Alrik Pieter

    2015-09-15

    The invention relates to a polypeptide comprising the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 93% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 93% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional polypeptide and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.

  2. Bodian's Silver Method Stains Neurofilament Polypeptides

    NASA Astrophysics Data System (ADS)

    Gambetti, P.; Autilio-Gambetti, L.; Papasozomenos, S. Ch.

    1981-09-01

    Bodian's silver method was used to stain polypeptides of rat spinal cord or peripheral nerve separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The bands corresponding to the three polypeptide subunits of the neurofilaments were intensely impregnated. Two other polypeptides were stained inconsistently and less intensely. The tubulin band was stained weakly or not at all; other polypeptides, including glial fibrillary acidic protein, actin, and vimentin, remained unstained. This novel application of Bodian's method provides indirect proof that neurofilaments are the neuronal subcellular structure stained by the technique.

  3. Methods for engineering polypeptide variants via somatic hypermutation and polypeptide made thereby

    SciTech Connect

    Tsien, Roger Y; Wang, Lei

    2015-01-13

    Methods using somatic hypermutation (SHM) for producing polypeptide and nucleic acid variants, and nucleic acids encoding such polypeptide variants are disclosed. Such variants may have desired properties. Also disclosed are novel polypeptides, such as improved fluorescent proteins, produced by the novel methods, and nucleic acids, vectors, and host cells comprising such vectors.

  4. Ordered biological nanostructures formed from chaperonin polypeptides

    NASA Technical Reports Server (NTRS)

    Trent, Jonathan D. (Inventor); McMillan, R. Andrew (Inventor); Kagawa, Hiromi (Inventor); Paavola, Chad D. (Inventor)

    2010-01-01

    The following application relates to nanotemplates, nanostructures, nanoarrays and nanodevices formed from wild-type and mutated chaperonin polypeptides, methods of producing such compositions, methods of using such compositions and particular chaperonin polypeptides that can be utilized in producing such compositions.

  5. Polypeptide vesicles with densely packed multilayer membranes.

    PubMed

    Song, Ziyuan; Kim, Hojun; Ba, Xiaochu; Baumgartner, Ryan; Lee, Jung Seok; Tang, Haoyu; Leal, Cecilia; Cheng, Jianjun

    2015-05-28

    Multilamellar membranes are important building blocks for constructing self-assembled structures with improved barrier properties, such as multilamellar lipid vesicles. Polymeric vesicles (polymersomes) have attracted growing interest, but multilamellar polymersomes are much less explored. Here, we report the formation of polypeptide vesicles with unprecedented densely packed multilayer membrane structures with poly(ethylene glycol)-block-poly(γ-(4,5-dimethoxy-2-nitrobenzyl)-l-glutamate) (PEG-b-PL), an amphiphilic diblock rod-coil copolymer containing a short PEG block and a short hydrophobic rod-like polypeptide segment. The polypeptide rods undergo smectic ordering with PEG buried between the hydrophobic polypeptide layers. The size of both blocks and the rigidity of the hydrophobic polypeptide block are critical in determining the membrane structures. Increase of the PEG length in PEG-b-PL results in the formation of bilayer sheets, while using random-coil polypeptide block leads to the formation of large compound micelles. UV treatment causes ester bond cleavage of the polypeptide side chain, which induces helix-to-coil transition, change of copolymer amphiphilicity, and eventual disassembly of vesicles. These polypeptide vesicles with unique membrane structures provide a new insight into self-assembly structure control by precisely tuning the composition and conformation of polymeric amphiphiles.

  6. Purification of Theiler's murine encephalomyelitis virus and analysis of the structural virion polypeptides: correlation of the polypeptide profile with virulence.

    PubMed

    Lipton, H L; Friedmann, A

    1980-03-01

    Theiler's murine encephalomyelitis viruses (TMEV) are separable into two groups based on their biological behavior: those highly virulent isolates which are unable to cause persistent infection and the less virulent isolates which regularly produce persistent central nervous system infection in mice. Two highly virulent and five less virulent TMEV were found to have the same buoyant density (1.34 g/ml) on isopycnic centrifugation and virion structure by electron microscopy. Negatively stained virus particles purified in Cs(2)SO(4) gradients appeared to have icosahedral symmetry and measured 28 nm in diameter. Mature virions were found to possess three major structural polypeptides, VP1, VP2 and VP3, in the range of 25,000 to 35,000 daltons, and a smaller fourth major polypeptide, VP4, of 6,000 daltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The precursor of VP2 and VP4, VP0, which is a minor polypeptide of mature picornavirus particles, was also identified. However, a slight but consistent difference in several of the capsid polypeptides between the highly virulent and less virulent TMEV was found. VP1 was slightly larger (34,000 versus 33,500 daltons) and VP2 was slightly smaller (31,000 versus 32,000 daltons) for the highly virulent strains compared to the same polypeptide species in the less virulent viruses. VP0 was also slightly smaller (35,500 versus 36,000 daltons) for the highly virulent isolates compared to their less virulent counterparts. Finally, trypsin which was used initially in our purification procedure resulted in preferential cleavage of a 2,000-molecular-weight fragment or fragments from VP1 of only the less virulent isolates.

  7. Purification of Theiler's Murine Encephalomyelitis Virus and Analysis of the Structural Virion Polypeptides: Correlation of the Polypeptide Profile with Virulence

    PubMed Central

    Lipton, Howard L.; Friedmann, Adam

    1980-01-01

    Theiler's murine encephalomyelitis viruses (TMEV) are separable into two groups based on their biological behavior: those highly virulent isolates which are unable to cause persistent infection and the less virulent isolates which regularly produce persistent central nervous system infection in mice. Two highly virulent and five less virulent TMEV were found to have the same buoyant density (1.34 g/ml) on isopycnic centrifugation and virion structure by electron microscopy. Negatively stained virus particles purified in Cs2SO4 gradients appeared to have icosahedral symmetry and measured 28 nm in diameter. Mature virions were found to possess three major structural polypeptides, VP1, VP2 and VP3, in the range of 25,000 to 35,000 daltons, and a smaller fourth major polypeptide, VP4, of 6,000 daltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The precursor of VP2 and VP4, VP0, which is a minor polypeptide of mature picornavirus particles, was also identified. However, a slight but consistent difference in several of the capsid polypeptides between the highly virulent and less virulent TMEV was found. VP1 was slightly larger (34,000 versus 33,500 daltons) and VP2 was slightly smaller (31,000 versus 32,000 daltons) for the highly virulent strains compared to the same polypeptide species in the less virulent viruses. VP0 was also slightly smaller (35,500 versus 36,000 daltons) for the highly virulent isolates compared to their less virulent counterparts. Finally, trypsin which was used initially in our purification procedure resulted in preferential cleavage of a 2,000-molecular-weight fragment or fragments from VP1 of only the less virulent isolates. Images PMID:6245266

  8. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    DOEpatents

    Spodsberg, Nikolaj

    2014-07-15

    The present invention relates to isolated polypeptides having cellobiohydrolase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  9. Polypeptides having xylanase activity and polynucleotides encoding same

    DOEpatents

    Spodsberg, Nikolaj

    2014-06-24

    The present invention relates to isolated polypeptides having xylanase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  10. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    DOEpatents

    Spodsberg, Nikolaj

    2014-06-24

    The present invention relates to isolated polypeptides having cellobiohydrolase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  11. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    DOEpatents

    Spodsberg, Nikolaj

    2014-07-08

    The present invention relates to isolated polypeptides having cellobiohydrolase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  12. Polypeptides having endoglucanase activity and polynucleotides encoding same

    DOEpatents

    Spodsberg, Nikolaj

    2014-07-15

    The present invention relates to isolated polypeptides having endoglucanase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  13. Polypeptides having endoglucanse activity and polynucleotides encoding same

    DOEpatents

    Spodsberg, Nikolaj

    2014-07-08

    The present invention relates to isolated polypeptides having endoglucanase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  14. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    SciTech Connect

    Spodsberg, Nikolaj

    2015-03-31

    The present invention relates to isolated polypeptides having cellobiohydrolase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  15. Polypeptides having xylanase activity and polynucleotides encoding same

    DOEpatents

    Spodsberg, Nikolaj

    2015-10-27

    The present invention relates to isolated polypeptides having xylanase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  16. Polypeptides having endoglucanase activity and polynucleotides encoding same

    DOEpatents

    Spodsberg, Nikolaj

    2015-11-17

    The present invention relates to isolated polypeptides having endoglucanase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  17. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    DOEpatents

    Spodsberg, Nikolaj

    2015-11-17

    The present invention relates to isolated polypeptides having cellobiohydrolase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  18. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    SciTech Connect

    Spodsberg, Nikolaj

    2015-03-10

    The present invention relates to isolated polypeptides having cellobiohydrolase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  19. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    DOEpatents

    Spodsberg, Nikolaj

    2016-06-28

    The present invention relates to isolated polypeptides having cellobiohydrolase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  20. Polypeptides having xylanase activity and polynucleotides encoding same

    SciTech Connect

    Spodsberg, Nikolaj

    2014-10-14

    The present invention relates to isolated polypeptides having xylanase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  1. Polypeptides having cellobiohydrolase activitiy and polynucleotides encoding same

    SciTech Connect

    Liu, Ye; Tang, Lan; Duan, Junxin

    2015-12-15

    The present invention provides isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also provides nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  2. Polypeptides having endoglucanase activity and polynucleotides encoding same

    SciTech Connect

    Liu, Ye; Duan, Junxin; Tang, Lan

    2015-09-22

    The present invention provides isolated polypeptides having endoglucanase activity and isolated polynucleotides encoding the polypeptides. The invention also provides nucleic acid constructs, vectors, and host cell comprising the polynucleotides as well as methods of producing and using the polypeptides.

  3. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    SciTech Connect

    Spodsberg, Nikolaj

    2015-07-14

    The present invention relates to isolated polypeptides having cellobiohydrolase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  4. Polypeptides having endoglucanase activity and polynucleotides encoding same

    SciTech Connect

    Spodsberg, Nikolaj

    2015-02-10

    The present invention relates to isolated polypeptides having endoglucanase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  5. Isolation of Polypeptide Sample and Measurement of Its Concentration.

    ERIC Educational Resources Information Center

    Beanan, Maureen J.

    2000-01-01

    Introduces a laboratory experiment that isolates a bacterial polypeptide sample and measures the concentration of polypeptides in the sample. Uses Escherichia coli strain MM294 and performs a bio-rad assay to determine the concentration of polypeptides. (YDS)

  6. Polypeptides having endoglucanase activity and polynucleotides encoding same

    SciTech Connect

    Spodsberg, Nikolaj

    2015-07-14

    The present invention relates to isolated polypeptides having endoglucanase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  7. Polypeptides having xylanase activity and polynucleotides encoding same

    SciTech Connect

    Spodsberg, Nikolaj

    2015-08-18

    The present invention relates to isolated polypeptides having xylanase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  8. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    DOEpatents

    Zhang, Yu; Duan, Junxin; Tang, Lan; Wu, Wenping

    2015-06-09

    Provided are isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. Also provided are nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  9. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    DOEpatents

    Liu, Ye; Tang, Lan

    2015-07-14

    The present invention provides isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also provides nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  10. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    DOEpatents

    Zhang, Yu; Tang, Lan; Henriksen, Svend Hostgaard Bang

    2016-05-17

    The present invention provides isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also provides nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  11. Study of fluxes at low concentrations of l-tri-iodothyronine with rat liver cells and their plasma-membrane vesicles. Evidence for the accumulation of the hormone against a gradient

    PubMed Central

    Rao, Govind S.; Rao, Marie Luise; Thilmann, Astrid; Quednau, Hans D.

    1981-01-01

    1. Influx and efflux of l-tri-[125I]iodothyronine with isolated rat liver parenchymal cells and their plasma-membrane vesicles were studied by a rapid centrifugation technique. 2. At 23°C and in the concentration range that included the concentration of free l-tri-iodothyronine in rat plasma (3–5pm) influx into cells was saturable; an apparent Kt value of 8.6±1.6pm was obtained. 3. At 5pm-l-tri-[125I]iodothyronine in the external medium the ratios of the concentrations inside to outside in cells and plasma-membrane vesicles were 38:1 and 366:1 respectively after 7s of incubation. At equilibrium (60s at 23°C) uptake of l-tri-[125I]iodothyronine by cells was linear with the hormone concentration, whereas that by plasma-membrane vesicles exhibited an apparent saturation with a Kd value of 6.1±1.3pm. 4. Efflux of l-tri-[125I]iodothyronine from cells equilibrated with the hormone (5–123pm) was constant up to 21 s; the amount that flowed out was 17.7±3.8% when cells were equilibrated with 5pm-hormone. When plasma-membrane vesicles were equilibrated with l-tri-[125I]iodothyronine (556–1226pm) 66.8±5.8% flowed out after 21 s. 5. From a consideration of the data on efflux from cells and binding of l-tri-[125I]iodothyronine to the liver homogenate, as studied by the charcoal-adsorption and equilibrium-dialysis methods, it appears that 18–22% of the hormone exists in the free form in the cell. 6. Vinblastine and colchicine diminished the uptake of l-tri-[125I]iodothyronine by cells but not by plasma-membrane vesicles; binding to the cytosol fraction was not affected. Phenylbutazone, 6-n-propyl-2-thiouracil, methimazole and corticosterone diminished the uptake by cells, plasma-membrane vesicles and binding to the cytosol fraction to different extents. 7. These results suggest that at low concentrations of l-tri-[125I]iodothyronine rat liver cells and their plasma-membrane vesicles accumulated the hormone against an apparent gradient by a membrane-mediated process

  12. Fungitoxic and insecticidal plant polypeptides.

    PubMed

    Becker-Ritt, Arlete Beatriz; Carlini, Célia Regina

    2012-01-01

    According to the World Bank and FAO, the population grows worldwide and the poorest countries are expected to double their population within the next decades, reaching approximately 7.2 billion in 2015. Moreover, the food and financial crisis together with the global economic recession pushed the number of hungry and undernourished people in the world to unprecedented levels. The substitution of animal proteins by plant proteins in food and feed is a general trend because of the lower cost and better production efficiency. Pathogens and pests can reduce the crop yields up to 30%. In some places, the losses can reach 80% due to climate conditions, proliferation of insects, and fungal diseases. All together, the harvest and postharvest losses vary from 5% to 20% and depending on the commodity can be as high as 50%. Plants have a complex chemical armory for defense composed of low and high molecular mass compounds that can act over a variety of pests and pathogens, from micro-organisms to phytophagous insects or grazing animals. Among them, plant fungitoxic and insecticidal polypeptides represent promising alternatives to increase the supply of plant-derived proteins and tackle the hunger in a global scale. PMID:23193601

  13. Materials Assembly and Formation Using Engineered Polypeptides

    NASA Astrophysics Data System (ADS)

    Sarikaya, Mehmet; Tamerler, Candan; Schwartz, Daniel T.; Baneyx, Francois

    2004-08-01

    Molecular biomimetics can be defined as mimicking function, synthesis, or structure of materials and systems at the molecular scale using biological pathways. Here, inorganic-binding polypeptides are used as molecular building blocks to control assembly and formation of functional inorganic and hybrid materials and systems for nano- and nanobiotechnology applications. These polypeptides are selected via phage or cell surface display technologies and modified by molecular biology to tailor their binding and multifunctionality properties. The potential of this approach in creating new materials systems with useful physical and biological properties is enormous. This mostly stems from molecular recognition and self-assembly characteristics of the polypeptides plus the added advantage of genetic manipulation of their composition and structure. In this review, we highlight the basic premises of molecular biomimetics, describe the approaches in selecting and engineering inorganic-binding polypeptides, and present examples of their utility as molecular linkers in current and future applications.

  14. Restriction/modification polypeptides, polynucleotides, and methods

    DOEpatents

    Westpheling, Janet; Chung, DaeHwan; Huddleston, Jennifer; Farkas, Joel A

    2015-02-24

    The present invention relates to the discovery of a novel restriction/modification system in Caldicellulosiruptor bescii. The discovered restriction enzyme is a HaeIII-like restriction enzyme that possesses a thermophilic activity profile. The restriction/modification system also includes a methyltransferase, M.CbeI, that methylates at least one cytosine residue in the CbeI recognition sequence to m.sup.4C. Thus, the invention provides, in various aspects, isolated CbeI or M.CbeI polypeptides, or biologically active fragments thereof; isolated polynucleotides that encode the CbeI or M.CbeI polypeptides or biologically active fragments thereof, including expression vectors that include such polynucleotide sequences; methods of digesting DNA using a CbeI polypeptide; methods of treating a DNA molecule using a M.CbeI polypeptide; and methods of transforming a Caldicellulosiruptor cell.

  15. Polypeptide having an amino acid replaced with N-benzylglycine

    SciTech Connect

    Mitchell, Alexander R.; Young, Janis D.

    1996-01-01

    The present invention relates to one or more polypeptides having useful biological activity in a mammal, which comprise: a polypeptide related to bradykinin of four to ten amino acid residues wherein one or more specific amino acids in the polypeptide chain are replaced with achiral N-benzylglycine. These polypeptide analogues have useful potent agonist or antagonist pharmacological properties depending upon the structure. A preferred polypeptide is (N-benzylglycine.sup.7)-bradykinin.

  16. Ectocytosis caused by sublytic autologous complement attack on human neutrophils. The sorting of endogenous plasma-membrane proteins and lipids into shed vesicles.

    PubMed Central

    Stein, J M; Luzio, J P

    1991-01-01

    During sublytic complement attack on human neutrophils, plasma-membrane vesicles are shed from the cell surface as a cell-protection mechanism. By using surface-iodinated neutrophils it was found that less than 2% of surface label was recovered in shed vesicles under conditions where 40% of complement component C9 was shed. SDS/PAGE of 125I-labelled shed vesicles and plasma membranes showed differences in iodination pattern, demonstrating the sorting of membrane proteins into the shed vesicles. Analysis of 32P-labelled phospholipids after labeling of neutrophils with [32P]Pi before sublytic complement attack showed the presence of phosphatidic acid, phosphatidylcholine, phosphatidyl-ethanolamine, phosphatidylinositol and polyphosphoinositides in shed vesicles. Quantitative analysis using [3H]acetic anhydride-labelling method showed that the molar proportions of phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine and sphingomyelin were the same in shed vesicles as in plasma membranes. In contrast, the molar proportions of cholesterol and diacylglycerol relative to sphingomyelin were almost twice those found in plasma membranes. The data demonstrate the existence of protein and lipid sorting mechanisms during the formation of shed vesicles when neutrophils are subject to sublytic complement attack. The term 'ectocytosis' is proposed to describe triggered shedding of right-side-out membrane vesicles from the surface of eukaryotic cells. Images Fig. 2. Fig. 3. Fig. 4. PMID:1848755

  17. Urinary polypeptides related to collagen synthesis

    PubMed Central

    Krane, Stephen M.; Muñoz, Alberto J.; Harris, Edward D.

    1970-01-01

    Of the total urinary hydroxyproline in normal subjects and those with skeletal disorders, between 4 and 20% was nondialyzable. In some patients with Paget's disease of bone, hyperparathyroidism with osteitis fibrosa, hyperphosphatasia, and extensive fibrous dysplasia the total urinary hydroxyproline was sufficiently high to permit purification of this polypeptide hydroxyproline by gel filtration and ion exchange chromatography. The partially purified polypeptides had molecular weights between 4500 and 10,000 and amino acid compositions and physical properties resembling those of gelatin. The polypeptide fractions also contained neutral sugar and glucosamine. These fragments had been shown to be susceptible to cleavage by purified bacterial collagenase suggesting the presence of the sequence-Pro-X-Gly-Pro-Y-. After administration of proline-14C to patients with Paget's disease hydroxyproline-14C was excreted in the urine. The hydroxyproline-14C specific activity reached a peak in 2-4 hr and declined rapidly. The specific activity of the polypeptide (retentate) portion was severalfold greater than that of the raw urine and diffusate. When the labeled urines were subjected to gel filtration the hydroxyproline-14C fractions of highest molecular weight which were eluted first from the columns had the highest specific activities. Exposure of the hydroxyproline-14C-containing polypeptides to bacterial collagenase rendered them dialyzable. Four patients with hyperparathyroidism and osteitis fibrosa were studied before and after removal of a parathyroid adenoma, a period of transition from a predominance of bone collagen resorption to one of relatively increased bone collagen synthesis. The total urinary hydroxyproline fell rapidly after operation whereas the ratio of the polypeptide fraction to the total rose three- to fourfold. The results of these studies suggest that the urinary polypeptides represent fragments of collagen related to collagen synthesis. Changes in the

  18. Coulomb repulsion in short polypeptides.

    PubMed

    Norouzy, Amir; Assaf, Khaleel I; Zhang, Shuai; Jacob, Maik H; Nau, Werner M

    2015-01-01

    Coulomb repulsion between like-charged side chains is presently viewed as a major force that impacts the biological activity of intrinsically disordered polypeptides (IDPs) by determining their spatial dimensions. We investigated short synthetic models of IDPs, purely composed of ionizable amino acid residues and therefore expected to display an extreme structural and dynamic response to pH variation. Two synergistic, custom-made, time-resolved fluorescence methods were applied in tandem to study the structure and dynamics of the acidic and basic hexapeptides Asp6, Glu6, Arg6, Lys6, and His6 between pH 1 and 12. (i) End-to-end distances were obtained from the short-distance Förster resonance energy transfer (sdFRET) from N-terminal 5-fluoro-l-tryptophan (FTrp) to C-terminal Dbo. (ii) End-to-end collision rates were obtained for the same peptides from the collision-induced fluorescence quenching (CIFQ) of Dbo by FTrp. Unexpectedly, the very high increase of charge density at elevated pH had no dynamical or conformational consequence in the anionic chains, neither in the absence nor in the presence of salt, in conflict with the common view and in partial conflict with accompanying molecular dynamics simulations. In contrast, the cationic peptides responded to ionization but with surprising patterns that mirrored the rich individual characteristics of each side chain type. The contrasting results had to be interpreted, by considering salt screening experiments, N-terminal acetylation, and simulations, in terms of an interplay of local dielectric constant and peptide-length dependent side chain charge-charge repulsion, side chain functional group solvation, N-terminal and side chain charge-charge repulsion, and side chain-side chain as well as side chain-backbone interactions. The common picture that emerged is that Coulomb repulsion between water-solvated side chains is efficiently quenched in short peptides as long as side chains are not in direct contact with each

  19. Helical antimicrobial polypeptides with radial amphiphilicity

    PubMed Central

    Xiong, Menghua; Lee, Michelle W.; Mansbach, Rachael A.; Song, Ziyuan; Bao, Yan; Peek, Richard M.; Yao, Catherine; Chen, Lin-Feng; Ferguson, Andrew L.; Wong, Gerard C. L.; Cheng, Jianjun

    2015-01-01

    α-Helical antimicrobial peptides (AMPs) generally have facially amphiphilic structures that may lead to undesired peptide interactions with blood proteins and self-aggregation due to exposed hydrophobic surfaces. Here we report the design of a class of cationic, helical homo-polypeptide antimicrobials with a hydrophobic internal helical core and a charged exterior shell, possessing unprecedented radial amphiphilicity. The radially amphiphilic structure enables the polypeptide to bind effectively to the negatively charged bacterial surface and exhibit high antimicrobial activity against both gram-positive and gram-negative bacteria. Moreover, the shielding of the hydrophobic core by the charged exterior shell decreases nonspecific interactions with eukaryotic cells, as evidenced by low hemolytic activity, and protects the polypeptide backbone from proteolytic degradation. The radially amphiphilic polypeptides can also be used as effective adjuvants, allowing improved permeation of commercial antibiotics in bacteria and enhanced antimicrobial activity by one to two orders of magnitude. Designing AMPs bearing this unprecedented, unique radially amphiphilic structure represents an alternative direction of AMP development; radially amphiphilic polypeptides may become a general platform for developing AMPs to treat drug-resistant bacteria. PMID:26460016

  20. A review of polypeptide-based polymersomes.

    PubMed

    Zhao, Lanxia; Li, Nuannuan; Wang, Kaiming; Shi, Chunhuan; Zhang, Longlong; Luan, Yuxia

    2014-01-01

    Self-assembled systems from biodegradable amphiphilic polymers at the nanometer scale, such as nanotubes, nanoparticles, polymer micelles, nanogels, and polymersomes, have attracted much attention especially in biomedical fields. Among these nano-aggregates, polymersomes have attracted tremendous interests as versatile carriers due to their colloidal stability, tunable membrane properties and ability of encapsulating or integrating a broad range of drugs and molecules. Biodegradable block polymers, especially aliphatic polyesters such as polylactide, polyglycolide and poly (ε-caprolactone) have been widely used as biomedical materials for a long time to well fit the requirement of biomedical drug carriers. To have a precise control of the aggregation behavior of nano-aggregates, the more ordered polypeptide has been used to self-assemble into the drug carriers. In this review we focus on the study of polymersomes which also named pepsomes formed by polypeptide-based copolymers and attempt to clarify the polypeptide-based polymersomes from following aspects: synthesis and characterization of the polypeptide-based copolymers, preparation, multifunction and application of polypeptide-based polymersomes.

  1. Carbohydrate degrading polypeptide and uses thereof

    DOEpatents

    Sagt, Cornelis Maria Jacobus; Schooneveld-Bergmans, Margot Elisabeth Francoise; Roubos, Johannes Andries; Los, Alrik Pieter

    2015-10-20

    The invention relates to a polypeptide having carbohydrate material degrading activity which comprises the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 4, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 96% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 96% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional protein and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.

  2. Anharmonic Theoretical Vibrational Spectroscopy of Polypeptides.

    PubMed

    Panek, Paweł T; Jacob, Christoph R

    2016-08-18

    Because of the size of polypeptides and proteins, the quantum-chemical prediction of their vibrational spectra presents an exceptionally challenging task. Here, we address one of these challenges, namely, the inclusion of anharmonicities. By performing the expansion of the potential energy surface in localized-mode coordinates instead of the normal-mode coordinates, it becomes possible to calculate anharmonic vibrational spectra of polypeptides efficiently and reliably. We apply this approach to calculate the infrared, Raman, and Raman optical activity spectra of helical alanine polypeptides consisting of up to 20 amino acids. We find that while anharmonicities do not alter the band shapes, simple scaling procedures cannot account for the different shifts found for the individual bands. This closes an important gap in theoretical vibrational spectroscopy by making it possible to quantify the anharmonic contributions and opens the door to a first-principles calculation of multidimensional vibrational spectra. PMID:27472016

  3. Biodegradable Epoxy Networks Cured with Polypeptides

    NASA Astrophysics Data System (ADS)

    Nakamura, Shigeo; Kramer, Edward J.

    2006-03-01

    Epoxy resins are used widely for adhesives as well as coatings. However, once cured they are usually highly cross-linked and are not biodegradable. To obtain potentially biodegradable polypeptides that can cure with epoxy resins and achieve as good properties as the conventional phenol novolac hardeners, poly(succinimide-co-tyrosine) was synthesized by thermal polycondensation of L-aspartic acid and L-tyrosine with phosphoric acid under reduced pressure. The tyrosine/succinimide ratio in the polypeptide was always lower than the tyrosine/(aspartic acid) feed ratio and was influenced by the synthesis conditions. Poly(succinimide-tyrosine- phenylalanine) was also synthesized from L-aspartic acid, L- tyrosine and L-phenylalanine. The thermal and mechanical properties of epoxy resins cured with these polypeptides are comparable to those of similar resins cured with conventional hardeners. In addition, enzymatic degradability tests showed that Chymotrypsin or Subtilisin A could cleave cured films in an alkaline borate buffer.

  4. Maize mitochondria synthesize organ-specific polypeptides

    SciTech Connect

    Newton, K.J.; Walbot, V.

    1985-10-01

    The authors detected both quantitative and qualitative organ-specific differences in the total protein composition of mitochondria of maize. Labeling of isolated mitochondria from each organ demonstrated that a few protein differences are due to changes in the polypeptides synthesized by the organelle. The synthesis of developmental stage-specific mitochondrial polypeptides was found in the scutella of developing and germinating kernels. The approximately 13-kDa polypeptide synthesized by mitochondria from seedlings of the Texas (T) male-sterile cytoplasm was shown to be constitutively expressed in all organs of line B37T tested. Methomyl, an insecticide known to inhibit the growth of T sterile plants, was shown to be an effective inhibitor of protein synthesis in mitochondria from T plants.

  5. Peppytides: Interactive Models of Polypeptide Chains

    SciTech Connect

    Zuckermann, Ron; Chakraborty, Promita; Derisi, Joe

    2014-01-21

    Peppytides are scaled, 3D-printed models of polypeptide chains that can be folded into accurate protein structures. Designed and created by Berkeley Lab Researcher, Promita Chakraborty, and Berkeley Lab Senior Scientist, Dr. Ron Zuckermann, Peppytides are accurate physical models of polypeptide chains that anyone can interact with and fold intro various protein structures - proving to be a great educational tool, resulting in a deeper understanding of these fascinating structures and how they function. Build your own Peppytide model and learn about how nature's machines fold into their intricate architectures!

  6. Peppytides: Interactive Models of Polypeptide Chains

    ScienceCinema

    Zuckermann, Ron; Chakraborty, Promita; Derisi, Joe

    2016-07-12

    Peppytides are scaled, 3D-printed models of polypeptide chains that can be folded into accurate protein structures. Designed and created by Berkeley Lab Researcher, Promita Chakraborty, and Berkeley Lab Senior Scientist, Dr. Ron Zuckermann, Peppytides are accurate physical models of polypeptide chains that anyone can interact with and fold intro various protein structures - proving to be a great educational tool, resulting in a deeper understanding of these fascinating structures and how they function. Build your own Peppytide model and learn about how nature's machines fold into their intricate architectures!

  7. Tuning Ice Nucleation with Supercharged Polypeptides.

    PubMed

    Yang, Huige; Ma, Chao; Li, Kaiyong; Liu, Kai; Loznik, Mark; Teeuwen, Rosalie; van Hest, Jan C M; Zhou, Xin; Herrmann, Andreas; Wang, Jianjun

    2016-07-01

    Supercharged unfolded polypeptides (SUPs) are exploited for controlling ice nucleation via tuning the nature of charge and charge density of SUPs. The results show that positively charged SUPs facilitate ice nucleation, while negatively charged ones suppress it. Moreover, the charge density of the SUP backbone is another parameter to control it. PMID:27119590

  8. Polypeptides having xylanase activity and polynucleotides encoding same

    DOEpatents

    Lopez de Leon, Alfredo; Rey, Michael

    2016-05-31

    The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  9. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    DOEpatents

    Zhang, Yu; Duan, Junxin; Tang, Lan; Wu, Wenping

    2016-06-14

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  10. Polypeptides having beta-glucosidase activity and polynucleotides encoding same

    DOEpatents

    Harris, Paul; Golightly, Elizabeth

    2011-06-14

    The present invention relates to isolated polypeptides having beta-glucosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

  11. Polypeptides having beta-glucosidase activity and polynucleotides encoding same

    DOEpatents

    Morant, Marc D; Patkar, Shamkant; Ding, Hanshu

    2013-11-12

    The present invention relates to isolated polypeptides having beta-glucosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  12. Polypeptides having beta-glucosidase activity and polynucleotides encoding same

    DOEpatents

    Harris, Paul; Golightly, Elizabeth

    2007-07-17

    The present invention relates to isolated polypeptides having beta-glucosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

  13. Polypeptides having beta-glucosidase activity and polynucleotides encoding same

    DOEpatents

    Harris, Paul; Golightly, Elizabeth

    2012-11-27

    The present invention relates to isolated polypeptides having beta-glucosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

  14. Polynucleotides encoding polypeptides having beta-glucosidase activity

    DOEpatents

    Harris, Paul; Golightly, Elizabeth

    2010-03-02

    The present invention relates to isolated polypeptides having beta-glucosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

  15. Polypeptides having endoglucanase activity and polynucleotides encoding same

    DOEpatents

    Lopez de Leon, Alfredo; Rey, Michael

    2015-03-10

    The present invention relates to isolated polypeptides having endoglucanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  16. Polypeptides having xylanase activity and polynucleotides encoding same

    DOEpatents

    Lopez de Leon, Alfredo; Rey, Michael

    2015-01-27

    The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  17. Polypeptides having beta-glucosidase activity and polynucleotides encoding same

    DOEpatents

    Morant, Marc

    2014-01-14

    The present invention relates to isolated polypeptides having beta-glucosidase activity, beta-xylosidase, or beta-glucosidase activity and isolated polynucleotides encoding polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  18. Polypeptides having xylanase activity and polynucleotides encoding the same

    DOEpatents

    Spodsberg, Nikolaj [Bagsvaed, DK

    2014-01-07

    The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The inventino also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  19. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    SciTech Connect

    Liu, Ye; Harris, Paul; Tang, Lan; Wu, Wenping

    2013-11-19

    The present invention relates to isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  20. Polypeptides having cellulolytic enhancing activity and nucleic acids encoding same

    SciTech Connect

    Brown, Kimberly; Harris, Paul; Zaretsky, Elizabeth; Re, Edward; Vlasenko, Elena; McFarland, Keith; Lopez de Leon, Alfredo

    2012-10-16

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

  1. Polypeptides having cellulolytic enhancing activity and nucleic acids encoding same

    SciTech Connect

    Brown, Kimberly; Harris, Paul; Zaretsky, Elizabeth; Re, Edward; Vlasenko, Elena; McFarland, Keith; Lopez de Leon, Alfredo

    2014-09-30

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

  2. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    DOEpatents

    Brown, Kimberly; Harris, Paul; Lopez De Leon, Alfredo; Merino, Sandra

    2007-05-22

    The present invention relates to isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

  3. Polypeptides having endoglucanase activity and polynucleotides encoding same

    SciTech Connect

    Spodsberg, Nikolaj; Shagasi, Tarana

    2015-06-30

    The present invention relates to isolated polypeptides having endoglucanase activity, catalytic domains, cellulose binding domains and polynucleotides encoding the polypeptides, catalytic domains or cellulose binding domains. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides, catalytic domains or cellulose binding domains.

  4. Polypeptides having endoglucanase activity and polynucleotides encoding same

    SciTech Connect

    Lopez de Leon, Alfredo; Rey, Michael

    2013-06-18

    The present invention relates to isolated polypeptides having endoglucanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  5. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    SciTech Connect

    Maiyuran, Suchindra; Kramer, Randall; Harris, Paul

    2013-10-29

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  6. Polypeptides having xylanase activity and polynucleotides encoding same

    SciTech Connect

    Spodsberg, Nikolaj

    2014-10-21

    The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  7. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    SciTech Connect

    Liu, Ye; Tang, Lan

    2015-11-20

    The present invention relates to isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  8. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    SciTech Connect

    Morant, Marc D.; Harris, Paul

    2015-10-13

    The present invention relates to isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  9. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    SciTech Connect

    Spodsberg, Nikolaj

    2015-11-17

    The present invention relates to isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  10. Polypeptides having beta-glucosidase activity and polynucleotides encoding same

    SciTech Connect

    Morant, Marc Dominique

    2014-10-14

    The present invention relates to isolated polypeptides having beta-glucosidase activity, beta-xylosidase activity, or beta-glucosidase and beta-xylosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  11. Polypeptides having xylanase activity and polynucleotides encoding same

    SciTech Connect

    Tang, Lan; Liu, Ye; Duan, Junxin; Hanshu, Ding

    2012-10-30

    The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  12. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    DOEpatents

    Dotson, William D.; Greenier, Jennifer; Ding, Hanshu

    2009-05-19

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated nucleic acids encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acids as well as methods for producing and using the polypeptides.

  13. Polypeptides having endoglucanase activity and polynucleotides encoding same

    DOEpatents

    Harris, Paul; Lopez de Leon, Alfredo; Rey, Michael; Ding, Hanshu; Vlasenko, Elena

    2010-11-02

    The present invention relates to isolated polypeptides having endoglucanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

  14. Polypeptides having xylanase activity and polynucleotides encoding same

    DOEpatents

    Lopez de Leon, Alfredo; Rey, Michael

    2010-12-14

    The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  15. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    DOEpatents

    Dotson, William D.; Greenier, Jennifer; Ding, Hanshu

    2007-09-18

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated nucleic acids encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acids as well as methods for producing and using the polypeptides.

  16. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    SciTech Connect

    Lopez de Leon, Alfredo; Ding, Hanshu; Brown, Kimberly

    2012-06-26

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  17. Polypeptide from a cellulolytic fungus having cellulolytic enhancing activity

    DOEpatents

    Brown, Kimberly; Harris, Paul; Zaretsky, Elizabeth; Re, Edward; Vlasenko, Elena; McFarland, Keith; Lopez de Leon, Alfredo

    2008-04-22

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

  18. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    SciTech Connect

    Lopez de Leon, Alfredo; Ding, Hanshu; Brown, Kimberly

    2011-10-25

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  19. Polypeptides having endoglucanase activity and polynucleotides encoding same

    DOEpatents

    Lopez de Leon, Alfredo; Rey, Michael

    2012-09-18

    The present invention relates to isolated polypeptides having endoglucanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  20. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    DOEpatents

    Schnorr, Kirk; Kramer, Randall

    2016-08-09

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  1. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    DOEpatents

    Schnorr, Kirk; Kramer, Randall

    2016-04-05

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  2. Polypeptides having endoglucanase activity and polynucleotides encoding same

    DOEpatents

    Spodsberg, Nikolaj

    2016-02-23

    The present invention relates to isolated polypeptides having endoglucanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  3. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    DOEpatents

    Tang, Lan; Liu, Ye; Duan, Junxin; Zhang, Yu; Joergensen, Christian; Kramer, Randall

    2014-09-16

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  4. Polypeptides having xylanase activity and polynucleotides encoding same

    DOEpatents

    Tang, Lan; Liu, Ye; Duan, Junxin; Ding, Hanshu

    2013-04-30

    The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  5. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding the same

    DOEpatents

    Tang, Lan; Liu, Ye; Duan, Junxin; Zhang, Yu; Jorgensen, Christian Isak; Kramer, Randall

    2013-12-24

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  6. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    DOEpatents

    Tang, Lan; Liu, Ye; Duan, Junxin; Zhang, Yu; Jorgensen, Christian Isak; Kramer, Randall

    2012-04-03

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  7. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding the same

    DOEpatents

    Tang, Lan; Liu, Ye; Duan, Junxin; Wu, Wenping; Kramer, Randall

    2013-11-19

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  8. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    DOEpatents

    Liu, Ye; Tang, Lan; Harris, Paul; Wu, Wenping

    2012-10-02

    The present invention relates to isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  9. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    DOEpatents

    Tang, Lan; Liu, Ye; Duan, Junxin; Zhang, Yu; Jorgensen, Christian Isak; Kramer, Randall

    2013-04-16

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  10. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    DOEpatents

    Duan, Junxin; Tang, Lan; Liu, Ye; Wu, Wenping; Quinlan, Jason; Kramer, Randall

    2013-06-18

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  11. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    DOEpatents

    Duan, Junxin; Liu, Ye; Tang, Lan; Wu, Wenping; Quinlan, Jason; Kramer, Randall

    2012-03-27

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  12. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    DOEpatents

    Tang, Lan; Liu, Ye; Duan, Junxin; Wu, Wenping; Kramer, Randall

    2014-10-21

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  13. Polypeptide-Coated Silica Particles Dispersed in Lyotropic Liquid Crystals of the Same Polypeptide.

    PubMed

    Rosu, Cornelia; Balamurugan, Sreelatha; Cueto, Rafael; Roy, Amitava; Russo, Paul S

    2016-07-28

    When a particle is introduced into a liquid crystal (LC), it distorts the LC director field, leading to new arrangements of the particles. This phenomenon is ordinarily studied using >100 nm particles and ∼2 nm mesogens. Usually the particle surface and mesogens are chemically distinct, which adds an enthalpic effect, even though the more interesting interactions are entropic. To raise the structures to the visible regime, while minimizing chemical differences between the particle surface and mesogen, silica particles coated with an α-helical polypeptide have been prepared and dispersed in lyotropic polypeptide LCs. The polypeptide is poly(γ-stearyl-α,l-glutamate) or PSLG. To make the particles easy to manipulate and easy to find, the silica core included superparamagnetic magnetite (Fe3O4) and covalently attached dye. Two methods were used to place polypeptides on these magnetic, fluorescent particles: a multistep grafting-to approach in which whole polypeptides were attached and a one-pot grafting-from approach in which the polymerization of the monomers was initiated from the particle surface. These approaches resulted in sparse and dense surface coverages, respectively. The influence of surface curvature and polypeptide molecular weight on the design of sparsely covered particles was investigated using the grafting-to approach. The aggregated grafting-from particles when freshly dispersed in a PSLG/solvent matrix disrupted the orientation of the characteristic cholesteric LC (ChLC) phase directors. In time, the hybrid particles were expelled from some domains, enabling the return of the familiar helical twist of the cholesteric mesophase. The sparsely coated grafting-to hybrid particles when inserted in the PSLG/solvent matrix assembled into stable islet-like formations that could not be disrupted even by an external magnetic field. The bulk particles aligned in chains that were easily manipulated by a magnetic field. These results indicate that

  14. POLYPEPTIDE AND POLYSACCHARIDE PROCESSING IN HYPERTHERMOPHILIC MICROORGANISMS

    SciTech Connect

    KELLY, ROBERT M.

    2008-12-22

    This project focused on the microbial physiology and biochemistry of heterotrophic hyperthermophiles with respect to mechanisms by which these organisms process polypeptides and polysaccharides under normal and stressed conditions. Emphasis is on two model organisms, for which completed genome sequences are available: Pyrococcus furiosus (growth Topt of 98°C), an archaeon, and Thermotoga maritima (growth Topt of 80°C), a bacterium. Both organisms are obligately anaerobic heterotrophs that reduce sulfur facultatively. Whole genome cDNA spotted microarrays were used to follow transcriptional response to a variety of environmental conditions in order to identify genes encoding proteins involved in the acquisition, synthesis, processing and utilization of polypeptides and polysaccharides. This project provided new insights into the physiological aspects of hyperthermophiles as these relate to microbial biochemistry and biological function in high temperature habitats. The capacity of these microorganisms to produce biohydrogen from renewable feedstocks makes them important for future efforts to develop biofuels.

  15. Analysis of adenovirus early region 4-encoded polypeptides synthesized in productively infected cells.

    PubMed Central

    Cutt, J R; Shenk, T; Hearing, P

    1987-01-01

    Peptide-specific antisera were developed to analyze the products encoded by adenovirus type 5 early region 4 (E4) open reading frames 6 and 7. Reading frame 6 previously was shown to encode a 34-kilodalton polypeptide (34K polypeptide) that forms a complex with the early region 1B (E1B)-55K antigen and is required for efficient viral growth in lytic infection. Antisera that were generated recognized the E4-34K protein as well as a family of related polypeptides generated by the fusion of open reading frames 6 and 7. These polypeptides shared amino-terminal sequences with the 34K protein. Short-pulse analysis suggested that the heterogeneity observed with the 6/7 fusion products resulted from differential splicing patterns of related E4 mRNAs. An antiserum directed against the amino terminus of reading frame 6 recognized only the free form of the 34K antigen that was not associated with the E1B-55K protein. This observation allowed the determination of the stability of the free and complexed form of this polypeptide. Pulse-chase analyses demonstrated that both forms of the 34K protein had half-lives greater than 24 h, suggesting that complex formation did not result in stabilization of this gene product. The half-lives of the 6/7 fusion products were approximately 4 h. The 34K protein also was shown to have a nuclear localization within infected cells. Finally, analysis of a mutant carrying deletions in both the E4-34K and E1B-55K polypeptides indicated that the complex formed between these two proteins was a functional unit in lytic infection. Images PMID:2949089

  16. Polypeptide having beta-glucosidase activity and uses thereof

    DOEpatents

    Schooneveld-Bergmans, Margot Elisabeth Francoise; Heijne, Wilbert Herman Marie; De Jong, Rene Marcel; Damveld, Robbertus Antonius

    2015-09-01

    The invention relates to a polypeptide comprising the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 70% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 70% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional polypeptide and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.

  17. Polypeptide having acetyl xylan esterase activity and uses thereof

    DOEpatents

    Schoonneveld-Bergmans, Margot Elisabeth Francoise; Heijne, Wilbert Herman Marie; Los, Alrik Pieter

    2015-10-20

    The invention relates to a polypeptide comprising the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 82% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 82% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional polypeptide and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.

  18. Polypeptide having beta-glucosidase activity and uses thereof

    DOEpatents

    Schoonneveld-Bergmans, Margot Elisabeth Francoise; Heijne, Wilbert Herman Marie; De Jong, Rene Marcel; Damveld, Robbertus Antonius

    2016-09-13

    The invention relates to a polypeptide comprising the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 96% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 96% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional polypeptide and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.

  19. Polypeptide having carbohydrate degrading activity and uses thereof

    DOEpatents

    Schooneveld-Bergmans, Margot Elisabeth Francoise; Heijne, Wilbert Herman Marie; Vlasie, Monica Diana; Damveld, Robbertus Antonius

    2015-08-18

    The invention relates to a polypeptide comprising the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 73% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 73% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional polypeptide and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.

  20. Toxicity study of isolated polypeptide from wool hydrolysate.

    PubMed

    Li, Jiashen; Li, Yi; Zhang, Yu; Liu, Xuan; Zhao, Zheng; Zhang, Jing; Han, Yanxia; Zhou, Dangxia

    2013-07-01

    The cytotoxicity of wool polypeptide has been evaluated by both cell and animal models. Wool was dissolved in sodium hydroxide solution, the pH value of the solution was adjusted to 5.55 and the precipitate was harvested as wool polypeptide. The spray-dried polypeptide was collected as powders and characterized by SEM, FTIR and TG-DSC. The cell culturing results showed that wool polypeptide had no obvious negative effect on cell viability in vitro. Both acute oral toxicity and subacute 30-day oral toxicology studies showed that wool polypeptide had no influence on body weight, feed consumption, blood chemistry, and hematology at any dose levels. There were no treatment related findings on gross or detailed necroscopy, organ weights, organ/body weight ratios and histology. Our study indicated the absence of toxicity in wool polypeptide and supported its safe use as a food ingredient or drug carrier.

  1. Toxicity study of isolated polypeptide from wool hydrolysate.

    PubMed

    Li, Jiashen; Li, Yi; Zhang, Yu; Liu, Xuan; Zhao, Zheng; Zhang, Jing; Han, Yanxia; Zhou, Dangxia

    2013-07-01

    The cytotoxicity of wool polypeptide has been evaluated by both cell and animal models. Wool was dissolved in sodium hydroxide solution, the pH value of the solution was adjusted to 5.55 and the precipitate was harvested as wool polypeptide. The spray-dried polypeptide was collected as powders and characterized by SEM, FTIR and TG-DSC. The cell culturing results showed that wool polypeptide had no obvious negative effect on cell viability in vitro. Both acute oral toxicity and subacute 30-day oral toxicology studies showed that wool polypeptide had no influence on body weight, feed consumption, blood chemistry, and hematology at any dose levels. There were no treatment related findings on gross or detailed necroscopy, organ weights, organ/body weight ratios and histology. Our study indicated the absence of toxicity in wool polypeptide and supported its safe use as a food ingredient or drug carrier. PMID:23597444

  2. Ordered Nanostructures Made Using Chaperonin Polypeptides

    NASA Technical Reports Server (NTRS)

    Trent, Jonathan; McMillan, Robert; Paavola, Chad; Mogul, Rakesh; Kagawa, Hiromi

    2004-01-01

    A recently invented method of fabricating periodic or otherwise ordered nanostructures involves the use of chaperonin polypeptides. The method is intended to serve as a potentially superior and less expensive alternative to conventional lithographic methods for use in the patterning steps of the fabrication of diverse objects characterized by features of the order of nanometers. Typical examples of such objects include arrays of quantum dots that would serve as the functional building blocks of future advanced electronic and photonic devices. A chaperonin is a double-ring protein structure having a molecular weight of about 60 plus or minus 5 kilodaltons. In nature, chaperonins are ubiquitous, essential, subcellular structures. Each natural chaperonin molecule comprises 14, 16, or 18 protein subunits, arranged as two stacked rings approximately 16 to 18 nm tall by approximately 15 to 17 nm wide, the exact dimensions depending on the biological species in which it originates. The natural role of chaperonins is unknown, but they are believed to aid in the correct folding of other proteins, by enclosing unfolded proteins and preventing nonspecific aggregation during assembly. What makes chaperonins useful for the purpose of the present method is that under the proper conditions, chaperonin rings assemble themselves into higher-order structures. This method exploits such higher-order structures to define nanoscale devices. The higher-order structures are tailored partly by choice of chemical and physical conditions for assembly and partly by using chaperonins that have been mutated. The mutations are made by established biochemical techniques. The assembly of chaperonin polypeptides into such structures as rings, tubes, filaments, and sheets (two-dimensional crystals) can be regulated chemically. Rings, tubes, and filaments of some chaperonin polypeptides can, for example, function as nano vessels if they are able to absorb, retain, protect, and release gases or

  3. Photodynamic induction of a bacterial cell surface polypeptide.

    PubMed Central

    Hoober, J K

    1977-01-01

    The photodynamic action of several dyes on cells of a bacterium, tentatively identified as a species of Arthrobacter, resulted in remarkable stimulation of synthesis of a polypeptide 21,000 daltons in mass. This polypeptide resides on the cell surface and can be solubilized by sodium dodecyl sulfate without lysis of the cells. Chlorophyllin and rose bengal are effective in inducing synthesis of the polypeptide in proportion to their ability to sensitize the photooxidation of histidine. Etiolated cells of the alga Chlamydomonas reinhardtii y-1 excrete a substance into the medium that also sensitized the photoinduction of the polypeptide. Images PMID:885841

  4. Computer simulation of polypeptides in a confinement.

    PubMed

    Sikorski, Andrzej; Romiszowski, Piotr

    2007-02-01

    A coarse-grained model of polypeptide chains confined in a slit formed by two parallel impenetrable surfaces was studied. The chains were flexible heteropolymers (polypeptides) built of two kinds of united atoms-hydrophobic and hydrophilic. The positions of the united atoms were restricted to the vertices of a [310] lattice. The force field consisted of a rigorous excluded volume, a long-distance potential between a pair of amino-acid residues and a local preference for forming secondary structure (helices). The properties of the chains were studied at a wide range of temperatures from good to bad solvent conditions. Monte-Carlo simulations were carried out using the algorithm based on the chain's local changes of conformation and employing the Replica Exchange technique. The influence of the chain length, the distances between the confining surfaces, the temperature and the force field on the dimension and the structure of chains were studied. It was shown that the presence of the confinement chain complicates the process of the chain collapse to low-temperature structures. For some conditions, one can find a rapid decrease of chain size and a second transition indicated by the rapid decrease of the total energy of the system.

  5. Caffeine-water-polypeptide interaction in aqueous solution

    NASA Astrophysics Data System (ADS)

    Ghabi, Habib; Dhahbi, Mahmoud

    1999-04-01

    The interaction of caffeine monomer with the synthetic polypeptides polyasparagine (pAg) and polyaspartic acid (pAsp) was studied by UV spectrophotometry. The results show that different types of interactions are possible depending on the nature of polypeptide. The form of the complex was discussed.

  6. Enzyme-catalyzed synthesis of polyamides and polypeptides

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polyamides and polypeptides are important polymers in biological systems and industrial processes. Usually polyamides are produced via chemical synthesis, whereas polypeptides and proteins are isolated from living systems or produced from Merrifield synthesis. An area of active research is to use ...

  7. Fibrillar dimer formation of islet amyloid polypeptides

    SciTech Connect

    Chiu, Chi -cheng; de Pablo, Juan J.

    2015-05-08

    Amyloid deposits of human islet amyloid polypeptide (hIAPP), a 37-residue hormone co-produced with insulin, have been implicated in the development of type 2 diabetes. Residues 20 – 29 of hIAPP have been proposed to constitute the amyloidogenic core for the aggregation process, yet the segment is mostly unstructured in the mature fibril, according to solid-state NMR data. Here we use molecular simulations combined with bias-exchange metadynamics to characterize the conformational free energies of hIAPP fibrillar dimer and its derivative, pramlintide. We show that residues 20 – 29 are involved in an intermediate that exhibits transient β-sheets, consistent with recent experimental and simulation results. By comparing the aggregation of hIAPP and pramlintide, we illustrate the effects of proline residues on inhibition of the dimerization of IAPP. The mechanistic insights presented here could be useful for development of therapeutic inhibitors of hIAPP amyloid formation.

  8. Fibrillar dimer formation of islet amyloid polypeptides

    NASA Astrophysics Data System (ADS)

    Chiu, Chi-cheng; de Pablo, Juan J.

    2015-09-01

    Amyloid deposits of human islet amyloid polypeptide (hIAPP), a 37-residue hormone co-produced with insulin, have been implicated in the development of type 2 diabetes. Residues 20 - 29 of hIAPP have been proposed to constitute the amyloidogenic core for the aggregation process, yet the segment is mostly unstructured in the mature fibril, according to solid-state NMR data. Here we use molecular simulations combined with bias-exchange metadynamics to characterize the conformational free energies of hIAPP fibrillar dimer and its derivative, pramlintide. We show that residues 20 - 29 are involved in an intermediate that exhibits transient β-sheets, consistent with recent experimental and simulation results. By comparing the aggregation of hIAPP and pramlintide, we illustrate the effects of proline residues on inhibition of the dimerization of IAPP. The mechanistic insights presented here could be useful for development of therapeutic inhibitors of hIAPP amyloid formation.

  9. The superfamily of organic anion transporting polypeptides.

    PubMed

    Hagenbuch, B; Meier, P J

    2003-01-10

    Organic anion transporting polypeptides (Oatps/OATPs) form a growing gene superfamily and mediate transport of a wide spectrum of amphipathic organic solutes. Different Oatps/OATPs have partially overlapping and partially distinct substrate preferences for organic solutes such as bile salts, steroid conjugates, thyroid hormones, anionic oligopeptides, drugs, toxins and other xenobiotics. While some Oatps/OATPs are preferentially or even selectively expressed in one tissue such as the liver, others are expressed in multiple organs including the blood-brain barrier (BBB), choroid plexus, lung, heart, intestine, kidney, placenta and testis. This review summarizes the actual state of the rapidly expanding OATP superfamily and covers the structural properties, the genomic classification, the phylogenetic relationships and the functional transport characteristics. In addition, we propose a new species independent and open ended nomenclature and classification system, which is based on divergent evolution and agrees with the guidelines of the Human Genome Nomenclature Committee.

  10. Chirality-selected phase behaviour in ionic polypeptide complexes

    PubMed Central

    Perry, Sarah L.; Leon, Lorraine; Hoffmann, Kyle Q.; Kade, Matthew J.; Priftis, Dimitrios; Black, Katie A.; Wong, Derek; Klein, Ryan A.; Pierce, Charles F.; Margossian, Khatcher O.; Whitmer, Jonathan K.; Qin, Jian; de Pablo, Juan J.; Tirrell, Matthew

    2015-01-01

    Polyelectrolyte complexes present new opportunities for self-assembled soft matter. Factors determining whether the phase of the complex is solid or liquid remain unclear. Ionic polypeptides enable examination of the effects of stereochemistry on complex formation. Here we demonstrate that chirality determines the state of polyelectrolyte complexes, formed from mixing dilute solutions of oppositely charged polypeptides, via a combination of electrostatic and hydrogen-bonding interactions. Fluid complexes occur when at least one of the polypeptides in the mixture is racemic, which disrupts backbone hydrogen-bonding networks. Pairs of purely chiral polypeptides, of any sense, form compact, fibrillar solids with a β-sheet structure. Analogous behaviour occurs in micelles formed from polypeptide block copolymers with polyethylene oxide, where assembly into aggregates with either solid or fluid cores, and eventually into ordered phases at high concentrations, is possible. Chirality is an exploitable tool for manipulating material properties in polyelectrolyte complexation. PMID:25586861

  11. Nucleic acids encoding antifungal polypeptides and uses thereof

    DOEpatents

    Altier, Daniel J.; Ellanskaya, I. A.; Gilliam, Jacob T.; Hunter-Cevera, Jennie; Presnail, James K; Schepers, Eric; Simmons, Carl R.; Torok, Tamas; Yalpani, Nasser

    2010-11-02

    Compositions and methods for protecting a plant from a pathogen, particularly a fungal pathogen, are provided. Compositions include an amino acid sequence, and variants and fragments thereof, for an antipathogenic polypeptide that was isolated from a fungal fermentation broth. Nucleic acid molecules that encode the antipathogenic polypeptides of the invention, and antipathogenic domains thereof, are also provided. A method for inducing pathogen resistance in a plant using the nucleotide sequences disclosed herein is further provided. The method comprises introducing into a plant an expression cassette comprising a promoter operably linked to a nucleotide sequence that encodes an antipathogenic polypeptide of the invention. Compositions comprising an antipathogenic polypeptide or a transformed microorganism comprising a nucleic acid of the invention in combination with a carrier and methods of using these compositions to protect a plant from a pathogen are further provided. Transformed plants, plant cells, seeds, and microorganisms comprising a nucleotide sequence that encodes an antipathogenic polypeptide of the invention are also disclosed.

  12. Isolated nucleic acids encoding antipathogenic polypeptides and uses thereof

    DOEpatents

    Altier, Daniel J.; Crane, Virginia C.; Ellanskaya, Irina; Ellanskaya, Natalia; Gilliam, Jacob T.; Hunter-Cevera, Jennie; Presnail, James K.; Schepers, Eric J.; Simmons, Carl R.; Torok, Tamas; Yalpani, Nasser

    2010-04-20

    Compositions and methods for protecting a plant from a pathogen, particularly a fungal pathogen, are provided. Compositions include amino acid sequences, and variants and fragments thereof, for antipathogenic polypeptides that were isolated from fungal fermentation broths. Nucleic acids that encode the antipathogenic polypeptides are also provided. A method for inducing pathogen resistance in a plant using the nucleotide sequences disclosed herein is further provided. The method comprises introducing into a plant an expression cassette comprising a promoter operably linked to a nucleotide sequence that encodes an antipathogenic polypeptide of the invention. Compositions comprising an antipathogenic polypeptide or a transformed microorganism comprising a nucleic acid of the invention in combination with a carrier and methods of using these compositions to protect a plant from a pathogen are further provided. Transformed plants, plant cells, seeds, and microorganisms comprising a nucleotide sequence that encodes an antipathogenic polypeptide of the invention are also disclosed.

  13. Chirality-selected phase behaviour in ionic polypeptide complexes

    SciTech Connect

    Perry, Sarah L.; Leon, Lorraine; Hoffmann, Kyle Q.; Kade, Matthew J.; Priftis, Dimitrios; Black, Katie A.; Wong, Derek; Klein, Ryan A.; Pierce, III, Charles F.; Margossian, Khatcher O.; Whitmer, Jonathan K.; Qin, Jian; de Pablo, Juan J.; Tirrell, Matthew

    2015-01-14

    In this study, polyelectrolyte complexes present new opportunities for self-assembled soft matter. Factors determining whether the phase of the complex is solid or liquid remain unclear. Ionic polypeptides enable examination of the effects of stereochemistry on complex formation. Here we demonstrate that chirality determines the state of polyelectrolyte complexes, formed from mixing dilute solutions of oppositely charged polypeptides, via a combination of electrostatic and hydrogen-bonding interactions. Fluid complexes occur when at least one of the polypeptides in the mixture is racemic, which disrupts backbone hydrogen-bonding networks. Pairs of purely chiral polypeptides, of any sense, form compact, fibrillar solids with a β-sheet structure. Analogous behaviour occurs in micelles formed from polypeptide block copolymers with polyethylene oxide, where assembly into aggregates with either solid or fluid cores, and eventually into ordered phases at high concentrations, is possible. Chirality is an exploitable tool for manipulating material properties in polyelectrolyte complexation.

  14. Chirality-selected phase behaviour in ionic polypeptide complexes

    DOE PAGESBeta

    Perry, Sarah L.; Leon, Lorraine; Hoffmann, Kyle Q.; Kade, Matthew J.; Priftis, Dimitrios; Black, Katie A.; Wong, Derek; Klein, Ryan A.; Pierce, III, Charles F.; Margossian, Khatcher O.; et al

    2015-01-14

    In this study, polyelectrolyte complexes present new opportunities for self-assembled soft matter. Factors determining whether the phase of the complex is solid or liquid remain unclear. Ionic polypeptides enable examination of the effects of stereochemistry on complex formation. Here we demonstrate that chirality determines the state of polyelectrolyte complexes, formed from mixing dilute solutions of oppositely charged polypeptides, via a combination of electrostatic and hydrogen-bonding interactions. Fluid complexes occur when at least one of the polypeptides in the mixture is racemic, which disrupts backbone hydrogen-bonding networks. Pairs of purely chiral polypeptides, of any sense, form compact, fibrillar solids with amore » β-sheet structure. Analogous behaviour occurs in micelles formed from polypeptide block copolymers with polyethylene oxide, where assembly into aggregates with either solid or fluid cores, and eventually into ordered phases at high concentrations, is possible. Chirality is an exploitable tool for manipulating material properties in polyelectrolyte complexation.« less

  15. Polypeptide heterogeneity of hamster and calf fibronectins.

    PubMed Central

    Pena, S D; Mills, G; Hughes, R C; Aplin, J D

    1980-01-01

    The adhesive glycoprotein fibronectin has been isolated from fresh hamster plasma by affinity chromatography on gelatin coupled to Sepharose beads by the method of Engvall & Ruoslahti [Int. J. Cancer (1979) 20, 1-5]. Polyacrylamide-gel electrophoresis of material heated in sodium dodecyl sulphate and 2-mercaptoethanol shows two prominent polypeptide subunits of approx. mol.wts. 215 000 and 200 000, with variable amounts of lower-molecular-weight fragments. The unexpected polypeptide heterogeneity of different preparations of hamster fibronectins and bovine serum fibronectin is shown to be partly an artefact and is generated during isolation and storage of purified fibronectin. Treatment of each hamster fibronectin subunit or a smaller fragment of approx. mol.wt. 140 000 with thermolysin or trypsin after radioiodination produces similar patterns of tyrpsine-containing peptides, indicating similar primary amino-acid sequences. Antibodies raised against the major subunits of hamster plasma fibronectin were coupled to Sepharose beads and used in conjunction with gelatin affinity chromatography to isolate fibronectins extracted with urea from baby-hamster kidney (BHK) cells and present in the long-term culture medium of these cells. The cell and medium fibronectins are similar to hamster plasma fibronectin in amino-acid and carbohydrate composition and also produce very similar peptide 'maps'. We conclude that the various forms of hamster fibronectins are structurally analogous in agreement with indistinguishable biological properties in mediating the substance adhesion of BKH cells [Pena & Hughes (1978) Cell Biol. Int. Rep. 3, 339-344]. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 6. Fig. 7. PMID:7458916

  16. HLA-D region antigen-associated invariant polypeptides as revealed by two-dimensional gel analysis. Glycosylation and structural inter-relationships.

    PubMed

    Rudd, C E; Bodmer, J G; Bodmer, W F; Crumpton, M J

    1985-02-10

    Two-dimensional polyacrylamide gel analyses of immunoprecipitates of HLA-D region antigens prepared from [35S]methionine-labeled B lymphoblastoid cells revealed a number of invariant polypeptides (Ii and theta) that co-precipitate with the alpha and beta polypeptides of the class II (Ia) antigens. The invariant polypeptides comprised at least three Ii spots of Mr = 31,000 (Ii1-Ii3) and a series of six theta spots of Mr = 34,000 (theta 1-theta 6). The structural inter-relationships of these polypeptides have been investigated. Tryptic peptide fingerprints showed that Ii and theta have closely related amino acid sequences. In contrast, the fingerprints of the HLA-DR alpha and beta polypeptides clearly differed from those of theta and Ii as well as from each other. Analyses of immunoprecipitates prepared from cells cultured in the presence of tunicamycin revealed the presence of two N-linked oligosaccharides on each invariant polypeptide and suggested that the more acidic theta polypeptides (theta 1 and theta 2) differed from the other invariant polypeptides by the presence of sialic acid on one or both N-linked oligosaccharides. Removal of sialic acid by neuraminidase simplified the pattern of theta spots into three distinct Ii-related polypeptides. Endo-beta-N-acetylglycosaminidase H digestion indicated that the individual theta polypeptides represent stages in carbohydrate processing whereby Ii with two N-linked immature oligosaccharides are converted initially to theta 6-theta 3 with one immature and one complex, but nonsialylated, oligosaccharide and finally to theta 2-theta 1 with two complex oligosaccharides. Digestion of the theta polypeptides with N-acetylgalactosamine oligosaccharidase indicated that the theta spots are also derived by O-glycosylation from the Ii polypeptides. This assignment is supported by results obtained using monensin to block glycosylation within the Golgi. At least three spots persisted after complete removal of the N- and O

  17. Discovery of Human sORF-Encoded Polypeptides (SEPs) in Cell Lines and Tissue

    PubMed Central

    2015-01-01

    The existence of nonannotated protein-coding human short open reading frames (sORFs) has been revealed through the direct detection of their sORF-encoded polypeptide (SEP) products. The discovery of novel SEPs increases the size of the genome and the proteome and provides insights into the molecular biology of mammalian cells, such as the prevalent usage of non-AUG start codons. Through modifications of the existing SEP-discovery workflow, we discover an additional 195 SEPs in K562 cells and extend this methodology to identify novel human SEPs in additional cell lines and human tissue for a final tally of 237 new SEPs. These results continue to expand the human genome and proteome and demonstrate that SEPs are a ubiquitous class of nonannotated polypeptides that require further investigation. PMID:24490786

  18. Simulating Massive Conformation Changes within Polypeptide Systems

    NASA Astrophysics Data System (ADS)

    Singh, Jaspinder Paul

    In this dissertation I employ all-atom structure based models with stable energy basins to several existing and novel polypeptide systems (postulated conformation changes of the mammalian prion protein and structurally dual proteins). The common themes are finding unfolding and refolding pathways between highly dissimilar protein structures as a means of understanding exactly how and why a protein may misfold. The modeling is based on the energy funnel landscape theory of protein conformation space. The principle of minimal frustration is considered as the model includes parameters which vary the roughness of the landscape and give rise to off-pathway misfoldings. The dual basin model is applied to the C-terminal (residues 166-226) of the mammalian prion protein. One basin represents the known alpha-helical (aH) structure while the other represents the same residues in a lefthanded beta-helical (LHBH) conformation. The LHBH structure has been proposed to help describe one class of in vitro grown fibrils, as well as possibly self-templating the conversion of normal cellular prion protein to the infectious form. Yet, it is unclear how the protein may make this global rearrangement. Our results demonstrate that the conformation changes are not strongly limited by large-scale geometry modification and that there may exist an overall preference for the LHBH conformation. Furthermore, our model presents novel intermediate trapping conformations with twisted LHBH structure. Polypeptides that display structural duality have primary structures that can give rise to different potential native conformations. We apply the structure-based all-atom model to a leucine zipper protein template with a stable aH structure that has been shown in experiment to switch to a β hairpin structure when exposed to a low-pH environment. We show that the model can be used to perform large-scale temperature-dependent conformational switching by simulating this switching behavior. We augmented

  19. Aspects of structural landscape of human islet amyloid polypeptide.

    PubMed

    He, Jianfeng; Dai, Jin; Li, Jing; Peng, Xubiao; Niemi, Antti J

    2015-01-28

    The human islet amyloid polypeptide (hIAPP) co-operates with insulin to maintain glycemic balance. It also constitutes the amyloid plaques that aggregate in the pancreas of type-II diabetic patients. We have performed extensive in silico investigations to analyse the structural landscape of monomeric hIAPP, which is presumed to be intrinsically disordered. For this, we construct from first principles a highly predictive energy function that describes a monomeric hIAPP observed in a nuclear magnetic resonance experiment, as a local energy minimum. We subject our theoretical model of hIAPP to repeated heating and cooling simulations, back and forth between a high temperature regime where the conformation resembles a random walker and a low temperature limit where no thermal motions prevail. We find that the final low temperature conformations display a high level of degeneracy, in a manner which is fully in line with the presumed intrinsically disordered character of hIAPP. In particular, we identify an isolated family of α-helical conformations that might cause the transition to amyloidosis, by nucleation. PMID:25638009

  20. Aspects of structural landscape of human islet amyloid polypeptide

    NASA Astrophysics Data System (ADS)

    He, Jianfeng; Dai, Jin; Li, Jing; Peng, Xubiao; Niemi, Antti J.

    2015-01-01

    The human islet amyloid polypeptide (hIAPP) co-operates with insulin to maintain glycemic balance. It also constitutes the amyloid plaques that aggregate in the pancreas of type-II diabetic patients. We have performed extensive in silico investigations to analyse the structural landscape of monomeric hIAPP, which is presumed to be intrinsically disordered. For this, we construct from first principles a highly predictive energy function that describes a monomeric hIAPP observed in a nuclear magnetic resonance experiment, as a local energy minimum. We subject our theoretical model of hIAPP to repeated heating and cooling simulations, back and forth between a high temperature regime where the conformation resembles a random walker and a low temperature limit where no thermal motions prevail. We find that the final low temperature conformations display a high level of degeneracy, in a manner which is fully in line with the presumed intrinsically disordered character of hIAPP. In particular, we identify an isolated family of α-helical conformations that might cause the transition to amyloidosis, by nucleation.

  1. Aspects of structural landscape of human islet amyloid polypeptide

    SciTech Connect

    He, Jianfeng Dai, Jin; Li, Jing; Peng, Xubiao; Niemi, Antti J.

    2015-01-28

    The human islet amyloid polypeptide (hIAPP) co-operates with insulin to maintain glycemic balance. It also constitutes the amyloid plaques that aggregate in the pancreas of type-II diabetic patients. We have performed extensive in silico investigations to analyse the structural landscape of monomeric hIAPP, which is presumed to be intrinsically disordered. For this, we construct from first principles a highly predictive energy function that describes a monomeric hIAPP observed in a nuclear magnetic resonance experiment, as a local energy minimum. We subject our theoretical model of hIAPP to repeated heating and cooling simulations, back and forth between a high temperature regime where the conformation resembles a random walker and a low temperature limit where no thermal motions prevail. We find that the final low temperature conformations display a high level of degeneracy, in a manner which is fully in line with the presumed intrinsically disordered character of hIAPP. In particular, we identify an isolated family of α-helical conformations that might cause the transition to amyloidosis, by nucleation.

  2. Selective posttranslational modification of phage-displayed polypeptides

    DOEpatents

    Tsao, Meng-Lin; Tian, Feng; Schultz, Peter

    2013-02-05

    The invention relates to posttranslational modification of phage-displayed polypeptides. These displayed polypeptides comprise at least one unnatural amino acid, e.g., an aryl-azide amino acid such as p-azido-L-phenylalanine, or an alkynyl-amino acid such as para-propargyloxyphenylalanine, which are incorporated into the phage-displayed fusion polypeptide at a selected position by using an in vivo orthogonal translation system comprising a suitable orthogonal aminoacyl-tRNA synthetase and a suitable orthogonal tRNA species. These unnatural amino acids advantageously provide targets for posttranslational modifications such as azide-alkyne [3+2]cycloaddition reactions and Staudinger modifications.

  3. Selective posttranslational modification of phage-displayed polypeptides

    DOEpatents

    Tsao, Meng-Lin; Tian, Feng; Schultz, Peter

    2013-11-19

    The invention relates to posttranslational modification of phage-displayed polypeptides. These displayed polypeptides comprise at least one unnatural amino acid, e.g., an aryl-azide amino acid such as p-azido-L-phenylalanine, or an alkynyl-amino acid such as para-propargyloxyphenylalanine, which are incorporated into the phage-displayed fusion polypeptide at a selected position by using an in vivo orthogonal translation system comprising a suitable orthogonal aminoacyl-tRNA synthetase and a suitable orthogonal tRNA species. These unnatural amino acids advantageously provide targets for posttranslational modifications such as azide-alkyne [3+2] cycloaddition reactions and Staudinger modifications.

  4. Amino acids of ricin and its polypeptides.

    PubMed

    Chakravartula, Srinivas V S; Guttarla, Nagaraj

    2008-02-15

    Ricin and its corresponding polypeptides (A & B chain) were purified from castor seed. The molecular weight of ricin subunits were 29,000 and 28,000 daltons. The amino acids in ricin determined were Asp45 The22 Ser40 Glu53 Cys4 Gly96 His5 Ile21 Leu33 Lys20 Met4 Phe13 Pro37 Tyr11 Ala45 Val23 Arg20 indicating that ricin contains approximately 516 amino acid residues. The amino acids of the two subunits of ricin A and B chains were Asp23 The12 Ser21 Glu29 Cys2 Gly48 His3 Ile12, Leu17 Lys10 Met2 Phe6 Pro17 Tyr7 Ala35 Val13 Arg13 while in B chain the amino acids were Asp22 The10 Ser19 Glu25 Cys2 Gly47 His1 Ile10, Leu15 Lys11 Met1 Phe7 Pro6 Tyr5 Ala32Val11 Arg10. The total helical content of ricin came around 53.6% which is a new observation. PMID:18266157

  5. Mechanisms of stability of electrospun polypeptide fibers

    NASA Astrophysics Data System (ADS)

    Gitnik, Alina; Khadka, Dhan; Cross, Michael; Le, Nicole; Haynie, Donald

    2013-03-01

    Electrospun nano- and microfibers made of biodegradable and absorbable polymers are of great interest in biomedical engineering for tissue engineering, wound healing and other purposes. We have investigated physical properties of fibers made of the synthetic organic polymer co-poly(L-glutamic acid4, L-tyrosine1) (PLEY). This water-soluble polypeptide has a net negative charge at neutral pH. Dehydrated fibers are crosslinked with a diimide reagent dissolved in ethanol, giving a maximum average number of crosslinks of 1 per polymer molecule. Fiber integrity has been assessed in an aqueous medium at pH 2, 7 and 12, before and after crosslinking. Non-crosslinked fibers dissolved rapidly at all pH values, on a timescale of seconds to minutes. Crosslinked fibers dissolved completely at pH 12, but not at pH 2 or pH 7, the rate depending on the concentration of crosslinking reagent and therefore the density of crosslinks. Dissolution at pH 12 is attributable to ionization of the tyrosine side chain, which has a nominal pKa of 10.4, an increase in electrostatic repulsion between side chains and the migration of counterions into the fiber. Fibers crosslinked in 50 mM EDC buckled on a timescale of minutes at pH 12 and dissolved shortly thereafter. Funding provided by the National Science Foundation

  6. Fibril structure of human islet amyloid polypeptide.

    PubMed

    Bedrood, Sahar; Li, Yiyu; Isas, J Mario; Hegde, Balachandra G; Baxa, Ulrich; Haworth, Ian S; Langen, Ralf

    2012-02-17

    Misfolding and amyloid fibril formation by human islet amyloid polypeptide (hIAPP) are thought to be important in the pathogenesis of type 2 diabetes, but the structures of the misfolded forms remain poorly understood. Here we developed an approach that combines site-directed spin labeling with continuous wave and pulsed EPR to investigate local secondary structure and to determine the relative orientation of the secondary structure elements with respect to each other. These data indicated that individual hIAPP molecules take up a hairpin fold within the fibril. This fold contains two β-strands that are much farther apart than expected from previous models. Atomistic structural models were obtained using computational refinement with EPR data as constraints. The resulting family of structures exhibited a left-handed helical twist, in agreement with the twisted morphology observed by electron microscopy. The fibril protofilaments contain stacked hIAPP monomers that form opposing β-sheets that twist around each other. The two β-strands of the monomer adopt out-of-plane positions and are staggered by about three peptide layers (∼15 Å). These results provide a mechanism for hIAPP fibril formation and could explain the remarkable stability of the fibrils. Thus, the structural model serves as a starting point for understanding and preventing hIAPP misfolding. PMID:22187437

  7. Fibrillar dimer formation of islet amyloid polypeptides

    DOE PAGESBeta

    Chiu, Chi -cheng; de Pablo, Juan J.

    2015-05-08

    Amyloid deposits of human islet amyloid polypeptide (hIAPP), a 37-residue hormone co-produced with insulin, have been implicated in the development of type 2 diabetes. Residues 20 – 29 of hIAPP have been proposed to constitute the amyloidogenic core for the aggregation process, yet the segment is mostly unstructured in the mature fibril, according to solid-state NMR data. Here we use molecular simulations combined with bias-exchange metadynamics to characterize the conformational free energies of hIAPP fibrillar dimer and its derivative, pramlintide. We show that residues 20 – 29 are involved in an intermediate that exhibits transient β-sheets, consistent with recent experimentalmore » and simulation results. By comparing the aggregation of hIAPP and pramlintide, we illustrate the effects of proline residues on inhibition of the dimerization of IAPP. The mechanistic insights presented here could be useful for development of therapeutic inhibitors of hIAPP amyloid formation.« less

  8. Islet Amyloid Polypeptide: Structure, Function, and Pathophysiology

    PubMed Central

    Akter, Rehana; Cao, Ping; Noor, Harris; Ridgway, Zachary; Tu, Ling-Hsien; Wang, Hui; Wong, Amy G.; Zhang, Xiaoxue; Abedini, Andisheh; Schmidt, Ann Marie; Raleigh, Daniel P.

    2016-01-01

    The hormone islet amyloid polypeptide (IAPP, or amylin) plays a role in glucose homeostasis but aggregates to form islet amyloid in type-2 diabetes. Islet amyloid formation contributes to β-cell dysfunction and death in the disease and to the failure of islet transplants. Recent work suggests a role for IAPP aggregation in cardiovascular complications of type-2 diabetes and hints at a possible role in type-1 diabetes. The mechanisms of IAPP amyloid formation in vivo or in vitro are not understood and the mechanisms of IAPP induced β-cell death are not fully defined. Activation of the inflammasome, defects in autophagy, ER stress, generation of reactive oxygen species, membrane disruption, and receptor mediated mechanisms have all been proposed to play a role. Open questions in the field include the relative importance of the various mechanisms of β-cell death, the relevance of reductionist biophysical studies to the situation in vivo, the molecular mechanism of amyloid formation in vitro and in vivo, the factors which trigger amyloid formation in type-2 diabetes, the potential role of IAPP in type-1 diabetes, the development of clinically relevant inhibitors of islet amyloidosis toxicity, and the design of soluble, bioactive variants of IAPP for use as adjuncts to insulin therapy. PMID:26649319

  9. Common spectrum of polypeptides occurs in secretion granule membranes of different exocrine glands

    SciTech Connect

    Cameron, R.S.; Cameron, P.L.; Castle, J.D.

    1986-10-01

    A highly purified membrane preparation from rat parotid secretion granules has been used as a comparative probe to examine the extent of compositional overlap in granule membranes of three other exocrine secretory tissues - pancreatic, lacrimal, and submandibular - from several standpoints. First, indirect immunofluorescent studies using a polyclonal polyspecific anti-parotid granule membrane antiserum has indicated a selective staining of granule membrane profiles in all acinar cells of all tissues. Second, highly purified granule membrane subfractions have been isolated from each exocrine tissue; comparative two-dimensional (isoelectric focusing; SDS) PAGE of radioiodinated granule membranes has identified 10-15 polypeptides of identical pI and apparent molecular mass. These species are likely to be integral membrane components since they are not extracted by either saponin-sodium sulfate or sodium carbonate (pH 11.5) treatments, and they do not have counterparts in the granule content. Finally, the identity among selected parotid and pancreatic radioiodinated granule membrane polypeptides has been documented using two-dimensional peptide mapping of chymotryptic and tryptic digests. These findings clearly indicate that exocrine secretory granules, irrespective of the nature of stored secretion, comprise a type of vesicular carrier with a common (and probably refined) membrane composition. Conceivably, the polypeptides identified carry out general functions related to exocrine secretion.

  10. Polypeptide composition of spleen necrosis virus, a reticuloendotheliosis virus.

    PubMed Central

    Mosser, A G

    1975-01-01

    The polypeptide composition of virions of spleen necrosis virus, a reticuloendotheliosis virus, was determined using electrophoresis on sodium dodecyl sulfate-containing, 10 percent polyacrylamide gels. Ten polypeptides were resolved. Four of these were present in minor and somewhat variable amounts. Two proteins, gp71 and gp22, contained D-glucosamine and were located on the outer surface of the lipid envelope, as demonstrated by lactoperoxidase-catalyzed iodination and by bromelain digestion. The results suggest that two of the minor proteins, p36 and p26, were also located on the outer surface, although they lacked D-glucosamine. Treatment of the virus with 0.25 percent Nonidet P-40 and 1 percent dithiothreitol produced a subparticle with a buoyant density of approximately 1.31 g/cm-3. This particle was relatively enriched with polypeptides p77, p62, and p50 and contained small amounts of three other polypeptides. PMID:1142473

  11. Photocleavage of the Polypeptide Backbone by 2-Nitrophenylalanine

    PubMed Central

    Peters, Francis B.; Brock, Ansgar; Wang, Jiangyun; Schultz, Peter G.

    2009-01-01

    Summary Photocleavage of the polypeptide backbone is potentially a powerful and general method to activate or deactivate functional peptides and proteins with high spatial and temporal resolution. Here we show that 2-nitrophenylalanine is able to photochemically cleave the polypeptide backbone by an unusual cinnoline forming reaction. This unnatural amino acid was genetically encoded in E. coli, and protein containing 2-nitrophenylalanine was expressed and site specifically photocleaved. PMID:19246005

  12. Islet amyloid polypeptide toxicity and membrane interactions.

    PubMed

    Cao, Ping; Abedini, Andisheh; Wang, Hui; Tu, Ling-Hsien; Zhang, Xiaoxue; Schmidt, Ann Marie; Raleigh, Daniel P

    2013-11-26

    Islet amyloid polypeptide (IAPP) is responsible for amyloid formation in type 2 diabetes and contributes to the failure of islet cell transplants, however the mechanisms of IAPP-induced cytotoxicity are not known. Interactions with model anionic membranes are known to catalyze IAPP amyloid formation in vitro. Human IAPP damages anionic membranes, promoting vesicle leakage, but the features that control IAPP-membrane interactions and the connection with cellular toxicity are not clear. Kinetic studies with wild-type IAPP and IAPP mutants demonstrate that membrane leakage is induced by prefibrillar IAPP species and continues over the course of amyloid formation, correlating additional membrane disruption with fibril growth. Analyses of a set of designed mutants reveal that membrane leakage does not require the formation of β-sheet or α-helical structures. A His-18 to Arg substitution enhances leakage, whereas replacement of all of the aromatic residues via a triple leucine mutant has no effect. Biophysical measurements in conjunction with cytotoxicity studies show that nonamyloidogenic rat IAPP is as effective as human IAPP at disrupting standard anionic model membranes under conditions where rat IAPP does not induce cellular toxicity. Similar results are obtained with more complex model membranes, including ternary systems that contain cholesterol and are capable of forming lipid rafts. A designed point mutant, I26P-IAPP; a designed double mutant, G24P, I26P-IAPP; a double N-methylated variant; and pramlintide, a US Food and Drug Administration-approved IAPP variant all induce membrane leakage, but are not cytotoxic, showing that there is no one-to-one relationship between disruption of model membranes and induction of cellular toxicity. PMID:24218607

  13. A recombinant hepatitis B core antigen polypeptide with the protamine-like domain deleted self-assembles into capsid particles but fails to bind nucleic acids.

    PubMed Central

    Gallina, A; Bonelli, F; Zentilin, L; Rindi, G; Muttini, M; Milanesi, G

    1989-01-01

    We have cloned in Escherichia coli both the complete core gene of hepatitis B virus and a truncated version of it, leading to the synthesis of high levels of a core-antigen-equivalent polypeptide (r-p22) and of an e-antigen-equivalent polypeptide (r-p16), respectively. We then compared the structural and antigenic properties of the two polypeptides, as well as their ability to bind viral nucleic acids. r-p16 was found to self-assemble into capsid-like particles that appeared similar, when observed under the electron microscope, to those formed by r-p22. In r-p16 particles, disulfide bonds linked the truncated polypeptides in dimers, assembled in the particle by noncovalent interactions. In r-p22 capsids, further disulfide bonds, conceivably involving the carboxy-terminal cysteines of r-p22 polypeptides, joined the dimers together, converting the structure into a covalently closed lattice. The protamine-like domain was at least partly exposed on the surface of r-p22 particles, since it was accessible to selective proteolysis. Finally, r-p22, but not r-p16, was shown to bind native and denatured DNA as well as RNA. Taken together, these results suggest that the protamine-like domain in core polypeptides is a nucleic acid-binding domain and is dispensable for the correct folding and assembly of amino-terminal and central regions. Images PMID:2677399

  14. Chemical synthesis of a polypeptide backbone derived from the primary sequence of the cancer protein NY-ESO-1 enabled by kinetically controlled ligation and pseudoprolines.

    PubMed

    Harris, Paul W R; Brimble, Margaret A

    2015-03-01

    The cancer protein NY-ESO-1 has been shown to be one of the most promising vaccine candidates although little is known about its cellular function. Using a chemical protein strategy, the 180 amino acid polypeptide, tagged with an arginine solubilizing tail, was assembled in a convergent manner from four unprotected peptide α-thioester peptide building blocks and one cysteinyl polypeptide, which were in turn prepared by Boc and Fmoc solid phase peptide synthesis (SPPS) respectively. To facilitate the assembly by ligation chemistries, non-native cysteines were introduced as chemical handles into the polypeptide fragments; pseudoproline dipeptides and microwave assisted Fmoc SPPS were crucial techniques to prepare the challenging hydrophobic C-terminal fragment. Three sequential kinetically controlled ligations, which exploited the reactivity between peptide arylthioesters and peptide alkylthioesters, were then used in order to assemble the more tractable N-terminal region of NY-ESO-1. The ensuing 147 residue polypeptide thioester then underwent successful final native chemical ligation with the very hydrophobic C-terminal polypeptide bearing an N-terminal cysteine affording the 186 residue polypeptide as an advanced intermediate en route to the native NY-ESO-1 protein.

  15. Structural and functional properties of a multi-enzyme complex from spinach chloroplasts. 1. Stoichiometry of the polypeptide chains.

    PubMed

    Rault, M; Giudici-Orticoni, M T; Gontero, B; Ricard, J

    1993-11-01

    Antibodies have been raised specifically against chloroplast phosphoribulokinase, glyceraldehyde-3-phosphate dehydrogenase and ribulose 1,5-bisphosphate carboxylase-oxygenase. Each of these antibodies recognizes the same macromolecular entity isolated and purified from chloroplasts. This entity is a multi-enzyme complex, previously isolated and made up of ribose-phosphate isomerase, phosphoribulokinase, ribulose 1,5-bisphosphate carboxylase-oxygenase, phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase. Under denaturing conditions the multi-enzyme complex contains two polypeptides of 54 kDa and 15 kDa corresponding to the large and the small subunits of ribulose 1,5-bisphosphate carboxylase-oxygenase, the two polypeptides of the glyceraldehyde-3-phosphate dehydrogenase of 39 kDa and 37 kDa, one polypeptide of 40 kDa pertaining to phosphoribulokinase and one polypeptide of 30 kDa very likely pertaining to ribose-phosphate isomerase. The combined use of immunochemical and densitometric techniques allows one to determine the number and the stoichiometry of the various types of polypeptide chains and to compare them with the quaternary structure of the corresponding isolated enzymes. Ribulose 1,5-bisphosphate carboxylase-oxygenase of higher plants consists of eight large and eight small subunits. Glyceraldehyde-3-phosphate dehydrogenase is made up of two types of polypeptide chains called A and B and its simplest quaternary structure is A2B2. Finally, phosphoribulokinase is a dimer made up of two identical subunits. Therefore, for the three isolated enzymes, the stoichiometry of the polypeptide chains is always 1:1. Within this multi-enzyme complex, there are two subunits of phosphoribulokinase, two A and B subunits of glyceraldehyde-3-phosphate dehydrogenase and two large and four small subunits of ribulose 1,5-bisphosphate carboxylase-oxygenase. Therefore the number and the stoichiometry of the polypeptide chains of phosphoribulokinase and

  16. RUBIDIUM, a program for computer-aided assignment of two-dimensional NMR spectra of polypeptides.

    PubMed

    Yu, C; Hwang, J F; Chen, T B; Soo, V W

    1992-01-01

    Taking advantage of the rule-based expert system technology, a program named RUBIDIUM (Rule-Based Identification In 2D NMR Spectrum) was developed to accomplish the automatic 1H NMR resonance assignments of polypeptides. Besides noise elimination and peak selection capabilities, RUBIDIUM detects the cross-peak patterns of amino acid residues in the COSY spectrum, assigning these patterns to amino acid types, performing sequential assignments using combined COSY/NOESY spectra, and finally, achieving the total assignment of the 1H NMR spectrum.

  17. Drug delivery to solid tumors by elastin-like polypeptides

    PubMed Central

    McDaniel, Jonathan R.; Callahan, Daniel J.; Chilkoti, Ashutosh

    2010-01-01

    Thermally responsive elastin-like polypeptides (ELPs) are a promising class of recombinant biopolymers for the delivery of drugs and imaging agents to solid tumors via systemic or local administration. This article reviews four applications of ELPs to drug delivery, with each delivery mechanism designed to best exploit the relationship between the characteristic transition temperature (Tt) of the ELP and body temperature (Tb). First, when Tt >> Tb, small hydrophobic drugs can be conjugated to the C-terminus of the ELP to impart the amphiphilicity needed to mediate the self-assembly of nanoparticles. These systemically delivered ELP-drug nanoparticles preferentially localize to the tumor site via the EPR effect, resulting in reduced toxicity and enhanced treatment efficacy. The remaining three approaches take direct advantage of the thermal responsiveness of ELPs. In the second strategy, where Tb < Tt < 42 °C, an ELP-drug conjugate can be injected in conjunction with external application of mild hyperthermia to the tumor to induce ELP coacervation and an increase in concentration within the tumor vasculature. The third approach utilizes hydrophilic-hydrophobic ELP block copolymers that have been designed to assemble into nanoparticles in response to hyperthermai due to the independent thermal transition of the hydrophobic block, thus resulting in multivalent ligand display of a ligand for spatially enhanced vascular targeting. In the final strategy, ELPs with Tt < Tb are conjugated with radiotherapeutics, injtect intioa tumor where they undergo coacervation to form an injectable drug depot for intratumoral delivery. These injectable coacervate ELP-radionuclide depots display a long residence in the tumor and result in inhibition of tumor growth. PMID:20546809

  18. Rational design and investigation of polypeptide multilayer films and capsules

    NASA Astrophysics Data System (ADS)

    Zhang, Ling

    Three major concerns in the science of materials today are control over structure and function at the molecular level, biodegradability, and scalability of production. Polymeric materials, notably polyelectrolyte multilayer films, have shown considerable promise in all these areas, and for rational development of multifunctionality. Polypeptides constitute an especially interesting class of polyelectrolyte, given their inherent biodegradability, means of control over structure, methods of large-scale synthesis, and ability to encode biological information. Relatively little is known, however, about polypeptide multilayer films, despite recent advances in the general area. In this dissertation, ten heteropolypeptides were designed and synthesized by Fmoc chemistry. These peptides and homopolypeptides available from a commercial source have been used to carry out a systematic study of the physical basis of polypeptide multilayer film assembly, structure, and stability, in order to gain a greater grasp of the roles of different kinds of non-covalent interaction, degree of polymerization, and polydispersity. The data show that amino acid composition, sequence, and specific combination of anionic and cationic polypeptides together determine film growth behavior, secondary structure content, overall density, surface morphology, and susceptibility to environmental perturbations. The peptides are largely unstructured in solution but tend to form beta sheets in a multilayer film at neutral pH. Electrostatic interactions dominate polypeptide adsorption and film stability, but hydrophobic interactions and hydrogen bonding have a significant influence on internal structure and surface morphology, decreasing film density and increasing film thickness and roughness. Experimental results of polypeptide multilayer films correlate well with molecular dynamics (MD) simulation results of interpolyelectrolyte complexes (IPECs) of the same polypeptide designs. Microcrystals of pyrene

  19. Sugar-nucleotide-binding and autoglycosylating polypeptide(s) from nasturtium fruit: biochemical capacities and potential functions.

    PubMed

    Faik, A; Desveaux, D; MacLachlan, G

    2000-05-01

    Polypeptide assemblies cross-linked by S-S bonds (molecular mass>200 kDa) and single polypeptides folded with internal S-S cross-links (<41 kDa) have been detected by SDS/PAGE in particulate membranes and soluble extracts of developing cotyledons of nasturtium (Tropaeolum majus L.). When first prepared from fruit homogenates, these polypeptides were found to bind reversibly to UDP-Gal (labelled with [(14)C]Gal or [(3)H]uridine), and to co-precipitate specifically with added xyloglucan from solutions made with 67% ethanol. Initially, the bound UDP-[(14)C]Gal could be replaced (bumped) by adding excess UDP, or exchanged (chased) with UDP-Gal, -Glc, -Man or -Xyl. However, this capacity for turnover was lost during incubation in reaction media, or during SDS/PAGE under reducing conditions, even as the glycone moiety was conserved by autoglycosylation to form a stable 41 kDa polypeptide. Polyclonal antibodies raised to a similar product purified from Arabidopsis bound to all the labelled nasturtium polypeptides in immunoblotting tests. The antibodies also inhibited the binding of nasturtium polypeptides to UDP-Gal, the uptake of UDP-[(14)C]Gal into intact nasturtium membrane vesicles and the incorporation of [(14)C]Gal into nascent xyloglucan within these vesicles. This is the first direct evidence that these polypeptides facilitate the channelling of UDP-activated sugars from the cytoplasm through Golgi vesicle membranes to lumenal sites, where they can be used as substrates for glycosyltransferases to synthesize products such as xyloglucan.

  20. Biochemistry of the erythrocyte Rh polypeptides: a review.

    PubMed Central

    Agre, P.; Smith, B. L.; Hartel-Schenk, S.

    1990-01-01

    The clinically important Rh blood group system is complex, consisting of multiple distinct antigens. Despite clinical recognition for over 50 years, the Rh blood group antigens have remained poorly understood on a molecular level until the recent identification and characterization of the "Rh polypeptides," the core structural proteins of the Rh antigens. This group of erythrocyte membrane proteins of molecular weight 30,000-35,000 daltons was first recognized by employing Rh-specific antibodies to immunoprecipitate radiolabeled components of erythrocyte membranes. By using antibodies specific for the Rh D, c, and E antigens, a series of highly related non-identical proteins were immunoprecipitated, indicating that the Rh antigens are composed of multiple related proteins. The Rh polypeptides have been purified and characterized, and they were found to have several unusual biochemical characteristics. The Rh polypeptides penetrate the membrane bilayer; they are linked to the underlying membrane skeleton; they are covalently fatty acid acylated with palmitate. While the Rh antigenic reactivity is unique to human erythrocytes, the Rh polypeptides have been isolated from erythrocytes of diverse species and are thought to be fundamental components of all mammalian erythrocyte membranes. The functional role of the Rh polypeptides remains undefined, but a role in the organization of membrane phospholipid is suspected. PMID:2127333

  1. Preparation process of active enzymolysis polypeptides from seahorse bone meal.

    PubMed

    Jiang, Zhanzhi; Xu, Yongjian; Su, Yuting

    2014-09-01

    The preparation process of protein enzymolysis technology for the three-spot seahorse (Hippocampus trimaculatus Leach) degreased bone meal was developed. Two enzymes with better enzymolysis effect were selected from the five proteases, and the optimization condition of the Alkaline Protease is temperature - 54.7°C, pH - 9, duration of 6 h, the acquired rate of polypeptides was 11.77%; and that of Trypsin is temperature - 45°C, pH - 8.8, and duration of 4 h, and the rate was 11.49% by Response Surface Methodology. The strategy of compound enzymes was "Trypsin + Alkaline Protease". The selected preparation process of active polypeptides by the compound enzymolysis technology acquired rate of polypeptides was 14.41 ± 0.16%, increased about 3% of acquired rate of polypeptides and 2.6-4.5% of the free radical scavenging rate than those of the single enzyme. The increased antioxidant capacity mainly came from the increased concentration of polypeptide in II(#) peak, which increased about 10% of the free radical scavenging rate. The strategy of selected compound enzymes can effectively improve the utilization rate of seahorse protein.

  2. Abnormal ghrelin and pancreatic polypeptide responses in gastroparesis.

    PubMed

    Gaddipati, Kishore V; Simonian, Hrair P; Kresge, Karen M; Boden, Guenther H; Parkman, Henry P

    2006-08-01

    Vagal nerve dysfunction has been implicated in the pathogenesis of diabetic gastroparesis, but its role in idiopathic gastroparesis remains uncertain. The increase in pancreatic polypeptide with sham feeding is often used as a measure of vagal integrity. Ghrelin has been suggested to function as an appetite-stimulating hormone from the gut to the brain acting through vagal afferent pathways. Systemic ghrelin also rises in part due to vagal efferent pathways. Alterations in ghrelin and its effects on appetite could play a role in gastroparesis. In this study we aimed [1] to investigate the presence of vagal nerve dysfunction in patients with idiopathic and diabetic gastroparesis and [2] to determine if alterations in ghrelin concentrations occur in gastroparesis. Normal subjects and patients with diabetic, idiopathic, or postsurgical gastroparesis underwent a sham feeding protocol. Serial blood samples were obtained for plasma ghrelin and pancreatic polypeptide. Sham feeding was characterized by an increase in pancreatic polypeptide and ghrelin in normal controls and patients with idiopathic gastroparesis. The changes in pancreatic polypeptide and ghrelin levels in diabetic and postsurgical gastroparesis were significantly less than those in normal subjects. Vagal nerve dysfunction, as evidenced by an impaired pancreatic polypeptide response with sham feeding, is present in diabetic gastroparesis but not idiopathic gastroparesis. Systemic ghrelin concentrations increased with sham feeding in normal subjects and patients with idiopathic gastroparesis but not in diabetic or postsurgical gastroparesis. Vagal function and regulation of ghrelin levels are impaired in diabetic gastroparesis. PMID:16868831

  3. Structural Polypeptides of the Granulosis Virus of Plodia interpunctella.

    PubMed

    Tweeten, K A; Bulla, L A; Consigli, R A

    1980-02-01

    Techniques were developed for the isolation and purification of three structural components of Plodia interpunctella granulosis virus: granulin, enveloped nucleocapsids, and nucleocapsids. The polypeptide composition and distribution of protein in each viral component were determined by sodium dodecyl sulfate discontinuous and gradient polyacrylamide slab gel electrophoresis. Enveloped nucleocapsids consisted of 15 structural proteins ranging in molecular weight from 12,600 to 97,300. Five of these proteins, having approximate molecular weights of 17,800, 39,700, 42,400, 48,200, and 97,300, were identified as envelope proteins by surface radioiodination of the enveloped nucleocapsids. Present in purified nucleocapsids were eight polypeptides. The predominant proteins in this structural component had molecular weights of 12,500 and 31,000. Whereas no evidence of polypeptide glycosylation was obtained, six of the viral proteins were observed to be phosphorylated.

  4. Human jagged polypeptide, encoding nucleic acids and methods of use

    DOEpatents

    Li, Linheng; Hood, Leroy

    2000-01-01

    The present invention provides an isolated polypeptide exhibiting substantially the same amino acid sequence as JAGGED, or an active fragment thereof, provided that the polypeptide does not have the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6. The invention further provides an isolated nucleic acid molecule containing a nucleotide sequence encoding substantially the same amino acid sequence as JAGGED, or an active fragment thereof, provided that the nucleotide sequence does not encode the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6. Also provided herein is a method of inhibiting differentiation of hematopoietic progenitor cells by contacting the progenitor cells with an isolated JAGGED polypeptide, or active fragment thereof. The invention additionally provides a method of diagnosing Alagille Syndrome in an individual. The method consists of detecting an Alagille Syndrome disease-associated mutation linked to a JAGGED locus.

  5. Methods of increasing secretion of polypeptides having biological activity

    DOEpatents

    Merino, Sandra

    2014-05-27

    The present invention relates to methods for producing a secreted polypeptide having biological activity, comprising: (a) transforming a fungal host cell with a fusion protein construct encoding a fusion protein, which comprises: (i) a first polynucleotide encoding a signal peptide; (ii) a second polynucleotide encoding at least a catalytic domain of an endoglucanase or a portion thereof; and (iii) a third polynucleotide encoding at least a catalytic domain of a polypeptide having biological activity; wherein the signal peptide and at least the catalytic domain of the endoglucanase increases secretion of the polypeptide having biological activity compared to the absence of at least the catalytic domain of the endoglucanase; (b) cultivating the transformed fungal host cell under conditions suitable for production of the fusion protein; and (c) recovering the fusion protein, a component thereof, or a combination thereof, having biological activity, from the cultivation medium.

  6. Methods of increasing secretion of polypeptides having biological activity

    DOEpatents

    Merino, Sandra

    2013-10-01

    The present invention relates to methods for producing a secreted polypeptide having biological activity, comprising: (a) transforming a fungal host cell with a fusion protein construct encoding a fusion protein, which comprises: (i) a first polynucleotide encoding a signal peptide; (ii) a second polynucleotide encoding at least a catalytic domain of an endoglucanase or a portion thereof; and (iii) a third polynucleotide encoding at least a catalytic domain of a polypeptide having biological activity; wherein the signal peptide and at least the catalytic domain of the endoglucanase increases secretion of the polypeptide having biological activity compared to the absence of at least the catalytic domain of the endoglucanase; (b) cultivating the transformed fungal host cell under conditions suitable for production of the fusion protein; and (c) recovering the fusion protein, a component thereof, or a combination thereof, having biological activity, from the cultivation medium.

  7. Optimal screening of surface-displayed polypeptide libraries.

    PubMed

    Boder, E T; Wittrup, K D

    1998-01-01

    Cell surface display of polypeptide libraries combined with flow cytometric cell sorting presents remarkable potential for enhancement of protein-ligand recognition properties. To maximize the utility of this approach, screening and purification conditions must be optimized to take full advantage of the quantitative feature of this technique. In particular, discrimination of improved library mutants from an excess of wild-type polypeptides is dependent upon an effective screening methodology. Fluorescence discrimination profiles for improved library mutants were derived from a mathematical model of expected cell fluorescence intensities for polypeptide libraries screened with fluorescent ligand. Profiles for surface-displayed libraries under equilibrium or kinetic screening conditions demonstrate distinct discrimination optima from which optimal equilibrium and kinetic screening parameters were derived. In addition, a statistical model of low cytometrically analyzed cell populations indicates the importance of low-stringency sorting followed by amplification through regrowth and resorting at increased stringency. This analysis further yields quantitative recommendations for cell-sorting stringency.

  8. Comparison of polypeptides from cultured human fibroblasts and sarcoma cells.

    PubMed

    Vartio, T; Kaelin, H; Vaheri, A

    1978-10-23

    The proteins in cell layers of cultured normal diploid human skin (ES, ER) and lung (WI-38) fibroblasts were compared to those of SV40-transformed human fibroblasts (WI-38/VA-13), human rhabdomyosarcoma (RD) and fibrosarcoma (HT-1080) cells using metabolic amino acid and sugar labeling and surface labeling with tritiated sodium borohydride after oxidation with galactose oxidase. The labeled proteins were analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography (fluorography). A transformation-associated decrease in the pericellular glycoprotein fibronectin (subunit molecular weight, 220 000) and in the synthesis of a set of polypeptides in the 130 000--180 000 dalton region was seen. Synthesis of a glycosylated 160 000 dalton polypeptide was markedly reduced. In transformed cells distinct increases of several specific polypeptides was detected in both [35S]methionine and [3H] mannose incorporation experiments but not using the surface labeling method.

  9. Methods of increasing secretion of polypeptides having biological activity

    SciTech Connect

    Merino, Sandra

    2014-10-28

    The present invention relates to methods for producing a secreted polypeptide having biological activity, comprising: (a) transforming a fungal host cell with a fusion protein construct encoding a fusion protein, which comprises: (i) a first polynucleotide encoding a signal peptide; (ii) a second polynucleotide encoding at least a catalytic domain of an endoglucanase or a portion thereof; and (iii) a third polynucleotide encoding at least a catalytic domain of a polypeptide having biological activity; wherein the signal peptide and at least the catalytic domain of the endoglucanase increases secretion of the polypeptide having biological activity compared to the absence of at least the catalytic domain of the endoglucanase; (b) cultivating the transformed fungal host cell under conditions suitable for production of the fusion protein; and (c) recovering the fusion protein, a component thereof, or a combination thereof, having biological activity, from the cultivation medium.

  10. Methods of increasing secretion of polypeptides having biological activity

    SciTech Connect

    Merino, Sandra

    2015-04-14

    The present invention relates to methods for producing a secreted polypeptide having biological activity, comprising: (a) transforming a fungal host cell with a fusion protein construct encoding a fusion protein, which comprises: (i) a first polynucleotide encoding a signal peptide; (ii) a second polynucleotide encoding at least a catalytic domain of an endoglucanase or a portion thereof; and (iii) a third polynucleotide encoding at least a catalytic domain of a polypeptide having biological activity; wherein the signal peptide and at least the catalytic domain of the endoglucanase increases secretion of the polypeptide having biological activity compared to the absence of at least the catalytic domain of the endoglucanase; (b) cultivating the transformed fungal host cell under conditions suitable for production of the fusion protein; and (c) recovering the fusion protein, a component thereof, or a combination thereof, having biological activity, from the cultivation medium.

  11. Biological activity of a polypeptide modulator of TRPV1 receptor.

    PubMed

    Dyachenko, I A; Andreev, Ya A; Logashina, Yu A; Murashev, A N; Grishin, E V

    2015-11-01

    This paper presents data on the activity of a new APHC2 polypeptide modulator of TRPV1 receptors, which was isolated from the sea anemone Heteractis crispa. It has been shown that APHC2 has an analgesic activity, does not impair normal motor activity, and does not change body temperature of experimental animals, which has a great practical value for design of potent analgesics of a new generation. Further study of the characteristics of binding of the polypeptide to the TRPV1 receptor may show approaches to the development of other antagonists of this receptor that do not influence the body temperature. PMID:26725234

  12. Atomic Layer Deposition of L-Alanine Polypeptide

    SciTech Connect

    Fu, Yaqin; Li, Binsong; Jiang, Ying-Bing; Dunphy, Darren R.; Tsai, Andy; Tam, Siu-Yue; Fan, Hongyou Y.; Zhang, Hongxia; Rogers, David; Rempe, Susan; Atanassov, Plamen; Cecchi, Joseph L.; Brinker, C. Jeffrey

    2014-10-30

    L-Alanine polypeptide thin films were synthesized via atomic layer deposition (ALD). Rather, instead of using an amino acid monomer as the precursor, an L-alanine amino acid derivatized with a protecting group was used to prevent self-polymerization, increase the vapor pressure, and allow linear cycle-by-cycle growth emblematic of ALD. Moreover, the successful deposition of a conformal polypeptide film has been confirmed by FTIR, TEM, and Mass Spectrometry, and the ALD process has been extended to polyvaline.

  13. Analysis of polypeptide movement in the SecY channel during SecA-mediated protein translocation.

    PubMed

    Erlandson, Karl J; Or, Eran; Osborne, Andrew R; Rapoport, Tom A

    2008-06-01

    In bacteria most secretory proteins are transported across the plasma membrane by the interplay of the ATPase SecA with the translocation channel formed by the SecY complex; SecA uses cycles of ATP hydrolysis to "push" consecutive segments of a polypeptide substrate through the channel. Here we have addressed the mechanism of this process by following the fate of stalled translocation intermediates. These were generated by using a polypeptide substrate containing a bulky disulfide-bonded loop, thus preventing the final residues from passing through the channel. Protease protection experiments showed that the intermediates were stable in the presence of ATP and could complete translocation once the block was removed. The translocation intermediate was also stable when SecA associated with ATPgammaS, a poorly hydrolyzable ATP analog, or ADP plus AlF(4), which mimics the transition state during ATP hydrolysis. In contrast, when SecA was in its ADP-bound state, the translocating polypeptide moved back into the cytosol, as indicated by the disappearance of the protected fragment. Backsliding was not significantly altered by deletion of the plug domain, a short helix in the center of the SecY channel, but it was slowed down when changes were introduced into the pore ring, the constriction of the hourglass-shaped channel. In all cases, backsliding was significantly slower than forward translocation. Together, these data suggest that SecA binds the polypeptide chain in its ATP state and releases it in the ADP state. The channel itself does not bind the polypeptide chain but provides "friction" that minimizes backsliding when ADP-bound SecA resets to "grab" the next segment of the substrate. PMID:18359943

  14. Methods of using viral replicase polynucleotides and polypeptides

    DOEpatents

    Gordon-Kamm, William J.; Lowe, Keith S.; Bailey, Matthew A.; Gregory, Carolyn A.; Hoerster, George J.; Larkins, Brian A.; Dilkes, Brian R.; Burnett, Ronald; Woo, Young Min

    2007-12-18

    The invention provides novel methods of using viral replicase polypeptides and polynucleotides. Included are methods for increasing transformation frequencies, increasing crop yield, providing a positive growth advantage, modulating cell division, transiently modulating cell division, and for providing a means of positive selection.

  15. Experimental production of antibodies against stratum corneum keratin polypeptides.

    PubMed

    Viac, J; Staquet, M J; Thivolet, J; Goujon, C

    1980-01-01

    Anti-keratin polypeptide sera (K.P.S) were obtained by immunizing guinea pigs with fibrous proteins from stratum corneum, which were acquired from normal human epidermis by m eans of S.D.S. polyacrylamide gel electrophoresis. After absorption with red blood cells and liver powder the sera were tested by indirect immunofluorescence technique on different substrates. Antibodies against polypeptides P1 and P2 of M.W. 67,000 and 62,000 dalton, respectively, were directed toward cytoplasmic Ag of keratinocytes of spinous and graunular layer of normal human and rabbit epidermis. No labeling could be detected in the basal cell layer. This finding is in favor of various differentiation stages of the keratinizing cells. P3 of M.W. 53,000 dalton induced low titre anibodies which labelled the whole epidermis, including the basal cell layer. The fourth polypeptide of M.W. 49,000 dalton seemed not to be immunogenic in such experiences. In tumors, such as basal cell carcinom,a squamous cell carcinoma, and warts, the expression of keratin antigens is markedly diminished. No analogy could be drawn between experimental keratin polypeptide antibodies and the human epidermal cytoplasmic antibodies which were detected in some patient sera.

  16. Chronic, programmed polypeptide delivery from an implanted, multireservoir microchip device.

    PubMed

    Prescott, James H; Lipka, Sara; Baldwin, Samuel; Sheppard, Norman F; Maloney, John M; Coppeta, Jonathan; Yomtov, Barry; Staples, Mark A; Santini, John T

    2006-04-01

    Implanted drug delivery systems are being increasingly used to realize the therapeutic potential of peptides and proteins. Here we describe the controlled pulsatile release of the polypeptide leuprolide from microchip implants over 6 months in dogs. Each microchip contains an array of discrete reservoirs from which dose delivery can be controlled by telemetry.

  17. Catalytic and reactive polypeptides and methods for their preparation and use

    DOEpatents

    Schultz, Peter

    1993-01-01

    Catalytic and reactive polypeptides include a binding site specific for a reactant or reactive intermediate involved in a chemical reaction of interest. The polypeptides further include at least one active functionality proximate the bi.

  18. Principles Governing the Self Assembly of Polypeptide Nanoparticles

    NASA Astrophysics Data System (ADS)

    Wahome, Newton

    Self assembling systems on the nanometer scale afford the advantage of being able to control submicron level events. In this study, we focus on the self-assembling polypeptide nanoparticles (SAPN). The SAPN scaffold is made up of oligomerizing domains that align along the principle rotational axes of icosahedral symmetry. By aligning them along these axes, a particle with spherical geometry can be achieved. This particle can be utilized as a vaccine, as a drug delivery vehicle, or as a biomedical imaging device. This research will try to answer why the SAPN self-assembles into distinct molecular weight ranges while mostly maintaining a spherical morphology. The first means will be theoretical and computational, where we will utilize a mathematical formalism to find out how the packing of SAPN's monomeric units can occur within symmetric space. Then molecular dynamics will be run within this symmetric space to test the per amino acid residue susceptibility of SAPN towards becoming polymorphic in nature. Means for examining the aggregation propensity of SAPN will be also be tested. Specifically, the relationship of different sequences of SAPN with pH will be elucidated. Co-assembly of SAPN to reduce the surface density of an aggregation prone epitope will be tested. Also, aggregation reduction consisting of the exchange of an anionic denaturant with a positively charged suppressor in order to mitigate a priori peptide association and misfolding, will also be attempted. SAPN has been shown to be an immunogenic platform for the presentation of pathogen derived antigens. We will attempt to show the efficacy of presenting an antigen from HIV-1 which is structurally restrained to best match the native conformation on the virus. Immunological studies will be performed to test the effect of this approach, as well testing the antigenicity of the nanoparticle in the absence of adjuvant. Finally, the antigen presenting nanoparticles will undergo formulation testing, to measure

  19. Solvent-free liquid crystals and liquids based on genetically engineered supercharged polypeptides with high elasticity.

    PubMed

    Liu, Kai; Pesce, Diego; Ma, Chao; Tuchband, Michael; Shuai, Min; Chen, Dong; Su, Juanjuan; Liu, Qing; Gerasimov, Jennifer Y; Kolbe, Anke; Zajaczkowski, Wojciech; Pisula, Wojciech; Müllen, Klaus; Clark, Noel A; Herrmann, Andreas

    2015-04-17

    A series of solvent-free elastin-like polypeptide liquid crystals and liquids are developed by electrostatic complexation of supercharged elastin-like polypeptides with surfactants. The smectic mesophases exhibit a high elasticity and the values can be easily tuned by varying the alkyl chain lengths of the surfactants or the lengths of the elastin-like polypeptides.

  20. Polypeptides having beta-glucosidase and beta-xylosidase activity and polynucleotides encoding same

    SciTech Connect

    Morant, Marc Dominique

    2014-05-06

    The present invention relates to isolated polypeptides having beta-glucosidase activity, beta-xylosidase activity, or beta-glucosidase and beta-xylosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  1. Polypeptides having beta-glucosidase activity and beta-xylosidase activity and polynucleotides encoding same

    SciTech Connect

    Morant, Marc Dominique

    2014-04-29

    The present invention relates to isolated polypeptides having beta-glucosidase activity, beta-xylosidase activity, or beta-glucosidase and beta-xylosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  2. Polypeptides having beta-glucosidase activity and beta-xylosidase activity and polynucleotides encoding same

    SciTech Connect

    Morant, Marc Dominique

    2014-05-06

    The present invention relates to isolated polypeptides having beta-glucosidase activity, beta-xylosidase activity, or beta-glucosidase and beta-xylosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  3. Biosynthesis of the neural cell adhesion molecule: characterization of polypeptide C

    PubMed Central

    1985-01-01

    The biosynthesis of the neural cell adhesion molecule (N-CAM) was studied in primary cultures of rat cerebral glial cells, cerebellar granule neurons, and skeletal muscle cells. The three cell types produced different N-CAM polypeptide patterns. Glial cells synthesized a 135,000 Mr polypeptide B and a 115,000 Mr polypeptide C, whereas neurons expressed a 200,000 Mr polypeptide A as well as polypeptide B. Skeletal muscle cells produced polypeptide B. The polypeptides synthesized by the three cell types were immunochemically identical. The membrane association of polypeptide C was investigated with methods that distinguish peripheral and integral membrane proteins. Polypeptide C was found to be a peripheral membrane protein, whereas polypeptides A and B were integral membrane proteins with cytoplasmic domains of approximately 50,000 and approximately 25,000 Mr, respectively. The affinity of the membrane binding of polypeptide C increased during postnatal development. The posttranslational modifications of polypeptide C were investigated in glial cell cultures, and it was found to be N-linked glycosylated and sulfated. PMID:4066759

  4. Polypeptides having beta-glucosidase activity and polynucleotides encoding the same

    DOEpatents

    Brown, Kimberly; Harris, Paul

    2013-12-17

    The present invention relates to isolated polypeptides having beta-glucosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  5. Polypeptide composition of purified QH2:cytochrome c oxidoreductase from beef-heart mitochondria.

    PubMed

    Marres, C M; Slater, E C

    1977-12-23

    1. The polypeptide composition of purified QH2: cytochrome c oxidoreductase prepared by three different methods from beef-heart mitochondria has been determined. Polyacrylamide gel electrophoresis in the presence of dodecyl sulphate resolves eight intrinsic polypeptide bands; when, in addition, 8 M urea is present and a more highly cross-linked gel is used, the smallest polypeptide band is resolved into three different bands. 2. The identity of several polypeptide bands has been established by fractionation. The two heaviest polypeptides (bands 1 and 2) represent the so-called core proteins, band 3 the hemoprotein of cytochrome b, band 4 the hemoprotein of cytochrome c1, band 5 and Rieske Fe-S protein, band 6 a polypeptide associated with cytochrome c1 and identified with the so-called oxidation factor, and band 7 a polypeptide peptide associated with cytochrome b. 3. The validity of molecular weight estimate for the polypeptides of the enzyme based on their mobility on dodecyl sulphate gels has been examined. The polypeptides of bands 1, 2 and 3 showed anomalous migration rates. The molecular weights of the other polypeptides have been estimated from their relative mobilities on either dodecyl sulphate gels or 8 M urea-dodecyl sulphate gels as 29 000, 24 000, 12 000, 8000, 6000, 5000 and 4000, respectively. 4. The stoicheiometry of the different polypeptides in the intact complex was determined using separate staining factors for the individual polypeptide band.

  6. Comparison of intestinal brush-border 95-Kdalton polypeptide and alpha- actinins

    PubMed Central

    1980-01-01

    To explore the suggestion that alpha-actinin cross-links actin filaments to the microvillar membrane (Mooseker and Tilney, 1975, J. Cell Biol. 67:725--743; Mooseker, 1976, J. Cell Biol. 71-417--433), we have assessed the possible relatedness of alpha-actinin and the brush- border 95-kdalton protein by four independent criteria: antigenicity, mobility on SDS gels, extractability in nonionic detergents, and peptide maps. We have found that anti-chicken gizzard alpha-actinin stains the junctional complex region of intact cells (Craig and Pardo, 1979, J. Cell Biol. 80:203--210) but does not stain isolated brush borders even though these structures contain a 95-kdalton polypeptide. Lack of staining is not caused by failure of the antibody to penetrate, as antiactin stains both the terminal web and the microvilli of isolated brush borders. By the antibody SDS gel overlay technique, we have established that anti-gizzard alpha-actinin recognizes homologous molecules in chicken skeletal and cardiac muscles, as well as in intestinal epithelial cells, but fails to recognize the brush-border 95- kdalton polypeptide. Conversely, anti-95-kdalton polypeptide does not recognize gizzard alpha-actinin. On high-resolution SDS polyacrylamide gel electrophoresis, alpha-actinin and brush-border 95-kdalton protein exhibit distinct mobilities. The two proteins also differ in their ability to be extracted in nonionic mobilities. The two proteins also differ in their ability to be extracted in nonionic detergent: epithelial cell immunoreactive alpha-actinin is soluble in NP-40, whereas 95-kdalton protein is insoluble. Finally, two-dimensional peptide mapping of iodinated tryptic peptides, as well as one- dimensional fingerprinting of partial tryptic, chymotryptic, papain, and S. aureus V8 protease digests, have revealed less than 5% homology between gizzard alpha-actinin and brush-border 95-kdalton polypeptide. The data suggest that there is no major structural homology between gizzard

  7. Three-Dimensional Polypeptide Architectures Through Tandem Catalysis and Click Chemistry

    NASA Astrophysics Data System (ADS)

    Rhodes, Allison Jane

    -defined, high-density brushes for applications in drug delivery and imaging. Here, we also report a method for the synthesis of soluble, well-defined, azido functionalized polypeptides in a straightforward, 3-step synthesis. Homo and diblock azidopolypeptides were prepared with controlled segment lengths via living polymerization using Co(PMe3)4 initiator. Through copper azide alkyne click chemistry (CuAAC) in organic solvent, azidopolypeptides were regioselectively and quantitatively modified with carboxylic acid (pH-responsive), amino acid and sugar functional groups. Finally, the advances towards well-defined hyperbranched polypeptides through alpha-amino-acid-N-thiocarboxyanhydrides (NTAs) will be discussed. Within the past 10 years, controlled NCA (alpha-amino acid-N-carboxyanhydride) ring-opening polymerization (ROP) has emerged, expanding the application of copolypeptide polymers in various drug delivery and tissue engineering motifs. Modification of NCA monomers to the corresponding alpha-amino-acid-N-thiocarboxyanhydride (NTA) will diversify ROP reactions, leading to more complex polypeptides (such as hyperbranched polymers), in addition to the possibility of performing these polymerizations under ambient conditions, which would greatly expand their potential utility. The project focuses on the preparation of hyperbranched polypeptides with well-defined architectures and controlled branching density in a one-pot reaction. This will be accomplished by taking advantage of the different selectivities of Co(PMe3)4 and depeNi(COD) polymerization initiators, and by exploiting the reactivity difference between NCA and the more stable NTA monomers.

  8. An engineered coiled-coil polypeptide assembled onto quantum dots for targeted cell imaging

    NASA Astrophysics Data System (ADS)

    Yao, Ming-Hao; Yang, Jie; Song, Ji-Tao; Zhang, Lin; Fang, Bi-Yun; Zhao, Dong-Hui; Xia, Rui-Xue; Jin, Rui-Mei; Zhao, Yuan-Di; Liu, Bo

    2015-12-01

    Quantum dot (QD)-polypeptide probes have been developed through the specific metal-affinity interaction between polypeptides appended with N-terminal polyhistidine sequences and CdSe/ZnS core-shell QDs. The size and charge of a QD-polypeptide can be tuned by using different coiled-coil polypeptides. Compared to glutathione-capped QDs (QD-GSH), QD-polypeptide probes showed an approximately two- to three-fold luminescence increase, and the luminescence increase was not obviously related to the charge of the polypeptide. QD-polypeptide probes with different charge have a great effect on nonspecific cellular uptake. QD-polypeptide probes with negative charge exhibited lower nonspecific cellular uptake in comparison to the QD-GSH, while positively charged QD-polypeptide probes presented higher cellular uptake than the QD-GSH. A targeted QD-ARGD probe can obviously increase targeted cellular uptake in α v β 3 overexpressing HeLa cells compared to QD-A. In addition, QD-polypeptide probes showed lower in vitro cytotoxicity compared to the original QDs. These results demonstrate that these QD-polypeptide probes with high specific cellular uptake, high fluorescence intensity and low background noise are expected to have great potential applications in targeted cell imaging.

  9. Silicification of genipin-cross-linked polypeptide hydrogels toward biohybrid materials and mesoporous oxides.

    PubMed

    Jan, Jeng-Shiung; Chen, Pei-Shan; Hsieh, Ping-Lun; Chen, Bo-Yu

    2012-12-01

    A simple and versatile approach is proposed to use cross-linked polypeptide hydrogels as templates for silica mineralization, allowing the synthesis of polypeptide-silica hybrid hydrogels and mesoporous silica (meso-SiO(2)) by subsequent calcination. The experimental data revealed that the cross-linked polypeptide hydrogels comprised of interconnected, membranous network served as templates for the high-fidelity transcription of silica replicas spanning from nanoscale to microscale, resulting in hybrid network comprised of interpenetrated polypeptide nanodomains and silica. The mechanical properties of these as-prepared polypeptide-silica hybrid hydrogels were found to vary with polypeptide chain length and composition. The synergy between cross-link, hydrophobic interaction, and silica deposition can lead to the enhancement of their mechanical properties. The polypeptide-silica hybrid hydrogel with polypeptide and silica content as low as 1.1 wt% can achieve 114 kN/m(2) of compressive strength. By removing the polypeptide nanodomains, mesoporous silicas with average pore sizes ranged between 2 nm and 6 nm can be obtained, depending on polypeptide chain length and composition. The polypeptide-silica hybrid hydrogels demonstrated good cell compatibility and can support cell attachment/proliferation. With the versatility of polymer chemistry and feasibility of amine-catalyzed sol-gel chemistry, the present method is facile for the synthesis of green nanocomposites and biomaterials.

  10. The geometry of the ribosomal polypeptide exit tunnel.

    PubMed

    Voss, N R; Gerstein, M; Steitz, T A; Moore, P B

    2006-07-21

    The geometry of the polypeptide exit tunnel has been determined using the crystal structure of the large ribosomal subunit from Haloarcula marismortui. The tunnel is a component of a much larger, interconnected system of channels accessible to solvent that permeates the subunit and is connected to the exterior at many points. Since water and other small molecules can diffuse into and out of the tunnel along many different trajectories, the large subunit cannot be part of the seal that keeps ions from passing through the ribosome-translocon complex. The structure referred to as the tunnel is the only passage in the solvent channel system that is both large enough to accommodate nascent peptides, and that traverses the particle. For objects of that size, it is effectively an unbranched tube connecting the peptidyl transferase center of the large subunit and the site where nascent peptides emerge. At no point is the tunnel big enough to accommodate folded polypeptides larger than alpha-helices.

  11. Imparting large macroscopic changes with small changes in polypeptide composition

    NASA Astrophysics Data System (ADS)

    Sing, Michelle; McKinley, Gareth; Olsen, Bradley

    Block copolymers composed of polypeptides provide an excellent platform for exploring the underlying physics surrounding macroscopic associative network behavior. Previous work in our group has elucidated a difference in the mechanical properties of two nearly identical elastin-like polypeptide (ELP) endblocks. In poly(ELP)s, this substitution is known to result in tighter beta turns. These beta turns exhibit slower responses to changes in temperature within the material. Under shear, the modulus for the alanine-containing ELP triblock is almost three times higher than the glycine-containing ELP. Additionally, preliminary tensile tests show higher stress and strain at break for the alanine ELP triblock. We are able to explain the reasons for this behavior using a variety of spectroscopic and analytical techniques. Small angle neutron and x-ray scattering indicate differences in ordering between the alanine and glycine containing ELP materials both in shear and in stagnant flow.

  12. Complexes of Negatively Charged Polypeptides with Cationic Lipids

    NASA Astrophysics Data System (ADS)

    Subramanian, G.; Li, Youli; Safinya, Cyrus R.

    1997-03-01

    Complexes of cationic lipids with oppositely charged proteins are promising candidates for new biomolecular materials. In addition to being used as a direct vehicle for protein transfection, they also find applications as templates for synthesis of molecular sieves. In spite of these wide ranging applications, the structure and interactions in these complexes have largely remained unclear. Here we report on the study of complexes formed between the cationic lipid didodecyldimethylammonium bromide (DDAB) with negatively charged polypeptide poly glutamic acid (PGA) both in the presence and absence of the neutral lipid dilauroylglycerophosphocholine (DLPC). X-ray diffraction of the complexes indicates a condensed lamellar lipid structure with the polypeptide intercalated between the layers. We present a comprehensive phase diagram on this system based on X-ray diffraction data. This work is supported in part by grants NSF DMR-9624091, PRF-31352 AC7, and CU LAR STP/UC 96-118.

  13. Compositions and methods for making selenocysteine containing polypeptides

    DOEpatents

    Soll, Dieter; Aldag, Caroline; Hohn, Michael

    2016-10-11

    Non-naturally occurring tRNA.sup.Sec and methods of using them for recombinant expression of proteins engineered to include one or more selenocysteine residues are disclosed. The non-naturally occurring tRNA.sup.Sec can be used for recombinant manufacture of selenocysteine containing polypeptides encoded by mRNA without the requirement of an SECIS element. In some embodiments, selenocysteine containing polypeptides are manufactured by co-expressing a non-naturally occurring tRNA.sup.Sec a recombinant expression system, such as E. coli, with SerRS, EF-Tu, SelA, or PSTK and SepSecS, and an mRNA with at least one codon that recognizes the anticodon of the non-naturally occurring tRNA.sup.Sec.

  14. Supramolecular Polymerization from Polypeptide-Grafted Comb Polymers

    SciTech Connect

    Wang, Jing; Lu, Hua; Kamat, Ranjan K; Pingali, Sai Venkatesh; Urban, Volker S; Cheng, Jianjun; Lin, Yao

    2011-01-01

    The helical and tubular structures self-assembled from proteins have inspired scientists to design synthetic building blocks that can be 'polymerized' into supramolecular polymers through coordinated noncovalent interactions. However, cooperative supramolecular polymerization from large, synthetic macromolecules remains a challenge because of the difficulty of controlling the structure and interactions of macromolecular monomers. Herein we report the synthesis of polypeptide-grafted comb polymers and the use of their tunable secondary interactions in solution to achieve controlled supramolecular polymerization. The resulting tubular supramolecular structures, with external diameters of hundreds of nanometers and lengths of tens of micrometers, are stable and resemble to some extent biological superstructures assembled from proteins. This study shows that highly specific intermolecular interactions between macromolecular monomers can enable the cooperative growth of supramolecular polymers. The general applicability of this strategy was demonstrated by carrying out supramolecular polymerization from gold nanoparticles grafted with the same polypeptides on the surface.

  15. Polypeptide synthesis induced in Nicotiana clevelandii protoplasts by infection with raspberry ringspot nepovirus.

    PubMed

    Acosta, O; Mayo, M A

    1993-01-01

    Infection of Nicotiana clevelandii protoplasts by raspberry ringspot nepovirus resulted in the accumulation of about 24 polypeptides that differed in M(r) and pI from polypeptides accumulating in mock-inoculated protoplasts. Similar polypeptides accumulated in protoplasts infected with the S and E strains of RRV but different infection-specific polypeptides were detected in protoplasts infected with tobacco ringspot nepovirus. The M(r) of RRV-specific polypeptides ranged from 210,000 to 18,000 and most are presumed to be derived from others by proteolytic cleavage. No evidence was found for marked changes in polypeptide abundance with time after inoculation or for any virus-specific polypeptide becoming disproportionately abundant in the medium during culture.

  16. Polypeptide synthesis induced in Nicotiana clevelandii protoplasts by infection with raspberry ringspot nepovirus.

    PubMed

    Acosta, O; Mayo, M A

    1993-01-01

    Infection of Nicotiana clevelandii protoplasts by raspberry ringspot nepovirus resulted in the accumulation of about 24 polypeptides that differed in M(r) and pI from polypeptides accumulating in mock-inoculated protoplasts. Similar polypeptides accumulated in protoplasts infected with the S and E strains of RRV but different infection-specific polypeptides were detected in protoplasts infected with tobacco ringspot nepovirus. The M(r) of RRV-specific polypeptides ranged from 210,000 to 18,000 and most are presumed to be derived from others by proteolytic cleavage. No evidence was found for marked changes in polypeptide abundance with time after inoculation or for any virus-specific polypeptide becoming disproportionately abundant in the medium during culture. PMID:8470949

  17. Polypeptide transfer from Hsp40 to Hsp70 molecular chaperones

    PubMed Central

    Summers, Daniel W.; Douglas, Peter M.; Ramos, Carlos H.I.; Cyr, Douglas M.

    2015-01-01

    Heat shock protein 40 (Hsp40) co-chaperones assist in cellular protein folding and degradation through the binding and delivery of non-native proteins to heat shock protein 70 (Hsp70). The mechanism for substrate transfer from Hsp40s to Hsp70 is unknown. Two recent studies provide new details that shed light on novel mechanisms for substrate recognition by Hsp40s and a common mechanism for polypeptide transfer to Hsp70. PMID:19359181

  18. Resilin-Like Polypeptide Hydrogels Engineered for Versatile Biological Functions

    PubMed Central

    Li, Linqing; Tong, Zhixiang; Jia, Xinqiao; Kiick, Kristi L.

    2012-01-01

    Natural resilin, the rubber-like protein that exists in specialized compartments of most arthropods, possesses excellent mechanical properties such as low stiffness, high resilience and effective energy storage. Recombinantly-engineered resilin-like polypeptides (RLPs) that possess the favorable attributes of native resilin would be attractive candidates for the modular design of biomaterials for engineering mechanically active tissues. Based on our previous success in creating a novel RLP-based hydrogel and demonstrating useful mechanical and cell-adhesive properties, we have produced a suite of new RLP-based constructs, each equipped with 12 repeats of the putative resilin consensus sequence and a single, distinct biologically active domain. This approach allows independent control over the concentrations of cell-binding, MMP-sensitive, and polysaccharide-sequestration domains in hydrogels comprising mixtures of the various RLPs. The high purity, molecular weight and correct compositions of each new polypeptide have been confirmed via high performance liquid chromatography (HPLC), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), and amino acid analysis. These RLP-based polypeptides exhibit largely random-coil conformation, both in solution and in the cross-linked hydrogels, as indicated by circular dichroic and infrared spectroscopic analyses. Hydrogels of various compositions, with a range of elastic moduli (1kPa to 25kPa) can be produced from these polypeptides, and the activity of the cell-binding and matrix metalloproteinase (MMP) sensitive domains was confirmed. Tris(hydroxymethyl phosphine) cross-linked RLP hydrogels were able to maintain their mechanical integrity as well as the viability of encapsulated primary human mesenchymal stem cells (MSCs). These results validate the promising properties of these RLP-based elastomeric biomaterials. PMID:23505396

  19. Targeting of Cancer Cells Using Quantum Dot-Polypeptide Hybrid Assemblies that Function as Molecular Imaging Agents and Carrier Systems

    PubMed Central

    Atmaja, Bayu; Lui, Bertrand H.; Hu, Yuhua; Beck, Stayce E.; Frank, Curtis W.; Cochran, Jennifer R.

    2015-01-01

    We report a highly tunable quantum dot (QD)-polypeptide hybrid assembly system with potential uses for both molecular imaging and delivery of biomolecular cargo to cancer cells. In this work, we demonstrate the tunability of the assembly system, its application for imaging cancer cells, and its ability to carry a biomolecule. The assemblies are formed through the self-assembly of carboxyl-functionalized QDs and poly(diethylene glycol-L-lysine)-poly(L-lysine) (PEGLL-PLL) diblock copolypeptide molecules, and they are modified with peptide ligands containing a cyclic arginine-glycine-aspartate [c(RGD)] motif that has affinity for αvβ3 and αvβ5 integrins overexpressed on the tumor vasculature. To illustrate the tunability of the QD-polypeptide assembly system, we show that binding to U87MG glioblastoma cells can be modulated and optimized by changing either the conditions under which the assemblies are formed or the relative lengths of the PEGLL and PLL blocks in the PEGLL-PLL molecules. The optimized c(RGD)-modified assemblies bind integrin receptors on U87MG cells and are endocytosed, as demonstrated by flow cytometry and live-cell imaging. Binding specificity is confirmed by competition with an excess of free c(RGD) peptide. Finally, we show that the QD-polypeptide assemblies can be loaded with fluorescently labeled ovalbumin, as a proof-of-concept for their potential use in biomolecule delivery. PMID:25750609

  20. Birth, life and death of nascent polypeptide chains

    PubMed Central

    Jha, Sujata; Komar, Anton A

    2011-01-01

    The journey of nascent polypeptides from synthesis at the peptidyl transferase center of the ribosome (“birth”) to full function (“maturity”) involves multiple interactions, constraints, modifications and folding events. Each step of this journey impacts the ultimate expression level and functional capacity of the translated protein. It has become clear that the kinetics of protein translation is predominantly modulated by synonymous codon usage along the mRNA, and that this provides an active mechanism for coordinating the synthesis, maturation and folding of nascent polypeptides. Multiple quality control systems ensure that proteins achieve their native, functional form. Unproductive co-translational folding intermediates that arise during protein synthesis may undergo enhanced interaction with components of these systems, such as chaperones, and/or be subjects of co-translational degradation (“death”). This review provides an overview of our current understanding of the complex co-translational events that accompany the synthesis, maturation, folding and degradation of nascent polypeptide chains. PMID:21538896

  1. Folding induced assembly of polypeptide decorated gold nanoparticles.

    PubMed

    Aili, Daniel; Enander, Karin; Rydberg, Johan; Nesterenko, Irina; Björefors, Fredrik; Baltzer, Lars; Liedberg, Bo

    2008-04-30

    Reversible assembly of gold nanoparticles controlled by the homodimerization and folding of an immobilized de novo designed synthetic polypeptide is described. In solution at neutral pH, the polypeptide folds into a helix-loop-helix four-helix bundle in the presence of zinc ions. When immobilized on gold nanoparticles, the addition of zinc ions induces dimerization and folding between peptide monomers located on separate particles, resulting in rapid particle aggregation. The particles can be completely redispersed by removal of the zinc ions from the peptide upon addition of EDTA. Calcium ions, which do not induce folding in solution, have no effect on the stability of the peptide decorated particles. The contribution from folding on particle assembly was further determined utilizing a reference peptide with the same primary sequence but containing both D and L amino acids. Particles functionalized with the reference peptide do not aggregate, as the peptides are unable to fold. The two peptides, linked to the nanoparticle surface via a cysteine residue located in the loop region, form submonolayers on planar gold with comparable properties regarding surface density, orientation, and ability to interact with zinc ions. These results demonstrate that nanoparticle assembly can be induced, controlled, and to some extent tuned, by exploiting specific molecular interactions involved in polypeptide folding. PMID:18380430

  2. Artemia hemoglobins. Increase in net synthesis of the beta-polypeptide (relative to the alpha-polypeptide) in hypoxia.

    PubMed

    Ferry, J A; Nichols, R C; Condon, S J; Stubbs, J D; Bowen, S T

    1983-04-15

    Previous studies have shown that in the brine shrimp there are three dimeric hemoglobins with polypeptide composition alpha 2, alpha beta, beta 2. Concentrations of the alpha- and beta-polypeptides increase in hypoxia. We now report a two-dimensional electrophoretic method for assay of radiolabelled polypeptides in each hemoglobin. Net synthesis (synthesis minus degradation) of the beta-chain, relative to that of the alpha-chain, increases more than 3-fold (in male and female adults) within 3 days following a downshift in oxygen concentration from 0.2 to 0.1 mM in the culture medium. 3 days after downshift (2 days after in vivo incorporation of radiolabelled leucine), the beta-homodimer contained 10-20% of the radiolabel in the three hemoglobins although beta 2 was usually not detectable in the protein stain of an overloaded gel. The amount of radioactive leucine incorporated per unit amount of protein was more than 300-times greater in the beta 2 homodimer than in the beta-subunit of the heterodimer, suggesting that beta 2 does not dissociate rapidly during electrophoresis on the first dimension non-denaturing gel. This evidence for stable association of the two beta-monomers and the 5-8 heme-binding domains within each monomer (in vivo and during electrophoresis on non-denaturing gels) allows us to exclude one of two alternative interpretations of genetic data published previously. We present an independent line of evidence for the dimer model of the native hemoglobins (which states that each polypeptide has many heme-binding domains). PMID:6830806

  3. Activation of the heat-stable polypeptide of the ATP-dependent proteolytic system.

    PubMed Central

    Ciechanover, A; Heller, H; Katz-Etzion, R; Hershko, A

    1981-01-01

    It had been shown previously that the heat-stable polypeptide of the ATP-dependent proteolytic system of reticulocytes, designated APF-1, forms covalent conjugates with protein substrates in an ATP-requiring process. We now describe an enzyme that carries out the activation by ATP of the polypeptide with pyrophosphate displacement. The formation of AMP-polypeptide and transfer of the polypeptide to a secondary acceptor are suggested by an APF-1 requirement for ATP-PPi and ATP-AMP exchange reactions, respectively. With radiolabeled polypeptide, an ATP-dependent labeling of the enzyme was shown to be by a linkage that is acid stable but is labile to treatment with mild alkali, hydroxylamine, borohydride, or mercuric salts. It therefore appears that the AMP-polypeptide undergoes attack by an -SH group of the enzyme to form a thiolester. PMID:6262770

  4. Polypeptide having or assisting in carbohydrate material degrading activity and uses thereof

    DOEpatents

    Schooneveld-Bergmans, Margot Elisabeth Francoise; Heijne, Wilbert Herman Marie; Los, Alrik Pieter

    2016-02-16

    The invention relates to a polypeptide which comprises the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 76% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 76% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional polypeptide and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.

  5. Multifunctional polypeptide-PEO nanoreactors via the hydrophobic switch.

    PubMed

    Wu, Yuzhou; Wang, Tao; Ng, David Y W; Weil, Tanja

    2012-09-14

    We prepare various protein-derived amphiphilic polymers. By modifying the polypeptide backbone with a few (5-8) hydrophilic or lipophilic substituents, we are able to switch the hydrophobicity of the polymer and control the formation of stable nano-sized micelles. In the hydrophobic interior of these micelles, ethynyl groups are introduced to provide a nanoreactor environment for click reactions with lipophilic cargo molecules, such as 3-azidocoumarin, a hydrophobic fluorophore, and the anti-cancer drug doxorubicin. These protein-derived amphiphilic polymers reported herein offer a promising potential to design a delivery platform for biomedical applications.

  6. Factors Governing Fibrillogenesis of Polypeptide Chains Revealed by Lattice Models

    NASA Astrophysics Data System (ADS)

    Li, Mai Suan; Co, Nguyen Truong; Reddy, Govardhan; Hu, Chin-Kun; Straub, J. E.; Thirumalai, D.

    2010-11-01

    Using lattice models we explore the factors that determine the tendencies of polypeptide chains to aggregate by exhaustively sampling the sequence and conformational space. The morphologies of the fibril-like structures and the time scales (τfib) for their formation depend on a balance between hydrophobic and Coulomb interactions. The extent of population of an ensemble of N* structures, which are fibril-prone structures in the spectrum of conformations of an isolated protein, is the major determinant of τfib. This observation is used to determine the aggregating sequences by exhaustively exploring the sequence space, thus providing a basis for genome wide search of fragments that are aggregation prone.

  7. [Anti-Inflammatory Activity of the Polypeptide of the Sea Anemone, Heteractis crispa].

    PubMed

    Sintsova, O V; Monastyrnaya, M M; Pislyagin, E A; Menchinskaya, E S; Leychenko, E V; Aminin, D L; Kozlovskaya, E P

    2015-01-01

    The anti-inflammatory effect of the recombinant polypeptide HCGS 1.20, a Kunitz-type serine protease inhibitor of the sea anemone Heteractis crispa, was investigated. It was shown that the polypeptide inhibits the increase of the concentration of calcium ions in mouse bone marrow derived macrophages elicited by histamine, and reduces the content of NO in lipopolysaccharide stimulated macrophages. A presumable mechanism of anti-inflammatory action of the polypeptide was being discussed. PMID:27125018

  8. Compositions comprising a polypeptide having cellulolytic enhancing activity and a bicyclic compound and uses thereof

    DOEpatents

    Quinlan, Jason; Xu, Feng; Sweeney, Matthew

    2016-10-04

    The present invention relates to compositions comprising: a polypeptide having cellulolytic enhancing activity and a bicyclic compound. The present invention also relates to methods of using the compositions.

  9. Self-assembly of high molecular weight polypeptide copolymers studied via diffusion limited aggregation.

    PubMed

    Meier, Christoph; Wu, Yuzhou; Pramanik, Goutam; Weil, Tanja

    2014-01-13

    The assembly of high molecular weight polypeptides into complex architectures exhibiting structural complexity ranging from the nano- to the mesoscale is of fundamental importance for various protein-related diseases but also hold great promise for various nano- and biotechnological applications. Here, the aggregation of partially unfolded high molecular weight polypeptides into multiscale fractal structures is investigated by means of diffusion limited aggregation and atomic force microscopy. The zeta potential, the hydrodynamic radius, and the obtained fractal morphologies were correlated with the conformation of the polypeptide backbones as obtained from circular dichroism measurements. The polypeptides are modified with polyethylene oxide side chains to stabilize the polypeptides and to normalize intermolecular interactions. The modification with the hydrophobic thioctic acid alters the folding of the polypeptide backbone, resulting in a change in solution aggregation and fractal morphology. We found that a more compact folding results in dense and highly branched structures, whereas a less compact folded polypeptide chain yields a more directional assembly. Our results provide first evidence for the role of compactness of polypeptide folding on aggregation. Furthermore, the mesoscale-structured biofilms were used to achieve a hierarchical protein assembly, which is demonstrated by deposition of Rhodamine-labeled HSA with the preassembled fractal structures. These results contribute important insights to the fundamental understanding of the aggregation of high molecular weight polypeptides in general and provide opportunities to study nanostructure-related effects on biological systems such as adhesion, proliferation, and the development of, for example, neuronal cells. PMID:24354281

  10. Compositions comprising a polypeptide having cellulolytic enhancing activity and a bicycle compound and uses thereof

    SciTech Connect

    Xu, Feng; Sweeney, Matthew; Quinlan, Jason

    2015-06-16

    The present invention relates to compositions comprising: a polypeptide having cellulolytic enhancing activity and a bicyclic compound. The present invention also relates to methods of using the compositions.

  11. Compositions comprising a polypeptide having cellulolytic enhancing activity and a quinone compound and uses thereof

    DOEpatents

    Quinlan, Jason; Xu, Feng; Sweeney, Matthew

    2016-03-01

    The present invention relates to compositions comprising: a polypeptide having cellulolytic enhancing activity and a quinone compound. The present invention also relates to methods of using the compositions.

  12. Compositions comprising a polypeptide having cellulolytic enhancing activity and a heterocyclic compound and uses thereof

    DOEpatents

    Xu, Feng; Sweeney, Matthew; Quinlan, Jason

    2016-08-02

    The present invention relates to compositions comprising: a polypeptide having cellulolytic enhancing activity and a heterocyclic compound. The present invention also relates to methods of using the compositions.

  13. Compositions comprising a polypeptide having cellulolytic enhancing activity and a dioxy compound and uses thereof

    DOEpatents

    Sweeney, Matthew; Xu, Feng; Quinlan, Jason

    2016-07-19

    The present invention relates to compositions comprising: a polypeptide having cellulolytic enhancing activity and a dioxy compound. The present invention also relates to methods of using the compositions.

  14. Genetic analysis of thirty-three platelet polypeptides detected in two-dimensional polyacrylamide gels.

    PubMed Central

    Hanash, S M; Neel, J V; Baier, L J; Rosenblum, B B; Niezgoda, W; Markel, D

    1986-01-01

    Two-dimensional gel electrophoresis followed by silver-staining was utilized to visualize platelet polypeptides for genetic analysis. A subset of 33 polypeptides that were most suited for scoring was selected. Families consisting of father-mother-child trios were studied. Thirty-six polypeptides of a total of 1,413 scored in children's gels exhibited the combination of a normal and a variant polypeptide. The observed index of heterozygosity of 2.55% is comparable to our previously reported findings for red cell proteins. Images Fig. 1 Fig. 2 PMID:3953575

  15. Self-assembly of high molecular weight polypeptide copolymers studied via diffusion limited aggregation.

    PubMed

    Meier, Christoph; Wu, Yuzhou; Pramanik, Goutam; Weil, Tanja

    2014-01-13

    The assembly of high molecular weight polypeptides into complex architectures exhibiting structural complexity ranging from the nano- to the mesoscale is of fundamental importance for various protein-related diseases but also hold great promise for various nano- and biotechnological applications. Here, the aggregation of partially unfolded high molecular weight polypeptides into multiscale fractal structures is investigated by means of diffusion limited aggregation and atomic force microscopy. The zeta potential, the hydrodynamic radius, and the obtained fractal morphologies were correlated with the conformation of the polypeptide backbones as obtained from circular dichroism measurements. The polypeptides are modified with polyethylene oxide side chains to stabilize the polypeptides and to normalize intermolecular interactions. The modification with the hydrophobic thioctic acid alters the folding of the polypeptide backbone, resulting in a change in solution aggregation and fractal morphology. We found that a more compact folding results in dense and highly branched structures, whereas a less compact folded polypeptide chain yields a more directional assembly. Our results provide first evidence for the role of compactness of polypeptide folding on aggregation. Furthermore, the mesoscale-structured biofilms were used to achieve a hierarchical protein assembly, which is demonstrated by deposition of Rhodamine-labeled HSA with the preassembled fractal structures. These results contribute important insights to the fundamental understanding of the aggregation of high molecular weight polypeptides in general and provide opportunities to study nanostructure-related effects on biological systems such as adhesion, proliferation, and the development of, for example, neuronal cells.

  16. Elastin-like Polypeptides as Models of Intrinsically Disordered Proteins

    PubMed Central

    Roberts, Stefan; Dzuricky, Michael; Chilkoti, Ashutosh

    2015-01-01

    Elastin-like polypeptides (ELPs) are a class of stimuli-responsive biopolymers inspired by the intrinsically disordered domains of tropoelastin that are composed of repeats of the VPGXG pentapeptide motif, where X is a “guest residue”. They undergo a reversible, thermally triggered lower critical solution temperature (LCST) phase transition, which has been utilized for a variety of applications including protein purification, affinity capture, immunoassays, and drug delivery. ELPs have been extensively studied as protein polymers and as biomaterials, but their relationship to other disordered proteins has heretofore not been established. The biophysical properties of ELPs that lend them their unique material behavior are similar to the properties of many intrinsically disordered proteins (IDP). Their low sequence complexity, phase behavior, and elastic properties make them an interesting “minimal” artificial IDP, and the study of ELPs can hence provide insights into the behavior of other more complex IDPs. Motivated by this emerging realization of the similarities between ELPs and IDPs, this review discusses the biophysical properties of ELPs, their biomedical utility, and their relationship to other disordered polypeptide sequences. PMID:26325592

  17. Two-Dimensional Coherent Infrared Spectroscopy of Polypeptides

    NASA Astrophysics Data System (ADS)

    Piryatinski, Andrei

    2000-03-01

    We simulate the third order (2D) coherent infrared response of polypeptides in the amide I spectral region. Applied multidimensional techniques are conceptually similar to multiple-pulse NMR spectroscopies and provide new and complimentary information [1,2]. Taking into account the transition dipole moment coupling between peptide groups we use the nonlinear exciton equations [2] (NEE) to compute the relevant response function. This approach accounts for single exciton dynamics and exciton-exciton scattering due to the vibrational anharmonicity of the peptide groups. The signature of one- and two-vibrational-exciton dynamics associated with different models of spectral broadening (homogeneous as well as diagonal and off-diagonal static disorder) can be extracted by analyzing the real and imaginary parts of the signal or/and its absolute value and phase [3]. To account for the fast events in protein folding such as helix-coil transitions, we have performed molecular dynamics simulations at different temperatures of an α-helical polypeptide in water solvent. 2D photon echo (PE) signal has been calculated and analyzed for different ratios of conformational population. [1] S. Mukamel, A. Piryatinski, V. Chernyak, Acc. Chem. Res., 32, (1999), 145. [2] W. M. Zhang, V. Chernyak, and S. Mukamel, J. Chem. Phys., 110, (1999), 5011. [3] A. Piryatinski, S. Tretiak, V. Chernyak, and S. Mukamel, J. Raman Sectrosc., (in press).

  18. Structural analysis of photosystem I polypeptides using chemical crosslinking

    NASA Technical Reports Server (NTRS)

    Armbrust, T. S.; Odom, W. R.; Guikema, J. A.; Spooner, B. S. (Principal Investigator)

    1994-01-01

    Thylakoid membranes, obtained from leaves of 14 d soybean (Glycine max L. cv. Williams) plants, were treated with the chemical crosslinkers glutaraldehyde or 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) to investigate the structural organization of photosystem I. Polypeptides were resolved using lithium dodecyl sulfate polyacrylamide gel electrophoresis, and were identified by western blot analysis using a library of polyclonal antibodies specific for photosystem I subunits. An electrophoretic examination of crosslinked thylakoids revealed numerous crosslinked products, using either glutaraldehyde or EDC. However, only a few of these could be identified by western blot analysis using subunit-specific polyclonal antibodies. Several glutaraldehyde dependent crosslinked species were identified. A single band was identified minimally composed of PsaC and PsaD, documenting the close interaction between these two subunits. The most interesting aspect of these studies was a crosslinked species composed of the PsaB subunit observed following EDC treatment of thylakoids. This is either an internally crosslinked species, which will provide structural information concerning the topology of the complex PsaB protein, a linkage with a polypeptide for which we do not yet have an immunological probe, or a masking of epitopes by the EDC linkage at critical locations in the peptide which is linked to PsaB.

  19. Cell Penetrating Elastin-like Polypeptides for Therapeutic Peptide Delivery

    PubMed Central

    Bidwell, Gene L.; Raucher, Drazen

    2010-01-01

    Current treatment of solid tumors is limited by side effects that result from the nonspecific delivery of drugs to the tumor site. Alternative targeted therapeutic approaches for localized tumors would significantly reduce systemic toxicity. Peptide therapeutics are a promising new strategy for targeted cancer therapy because of the ease of peptide design and the specificity of peptides for their intracellular molecular targets. However, the utility of peptides is limited by their poor pharmacokinetic parameters and poor tissue and cellular membrane permeability in vivo. This review article summarizes the development of elastin-like polypeptide (ELP) as a potential carrier for thermally targeted delivery of therapeutic peptides (TP), and the use of cell penetrating peptides (CPP) to enhance the intracellular delivery of the ELP-fused TPs. CPP-fused ELPs have been used to deliver a peptide inhibitor of c-Myc function and a peptide mimetic of p21 in several cancer models in vitro, and both polypeptides are currently yielding promising results in in vivo models of breast and brain cancer. PMID:20478348

  20. cDNA encoding a polypeptide including a hevein sequence

    DOEpatents

    Raikhel, N.V.; Broekaert, W.F.; Chua, N.H.; Kush, A.

    1995-03-21

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1,018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74--79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli. 11 figures.

  1. Reverse transcriptase activity of an intron encoded polypeptide.

    PubMed Central

    Fassbender, S; Brühl, K H; Ciriacy, M; Kück, U

    1994-01-01

    A number of group II introns from eukaryotic organelles and prokaryotes contain open reading frames for polypeptides with homology to retroviral reverse transcriptases (RTs). We have used the yeast transposon (Ty) system to express ORFs for RTs from eukaryotic organelles. This includes the mitochondrial coxI intron i1 from the fungus Podospora anserina, the plastid petD intron from the alga Scenedesmus obliquus and the mitochondrial RTL gene from the alga Chlamydomonas reinhardtii. The ORFs were fused with the TYA ORF from the yeast retrotransposon Ty to produce virus-like particles in the recipient strains with detectable amounts of the RT-like polypeptides. Analysis of the heterologous gene products revealed biochemical evidence that the P. anserina intron encodes an RNA-directed DNA polymerase with properties typically found for RTs of viral or retrotransposable origin. In vitro assays showed that the intron encoded RT is sensitive to RT inhibitors such as N-ethylmaleimide and dideoxythymidine triphosphate but is insensitive against the DNA polymerase inhibitor aphidicolin. The direct biochemical evidence provided here supports the idea that intron encoded RTs are involved in intron transposition events. Images PMID:7514530

  2. Uncharged Helical Modular Polypeptide Hydrogels for Cellular Scaffolds.

    PubMed

    Ahrens, Caroline C; Welch, M Elizabeth; Griffith, Linda G; Hammond, Paula T

    2015-12-14

    Grafted synthetic polypeptides hold appeal for extending the range of biophysical properties achievable in synthetic extracellular matrix (ECM) hydrogels. Here, N-carboxyanhydride polypeptide, poly(γ-propargyl-l-glutamate) (PPLG) macromers were generated by fully grafting the "clickable" side chains with mixtures of short polyethylene glycol (PEG) chains terminated with inert (-OH) or reactive (maleimide and/or norbornene) groups, then reacting a fraction of these groups with an RGD cell attachment motif. A panel of synthetic hydrogels was then created by cross-linking the PPLG macromers with a 4-arm PEG star molecule. Compared to well-established PEG-only hydrogels, gels containing PPLG exhibited dramatically less dependence on swelling as a function of cross-link density. Further, PPLG-containing gels, which retain an α-helical chain conformation, were more effective than standard PEG gels in fostering attachment of a human mesenchymal stem cell (hMSC) line for a given concentration of RGD in the gel. These favorable properties of PPLG-containing PEG hydrogels suggest they may find broad use in synthetic ECM.

  3. Human pancreatic polypeptide in children and young adults.

    PubMed

    Hanukoglu, A; Chalew, S; Kowarski, A A

    1990-01-01

    Measurement of human pancreatic polypeptide may be useful for assessment of gastrointestinal function, integrity of the parasympathetic nervous system or screening for endocrine neoplasia. In adults hPP levels have been reported to increase with age. However hPP levels throughout childhood have not been well characterized in comparison with the adult range. We studied fasting human pancreatic polypeptide (hPP) from 45 pediatric patients, from infancy - 15 years, and 18 older adolescents and adults aged 16-45 years. The mean hPP level of children (233 +/- 147 pg/ml) was significantly higher than that (113 +/- 35 pg/ml) of adults (P less than .0001). There was no difference in mean hPP levels of children with normal growth hormone secretion compared to growth hormone deficient patients. There was no effect of gender or body mass index on hPP levels. We conclude that fasting hPP levels must be interpreted with respect to the age of the subject, children particularly, in that preteens may have higher fasting levels than older teenagers and adults.

  4. CDNA encoding a polypeptide including a hevein sequence

    DOEpatents

    Raikhel, Natasha V.; Broekaert, Willem F.; Chua, Nam-Hai; Kush, Anil

    1995-03-21

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74-79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.

  5. cDNA encoding a polypeptide including a hevein sequence

    DOEpatents

    Raikhel, Natasha V.; Broekaert, Willem F.; Chua, Nam-Hai; Kush, Anil

    1999-05-04

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74-79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.

  6. cDNA encoding a polypeptide including a hevein sequence

    DOEpatents

    Raikhel, N.V.; Broekaert, W.F.; Chua, N.H.; Kush, A.

    1999-05-04

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74--79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli. 12 figs.

  7. Membrane Permeation Induced by Aggregates of Human Islet Amyloid Polypeptides

    PubMed Central

    Poojari, Chetan; Xiao, Dequan; Batista, Victor S.; Strodel, Birgit

    2013-01-01

    Several neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases as well as nonneuropathic diseases such as type II diabetes and atrial amyloidosis are associated with aggregation of amyloid polypeptides into fibrillar structures, or plaques. In this study, we use molecular dynamics simulations to test the stability and orientation of membrane-embedded aggregates of the human islet amyloid polypeptide (hIAPP) implicated in type II diabetes. We find that in both monolayers and bilayers of dipalmitoylphosphatidylglycerol (DPPG) hIAPP trimers and tetramers remain inside the membranes and preserve their β-sheet secondary structure. Lipid bilayer-inserted hIAPP trimers and tetramers orient inside DPPG at 60° relative to the membrane/water interface and lead to water permeation and Na+ intrusion, consistent with ion-toxicity in islet β-cells. In particular, hIAPP trimers form a water-filled β-sandwich that induce water permeability comparable with channel-forming proteins, such as aquaporins and gramicidin-A. The predicted disruptive orientation is consistent with the amphiphilic properties of the hIAPP aggregates and could be probed by chiral sum frequency generation (SFG) spectroscopy, as predicted by the simulated SFG spectra. PMID:24268144

  8. Human pancreatic polypeptide in children and young adults.

    PubMed

    Hanukoglu, A; Chalew, S; Kowarski, A A

    1990-01-01

    Measurement of human pancreatic polypeptide may be useful for assessment of gastrointestinal function, integrity of the parasympathetic nervous system or screening for endocrine neoplasia. In adults hPP levels have been reported to increase with age. However hPP levels throughout childhood have not been well characterized in comparison with the adult range. We studied fasting human pancreatic polypeptide (hPP) from 45 pediatric patients, from infancy - 15 years, and 18 older adolescents and adults aged 16-45 years. The mean hPP level of children (233 +/- 147 pg/ml) was significantly higher than that (113 +/- 35 pg/ml) of adults (P less than .0001). There was no difference in mean hPP levels of children with normal growth hormone secretion compared to growth hormone deficient patients. There was no effect of gender or body mass index on hPP levels. We conclude that fasting hPP levels must be interpreted with respect to the age of the subject, children particularly, in that preteens may have higher fasting levels than older teenagers and adults. PMID:2307392

  9. Polypeptide profiles of South Indian isolate of Trypanosoma evansi.

    PubMed

    Sivajothi, S; Rayulu, V C; Bhaskar Reddy, B V; Malakondaiah, P; Sreenivasulu, D; Sudhakara Reddy, B

    2016-09-01

    The field isolates of Trypanosoma evansi was collected from the infected cattle and it was propagated in rats. Trypanosoma evansi parasites were separated from the blood of infected rats by using diethylaminoethyl cellulose column chromatography. Whole cell lysate antigen (WCL) was prepared from purified trypanosomes by ultrasonication and centrifugation. The prepared WCL antigen was further purified by 50 % ammonium sulphate precipitation. Protein concentration of WCL antigen of T. evansi was 60 mg/ml. Protein concentration was adjusted to 1.0 mg/ml in PBS, pH 8.0 and stored at -20(0) C.   Polypeptide profiles of WCL antigen of T. evansi was determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis. A total of eight polypeptide bands of the size ranging from 25 to 85 kDa in WCL antigen of T. evansi were obtained. Five prominent bands with molecular weight of 74, 60, 53, 42 and 37 kDa and three light bands with molecular weight of 85, 34 and 25 kDa were observed. PMID:27605761

  10. cDNA encoding a polypeptide including a hevein sequence

    SciTech Connect

    Raikhel, N.V.; Broekaert, W.F.; Chua, N.H.; Kush, A.

    2000-07-04

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74--79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.

  11. Two nonhomologus viruses of Cryphonectria (Endothia) parasitica reduce accumulation of specific virulence-associated polypeptides.

    PubMed Central

    Powell, W A; Van Alfen, N K

    1987-01-01

    Double-stranded RNA responsible for transmissible hypovirulence of Cryphonectria (Endothia) parasitica affected the accumulation of specific polypeptides. Nonhomologous hypovirulence-causing double-stranded RNAs, originating in Europe or North America, affected accumulation of the same polypeptides. Fewer than 5% of detectable proteins were affected, indicating that hypovirulence is probably not the result of general debilitation of the fungus. Images PMID:3667533

  12. Design of a single-chain polypeptide tetrahedron assembled from coiled-coil segments.

    PubMed

    Gradišar, Helena; Božič, Sabina; Doles, Tibor; Vengust, Damjan; Hafner-Bratkovič, Iva; Mertelj, Alenka; Webb, Ben; Šali, Andrej; Klavžar, Sandi; Jerala, Roman

    2013-06-01

    Protein structures evolved through a complex interplay of cooperative interactions, and it is still very challenging to design new protein folds de novo. Here we present a strategy to design self-assembling polypeptide nanostructured polyhedra based on modularization using orthogonal dimerizing segments. We designed and experimentally demonstrated the formation of the tetrahedron that self-assembles from a single polypeptide chain comprising 12 concatenated coiled coil-forming segments separated by flexible peptide hinges. The path of the polypeptide chain is guided by a defined order of segments that traverse each of the six edges of the tetrahedron exactly twice, forming coiled-coil dimers with their corresponding partners. The coincidence of the polypeptide termini in the same vertex is demonstrated by reconstituting a split fluorescent protein in the polypeptide with the correct tetrahedral topology. Polypeptides with a deleted or scrambled segment order fail to self-assemble correctly. This design platform provides a foundation for constructing new topological polypeptide folds based on the set of orthogonal interacting polypeptide segments.

  13. Nodule-Specific Polypeptides from Effective Alfalfa Root Nodules and from Ineffective Nodules Lacking Nitrogenase 1

    PubMed Central

    Lang-Unnasch, Naomi; Ausubel, Frederick M.

    1985-01-01

    In addition to leghemoglobin, at least nine nodule-specific polypeptides from the alfalfa (Medicago sativa L.)-Rhizobium meliloti symbiosis were identified by immune assay. Some of these polypeptides may be subunits of larger proteins but none appeared to be subunits of the same multimeric protein. All nine of the nodule-specific polypeptides were localized to within the plant cytosol; they were not found in extracts of bacteroids or in the peribacteroid space. At least one of these nodule-specific polypeptides was found to be antigenically related to nodule-specific polypeptides in pea and/or soybean. Ineffective nodules elicited by R. meliloti strains containing mutations in four different genes required for nitrogenase synthesis contained reduced concentrations of leghemoglobin and of several of the nodule-specific polypeptides. Other nodule-specific polypeptides were unaltered or actually enriched in the ineffective nodules. Many of the differences between the ineffective and effective nodules were apparent in nodules harvested shortly after the nodules became visible. These differences were greatly amplified in older nodules. When the four ineffective nodule types were compared to one another, there were clear quantitative differences in the concentrations of several of the nodule-specific polypeptides. These differences suggest that lack of a functional nitrogenase does not have a single direct effect on nodule development. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:16664146

  14. Identification and analysis of the major yolk polypeptide from the caribbean fruit fly, Anastrepha suspensa (Loew)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A single major yolk polypeptide (YP) having a molecular mass of approximately 48,000 daltons (Da), was identified in the ovaries and oviposited eggs of the Caribbean fruit fly, Anastrepha suspensa. The polypeptide was partially purified from oviposited eggs using gel permeation and ion-exchange chro...

  15. Directed evolution methods for improving polypeptide folding and solubility and superfolder fluorescent proteins generated thereby

    DOEpatents

    Waldo, Geoffrey S.

    2007-09-18

    The current invention provides methods of improving folding of polypeptides using a poorly folding domain as a component of a fusion protein comprising the poorly folding domain and a polypeptide of interest to be improved. The invention also provides novel green fluorescent proteins (GFPs) and red fluorescent proteins that have enhanced folding properties.

  16. Thymus Polypeptide Preparation Tactivin Restores Learning and Memory in Thymectomied Rats.

    PubMed

    Novoseletskaya, A V; Kiseleva, N M; Zimina, I V; Bystrova, O V; Belova, O V; Inozemtsev, A N; Arion, V Ya; Sergienko, V I

    2015-09-01

    We studied the effects of tactivin and splenic polypeptides on learning and memory of thymectomized animals. In 3-week rats, thymectomy blocked active avoidance conditioning. Injections of tactivin (0.5 mg/kg) during 1 month after surgery restored learning capacity; splenic polypeptides were ineffective.

  17. Applications of elastin-like polypeptides in drug delivery

    PubMed Central

    MacEwan, Sarah R; Chilkoti, Ashutosh

    2014-01-01

    Elastin-like polypeptides (ELPs) are biopolymers inspired by human elastin. Their lower critical solution temperature phase transition behavior and biocompatibility make them useful materials for stimulus-responsive applications in biological environments. Due to their genetically encoded design and recombinant synthesis, the sequence and size of ELPs can be exactly defined. These design parameters control the structure and function of the ELP with a precision that is unmatched by synthetic polymers. Due to these attributes, ELPs have been used extensively for drug delivery in a variety of different embodiments—as soluble macromolecular carriers, self-assembled nanoparticles, cross-linked microparticles, or thermally coacervated depots. These ELP systems have been used to deliver biologic therapeutics, radionuclides, and small molecule drugs to a variety of anatomical sites for the treatment of diseases including cancer, type 2 diabetes, osteoarthritis, and neuroinflammation. PMID:24979207

  18. Circadian rhythm of gastric inhibitory polypeptide (GIP) in man.

    PubMed

    Salera, M; Giacomoni, P; Pironi, L; Ustra, C; Capelli, M; Giorgi, A; Miglioli, M; Barbara, L

    1983-01-01

    The diurnal variations of serum gastric inhibitory polypeptide (GIP), serum insulin, plasma glucagon, plasma glucose and serum triglycerides were studied for 24 hr in 6 healthy young men, consuming three meals and performing their usual physical activities. Serum GIP levels peaked after each meal and stayed significantly elevated from the peak after lunch till late night. Glucose and insulin showed early and short-lasting postprandial peaks, declining thereafter to basal values within a short time. Plasma glucagon was inhibited by the meal ingestion and fluctuated around the basal levels in the interdigestive periods. On the other hand, serum triglycerides tended to parallel GIP changes for most of the day, being significantly elevated starting from lunch consumption to late night. The present results suggest that GIP may have effects other than the insulinogenic one, being probably involved in the control of lipid metabolism.

  19. Expansin polynucleotides, related polypeptides and methods of use

    DOEpatents

    Cosgrove, Daniel J.; Wu, Yajun

    2006-02-21

    The present invention relates to beta expansin polypeptides, nucleotide sequences encoding the same and regulatory elements and their use in altering cell wall structure in plants. Nucleic acid constructs comprising a beta expansin sequence operably linked to a promoter, or other regulatory sequence are disclosed as well as vectors, plant cells, plants, and transformed seeds containing such constructs are provided. Methods for the use of such constructs in repressing or inducing expression of a beta expansin sequences in a plant are also provided as well as methods for harvesting transgenic expansin proteins. In addition, methods are provided for inhibiting or improving cell wall structure in plants by repression or induction of expansin sequences in plants.

  20. Discovery and characterization of smORF-encoded bioactive polypeptides.

    PubMed

    Saghatelian, Alan; Couso, Juan Pablo

    2015-12-01

    Analysis of genomes, transcriptomes and proteomes reveals the existence of hundreds to thousands of translated, yet non-annotated, short open reading frames (small ORFs or smORFs). The discovery of smORFs and their protein products, smORF-encoded polypeptides (SEPs), points to a fundamental gap in our knowledge of protein-coding genes. Various studies have identified central roles for smORFs in metabolism, apoptosis and development. The discovery of these bioactive SEPs emphasizes the functional potential of this unexplored class of biomolecules. Here, we provide an overview of this emerging field and highlight the opportunities for chemical biology to answer fundamental questions about these novel genes. Such studies will provide new insights into the protein-coding potential of genomes and identify functional genes with roles in biology and disease. PMID:26575237

  1. Fractionation and Analysis of Polypeptides of Euglena gracilis Chloroplasts.

    PubMed

    Vasconcelos, A C; Mendiola-Morgenthaler, L R; Floyd, G L; Salisbury, J L

    1976-07-01

    Intact Euglena gracilis chloroplasts, purified on gradients of silica sol, were lysed osmotically and fractionated by centrifugation on discontinuous gradients of sucrose into their soluble, envelope membrane, and thylakoid membrane components. The proteins of the different subchloroplast fractions, as well as those of whole chloroplasts, were analyzed by electrophoresis on sodium dodecyl sulfate polyacrylamide gels. The polypeptide profile of each fraction was distinctive and was in general similar to the profile obtained for analogous fractions of the chloroplasts of higher plants.The envelope membranes were separated into two fractions in the gradients according to their banding densities. Electron micrographs showed that the light envelope fraction consisted mostly of single-membrane vesicles, whereas the heavy envelope fraction consisted of multiple layers of folded membranes. Both envelope fractions were ultrastructurally distinct from the thylakoid membranes. PMID:16659627

  2. Identification of antigenically related polypeptides at centrioles and basal bodies.

    PubMed

    Lin, W; Fung, B; Shyamala, M; Kasamatsu, H

    1981-04-01

    An antigen localized at the centriolar region has been identified by indirect immunofluorescence studies in African green monkey kidney, human, hamster, rat, and mouse cells. The antigen consists of two polypeptides of 14,000 and 17,000 daltons. A related antigen is also present at the basal body region in ciliated cells from chicken, cat, mouse, pig, steer, and rabbit trachea and from rabbit fimbria. Immunoelectron microscopy shows that the immunoreactive antigen is indeed located in the region around the basal bodies of ciliated cat tracheal cells. Thus, we have found an antigen that is common to a variety of cell types from many different animal sources and is specifically associated with both centrioles and basal bodies. The possible role of the antigen in differentiation is discussed.

  3. NMR and rotational angles in solution conformation of polypeptides

    NASA Astrophysics Data System (ADS)

    Bystrov, V. F.

    1985-01-01

    Professor San-Ichiro Mizushima and Professor Yonezo Morino's classical contributions provided unique means and firm basis for understanding of conformational states and internal rotation in polypeptide molecules. Now the NMR spectroscopy is the best choice to study molecular conformation, mechanism of action and structure-functional relationships of peptide and proteins in solution under conditions approaching those of their physiological environments. Crucial details of spatial structure and interactions of these molecules in solution are revealed by using proton-proton and carbon-proton vicinal coupling constants, proton nuclear Overhauser effect and spectral perturbation techniques. The results of NMR conformational analysis are presented for valinomycin "bracelet", gramicidin A double helices, honey-bee neurotoxin apamin, scorpion insectotoxins and snake neurotoxins of long and short types.

  4. Binding of netropsin to DNA in complexes with polypeptides containing repetitive lysine sequences.

    PubMed

    Votavová, H; Burckhardt, G; Sponar, J; Zimmer, C

    1981-07-27

    The interaction of the antibiotic netropsin with calf thymus DNA, T4 DNA and poly(dA-dT) . poly(dA-dT) in complexes with sequential polypeptides containing repetitive lysine sequences and histone H1 was investigated using circular dichroism spectroscopy and equilibrium dialysis. Both soluble DNA-polypeptide complexes and insoluble complexes showed binding of netropsin. The possibility of displacement of polypeptides from DNA binding sites by competition with netropsin molecules was eliminated by experiments using 14C-labelled polypeptides. From the analysis of CD titration behavior as well as from the results of equilibrium dialysis studies it follows that netropsin does not compete with polypeptides for DNA binding sites, which suggests that these two ligands occupy different sites. Various explanations for minor differences in the CD behavior of the bound netropsin in the saturation region are also discussed.

  5. Ice Growth Inhibition in Antifreeze Polypeptide Solution by Short-Time Solution Preheating

    PubMed Central

    Nishi, Naoto; Miyamoto, Takuya; Waku, Tomonori; Tanaka, Naoki; Hagiwara, Yoshimichi

    2016-01-01

    The objective of this study is to enhance the inhibition of ice growth in the aqueous solution of a polypeptide, which is inspired by winter flounder antifreeze protein. We carried out measurements on unidirectional freezing of the polypeptide solution. The thickness of the solution was 0.02 mm, and the concentration of polypeptide was varied from 0 to 2 mg/mL. We captured successive microscopic images of ice/solution interfaces, and measured the interface velocity from the locations of tips of the pectinate interface in the images. We also simultaneously measured the temperature by using a small thermocouple. The ice/solution interface temperature was defined by the temperature at the tips. It was found that the interface temperature was decreased with an increasing concentration of polypeptide. To try varying the activity of the polypeptide, we preheated the polypeptide solution and cooled it before carrying out the measurements. Preheating for 1–5 hours was found to cause a further decrease in the interface temperature. Furthermore, wider regions of solution and ice with inclined interfaces in the pectinate interface structure were observed, compared with the case where the solution was not preheated. Thus, the ice growth inhibition was enhanced by this preheating. To investigate the reason for this enhancement, we measured the conformation and aggregates of polypeptide in the solution. We also measured the local concentration of polypeptide. It was found that the polypeptide aggregates became larger as a result of preheating, although the polypeptide conformation was unchanged. These large aggregates caused both adsorption to the interface and the wide regions of supercooled solution in the pectinate interface structure. PMID:27152720

  6. Ice Growth Inhibition in Antifreeze Polypeptide Solution by Short-Time Solution Preheating.

    PubMed

    Nishi, Naoto; Miyamoto, Takuya; Waku, Tomonori; Tanaka, Naoki; Hagiwara, Yoshimichi

    2016-01-01

    The objective of this study is to enhance the inhibition of ice growth in the aqueous solution of a polypeptide, which is inspired by winter flounder antifreeze protein. We carried out measurements on unidirectional freezing of the polypeptide solution. The thickness of the solution was 0.02 mm, and the concentration of polypeptide was varied from 0 to 2 mg/mL. We captured successive microscopic images of ice/solution interfaces, and measured the interface velocity from the locations of tips of the pectinate interface in the images. We also simultaneously measured the temperature by using a small thermocouple. The ice/solution interface temperature was defined by the temperature at the tips. It was found that the interface temperature was decreased with an increasing concentration of polypeptide. To try varying the activity of the polypeptide, we preheated the polypeptide solution and cooled it before carrying out the measurements. Preheating for 1-5 hours was found to cause a further decrease in the interface temperature. Furthermore, wider regions of solution and ice with inclined interfaces in the pectinate interface structure were observed, compared with the case where the solution was not preheated. Thus, the ice growth inhibition was enhanced by this preheating. To investigate the reason for this enhancement, we measured the conformation and aggregates of polypeptide in the solution. We also measured the local concentration of polypeptide. It was found that the polypeptide aggregates became larger as a result of preheating, although the polypeptide conformation was unchanged. These large aggregates caused both adsorption to the interface and the wide regions of supercooled solution in the pectinate interface structure. PMID:27152720

  7. Mechanisms of fat-induced gastric inhibitory polypeptide/glucose-dependent insulinotropic polypeptide secretion from K cells.

    PubMed

    Yamane, Shunsuke; Harada, Norio; Inagaki, Nobuya

    2016-04-01

    Gastric inhibitory polypeptide/glucose-dependent insulinotropic polypeptide (GIP) is one of the incretins, which are gastrointestinal hormones released in response to nutrient ingestion and potentiate glucose-stimulated insulin secretion. Single fat ingestion stimulates GIP secretion from enteroendocrine K cells; chronic high-fat diet (HFD) loading enhances GIP secretion and induces obesity in mice in a GIP-dependent manner. However, the mechanisms of GIP secretion from K cells in response to fat ingestion and GIP hypersecretion in HFD-induced obesity are not well understood. We generated GIP-green fluorescent protein knock-in (GIP (gfp/+)) mice, in which K cells are labeled by enhanced GIP-green fluorescent protein. Microarray analysis of isolated K cells from GIP (gfp/+) mice showed that both fatty acid-binding protein 5 and G protein-coupled receptor 120 are highly expressed in K cells. Single oral administration of fat resulted in significant reduction of GIP secretion in both fatty acid-binding protein 5- and G protein-coupled receptor 120-deficient mice, showing that fatty acid-binding protein 5 and G protein-coupled receptor 120 are involved in acute fat-induced GIP secretion. Furthermore, the transcriptional factor, regulatory factor X6 (Rfx6), is highly expressed in K cells. In vitro experiments using the mouse enteroendocrine cell line, STC-1, showed that GIP messenger ribonucleic acid levels are upregulated by Rfx6. Expression levels of Rfx6 messenger ribonucleic acid as well as that of GIP messenger ribonucleic acid were augmented in the K cells of HFD-induced obese mice, in which GIP content in the small intestine is increased compared with that in lean mice fed a control diet. These results suggest that Rfx6 is involved in hypersecretion of GIP in HFD-induced obese conditions by increasing GIP gene expression.

  8. Peptides and polypeptides as scaffolds for optoelectronics and biomaterials applications

    NASA Astrophysics Data System (ADS)

    Charati, Manoj B.

    Peptides and polypeptides are emerging as a new class of biomaterials due to their unique structural, physiochemical, mechanical, and biological properties. The development of peptide and protein-based biomaterials is driven by the convergence of convenient techniques for peptide/protein engineering and its importance in applications as smart biomaterials. The thesis is divided in two parts; the first part highlights the importance of incorporation of non-natural amino acids into peptides and proteins. In particular, incorporation on p-bromophenylalanine in short alpha-helical peptide templates to control the association of chromophores is discussed. In the second part, design of a multi-component, biocompatible polypeptide with superior elasticity is discussed. Part 1. Novel peptide templates to control association of chromophores. Tailor made peptide and protein materials have many versatile applications, as both conformation and functional group position can be controlled. Such control may have intriguing applications in the development of hybrid materials for electroactive applications. A critical need in fabricating devices from organic semiconducting materials is to achieve control over the conformation and distance between two conjugated chains. Controlling chromophore spacing and orientation with required precision over nanometer length scale poses a greater challenge. Here we propose a peptide based template to control the alignment of the methylstilbene and Oxa-PPV chromophores with desired orientations and spacing. The hybrid peptides were characterized via CD, exciton coupled CD, 1H NMR and photoluminescence experiments. It is observed that slight change in the orientation of molecules has pronounced effect on the photo-physical behavior of the molecules. Characterization of the hybrid peptides via circular dichroism (CD) confirmed the helical character of the designed peptides and indicated that inclusion of non-natural amino acids has significant

  9. Multifunctional quantum dot-polypeptide hybrid nanogel for targeted imaging and drug delivery

    NASA Astrophysics Data System (ADS)

    Yang, Jie; Yao, Ming-Hao; Wen, Lang; Song, Ji-Tao; Zhang, Ming-Zhen; Zhao, Yuan-Di; Liu, Bo

    2014-09-01

    A new type of multifunctional quantum dot (QD)-polypeptide hybrid nanogel with targeted imaging and drug delivery properties has been developed by metal-affinity driven self-assembly between artificial polypeptides and CdSe-ZnS core-shell QDs. On the surface of QDs, a tunable sandwich-like microstructure consisting of two hydrophobic layers and one hydrophilic layer between them was verified by capillary electrophoresis, transmission electron microscopy, and dynamic light scattering measurements. Hydrophobic and hydrophilic drugs can be simultaneously loaded in a QD-polypeptide nanogel. In vitro drug release of drug-loaded QD-polypeptide nanogels varies strongly with temperature, pH, and competitors. A drug-loaded QD-polypeptide nanogel with an arginine-glycine-aspartic acid (RGD) motif exhibited efficient receptor-mediated endocytosis in αvβ3 overexpressing HeLa cells but not in the control MCF-7 cells as analyzed by confocal microscopy and flow cytometry. In contrast, non-targeted QD-polypeptide nanogels revealed minimal binding and uptake in HeLa cells. Compared with the original QDs, the QD-polypeptide nanogels showed lower in vitro cytotoxicity for both HeLa cells and NIH 3T3 cells. Furthermore, the cytotoxicity of the targeted QD-polypeptide nanogel was lower for normal NIH 3T3 cells than that for HeLa cancer cells. These results demonstrate that the integration of imaging and drug delivery functions in a single QD-polypeptide nanogel has the potential for application in cancer diagnosis, imaging, and therapy.A new type of multifunctional quantum dot (QD)-polypeptide hybrid nanogel with targeted imaging and drug delivery properties has been developed by metal-affinity driven self-assembly between artificial polypeptides and CdSe-ZnS core-shell QDs. On the surface of QDs, a tunable sandwich-like microstructure consisting of two hydrophobic layers and one hydrophilic layer between them was verified by capillary electrophoresis, transmission electron

  10. Photochromic polypeptides as synthetic models of biological photoreceptors: a spectroscopic study.

    PubMed Central

    Angelini, N; Corrias, B; Fissi, A; Pieroni, O; Lenci, F

    1998-01-01

    L-Glutamic acid polypeptides containing photochromic nitrospiropyran bound to the side chains at various percentages ("local" concentration) have been synthesized and investigated as possible artificial models of biological photoreceptors. Absorption and fluorescence spectroscopy have been utilized to investigate the photophysical and photochemical properties of nitrospiropyrans, both inserted in the polypeptide chain and in solution as "free" dye. Conformational variations produced by dark storage and light exposure of the photochromic polypeptides have been studied by means of circular dichroism. Dark-kept "free" dyes in hexafluoro-2-propanol solution in the merocyanine form ("open" form) give rise to molecular aggregates, which have been characterized as merocyanine dimers. The equilibrium constant between the monomer and the dimer, K, and their molar extinction coefficients, epsilon, at several wavelengths have been determined. Fluorescence measurements on "free" and polypeptide-bound nitrospiropyrans suggest that the dimerization process between merocyanines is favored when the photochromic units are inserted in the polypeptide chain and that under these conditions an efficient energy transfer from the monomer (donor) to the dimer (acceptor) occurs. By varying "local" as well as total nitrospiropyran concentration, it has been shown that the dimeric species result from intermolecular interactions between photochromic groups inserted in the same polypeptide chain. The alpha-helix --> random coil transition of the polypeptide structure after dark storage has eventually been shown to be the result of the dimerization process and not of the dark isomerization per se from the "closed" spiropyran form to the "open" merocyanine form of the dye. PMID:9591684

  11. Molecular Structure, Membrane Interactions, and Toxicity of the Islet Amyloid Polypeptide in Type 2 Diabetes Mellitus

    PubMed Central

    Caillon, Lucie; Hoffmann, Anais R. F.; Botz, Alexandra; Khemtemourian, Lucie

    2016-01-01

    Human islet amyloid polypeptide (hIAPP) is the major component of the amyloid deposits found in the pancreatic islets of patients with type 2 diabetes mellitus (T2DM). Mature hIAPP, a 37-aa peptide, is natively unfolded in its monomeric state but forms islet amyloid in T2DM. In common with other misfolded and aggregated proteins, amyloid formation involves aggregation of monomers of hIAPP into oligomers, fibrils, and ultimately mature amyloid deposits. hIAPP is coproduced and stored with insulin by the pancreatic islet β-cells and is released in response to the stimuli that lead to insulin secretion. Accumulating evidence suggests that hIAPP amyloid deposits that accompany T2DM are not just an insignificant phenomenon derived from the disease progression but that hIAPP aggregation induces processes that impair the functionality and the viability of β-cells. In this review, we particularly focus on hIAPP structure, hIAPP aggregation, and hIAPP-membrane interactions. We will also discuss recent findings on the mechanism of hIAPP-membrane damage and on hIAPP-induced cell death. Finally, the development of successful antiamyloidogenic agents that prevent hIAPP fibril formation will be examined. PMID:26636105

  12. Characterization of an amidated form of pancreatic polypeptide from the daddy sculpin (Cottus scorpius).

    PubMed

    Conlon, J M; Schmidt, W E; Gallwitz, B; Falkmer, S; Thim, L

    1986-12-30

    The primary structure of pancreatic polypeptide from the teleostean fish, Cottus scorpius (daddy sculpin) was established as: YPPQPESPGGNASPEDWAKYHAAVRHYVNLITRQRYNH2 The presence of a COOH-terminally alpha-amidated amino acid was established using an HPLC method of general applicability. Although the peptide shows strong homology towards anglerfish pancreatic polypeptide (86%), homology towards porcine peptide YY (PYY) (61%) and porcine neuropeptide Y (NPY) (61%) was greater than towards porcine pancreatic polypeptide (PP) (47%). This result supports suggestions that the gene duplication events which led to PP, NPY and PYY formation took place after the time of divergence of fish and mammals. PMID:3562898

  13. The Research on the Impact of Maca Polypeptide on Sport Fatigue.

    PubMed

    Miao, Hua

    2015-01-01

    In order to study the effect of maca polypeptide on sport fatigue, this paper selected 40 male mice, and they were randomly divided into group A, B, C and D. group A, B and C were fed food with different concentrations of maca polypeptide, and group D was control group. After two weeks of feeding, measured physiological indexes of mice, including blood glucose, urea nitrogen and creatinine. At last gived the experimental results, as well as the analysis. Experimental results show that maca polypeptide can improve the ability of anti-fatigue mice, and in a certain concentration range, the higher the concentration, the better the resistance to fatigue. PMID:26998182

  14. The Research on the Impact of Maca Polypeptide on Sport Fatigue

    PubMed Central

    Miao, Hua

    2015-01-01

    In order to study the effect of maca polypeptide on sport fatigue, this paper selected 40 male mice, and they were randomly divided into group A, B, C and D. group A, B and C were fed food with different concentrations of maca polypeptide, and group D was control group. After two weeks of feeding, measured physiological indexes of mice, including blood glucose, urea nitrogen and creatinine. At last gived the experimental results, as well as the analysis. Experimental results show that maca polypeptide can improve the ability of anti-fatigue mice, and in a certain concentration range, the higher the concentration, the better the resistance to fatigue. PMID:26998182

  15. Translational Modification of an 18 Kilodalton Polypeptide by Spermidine in Rice Cell Suspension Cultures

    PubMed Central

    Mehta, Arkesh M.; Saftner, Robert A.; Schaeffer, Gideon W.; Mattoo, Autar K.

    1991-01-01

    When rice (Oryza sativa) cell suspension cultures are grown in the presence of [terminal methylenes-3H]spermidine, label is incorporated in a single polypeptide with a molecular mass of 18 kilodaltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Preincubation of cell cultures with polyamine biosynthesis inhibitors difluoromethylarginine and difluoromethylornithine, resulted in increased incorporation of the label into the 18 kilodalton polypeptide. In cells in which protein synthesis was arrested by cycloheximide, no label was detected in the 18 kilodalton polypeptide, suggesting a requirement for de novo protein synthesis. Images Figure 2 Figure 3 PMID:16668128

  16. Fusions of elastin-like polypeptides to pharmaceutical proteins

    PubMed Central

    Hassouneh, Wafa; MacEwan, Sarah R; Chilkoti, Ashutosh

    2013-01-01

    Elastin-like polypeptides (ELPs) are a class of stimulus responsive biopolymers whose physicochemical properties and biocompatibility are particularly suitable for in vivo applications, such as drug delivery and tissue engineering. The lower critical solution temperature (LCST) behavior of ELPs allows them to be utilized as soluble macromolecules below their LCST, or as self-assembled nano-scale particles such as micelles, micron-scale coacervates, or viscous gels above their LCST, depending on the ELP architecture. As each ELP sequence is specified at its genetic level, functionalization of an ELP with peptides and proteins is simple to accomplish by the fusion of a gene encoding an ELP with that of the peptide or protein of interest. Protein ELP fusions, where the appended protein serves a therapeutic or targeting function, are suitable for applications in which the ELP can improve the systemic pharmacokinetics and biodistribution of the protein, or can be used as an injectable depot for sustained, local protein delivery. Here we describe considerations in the design of therapeutic protein ELP fusions and provide details of their gene design, expression, and purification. PMID:22208987

  17. Monoclonal antibodies to Alzheimer neurofibrillary tangles. 1. Identification of polypeptides.

    PubMed Central

    Yen, S. H.; Crowe, A.; Dickson, D. W.

    1985-01-01

    Ten monoclonal antibodies to Alzheimer neurofibrillary tangles (ANTs) were produced by immunizing mice with a brain homogenate from senile dementia of the Alzheimer type (SDAT). In methanol-fixed isolated neuronal perikarya, six of these antibodies reacted with nearly every ANT, three recognized 70-88% of ANTs, and one bound to less than 30% of ANT. In paraffin sections, three of the antibodies did not bind to tangles that had been fixed in formalin, three stained weekly, and four reacted with tangles in tissues that had been in formalin for more than a decade. Immunoblotting of brain homogenates showed that all but one antibody reacted with proteins from SDAT samples insoluble in SDS and too large to enter even the 3% polyacrylamide stacking gel. Polypeptides extractable by Tris buffer of molecular weight 58, 66, and 70 kd were detected in both normal and SDAT brains by two antibodies and only in SDAT brain by two other antibodies. One antibody did not show any reaction on the immunoblot. The results demonstrate that the epitopes recognized by these antibodies are not identical and that ANTs contain unique antigenic determinants as well as determinants in common with normal brain. Whether the unique determinants are acquired during tangle development or are essential in tangle formation remains to be investigated. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 PMID:2411142

  18. Bovine pancreatic polypeptide as an antagonist of muscarinic cholinergic receptors

    SciTech Connect

    Pan, G.Z.; Lu, L.; Qian, J.; Xue, B.G.

    1987-03-01

    In dispersed acini from rat pancreas, it was found that bovine pancreatic polypeptide (BPP) and its C-fragment hexapeptide amide (PP-6), at concentrations of 0.1 and 30 ..mu..M, respectively, could significantly inhibit amylase secretion stimulated by carbachol, and this inhibition by BPP was dose dependent. /sup 45/Ca outflux induced by carbachol was also inhibited by BPP or PP-6, but they had no effect on cholecystokinin octapeptide- (CCK-8) or A23187-stimulated /sup 45/Ca outflux. BPP was also capable of displacing the specific binding of (/sup 3/H)-quinuclidinyl benzilate to its receptors, and it possessed a higher affinity (K/sub i/35nM) than carbachol (K/sub i/ 1.8 ..mu..M) in binding with M-receptors. It is concluded from this study that BPP acts as an antagonist of muscarinic cholinergic receptors in rat pancreatic acini. In addition, BPP inhibited the potentiation of amylase secretion caused by the combination of carbachol plus secretin or vasoactive intestinal peptide. This may be a possible explanation of the inhibitory effect of BPP on secretin-induced pancreatic enzyme secretion shown in vivo, since pancreatic enzyme secretion stimulated by secretin under experimental conditions may be the result of potentiation of enzyme release produced by the peptide in combination with a cholinergic stimulant.

  19. How lamina-associated polypeptide 1 (LAP1) activates Torsin

    PubMed Central

    Sosa, Brian A; Demircioglu, F Esra; Chen, James Z; Ingram, Jessica; Ploegh, Hidde L; Schwartz, Thomas U

    2014-01-01

    Lamina-associated polypeptide 1 (LAP1) resides at the nuclear envelope and interacts with Torsins, poorly understood endoplasmic reticulum (ER)-localized AAA+ ATPases, through a conserved, perinuclear domain. We determined the crystal structure of the perinuclear domain of human LAP1. LAP1 possesses an atypical AAA+ fold. While LAP1 lacks canonical nucleotide binding motifs, its strictly conserved arginine 563 is positioned exactly where the arginine finger of canonical AAA+ ATPases is found. Based on modeling and electron microscopic analysis, we propose that LAP1 targets Torsin to the nuclear envelope by forming an alternating, heterohexameric (LAP1-Torsin)3 ring, in which LAP1 acts as the Torsin activator. The experimental data show that mutation of arginine 563 in LAP1 reduces its ability to stimulate TorsinA ATPase hydrolysis. This knowledge may help scientists understand the etiology of DYT1 primary dystonia, a movement disorder caused by a single glutamate deletion in TorsinA. DOI: http://dx.doi.org/10.7554/eLife.03239.001 PMID:25149450

  20. Prevention of Pleural Adhesions by Bioactive Polypeptides - A Pilot Study

    PubMed Central

    Åkerberg, D.; Posaric-Bauden, M.; Isaksson, K.; Andersson, R.; Tingstedt, B.

    2013-01-01

    Objective: Postoperative pleural adhesions lead to major problems in repeated thoracic surgery. To date, no antiadhesive product has been proven clinically effective. Previous studies of differently charged polypeptides, poly-L-lysine (PL) and poly-L-glutamate (PG) have shown promising results reducing postoperative abdominal adhesions in experimental settings. This pilot study examined the possible pleural adhesion prevention by using the PL+PG concept after pleural surgery and its possible effect on key parameters; plasmin activator inhibitor-1 (PAI-1) and tissue growth factor beta 1 (TGFb) in the fibrinolytic process. Methods: A total of 22 male rats were used in the study, one control group (n=10) and one experimental group (n=12). All animals underwent primary pleural surgery, the controls receiving saline in the pleural cavity and the experimental group the PL+PG solution administered by spray. The animals were evaluated on day 7. Macroscopic appearance of adhesions was evaluated by a scoring system. Histology slides of the adhesions and pleural biopsies for evaluation of PAI-1 and TGFb1 were taken on day 7. Results: A significant reduction of adhesions in the PL+PG group (p<0.05) was noted at day 7 both regarding the length and severity of adhesions. There were no significant differences in the concentration of PAI-1 and TGFb1 when comparing the two groups. Conclusions: PL+PG may be used to prevent pleural adhesions. The process of fibrinolysis, and fibrosis was though not affected after PLPG administration. PMID:24151443

  1. Polypeptide A9K at nanoscale carbon: a simulation study.

    PubMed

    Chaban, Vitaly V; Arruda, Andre; Fileti, Eudes Eterno

    2015-10-21

    The amphiphilic nature of surfactant-like peptides is responsible for their propensity to aggregate at the nanoscale. These peptides can be readily used for a non-covalent functionalization of nanoparticles and macromolecules. This work reports an observation of supramolecular ensembles consisting of ultrashort carbon nanotubes (USCNTs), graphene (GR) and A9K polypeptides formed by lysine and arginine. The potential of mean force (PMF) is used as a major descriptor of the CNT-A9K and GR-A9K binding process, supplementing structural data. The phase space sampling is performed by multiple equilibrium molecular dynamics simulations with position restraints, where applicable. Binding in all cases was found to be thermodynamically favorable. Encapsulation in the (10,10) USCNT is particularly favorable. The curvature of the external surface does not favor binding. Thus, binding of A9K at GR is stronger than its binding at the outer sidewall of USCNTs. Overall, the presented results favor non-covalent functionalization of nanoscale carbons that are considered interesting in the fields of biomaterials, biosensors, biomedical devices, and drug delivery systems.

  2. Coarse-grained, foldable, physical model of the polypeptide chain

    PubMed Central

    Chakraborty, Promita; Zuckermann, Ronald N.

    2013-01-01

    Although nonflexible, scaled molecular models like Pauling–Corey’s and its descendants have made significant contributions in structural biology research and pedagogy, recent technical advances in 3D printing and electronics make it possible to go one step further in designing physical models of biomacromolecules: to make them conformationally dynamic. We report here the design, construction, and validation of a flexible, scaled, physical model of the polypeptide chain, which accurately reproduces the bond rotational degrees of freedom in the peptide backbone. The coarse-grained backbone model consists of repeating amide and α-carbon units, connected by mechanical bonds (corresponding to φ and ψ) that include realistic barriers to rotation that closely approximate those found at the molecular scale. Longer-range hydrogen-bonding interactions are also incorporated, allowing the chain to readily fold into stable secondary structures. The model is easily constructed with readily obtainable parts and promises to be a tremendous educational aid to the intuitive understanding of chain folding as the basis for macromolecular structure. Furthermore, this physical model can serve as the basis for linking tangible biomacromolecular models directly to the vast array of existing computational tools to provide an enhanced and interactive human–computer interface. PMID:23898168

  3. Stimulation of growth hormone by vasoactive intestinal polypeptide in acromegaly.

    PubMed

    Chihara, K; Kaji, H; Minamitani, N; Kodama, H; Kita, T; Goto, B; Chiba, T; Coy, D H; Fujita, T

    1984-01-01

    Vasoactive intestinal polypeptide (VIP) was administered as an iv bolus of 1 micrograms/kg BW to 8 acromegalic patients and in doses of 0.5 and 1 microgram/kg BW to 15 normal volunteers. Both systolic and diastolic blood pressures decreased, and pulse rate increased transiently after VIP injection. VIP stimulated PRL release from the anterior pituitary in normal subjects. Plasma PRL responses to VIP in women were dose dependent and larger than those in men. On the other hand, plasma GH levels rose markedly after VIP injection in all 6 patients with untreated acromegaly. In 2 patients studied after transsphenoidal microadenomectomy, there was no plasma GH response to VIP. In 2 other patients with inactive acromegaly as well as in normal subjects, VIP failed to affect plasma GH levels. In all 6 patients with active acromegaly, LRH (1-2 micrograms/kg BW, iv) did not increase plasma GH levels, but TRH (5-10 micrograms/kg BW, iv) caused significant increases in plasma GH, the magnitude of which was not similar to that of increases seen after VIP injection. Paradoxical GH responses to TRH were not observed in patients in the inactive phase after transsphenoidal surgery. These findings suggest that VIP stimulates GH release in vivo in acromegalic patients. A VIP test as well as a TRH test offer promise as simple and reliable techniques to evaluate the activity of acromegaly, particularly after transsphenoidal surgery.

  4. Fiber-Dependent and -Independent Toxicity of Islet Amyloid Polypeptide

    PubMed Central

    Schlamadinger, Diana E.; Miranker, Andrew D.

    2014-01-01

    The 37-residue peptide hormone islet amyloid polypeptide (IAPP) plays a central role in diabetes pathology. Although its amyloid fiber aggregation kinetics and cytotoxicity to β-cells are well documented, few reports have directly assessed the role of fibers in cell-based toxicity experiments. Here, we report that amyloid formation of IAPP can be strongly inhibited by the extracellular environment of live cells. For example, fiber formation is more strongly suppressed in cell culture medium than in aqueous buffer. The serum component of the medium is responsible for this inhibition. Although amyloid formation was previously shown to be catalyzed by both synthetic and chloroform-extracted phospholipid surfaces, it is instead inhibited by membrane surfaces prepared directly from the plasma membranes of an immortal β-cell line. This disparity is reconciled by direct assessment of fibers in cell-culture-based toxicity experiments. We discovered that fibers are nontoxic if they are washed free of adsorbed nonfibrillar components. Moreover, toxicity is not only rescued when monomers are added back to fibers but is greater than what is observed from the precursor alone. Our results are interpreted in light of the capacity of the fiber surface to template amyloid nucleation. PMID:25468335

  5. Free radical scavenging abilities of polypeptide from Chlamys farreri

    NASA Astrophysics Data System (ADS)

    Han, Zhiwu; Chu, Xiao; Liu, Chengjuan; Wang, Yuejun; Mi, Sun; Wang, Chunbo

    2006-09-01

    We investigated the radical scavenging effect and antioxidation property of polypeptide extracted from Chlamys farreri (PCF) in vitro using chemiluminescence and electron spin resonance (ESR) methods. We examined the scavenging effects of PCF on superoxide anions (O{2/-}), hydroxyl radicals (OH·), peroxynitrite (ONOO-) and the inhibiting capacity of PCF on peroxidation of linoleic acid. Our experiment suggested that PCF could scavenge oxygen free radicals including superoxide anions (O{2/-}) (IC50=0.3 mg/ml), hydroxyl radicals (OH·) (IC50=0.2 μg/ml) generated from the reaction systems and effectively inhibit the oxidative activity of ONOO- (IC50=0.2 mg/ml). At 1.25 mg/ml of PCF, the inhibition ratio on lipid peroxidation of linoleic acid was 43%. The scavenging effect of PCF on O{2/-}, OH· and ONOO- free radicals were stronger than those of vitamin C but less on lipid peroxidation of linoleic acid. Thus PCF could scavenge free radicals and inhibit the peroxidation of linoleic acid in vitro. It is an antioxidant from marine products and potential for industrial production in future.

  6. Superantigen-Like Effects of a Candida albicans Polypeptide

    PubMed Central

    Devore-Carter, Denise; Kar, Sujata; Vellucci, Vincent; Bhattacherjee, Vasker; Domanski, Paul; Hostetter, Margaret K.

    2008-01-01

    The amino terminal sequence of the Candida albicans cell wall protein Int1 exhibited partial identity with the major histocompatibility complex (MHC) class II binding site of the Mycoplasma arthritidis superantigen MAM. Int1-positive C. albicans blastospores activated human T lymphocytes and expanded Vβ subsets 2, 3, and/or 14; Int1-negative strains were inactive. Release of interferon-γ (IFN-γ) but not of tumor necrosis factor–α or interleukin-6 was Int1 dependent; interleukin-4 and interleukin-10 were not detected. T lymphocyte activation, Vβ expansion, and IFN-γ release were associated with a soluble polypeptide that encompassed the first 263 amino acids of Int1 (Pep263). Monoclonal antibody 163.5, which recognizes an Int1 epitope that overlaps the region of identity with MAM, significantly inhibited these activities when triggered by Int1-positive blastospores or Pep263 but not by staphylococcal enterotoxin B. Histidine263 was required. Pep263 bound to T lymphocytes and MHC class II and was detected in the urine of a patient with C. albicans fungemia. These studies identify a candidal protein that displays superantigen-like activities. PMID:18419534

  7. Fibril Structure of Human Islet Amyloid Polypeptide*♦

    PubMed Central

    Bedrood, Sahar; Li, Yiyu; Isas, J. Mario; Hegde, Balachandra G.; Baxa, Ulrich; Haworth, Ian S.; Langen, Ralf

    2012-01-01

    Misfolding and amyloid fibril formation by human islet amyloid polypeptide (hIAPP) are thought to be important in the pathogenesis of type 2 diabetes, but the structures of the misfolded forms remain poorly understood. Here we developed an approach that combines site-directed spin labeling with continuous wave and pulsed EPR to investigate local secondary structure and to determine the relative orientation of the secondary structure elements with respect to each other. These data indicated that individual hIAPP molecules take up a hairpin fold within the fibril. This fold contains two β-strands that are much farther apart than expected from previous models. Atomistic structural models were obtained using computational refinement with EPR data as constraints. The resulting family of structures exhibited a left-handed helical twist, in agreement with the twisted morphology observed by electron microscopy. The fibril protofilaments contain stacked hIAPP monomers that form opposing β-sheets that twist around each other. The two β-strands of the monomer adopt out-of-plane positions and are staggered by about three peptide layers (∼15 Å). These results provide a mechanism for hIAPP fibril formation and could explain the remarkable stability of the fibrils. Thus, the structural model serves as a starting point for understanding and preventing hIAPP misfolding. PMID:22187437

  8. Extended structure of rat islet amyloid polypeptide in solution.

    PubMed

    Wei, Lei; Jiang, Ping; Manimekalai, Malathy Sony Subramanian; Hunke, Cornelia; Grüber, Gerhard; Pervushin, Konstantin; Mu, Yuguang

    2015-01-01

    The process of islet amyloid polypeptide (IAPP) formation and the prefibrillar oligomers are supposed to be one of the pathogenic agents causing pancreatic β-cell dysfunction. The human IAPP (hIAPP) aggregates easily and therefore, it is difficult to characterize its structural features by standard biophysical tools. The rat version of IAPP (rIAPP) that differs by six amino acids when compared with hIAPP, is not prone to aggregation and does not form amyloid fibrils. Similar to hIAPP it also demonstrates random-coiled nature in solution. The structural propensity of rIAPP has been studied as a hIAPP mimic in recent works. However, the overall shape of it in solution still remains elusive. Using small angle X-ray scattering (SAXS) measurements combined with nuclear magnetic resonance (NMR) and molecular dynamics simulations (MD) the solution structure of rIAPP was studied. An unambiguously extended structural model with a radius of gyration of 1.83 nm was determined from SAXS data. Consistent with previous studies, an overall random-coiled feature with residual helical propensity in the N-terminus was confirmed. Combined efforts are necessary to unambiguously resolve the structural features of intrinsic disordered proteins.

  9. Distribution of yolk polypeptides in the follicle cells during the differentiation of the follicular epithelium in Sarcophaga bullata egg follicles.

    PubMed

    Geysen, J; Cardoen, J; De Loof, A

    1987-09-01

    In S. bullata, the ovaries contribute to the synthesis of yolk polypeptides. A specific antiserum for yolk polypeptides was used to visualize the presence of yolk polypeptides in the follicle cells during their differentiation. After vitellogenesis has started, all follicle cells contain yolk polypeptides. The squamous follicle cells covering the nurse cells and the border cells lose yolk polypeptides before mid-vitellogenesis, whereas the follicle cells over the oocyte contain yolk polypeptides until after late vitellogenesis. All follicle cells are immunonegative afterwards. In vitro translation of poly(A)+ RNA demonstrated that the presence of yolk polypeptide mRNA correlates well with follicle cell immunopositivity for yolk polypeptides. This suggests that the follicle cells synthesize the ovarian yolk polypeptides. Differences in cellular and nuclear morphology, total and poly(A)+ RNA synthesis and the rate of yolk polypeptide synthesis were shown to be correlated with the presence or absence of yolk polypeptides in the differentiating follicular epithelium. The possible relationship between these different aspects of follicle cell differentiation, follicle cell polyploidy and the extracellular current pattern around follicles are discussed.

  10. Tuning calcium carbonate growth through physical confinement and templating with amyloid-like polypeptide aggregates

    NASA Astrophysics Data System (ADS)

    Colaco, Martin Francis

    that this methodology does not extend to three-dimensional confined systems, as the water has no method of escape. Through the addition of an insoluble hydroscopic polymer to our microreactors, amorphous calcium carbonate of controllable sizes can be grown. However, crystalline calcium carbonate cannot be grown without some type of templating. Studies of calcium carbonate templating have predominantly been performed on SAMs or in poorly characterized gels or protein films. The use of ordered protein or polypeptide aggregates for templating permits both geometry and charge surface density to be varied. We have studied the kinetics and final morphology of ordered aggregates of poly-L-glutamic acid and a copolymer of glutamic acid and alanine through experiments and simulations. Electrostatics, not structure, of the monomer appeared to be the dominating factor in the aggregation, as pH and salt concentration changes led to dramatic changes in the kinetics. Examining our experimental with existing models provided inconsistent results, so we developed a new model that yielded physically realistic rate constants, while generating better fits with longer lag phases and faster growths. However, despite the similarity of aggregation conditions, the two polypeptides yielded vastly different morphologies, with the PEA forming typical amyloid-like fibrils and PE forming larger, twisted lamellar aggregates. Templating with these aggregates also yielded dramatically different patterns. Polycrystalline rhombohedral calcite with smooth faces and edges grew on PEA fibrils, with minimal templating in evidence. However, on PE, numerous calcite crystals with triangular projections tracked the surface of the aggregate. The PE lamellae are characterized by extensive beta-sheet structure. In this conformation, the glutamic acid spacings on the surface of the aggregates can mimic the spacings of the carboxylates in the calcite lattice. In addition, the high negative charge density on the

  11. Simultaneous Polymerization and Polypeptide Particle Production via Reactive Spray-Drying.

    PubMed

    Glavas, Lidija; Odelius, Karin; Albertsson, Ann-Christine

    2016-09-12

    A method for producing polypeptide particles via in situ polymerization of N-carboxyanhydrides during spray-drying has been developed. This method was enabled by the development of a fast and robust synthetic pathway to polypeptides using 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) as an initiator for the ring-opening polymerization of N-carboxyanhydrides. The polymerizations finished within 5 s and proved to be very tolerant toward impurities such as amino acid salts and water. The formed particles were prepared by mixing the monomer, N-carboxyanhydride of l-glutamic acid benzyl ester (NCAGlu) and the initiator (DBU) during the atomization process in the spray-dryer and were spherical with a size of ∼1 μm. This method combines two steps; making it a straightforward process that facilitates the production of polypeptide particles. Hence, it furthers the use of spray-drying and polypeptide particles in the pharmaceutical industry.

  12. Theoretical investigations on model ternary polypeptides using genetic algorithm—Some new results

    NASA Astrophysics Data System (ADS)

    Arora, Vinita; Bakhshi, A. K.

    2011-04-01

    Using genetic algorithm (GA) model ternary polypeptides containing glycine, alanine and serine in β-pleated conformation have been theoretically investigated. In designing, the criterion to attain the optimum solution at the end of GA run is minimum band gap and maximum delocalization in the polypeptide chain. Ab initio results obtained using Clementi's minimal basis set are used as input. Effects of (i) change of basis set from minimal to double zeta, (ii) change in secondary structure from β-pleated to α-helical, (iii) presence of solvation shell and (iv) binding of H + and Li + ions to peptide group on the resulting solution as well as on electronic structure and conduction properties of polypeptides are investigated. A comparison is drawn between results obtained for the two cationic adducts. The protonated adduct is expected to withdraw more negative charge from the polypeptide chain due to smaller size of H + and is found to have high electron affinity compared to Li + adduct.

  13. Papain-Catalyzed Chemoenzymatic Synthesis of Telechelic Polypeptides Using Bis(Leucine Ethyl Ester) Initiator.

    PubMed

    Tsuchiya, Kousuke; Numata, Keiji

    2016-07-01

    In order to construct unique polypeptide architectures, a novel telechelic-type initiator with two leucine ethyl ester units is designed for chemoenzymatic polymerization. Glycine or alanine ethyl ester is chemoenzymatically polymerized using papain in the presence of the initiator, and the propagation occurs at each leucine ethyl ester unit to produce the telechelic polypeptide. The formation of the telechelic polypeptides is confirmed by (1) H NMR and MALDI-TOF mass spectroscopies. It is revealed by AFM observation that long nanofibrils are formed from the telechelic polyalanine, whereas a conventional linear polyalanine with a similar degree of polymerization shows granule-like structures. The telechelic polyglycine and polyalanine show the crystalline structures of Polyglycine II and antiparallel β-sheet, respectively. It is demonstrated that this method to synthesize telechelic-type polypeptides potentially opens up a pathway to construct novel hierarchical structures by self-assembly. PMID:26947148

  14. Neutralizing determinants defined by monoclonal antibodies on polypeptides specified by bovine herpesvirus 1.

    PubMed Central

    Collins, J K; Butcher, A C; Riegel, C A; McGrane, V; Blair, C D; Teramoto, Y A; Winston, S

    1984-01-01

    Monoclonal antibodies were used to study neutralizing determinants on polypeptides of bovine herpesvirus 1. Two of three monoclonal antibodies which recognized nonoverlapping epitopes on a glycoprotein of 82,000 daltons were found to neutralize. A second group of monoclonal antibodies that individually precipitated five viral glycopolypeptides ranging in size from 102,000 to 55,000 daltons also neutralized. Two monoclonal antibodies which were the most efficient in neutralization recognized a non-glycosylated protein of 115,000 daltons which was the major polypeptide on the virus. A fourth group of monoclonal antibodies precipitated a non-glycosylated polypeptide of 91,000 daltons and several smaller polypeptides, but these antibodies demonstrated only limited neutralizing activity. Images PMID:6208375

  15. Biosynthesis of metal-binding polypeptides and their precursors in response to cadmium in Datura innoxia

    SciTech Connect

    Jackson, P.J.; Delhaize, E.; Kuske, C.R.

    1991-01-01

    Metal-tolerant Datura innoxia cells synthesize large amounts of a class of metal-binding polypeptides, poly({gamma}-glutamylcysteinyl) glycines (({gamma}-EC){sub n}G, n=2-5), when exposed to Cd. These polypeptides have a high affinity for Cd (2) and certain other metal ions and are thought to play a role in metal tolerance in higher plants. ({gamma}-EC){sub n}G is biosynthetically derived from glutathione. Therefore, the response of Datura cells to Cd must include an increase in production of glutathione and its precursors, since cells rapidly accumulate very high concentrations of these metal-binding polypeptides. The biosynthesis of ({gamma}-EC){sub n}Gs, glutathione, and cysteine in response to Cd exposure is described. The physiological significance of the synthesis of these polypeptides and their precursors and its relevance to Cd tolerance and metal homeostasis are discussed. 34 refs., 6 figs., 1 tab.

  16. Vasoactive Intestinal Polypeptide and Muscarinic Receptors: Supersensitivity Induced by Long-Term Atropine Treatment

    NASA Astrophysics Data System (ADS)

    Hedlund, Britta; Abens, Janis; Bartfai, Tamas

    1983-04-01

    Long-term treatment of rats with atropine induced large increases in the numbers of muscarinic receptors and receptors for vasoactive intestinal polypeptide in the salivary glands. Since receptors for vasoactive intestinal polypeptide coexist with muscarinic receptors on the same neurons in this preparation, the results suggest that a drug that alters the sensitivity of one receptor may also affect the sensitivity of the receptor for a costored transmitter and in this way contribute to the therapeutic or side effects of the drug.

  17. EXPRESSED PROTEIN LIGATION. A NEW TOOL FOR THE BIOSYNTHESIS OF CYCLIC POLYPEPTIDES

    SciTech Connect

    Kimura, R; Camarero, J A

    2004-11-11

    The present paper reviews the use of expressed protein ligation for the biosynthesis of backbone cyclic polypeptides. This general method allows the in vivo and in vitro biosynthesis of cyclic polypeptides using recombinant DNA expression techniques. Biosynthetic access to backbone cyclic peptides opens the possibility to generate cell-based combinatorial libraries that can be screened inside living cells for their ability to attenuate or inhibit cellular processes.

  18. Metal ion-dependent, reversible, protein filament formation by designed beta-roll polypeptides

    PubMed Central

    Scotter, Andrew J; Guo, Meng; Tomczak, Melanie M; Daley, Margaret E; Campbell, Robert L; Oko, Richard J; Bateman, David A; Chakrabartty, Avijit; Sykes, Brian D; Davies, Peter L

    2007-01-01

    Background A right-handed, calcium-dependent β-roll structure found in secreted proteases and repeat-in-toxin proteins was used as a template for the design of minimal, soluble, monomeric polypeptides that would fold in the presence of Ca2+. Two polypeptides were synthesised to contain two and four metal-binding sites, respectively, and exploit stacked tryptophan pairs to stabilise the fold and report on the conformational state of the polypeptide. Results Initial analysis of the two polypeptides in the presence of calcium suggested the polypeptides were disordered. The addition of lanthanum to these peptides caused aggregation. Upon further study by right angle light scattering and electron microscopy, the aggregates were identified as ordered protein filaments that required lanthanum to polymerize. These filaments could be disassembled by the addition of a chelating agent. A simple head-to-tail model is proposed for filament formation that explains the metal ion-dependency. The model is supported by the capping of one of the polypeptides with biotin, which disrupts filament formation and provides the ability to control the average length of the filaments. Conclusion Metal ion-dependent, reversible protein filament formation is demonstrated for two designed polypeptides. The polypeptides form filaments that are approximately 3 nm in diameter and several hundred nm in length. They are not amyloid-like in nature as demonstrated by their behaviour in the presence of congo red and thioflavin T. A capping strategy allows for the control of filament length and for potential applications including the "decoration" of a protein filament with various functional moieties. PMID:17908326

  19. New Kunitz-Type HCRG Polypeptides from the Sea Anemone Heteractis crispa.

    PubMed

    Gladkikh, Irina; Monastyrnaya, Margarita; Zelepuga, Elena; Sintsova, Oksana; Tabakmakher, Valentin; Gnedenko, Oksana; Ivanov, Alexis; Hua, Kuo-Feng; Kozlovskaya, Emma

    2015-10-01

    Sea anemones are a rich source of Kunitz-type polypeptides that possess not only protease inhibitor activity, but also Kv channels toxicity, analgesic, antihistamine, and anti-inflammatory activities. Two Kunitz-type inhibitors belonging to a new Heteractis crispa RG (HCRG) polypeptide subfamily have been isolated from the sea anemone Heteractis crispa. The amino acid sequences of HCRG1 and HCRG2 identified using the Edman degradation method share up to 95% of their identity with the representatives of the HCGS polypeptide multigene subfamily derived from H. crispa cDNA. Polypeptides are characterized by positively charged Arg at the N-terminus as well as P1 Lys residue at their canonical binding loop, identical to those of bovine pancreatic trypsin inhibitor (BPTI). These polypeptides are shown by our current evidence to be more potent inhibitors of trypsin than the known representatives of the HCGS subfamily with P1Thr. The kinetic and thermodynamic characteristics of the intermolecular interactions between inhibitors and serine proteases were determined by the surface plasmon resonance (SPR) method. Residues functionally important for polypeptide binding to trypsin were revealed using molecular modeling methods. Furthermore, HCRG1 and HCRG2 possess anti-inflammatory activity, reducing tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6) secretions, as well as proIL-1β expression in lipopolysaccharide (LPS)-activated macrophages. However, there was no effect on nitric oxide (NO) generation. PMID:26404319

  20. Two small virus-specific polypeptides are produced during infection with Sindbis virus.

    PubMed Central

    Welch, W J; Sefton, B M

    1979-01-01

    We have identified and characterized two small virus-specific polypeptides which are produced during infection of cells with Sindbis virus, but which are not incorporated into the mature virion. The larger of these is a glycoprotein with an approximate molecular weight of 9,800 and is found predominantly in the medium of infected cells. Three independent lines of evidence demonstrate conclusively that this 9,800-dalton glycoprotein is produced during the proteolytic conversion of the precursor polypeptide, PE2, to the virion glycoprotein E2. This small glycoprotein is therefore analogous to the virion glycoprotein E3 of the very closely related alphavirus, Semliki Forest virus. The 9,800-dalton glycoprotein of Sindbis virus, unlike the E3 glycoprotein of Semliki Forest virus, is not, however, present in the viral particle. The other virus-specific polypeptide is 4,200 daltons in size, does not appear to be a glycoprotein, and is neither incorporated into the mature virus nor released into the culture medium. The gene for this small polypeptide is present in the viral 26S mRNA (the mRNA which encodes all the viral structural polypeptides) and appears to be located in the portion of the mRNA which encodes the two viral glycoproteins. The possibility that this 4,200-dalton polypeptide functions as a signal peptide during the synthesis of the viral membrane glycoproteins is discussed. Images PMID:448798

  1. New Kunitz-Type HCRG Polypeptides from the Sea Anemone Heteractis crispa

    PubMed Central

    Gladkikh, Irina; Monastyrnaya, Margarita; Zelepuga, Elena; Sintsova, Oksana; Tabakmakher, Valentin; Gnedenko, Oksana; Ivanov, Alexis; Hua, Kuo-Feng; Kozlovskaya, Emma

    2015-01-01

    Sea anemones are a rich source of Kunitz-type polypeptides that possess not only protease inhibitor activity, but also Kv channels toxicity, analgesic, antihistamine, and anti-inflammatory activities. Two Kunitz-type inhibitors belonging to a new Heteractis crispa RG (HCRG) polypeptide subfamily have been isolated from the sea anemone Heteractis crispa. The amino acid sequences of HCRG1 and HCRG2 identified using the Edman degradation method share up to 95% of their identity with the representatives of the HCGS polypeptide multigene subfamily derived from H. crispa cDNA. Polypeptides are characterized by positively charged Arg at the N-terminus as well as P1 Lys residue at their canonical binding loop, identical to those of bovine pancreatic trypsin inhibitor (BPTI). These polypeptides are shown by our current evidence to be more potent inhibitors of trypsin than the known representatives of the HCGS subfamily with P1Thr. The kinetic and thermodynamic characteristics of the intermolecular interactions between inhibitors and serine proteases were determined by the surface plasmon resonance (SPR) method. Residues functionally important for polypeptide binding to trypsin were revealed using molecular modeling methods. Furthermore, HCRG1 and HCRG2 possess anti-inflammatory activity, reducing tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6) secretions, as well as proIL-1β expression in lipopolysaccharide (LPS)-activated macrophages. However, there was no effect on nitric oxide (NO) generation. PMID:26404319

  2. Algorithm to design inhibitors using stereochemically mixed l,d polypeptides: Validation against HIV protease.

    PubMed

    Gupta, Pooja; Durani, Susheel

    2015-11-01

    Polypeptides have potential to be designed as drugs or inhibitors against the desired targets. In polypeptides, every chiral α-amino acid has enantiomeric structural possibility to become l or d amino acids and can be used as design monomer. Among the various possibilities, use of stereochemistry as a design tool has potential to determine both functional specificity and metabolic stability of the designed polypeptides. The polypeptides with mixed l,d amino acids are a class of peptidomimitics, an attractive drug like molecules and also less susceptible to proteolytic activities. Therefore in this study, a three step algorithm is proposed to design the polypeptides against desired drug targets. For this, all possible configurational isomers of mixed l,d polyleucine (Ac-Leu8-NHMe) structure were randomly modeled with simulated annealing molecular dynamics and the resultant library of discrete folds were scored against HIV protease as a model target. The best scored folds of mixed l,d structures were inverse optimized for sequences in situ and the resultant sequences as inhibitors were validated for conformational integrity using molecular dynamics. This study presents and validates an algorithm to design polypeptides of mixed l,d structures as drugs/inhibitors by inverse fitting them as molecular ligands against desired target.

  3. Global profiling of SRP interaction with nascent polypeptides.

    PubMed

    Schibich, Daniela; Gloge, Felix; Pöhner, Ina; Björkholm, Patrik; Wade, Rebecca C; von Heijne, Gunnar; Bukau, Bernd; Kramer, Günter

    2016-08-11

    Signal recognition particle (SRP) is a universally conserved protein-RNA complex that mediates co-translational protein translocation and membrane insertion by targeting translating ribosomes to membrane translocons. The existence of parallel co- and post-translational transport pathways, however, raises the question of the cellular substrate pool of SRP and the molecular basis of substrate selection. Here we determine the binding sites of bacterial SRP within the nascent proteome of Escherichia coli at amino acid resolution, by sequencing messenger RNA footprints of ribosome-nascent-chain complexes associated with SRP. SRP, on the basis of its strong preference for hydrophobic transmembrane domains (TMDs), constitutes a compartment-specific targeting factor for nascent inner membrane proteins (IMPs) that efficiently excludes signal-sequence-containing precursors of periplasmic and outer membrane proteins. SRP associates with hydrophobic TMDs enriched in consecutive stretches of hydrophobic and bulky aromatic amino acids immediately on their emergence from the ribosomal exit tunnel. By contrast with current models, N-terminal TMDs are frequently skipped and TMDs internal to the polypeptide sequence are selectively recognized. Furthermore, SRP binds several TMDs in many multi-spanning membrane proteins, suggesting cycles of SRP-mediated membrane targeting. SRP-mediated targeting is not accompanied by a transient slowdown of translation and is not influenced by the ribosome-associated chaperone trigger factor (TF), which has a distinct substrate pool and acts at different stages during translation. Overall, our proteome-wide data set of SRP-binding sites reveals the underlying principles of pathway decisions for nascent chains in bacteria, with SRP acting as the dominant triaging factor, sufficient to separate IMPs from substrates of the SecA-SecB post-translational translocation and TF-assisted cytosolic protein folding pathways. PMID:27487212

  4. Quantifying polypeptide conformational space: sensitivity to conformation and ensemble definition.

    PubMed

    Sullivan, David C; Lim, Carmay

    2006-08-24

    Quantifying the density of conformations over phase space (the conformational distribution) is needed to model important macromolecular processes such as protein folding. In this work, we quantify the conformational distribution for a simple polypeptide (N-mer polyalanine) using the cumulative distribution function (CDF), which gives the probability that two randomly selected conformations are separated by less than a "conformational" distance and whose inverse gives conformation counts as a function of conformational radius. An important finding is that the conformation counts obtained by the CDF inverse depend critically on the assignment of a conformation's distance span and the ensemble (e.g., unfolded state model): varying ensemble and conformation definition (1 --> 2 A) varies the CDF-based conformation counts for Ala(50) from 10(11) to 10(69). In particular, relatively short molecular dynamics (MD) relaxation of Ala(50)'s random-walk ensemble reduces the number of conformers from 10(55) to 10(14) (using a 1 A root-mean-square-deviation radius conformation definition) pointing to potential disconnections in comparing the results from simplified models of unfolded proteins with those from all-atom MD simulations. Explicit waters are found to roughen the landscape considerably. Under some common conformation definitions, the results herein provide (i) an upper limit to the number of accessible conformations that compose unfolded states of proteins, (ii) the optimal clustering radius/conformation radius for counting conformations for a given energy and solvent model, (iii) a means of comparing various studies, and (iv) an assessment of the applicability of random search in protein folding.

  5. Herpes simplex virus mutants defective in the virion-associated shutoff of host polypeptide synthesis and exhibiting abnormal synthesis of alpha (immediate early) viral polypeptides.

    PubMed

    Read, G S; Frenkel, N

    1983-05-01

    Six mutants isolated from herpes simplex virus type 1 were judged to be defective with respect to the virion-associated function acting to rapidly shut off host polypeptide synthesis in herpes simplex virus-infected cells. The mutants were capable of proper entry into the cells, but, unlike the parent wild-type virus, they failed to shut off host polypeptide syntehsis in the presence of actinomycin D. They were consequently designated as virion-associated host shutoff (vhs) mutants. In the presence of actinomycin D, three of the mutants, vhs1, -2, and -3, failed to shut off the host at both 34 and 39 degrees C, whereas vhs4, -5, and -6 exhibited a temperature-dependent vhs phenotype. Since the mutants were capable of growth at 34 degrees C, it appeared that the vhs function was not essential for virus replication in cultured cells. Temperature-shift experiments performed with the vhs4 mutant showed that an active vhs function was required throughout the shutoff process and that, once established, the translational shutoff could not be reversed. In the absence of actinomycin D, the mutants induced a generalized, secondary shutoff of host translation, which required the synthesis of beta (early) or gamma (late) viral polypeptide(s). The vhs mutants appeared to be defective also with respect to post-transcriptional shutoff of alpha (immediate early) viral gene expression, since (i) cells infected with mutant viruses overproduced alpha viral polypeptides, (ii) there was an increased functional stability of alpha mRNA in the vhs1 mutant virus-infected cells, and (iii) superinfection of vhs1-infected cells with wild-type virus, in the presence of actinomycin D, resulted in a more pronounced shutoff of alpha polypeptide synthesis from preformed alpha mRNA than equivalent superinfection with vhs1 virus. The data suggest that the synthesis of alpha polypeptides in wild-type virus infections is subject to a negative post-transcriptional control involving viral gene product

  6. Allosteric opening of the polypeptide-binding site when an Hsp70 binds ATP

    PubMed Central

    Qi, Ruifeng; Sarbeng, Evans Boateng; Liu, Qun; Le, Katherine Quynh; Xu, Xinping; Xu, Hongya; Yang, Jiao; Wong, Jennifer Li; Vorvis, Christina; Hendrickson, Wayne A.; Zhou, Lei; Liu, Qinglian

    2013-01-01

    The 70kD heat shock proteins (Hsp70s) are ubiquitous molecular chaperones essential for cellular protein folding and proteostasis. Each Hsp70 has two functional domains: a nucleotide-binding domain (NBD) that binds and hydrolyzes ATP, and a substrate-binding domain (SBD) that binds extended polypeptides. NBD and SBD interact little when in ADP; however, ATP binding allosterically couples the polypeptide- and ATP-binding sites. ATP binding promotes polypeptide release; polypeptide rebinding stimulates ATP hydrolysis. This allosteric coupling is poorly understood. Here we present the crystal structure of an intact Hsp70 from Escherichia coli in an ATP-bound state at 1.96 Å resolution. NBD-ATP adopts a unique conformation, forming extensive interfaces with a radically changed SBD that has its α-helical lid displaced and the polypeptide-binding channel of its β-subdomain restructured. These conformational changes together with our biochemical tests provide a long-sought structural explanation for allosteric coupling in Hsp70 activity. PMID:23708608

  7. Congenital deficiency of two polypeptide subunits of the iron-protein fragment of mitochondrial complex I.

    PubMed

    Moreadith, R W; Cleeter, M W; Ragan, C I; Batshaw, M L; Lehninger, A L

    1987-02-01

    Recently, we described a patient with severe lactic acidosis due to congenital complex I (NADH-ubiquinone oxidoreductase) deficiency. We now report further enzymatic and immunological characterizations. Both NADH and ferricyanide titrations of complex I activity (measured as NADH-ferricyanide reductase) were distinctly altered in the mitochondria from the patient's tissues. In addition, antisera against complex I immunoprecipitated NADH-ferricyanide reductase from the control but not the patient's mitochondria. However, immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of complex I polypeptides demonstrated that the majority of the 25 polypeptides comprising complex I were present in the affected mitochondria. A more detailed analysis using subunit selective antisera against the main polypeptides of the iron-protein fragments of complex I revealed a selective absence of the 75- and 13-kD polypeptides. These findings suggest that the underlying basis for this patient's disease was a congenital deficiency of at least two polypeptides comprising the iron-protein fragment of complex I, which resulted in the inability to correctly assemble a functional enzyme complex.

  8. Competition between surface adsorption and folding of fibril-forming polypeptides

    NASA Astrophysics Data System (ADS)

    Ni, Ran; Kleijn, J. Mieke; Abeln, Sanne; Cohen Stuart, Martien A.; Bolhuis, Peter G.

    2015-02-01

    Self-assembly of polypeptides into fibrillar structures can be initiated by planar surfaces that interact favorably with certain residues. Using a coarse-grained model, we systematically studied the folding and adsorption behavior of a β -roll forming polypeptide. We find that there are two different folding pathways depending on the temperature: (i) at low temperature, the polypeptide folds in solution into a β -roll before adsorbing onto the attractive surface; (ii) at higher temperature, the polypeptide first adsorbs in a disordered state and folds while on the surface. The folding temperature increases with increasing attraction as the folded β -roll is stabilized by the surface. Surprisingly, further increasing the attraction lowers the folding temperature again, as strong attraction also stabilizes the adsorbed disordered state, which competes with folding of the polypeptide. Our results suggest that to enhance the folding, one should use a weakly attractive surface. They also explain the recent experimental observation of the nonmonotonic effect of charge on the fibril formation on an oppositely charged surface [C. Charbonneau et al., ACS Nano 8, 2328 (2014), 10.1021/nn405799t].

  9. Residues in chaperonin GroEL required for polypeptide binding and release.

    PubMed

    Fenton, W A; Kashi, Y; Furtak, K; Horwich, A L

    1994-10-13

    Chaperonins are ring-shaped protein complexes that are essential in the cell, mediating ATP-dependent polypeptide folding in a variety of compartments. Recent studies suggest that they function through multiple rounds of binding and release of non-native proteins: with each round of ATP-driven release into the bulk solution, a substrate protein kinetically partitions between folding to the native state or rebinding to another chaperonin molecule. To gain further insight into the mechanism of polypeptide binding and release by the chaperonin GroEL from Escherichia coli, we have undertaken a mutational analysis that relates the functional properties of GroEL to its crystal structure. Our functional tests identify a putative polypeptide-binding site on the inside surface of the apical domain, facing the central channel, consisting of hydrophobic residues. These same residues are essential for binding of the co-chaperonin GroES, which is required for productive polypeptide release. A highly conserved residue, Asp 87, positioned within a putative nucleotide-binding pocket in the top of the equatorial domain, is essential for ATP hydrolysis and polypeptide release.

  10. Application of Statistical Thermodynamics To Predict the Adsorption Properties of Polypeptides in Reversed-Phase HPLC.

    PubMed

    Tarasova, Irina A; Goloborodko, Anton A; Perlova, Tatyana Y; Pridatchenko, Marina L; Gorshkov, Alexander V; Evreinov, Victor V; Ivanov, Alexander R; Gorshkov, Mikhail V

    2015-07-01

    The theory of critical chromatography for biomacromolecules (BioLCCC) describes polypeptide retention in reversed-phase HPLC using the basic principles of statistical thermodynamics. However, whether this theory correctly depicts a variety of empirical observations and laws introduced for peptide chromatography over the last decades remains to be determined. In this study, by comparing theoretical results with experimental data, we demonstrate that the BioLCCC: (1) fits the empirical dependence of the polypeptide retention on the amino acid sequence length with R(2) > 0.99 and allows in silico determination of the linear regression coefficients of the log-length correction in the additive model for arbitrary sequences and lengths and (2) predicts the distribution coefficients of polypeptides with an accuracy from 0.98 to 0.99 R(2). The latter enables direct calculation of the retention factors for given solvent compositions and modeling of the migration dynamics of polypeptides separated under isocratic or gradient conditions. The obtained results demonstrate that the suggested theory correctly relates the main aspects of polypeptide separation in reversed-phase HPLC. PMID:26023813

  11. DVL, a novel class of small polypeptides: overexpression alters Arabidopsis development.

    PubMed

    Wen, Jiangqi; Lease, Kevin A; Walker, John C

    2004-03-01

    Small polypeptides can act as important regulatory molecules that coordinate cellular responses required for differentiation, growth, and development. In a gain-of-function genetic screen for genes that influence fruit development in Arabidopsis, we identified a novel gene -DEVIL1 (DVL1) - encoding a small protein. Overexpression of DVL1 results in pleiotropic phenotypes featured by shortened stature, rounder rosette leaves, clustered inflorescences, shortened pedicles, and siliques with pronged tips. cDNA analysis indicates that DVL1 has a 153-nucleotide (nt) open-reading frame (ORF) encoding a 51-amino acid polypeptide that shares no significant similarity to previously identified proteins. Sequence alignment shows that DVL1 belongs to a family of related genes that are limited to angiosperm plants. Ectopic overexpression of each of the five closely related Arabidopsis DVL genes causes similar phenotypic changes, suggesting overlapping function in the DVL gene family. Point mutations of conserved amino acids in the C-terminal region of the DVL1 polypeptide reveal that these conserved residues are required for DVL1-overexpression phenotypes. Our results show that the DVL family is a novel class of small polypeptides and the overexpression phenotypes suggest that these polypeptides may have a role in plant development. PMID:14871303

  12. Fluorescence probe of polypeptide conformational dynamics in gas phase and in solution

    NASA Astrophysics Data System (ADS)

    Iavarone, Anthony T.; Meinen, Jan; Schulze, Susanne; Parks, Joel H.

    2006-07-01

    Fluorescence measurements of polypeptides derivatized with the fluorescent dye BODIPY TMR have been used to probe the polypeptide conformational dynamics as a function of temperature and charge state. Measurements of (BODIPY TMR)-[Pro]n-Arg-Trp and (BODIPY TMR)-[Gly-Ser]m-Arg-Trp have been performed for charge states 1+ and 2+ of n = 4 and 10 and m = 2 and 5. The 2+ charge states of both of these polypeptides exhibit similar temperature dependences for equal chain lengths (n = 4, m = 2 and n = 10, m = 5) and suggest conformations dominated by Coulomb repulsion. In the absence of such Coulomb repulsion, the 1+ charge state conformations appear to be characterized by the flexibility of the polypeptide chain for which [Gly-Ser]m > [Pro]n. Comparisons of these gas phase polypeptide measurements with corresponding measurements in solution provide a direct measure of the effects of solvent on the conformational dynamics. The change in fluorescence as a function of temperature in the gas phase is two orders of magnitude greater than that in solution, a dramatic result we attribute to the restrictions on intramolecular dynamics imposed by diffusion-limited kinetics and the lack of shielding by solvent. Measurements were also made of unsolvated Pron peptides without the tryptophan (Trp) residue to isolate the interaction of the fluorescent dye with charges.

  13. Positive charges on the translocating polypeptide chain arrest movement through the translocon.

    PubMed

    Fujita, Hidenobu; Yamagishi, Marifu; Kida, Yuichiro; Sakaguchi, Masao

    2011-12-15

    Polypeptide chains synthesized by membrane-bound ribosomes are translocated through, and integrated into, the endoplasmic reticulum (ER) membrane by means of the protein translocation channel, the translocon. Positive charges on the nascent chain determine the orientation of the hydrophobic segment as it is inserted into the translocon and enhance the stop-translocation of translocating hydrophobic segments. Here we show that positive charges temporarily arrested ongoing polypeptide chain movement through the ER translocon by electrostatic interaction, even in the absence of a hydrophobic segment. The C-terminus of the polypeptide chain was elongated during the arrest, and then the full-length polypeptide chain moved through the translocon. The translocation-arrested polypeptide was not anchored to the membrane and the charges were on the cytoplasmic side of the membrane. The arrest effect was prevented by negatively charged residues inserted into the positive-charge cluster, and it was also suppressed by high salt conditions. We propose that positive charges are independent translocation regulators that are more active than previously believed. PMID:22223880

  14. Application of Statistical Thermodynamics To Predict the Adsorption Properties of Polypeptides in Reversed-Phase HPLC.

    PubMed

    Tarasova, Irina A; Goloborodko, Anton A; Perlova, Tatyana Y; Pridatchenko, Marina L; Gorshkov, Alexander V; Evreinov, Victor V; Ivanov, Alexander R; Gorshkov, Mikhail V

    2015-07-01

    The theory of critical chromatography for biomacromolecules (BioLCCC) describes polypeptide retention in reversed-phase HPLC using the basic principles of statistical thermodynamics. However, whether this theory correctly depicts a variety of empirical observations and laws introduced for peptide chromatography over the last decades remains to be determined. In this study, by comparing theoretical results with experimental data, we demonstrate that the BioLCCC: (1) fits the empirical dependence of the polypeptide retention on the amino acid sequence length with R(2) > 0.99 and allows in silico determination of the linear regression coefficients of the log-length correction in the additive model for arbitrary sequences and lengths and (2) predicts the distribution coefficients of polypeptides with an accuracy from 0.98 to 0.99 R(2). The latter enables direct calculation of the retention factors for given solvent compositions and modeling of the migration dynamics of polypeptides separated under isocratic or gradient conditions. The obtained results demonstrate that the suggested theory correctly relates the main aspects of polypeptide separation in reversed-phase HPLC.

  15. Export is the default pathway for soluble unfolded polypeptides that accumulate during expression in Escherichia coli

    SciTech Connect

    Scotto-Lavino, E.; Freimuth, P.; Bai, M.; Zhang, Y.-B.

    2011-09-01

    Several E. coli endogenous, cytoplasmic proteins that are known clients of the chaperonin GroEL were overexpressed to examine the fate of accumulated unfolded polypeptides. Substantial fractions of about half of the proteins formed insoluble aggregates, consistent with the hypothesis that these proteins were produced at rates or in amounts that exceeded the protein-folding capacity of GroEL. In addition, large fractions of three overexpressed GroEL client proteins were localized in an extra-cytoplasmic, osmotically-sensitive compartment, suggesting they had initially accumulated in the cytoplasm as soluble unfolded polypeptides and thus were able to access a protein export pathway. Consistent with this model, an intrinsically unfoldable, hydrophilic, non-secretory polypeptide was quantitatively exported from the E. coli cytoplasm into an osmotically-sensitive compartment. Our results support the conclusion that a soluble, unfolded conformation alone may be sufficient to direct non-secretory polypeptides into a protein export pathway for signal peptide-independent translocation across the inner membrane, and that export rather than degradation by cytoplasmic proteases is the preferred fate for newly-synthesized, soluble, unfolded polypeptides that accumulate in the cytoplasm. The stable folded conformation of exported GroEL client proteins further suggests that the requirement for GroEL may be conditional on protein folding in the molecularly-crowded environment of the cytoplasm.

  16. Augmented cystine-glutamate exchange by pituitary adenylate cyclase-activating polypeptide signaling via the VPAC1 receptor.

    PubMed

    Resch, Jon M; Albano, Rebecca; Liu, XiaoQian; Hjelmhaug, Julie; Lobner, Doug; Baker, David A; Choi, SuJean

    2014-07-28

    In the central nervous system, cystine import in exchange for glutamate through system xc(-) is critical for the production of the antioxidant glutathione by astrocytes, as well as the maintenance of extracellular glutamate. Therefore, regulation of system xc(-) activity affects multiple aspects of cellular physiology and may contribute to disease states. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuronally-derived peptide that has already been demonstrated to modulate multiple aspects of glutamate signaling suggesting PACAP may also target activity of cystine-glutamate exchange via system xc(-) . In the current study, 24-hour treatment of primary cortical cultures containing neurons and glia with PACAP concentration-dependently increased system xc(-) function as measured by radiolabeled cystine uptake. Furthermore, the increase in cystine uptake was completely abolished by the system xc(-) inhibitor, (S)-4-carboxyphenylglycine (CPG), attributing increases in cystine uptake specifically to system xc(-) activity. Time course and quantitative PCR results indicate that PACAP signaling may increase cystine-glutamate exchange by increasing expression of xCT, the catalytic subunit of system xc(-) . Furthermore, the potentiation of system xc(-) activity by PACAP occurs via a PKA-dependent pathway that is not mediated by the PAC1R, but rather the shared vasoactive intestinal polypeptide receptor VPAC1R. Finally, assessment of neuronal, astrocytic, and microglial-enriched cultures demonstrated that only astrocyte-enriched cultures exhibit enhanced cystine uptake following both PACAP and VIP treatment. These data introduce a novel mechanism by which both PACAP and VIP regulate system xc(-) activity. Synapse, 2014. © 2014 Wiley Periodicals, Inc.

  17. Effect of oxygen on morphogenesis and polypeptide expression by Mucor racemosus

    SciTech Connect

    Phillips, G.J.; Borgia, P.T.

    1985-12-01

    The morphology of Mucor racemosus in cultures continuously sparged with nitrogen gas was investigated. When appropriate precautions were taken to prevent oxygen from entering the cultures, the morphology of the cells was uniformly yeastlike irrespective of the N/sub 2/ flow rate. When small amounts of oxygen entered the cultures the resulting microaerobic conditions evoked mycelial development. Polypeptides synthesized by aerobic mycelia, microaerobic mycelia, anaerobic yeasts, and yeasts grown in a CO/sub 2/ atmosphere were compared by two-dimensional gel electrophoresis. The results indicated that a large number of differences in polypeptide expression exist when microaerobic mycelia or anaerobic yeasts are compared with aerobic mycelia and that these alterations correlate with a change from an oxidative to a fermentative metabolic mode. The authors hypothesize that oxygen regulates the expression of polypeptides involved in both the metabolic mode and in morphogenesis.

  18. Comparison between the polypeptide profile of halophilic bacteria and salt tolerant plants.

    PubMed

    Muñoz, G; González, C; Flores, P; Prado, B; Campos, V

    1997-12-01

    Changes in the polypeptide profile induced by salt stress in halotolerant and halophilic bacteria, isolated from the Atacama desert (northern Chile), were compared with those in the cotyledons of Prosopis chilensis (Leguminoseae) seedlings, a salt tolerant plant. SDS-PAGE analyses show the presence of four predominant polypeptides, with molecular weights around 78, 70, 60 and 44 kDa respectively, both in bacteria and in cotyledons from P. chilensis seedlings raised under salt stress conditions. Moreover, the 60 and 44 kDa polypeptides seem to be salt responsive, since their concentration increases with increasing NaCl in the growth medium. Our results suggest a common mechanism for salt tolerance in prokaryotes and in eukaryotes.

  19. GAWK, a novel human pituitary polypeptide: isolation, immunocytochemical localization and complete amino acid sequence.

    PubMed

    Benjannet, S; Leduc, R; Lazure, C; Seidah, N G; Marcinkiewicz, M; Chrétien, M

    1985-01-16

    During the course of reverse-phase high pressure liquid chromatography (RP-HPLC) purification of a postulated big ACTH (1) from human pituitary gland extracts, a highly purified peptide bearing no resemblance to any known polypeptide was isolated. The complete sequence of this 74 amino acid polypeptide, called GAWK, has been determined. Search on a computer data bank on the possible homology to any known protein or fragment, using a mutation data matrix, failed to reveal any homology greater than 30%. An antibody produced against a synthetic fragment allowed us to detect several immunoreactive forms. The antisera also enabled us to localize the polypeptide, by immunocytochemistry, in the anterior lobe of the pituitary gland.

  20. [POLYPEPTIDES INFLUENCE ON TISSUE CELL CULTURES REGENERATION OF VARIOUS AGE RATS].

    PubMed

    Ryzhak, A P; Chalisova, N I; Lin'kova, N S; Khalimov, R I; Ryzhak, G A; Zhekalov, A N

    2015-01-01

    A comparative study of polypeptides extracted from the tissues of calves: Cortexin (from brain cortex), Epinorm (from pineal gland), Ventvil (from liver), Prostatilen (from prostate), Thymalin (from thymus), Chelohart (from heart), Chondrolux (from cartilage) on the relevant organotypic tissue cultures of young and old rats, in concentration 0,01-100 ng/ml was performed. Polypeptides specifically stimulated "young" and "old" cell cultures growth in concentration 20-50 ng/ml. This effect correlates with increasing of PCNA and decreasing of p53 expression in brain cortex, pineal gland, liver, prostate, heart, cartilage. Moreover, Thymalin activated CD5, CD20 expression--markers of B-cells differentiation. These data show that polypeptides isolated from different tissues have selective molecular activity on the regeneration of suitable tissues in aging.

  1. Beaded nanofibers assembled from double-hydrophobic elastin-like block polypeptides: Effects of trifluoroethanol.

    PubMed

    Le, Duc H T; Okubo, Tatsuya; Sugawara-Narutaki, Ayae

    2015-03-01

    A "double-hydrophobic" elastin-like triblock polypeptide GPG has been constructed by mimicking the localization of proline- and glycine-rich hydrophobic domains of native elastin, a protein that provides elasticity and resilience to connective tissues. In this study, the effects of trifluoroethanol (TFE), an organic solvent that strongly affects secondary structures of polypeptides on self-assembly of GPG in aqueous solutions were systematically studied. Beaded nanofiber formation of GPG, where nanoparticles are initially formed by coacervation of the polypeptides followed by their connection into one-dimensional nanostructures, is accelerated by the addition of TFE at the concentrations up to 30% (v/v), whereas aggregates of nanoparticles are formed at 60% TFE. The concentration-dependent assembly pattern discussed is based on the influence of TFE on the secondary structures of GPG. Well-defined nanofibers whose diameter and secondary structures are controlled by TFE concentration may be ideal building blocks for constructing bioelastic materials in tissue engineering.

  2. Immunohistochemical localization of polypeptide hormones in pancreatic endocrine cells of a dipnoan fish, Protopterus aethiopicus.

    PubMed

    Scheuermann, D W; Adriaensen, D; Timmermans, J P; De Groodt-Lasseel, M H

    1991-01-01

    Light microscopical immunohistochemistry was used to demonstrate the regulatory peptides present in the endocrine pancreas of Protopterus aethiopicus. The peptides studied included insulin, glucagon, pancreatic polypeptide and somatostatin. The results showed that the 4 regulatory peptides commonly detected in the mammalian endocrine pancreas were immunologically discernible in this dipnoan fish. Large amounts of insulin-immunoreactive cells, in the centre of the pancreatic islets, were surrounded by a small rim of glucagon-or pancreatic polypeptide-immunoreactive cells. In addition, adjacent sections stained with anti-glucagon and anti-pancreatic polypeptide revealed that these hormones could be found in the same cells. Somatostatin-positive cells were scattered throughout the islets. Their processes were seen to contact many different endocrine pancreatic cells, suggesting that the somatostatin-immunoreactive cells control the functions of other endocrine pancreatic cells. PMID:1687100

  3. Identification, synthesis, and characterization of the yolk polypeptides of Plodia interpunctella.

    PubMed

    Shirk, P D; Bean, D; Millemann, A M; Brookes, V J

    1984-10-01

    The mature eggs of Plodia interpunctella were found to contain four major polypeptides. These yolk polypeptides (YPs) were found to have approximate molecular weights of 153,000 daltons (YP1), 69,000 daltons (YP2), 43,000 daltons (YP3), and 33,000 daltons (YP4) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In addition, we found YP1 was resolved by a 5% polyacrylamide gel into two separate polypeptides of 153,000 and 147,000 daltons. All of the YPs could be labeled in vivo or in vitro with [35S]-methionine. Yolk peptide 1 and YP3 were synthesized by fat body of pharate adult and adult females and secreted into the hemolymph. Yolk peptide 2 and YP4 were synthesized and secreted into incubation medium by ovaries that contained vitellogenic oocytes, but these polypeptides were not found in the hemolymph. Fat bodies of males synthesized and secreted an immunoprecipitable polypeptide similar to YP3 as well as immunoprecipitable polypeptides larger than 200,000 daltons that had no counterparts in the oocytes. Peptide mapping by protease digestion showed each YP to be cleaved into unique fragments, suggesting that no precursor-product relationship exists between the YPs. Ion exchange chromatography and gel permeation chromatography separated that yolk proteins into two groups with approximate molecular weights of 462,000 and 264,000 daltons. By resolving these peaks on SDS-PAGE, it was found that YP1 and YP3 formed the 462,000-dalton yolk protein and YP2 and YP4 formed the 264,000-dalton yolk protein.

  4. Structure and expression of a pea nuclear gene encoding a chlorophyll a/b-binding polypeptide

    SciTech Connect

    Cashmore, A.R.

    1984-05-01

    A nuclear gene AB80 has been isolated from a phage lambda Charon 4 library of pea DNA. The sequence of the gene has been determined and it has been shown to contain an interrupted reading frame of 269 amino acids, corresponding to a precursor to a constituent polypeptide of the light-harvesting chlorophyll a/b-protein complex. Primer extension and S1 nuclease studies defined a cap site for AB80. The first methionine codon 3' from this site is 69 nucleotides away and is the initiating codon of the open reading frame. A TATA sequence occurs 31 nucleotides 5' from the cap site. A second TATA sequence is found 7 nucleotides on the 5' side of the initiating methionine codon and the sequences surrounding this TATA sequence are strikingly similar to those surrounding the first TATA sequence. The mature polypeptide encoded by AB80 differs by 5 amino acids from the polypeptide corresponding to a previously characterized cDNA sequence pAB96. This result is indicative of heterogeneity within the constituent polypeptides of the light-harvesting chlorophyll a/b-protein complex. The sequence Arg-Lys-Ser-Ala-Thr-Thr-Lys-Lys occurs at, or near, the NH/sub 2/-terminus of the mature polypeptide encoded by AB80. This basic peptide is of interest because of its apparent involvement in changes in excitation-energy distribution in chloroplast membranes. Some general similarities, but no extensive sequence homology, is found on comparing the transit sequence for the precursor to the chlorophyll a/b-binding polypeptide with the transit sequences previously determined for the precursors to the small subunit of ribulose-1,5-bisphosphate carboxylase. 40 references, 3 figures.

  5. Catalytic and reactive polypeptides and methods for their preparation and use

    DOEpatents

    Schultz, Peter

    1994-01-01

    Catalytic and reactive polypeptides include a binding site specific for a reactant or reactive intermediate involved in a chemical reaction of interest. The polypeptides further include at least one active functionality proximate the binding site, where the active functionality is capable of catalyzing or chemically participating in the chemical reaction in such a way that the reaction rate is enhanced. Methods for preparing the catalytic peptides include chemical synthesis, site-directed mutagenesis of antibody and enzyme genes, covalent attachment of the functionalities through particular amino acid side chains, and the like.

  6. Ionic α-Helical Polypeptides towards Non-Viral Gene Delivery

    PubMed Central

    Zhang, Rujing; Song, Ziyuan; Yin, Lichen; Zheng, Nan; Tang, Haoyu; Lu, Hua; Gabrielson, Nathan P.; Lin, Yao; Kim, Kyung; Cheng, Jianjun

    2014-01-01

    The advent of polymeric materials has significantly promoted the development and rapid growth of various technologies in biomedical applications, such as tissue engineering and controlled drug and gene delivery. Water-soluble polypeptides bearing functional side chains and adopting stable secondary structures are a new class of functional polymeric materials of potentially broad applications in medicine and biotechnology. In this article, we summarize our recent effort on the design and synthesis of the water-soluble α-helical ionic polypeptides originally developed in our laboratory and highlight their applications in cell membrane penetration and non-viral gene/siRNA delivery. PMID:25377262

  7. Isolation of vulgin, a new antifungal polypeptide with mitogenic activity from the pinto bean.

    PubMed

    Ye, X Y; Ng, T B

    2003-02-01

    An antifungal polypeptide bearing an N-termnial sequence with some homology to chitinases was purified from an extract of pinto beans. The polypeptide, designated vulgin, exerted antifungal activity toward Mycosphaerella arachidicola, Coprinus cornatus, Fusarium oxysporum and Botrytis cinerea. Vulgin inhibited translation in a rabbit reticulocyte lysate system with an IC50 of 4.3 microM and HIV-1 reverse transcriptase activity with an IC50 of 58 microM. Vulgin stimulated in vitro incorporation of methyl [3H] thymidine into mouse splenocytes. PMID:12630696

  8. Mining the Melanosome for Tumor Vaccine Targets: P.polypeptide Is a Novel Tumor-associated Antigen

    PubMed Central

    Touloukian, Christopher E.; Leitner, Wolfgang W.; Robbins, Paul F.; Rosenberg, Steven A.; Restifo, Nicholas P.

    2008-01-01

    To identify novel, tumor-specific target antigens for vaccine development, we studied immune responses to P.polypeptide, an Mr 110,000 integral melanosomal membrane protein associated with the Prader-Willi syndrome. Together with expressed sequence tag (EST) and serial analyses of gene expression (SAGE) library analyses, reverse transcription-PCR and Northern blotting verified that P.polypeptide expression was limited to melanoma and melanocytes. A single dominant epitope corresponding to positions 427–435 (IMLCLIAAV) was identified using allele-specific epitope forecasting combined with work in HLA-A*0201/Kb transgenic mice. This epitope was then used to generate de novo human P.polypeptide-specific CD8+ T cells capable of recognizing P.polypeptide expressing human tumor cell lines in an HLA-A*0201-restricted fashion. Thus, P.polypeptide may be valuable in the creation of novel therapeutic anticancer vaccines. PMID:11719435

  9. Antipeptide antibodies that can distinguish specific subunit polypeptides of glutamine synthetase from bean (Phaseolus vulgaris L.)

    NASA Technical Reports Server (NTRS)

    Cai, X.; Henry, R. L.; Takemoto, L. J.; Guikema, J. A.; Wong, P. P.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    The amino acid sequences of the beta and gamma subunit polypeptides of glutamine synthetase from bean (Phaseolus vulgaris L.) root nodules are very similar. However, there are small regions within the sequences that are significantly different between the two polypeptides. The sequences between amino acids 2 and 9 and between 264 and 274 are examples. Three peptides (gamma 2-9, gamma 264-274, and beta 264-274) corresponding to these sequences were synthesized. Antibodies against these peptides were raised in rabbits and purified with corresponding peptide-Sepharose affinity chromatography. Western blot analysis of polyacrylamide gel electrophoresis of bean nodule proteins demonstrated that the anti-beta 264-274 antibodies reacted specifically with the beta polypeptide and the anti-gamma 264-274 and anti-gamma 2-9 antibodies reacted specifically with the gamma polypeptide of the native and denatured glutamine synthetase. These results showed the feasibility of using synthetic peptides in developing antibodies that are capable of distinguishing proteins with similar primary structures.

  10. Noncanonical Self-Assembly of Highly Asymmetric Genetically Encoded Polypeptide Amphiphiles into Cylindrical Micelles

    PubMed Central

    2015-01-01

    Elastin-like polypeptides (ELPs) are a class of biopolymers consisting of the pentameric repeat (VPGαG)n based on the sequence of mammalian tropoelastin that display a thermally induced soluble-to-insoluble phase transition in aqueous solution. We have discovered a remarkably simple approach to driving the spontaneous self-assembly of high molecular weight ELPs into nanostructures by genetically fusing a short 1.5 kDa (XGy)z assembly domain to one end of the ELP. Classical theories of self-assembly based on the geometric mass balance of hydrophilic and hydrophobic block copolymers suggest that these highly asymmetric polypeptides should form spherical micelles. Surprisingly, when sufficiently hydrophobic amino acids (X) are presented in a periodic sequence such as (FGG)8 or (YG)8, these highly asymmetric polypeptides self-assemble into cylindrical micelles whose length can be tuned by the sequence of the morphogenic tag. These nanostructures were characterized by light scattering, tunable resistive pulse sensing, fluorescence spectrophotometry, and thermal turbidimetry, as well as by cryogenic transmission electron microscopy (cryo-TEM) and small-angle neutron scattering (SANS). These short assembly domains provide a facile strategy to control the size, shape, and stability of stimuli responsive polypeptide nanostructures. PMID:25268037

  11. Acetylation in vitro of constituent polypeptides by smooth endoplasmic reticulum (SER) and Golgi membrane fractions

    SciTech Connect

    Sambasivam, H.; Murray, R.K.

    1986-05-01

    Many polypeptides of the membranes of the ER are phosphorylated. To determine if any such polypeptides are acetylated, microsomal and other classical subcellular fractions were incubated with (/sup 3/H) acetyl-CoA; the specific activity of the microsomal fraction (MF) was the greatest. SDS-PAGE revealed that some 20 polypeptides of the MF were acetylated; 2-D electrophoretograms extended this number to approximately 60. Separation of the MF into smooth (S) and rough (R) fractions showed that the great majority of the labelled polypeptides belonged to the former. Isolation of a Golgi fraction revealed that its acetylation activity was approximately 3-fold greater than the SER fraction. Extensive proteolytic digestion of the MF followed by radiochromatography disclosed some 9 components whose precise nature (acetylated amino acids and/or sialic acids, etc.) is under study. Assuming that the majority of the radioactivity is in the former components and that a similar process occurs in vivo, the authors suggest that the Golgi apparatus may be a major site of acetylation of membrane and possibly other proteins.

  12. Ectomycorrhizin Synthesis and Polypeptide Changes during the Early Stage of Eucalypt Mycorrhiza Development 1

    PubMed Central

    Hilbert, Jean-Louis; Costa, Guy; Martin, Francis

    1991-01-01

    In functioning eucalypt ectomycorrhizas, biochemical alterations are accompanied by a differential accumulation of polypeptides including the synthesis of symbiosis-related proteins (JL Hilbert, Martin FM [1988] New Phytol 110: 339-346). In the present study, protein biosynthesis in the early stages of ectomycorrhiza formation on Eucalyptus globulus subsp. bicostata Kirkp. was examined using compatible and incompatible isolates of the basidiomycete Pisolithus tinctorius (Coker & Couch). Changes in polypeptide composition were observed within hours following contact of the compatible mycelium with the roots, well before the differentiation of typical symbiotic tissues. At this stage, at least seven symbiosis-related proteins (ectomycorrhizins) accumulated in root tissues. In vivo incorporation of [35S]methionine by ectomycorrhizas followed by electrophoresis of the labeled proteins revealed that most of these differences in polypeptide concentrations, including the ectomycorrhizin accumulation, are the result of differential protein biosynthesis rather than posttranslational modifications of the polypeptides. The initial development of eucalypt ectomycorrhizas, therefore, coincides with the synthesis of symbiosis-related proteins and the data presented here provide essential evidence to ascribe a functional developmental role to these proteins. ImagesFigure 2Figure 3Figure 4Figure 5 PMID:16668539

  13. A role for helical intermediates in amyloid formation by natively unfolded polypeptides?

    NASA Astrophysics Data System (ADS)

    Abedini, Andisheh; Raleigh, Daniel P.

    2009-03-01

    Amyloid formation and aberrant protein aggregation have been implicated in more than 15 different human diseases and an even wider range of proteins form amyloid in vitro. From a structural perspective the proteins which form amyloid can be divided into two classes: those which adopt a compact globular fold and must presumably at least partially unfold to form amyloid and those which are unstructured in their monomeric state. Important examples of the latter include the Aβ peptide of Alzheimer's disease, atrial natriuretic factor, calcitonin, pro-calcitonin, islet amyloid polypeptide (IAPP, amylin), α-synuclein and the medin polypeptide. The kinetics of amyloid assembly are complex and typically involve a lag phase during which little or no fibril material is formed, followed by a rapid growth stage leading to the β-sheet-rich amyloid structure. Increasing evidence suggests that some natively unfolded polypeptides populate a helical intermediate during the lag phase. We propose a model in which early oligomerization is linked to helix formation and is promoted by helix-helix association. Recent work has highlighted the potential importance of polypeptide membrane interactions in amyloid formation and helical intermediates appear to play an important role here as well. Characterization of helical intermediates is experimentally challenging but new spectroscopic techniques are emerging which hold considerable promise and even have the potential to provide residue specific information.

  14. Antibody produced against isolated Rh(D) polypeptide reacts with other Rh-related antigens.

    PubMed

    Suyama, K; Goldstein, J

    1988-11-01

    Rh(D) antigen-containing polypeptide was prepared by immune precipitation of intact cDE/cDE erythrocytes by using a high-titer preparation of polyclonal anti-D. when isolated Rh(D) polypeptide was administered to rabbits, antibody was produced that was unresponsive toward Rh-positive and -negative cells but reacted strongly with the immunogen in enzyme-linked immunosorbent assay-type immunobinding and Western blot immunostaining assays. Rabbit antibody also immunostains isolated Rh(c) polypeptide as well as the Rh antigen-containing components of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated membrane proteins from Rh(D)-positive (cDE/cDE,CDe/CDe), Rh(D)-negative (cde/cde,Cde/Cde), and -D-/-D- cells. It does not react with any membrane protein from Rh-null regulator type cells, thus indicating a specificity for Rh-related proteins. We have also been able to demonstrate that polyclonal and monoclonal anti-D preparations that do not immunostain isolated Rh(D) polypeptide will react with it in our immunobinding assay.

  15. AGGRESCAN: a server for the prediction and evaluation of "hot spots" of aggregation in polypeptides

    PubMed Central

    Conchillo-Solé, Oscar; de Groot, Natalia S; Avilés, Francesc X; Vendrell, Josep; Daura, Xavier; Ventura, Salvador

    2007-01-01

    Background Protein aggregation correlates with the development of several debilitating human disorders of growing incidence, such as Alzheimer's and Parkinson's diseases. On the biotechnological side, protein production is often hampered by the accumulation of recombinant proteins into aggregates. Thus, the development of methods to anticipate the aggregation properties of polypeptides is receiving increasing attention. AGGRESCAN is a web-based software for the prediction of aggregation-prone segments in protein sequences, the analysis of the effect of mutations on protein aggregation propensities and the comparison of the aggregation properties of different proteins or protein sets. Results AGGRESCAN is based on an aggregation-propensity scale for natural amino acids derived from in vivo experiments and on the assumption that short and specific sequence stretches modulate protein aggregation. The algorithm is shown to identify a series of protein fragments involved in the aggregation of disease-related proteins and to predict the effect of genetic mutations on their deposition propensities. It also provides new insights into the differential aggregation properties displayed by globular proteins, natively unfolded polypeptides, amyloidogenic proteins and proteins found in bacterial inclusion bodies. Conclusion By identifying aggregation-prone segments in proteins, AGGRESCAN shall facilitate (i) the identification of possible therapeutic targets for anti-depositional strategies in conformational diseases and (ii) the anticipation of aggregation phenomena during storage or recombinant production of bioactive polypeptides or polypeptide sets. PMID:17324296

  16. The Beads of Translation: Using Beads to Translate mRNA into a Polypeptide Bracelet

    ERIC Educational Resources Information Center

    Dunlap, Dacey; Patrick, Patricia

    2012-01-01

    During this activity, by making beaded bracelets that represent the steps of translation, students simulate the creation of an amino acid chain. They are given an mRNA sequence that they translate into a corresponding polypeptide chain (beads). This activity focuses on the events and sites of translation. The activity provides students with a…

  17. Targeting a c-Myc inhibitory polypeptide to specific intracellular compartments using cell penetrating peptides.

    PubMed

    Bidwell, Gene L; Davis, Aisha N; Raucher, Drazen

    2009-04-01

    The therapeutic index of current anti-cancer chemotherapeutics can be improved by two major mechanisms: 1) developing drugs which are specifically toxic to the cancer cells and 2) developing methods to deliver drugs to the tumor site. In an attempt to combine these approaches, we developed a thermally responsive polypeptide inhibitor of c-Myc. This polypeptide is based on the thermally responsive Elastin-like polypeptide (ELP). When injected systemically, ELP-fused drugs will aggregate and accumulate at the tumor site where local hyperthermia is applied. ELP was fused to a peptide which blocks c-Myc/Max dimerization (H1), thereby inhibiting transcription activation by c-Myc (ELP-H1). In this study, the cellular uptake, intracellular distribution, and potency of the Pen, Tat and Bac cell penetrating peptides fused to ELP-H1 were evaluated. While Pen-ELP-H1 and Tat-ELP-H1 were localized in the cytoplasm, Bac-ELP-H1 localized to the nucleus in a subset of the cells and was the most potent inhibitor of MCF-7 cell proliferation. This data demonstrates that ELP can be targeted to the desired cellular compartment simply by choice of the CPP used, resulting in a more potent nuclear targeted c-Myc inhibitory polypeptide which may be beneficial in cancer therapy.

  18. Dynamics of the formation of a hydrogel by a pathogenic amyloid peptide: islet amyloid polypeptide

    PubMed Central

    Jean, Létitia; Lee, Chiu Fan; Hodder, Peter; Hawkins, Nick; Vaux, David J.

    2016-01-01

    Many chronic degenerative diseases result from aggregation of misfolded polypeptides to form amyloids. Many amyloidogenic polypeptides are surfactants and their assembly can be catalysed by hydrophobic-hydrophilic interfaces (an air-water interface in-vitro or membranes in-vivo). We recently demonstrated the specificity of surface-induced amyloidogenesis but the mechanisms of amyloidogenesis and more specifically of adsorption at hydrophobic-hydrophilic interfaces remain poorly understood. Thus, it is critical to determine how amyloidogenic polypeptides behave at interfaces. Here we used surface tensiometry, rheology and electron microscopy to demonstrate the complex dynamics of gelation by full-length human islet amyloid polypeptide (involved in type II diabetes) both in the bulk solution and at hydrophobic-hydrophilic interfaces (air-water interface and phospholipids). We show that the hydrogel consists of a 3D supramolecular network of fibrils. We also assessed the role of solvation and dissected the evolution over time of the assembly processes. Amyloid gelation could have important pathological consequences for membrane integrity and cellular functions. PMID:27535008

  19. The capsid polypeptides of the 190S virus of Helminthosporium victoriae.

    PubMed

    Ghabrial, S A; Bibb, J A; Price, K H; Havens, W M; Lesnaw, J A

    1987-07-01

    SDS-PAGE of the 190S virus of Helminthosporium victoriae, using a discontinuous buffer system, revealed two major capsid polypeptides of mol. wt. 88K and 83K (p88 and p83) and a minor polypeptide, p78. Peptide mapping by both limited proteolysis and selective chemical cleavage showed p83 and p78 to be closely related to p88. The origin of p83/p78 could not be explained by proteolysis of p88 during virus preparation and storage. In rabbit reticulocyte lysates, denatured dsRNA directed the synthesis of a single major translation product which was identical to capsid polypeptide p88 on the basis of coelectrophoresis, immunoprecipitation and peptide mapping. No translation products comparable in size to p83 or p78 were detected in vitro. These data indicated that the capsid of the 190S virus is encoded by a single gene and verified the classification of the virus as a member of the family Totiviridae. Radioiodination of intact virus under conditions considered optimum for surface-specific iodination showed p88 to be more readily available for labelling than p83 or p78. Furthermore, when Western blots of capsid polypeptides were reacted with an antiserum to glutaraldehyde-stabilized virus (190S-G), p88 was more reactive to 190S-G antibodies than was p83/p78. These results suggest p88 is external to p83/p78 in the capsid.

  20. Simultaneous Polymerization and Polypeptide Particle Production via Reactive Spray-Drying.

    PubMed

    Glavas, Lidija; Odelius, Karin; Albertsson, Ann-Christine

    2016-09-12

    A method for producing polypeptide particles via in situ polymerization of N-carboxyanhydrides during spray-drying has been developed. This method was enabled by the development of a fast and robust synthetic pathway to polypeptides using 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) as an initiator for the ring-opening polymerization of N-carboxyanhydrides. The polymerizations finished within 5 s and proved to be very tolerant toward impurities such as amino acid salts and water. The formed particles were prepared by mixing the monomer, N-carboxyanhydride of l-glutamic acid benzyl ester (NCAGlu) and the initiator (DBU) during the atomization process in the spray-dryer and were spherical with a size of ∼1 μm. This method combines two steps; making it a straightforward process that facilitates the production of polypeptide particles. Hence, it furthers the use of spray-drying and polypeptide particles in the pharmaceutical industry. PMID:27445061

  1. Cellular Interactions and Biocompatibility of Self-Assembling Diblock Polypeptide Hydrogels

    NASA Astrophysics Data System (ADS)

    Pakstis, Lisa; Ozbas, Bulent; Pochan, Darrin; Robinson, Clifford; Nowak, Andrew; Deming, Timothy

    2002-03-01

    Self-assembling peptide based hydrogels having a unique nano- and microscopic morphology are being studied for potential use as tissue engineering scaffolds. Low molecular weight ( ~20 kg/mol), amphiphilic, diblock polypeptides of hydrophilic lysine (K) or glutamic acid (E) and hydrophobic leucine (L) or valine (V) form hydrogels in aqueous solution at neutral pH and at very low volume fraction of polymer (vol. fraction polypeptide >=0.5 wt%). The morphology of these hydrogels has been characterized using laser confocal microscopy (LCM), small angle neutron scattering (SANS), and cryogenic transmission electron microscopy (cryoTEM) imaging. Studies of the interactions of the hydrogels with bacterial and mammalian cells reveal that these materials are non-cytotoxic and biocompatible. Hence, the chemistry of the assembled diblock polypeptides allows for cellular proliferation whereas the same chemistry in the homopolyeric form is cytotoxic. Current research is directed at the design and incorporation of binding sites within the polypeptide to specifically target interactions of the hydrogel with desired cells types.

  2. A few positively charged residues slow movement of a polypeptide chain across the endoplasmic reticulum membrane.

    PubMed

    Yamagishi, Marifu; Onishi, Yukiko; Yoshimura, Shotaro; Fujita, Hidenobu; Imai, Kenta; Kida, Yuichiro; Sakaguchi, Masao

    2014-08-26

    Many polypeptide chains are translocated across and integrated into the endoplasmic reticulum membrane through protein-conducting channels. During the process, amino acid sequences of translocating polypeptide chains are scanned by the channels and classified to be retained in the membrane or translocated into the lumen. We established an experimental system with which the kinetic effect of each amino acid residue on the polypeptide chain movement can be analyzed with a time resolution of tens of seconds. Positive charges greatly slow movement; only two lysine residues caused a remarkable slow down, and their effects were additive. The lysine residue was more effective than arginine. In contrast, clusters comprising three residues of each of the other 18 amino acids had little effect on chain movement. We also demonstrated that a four lysine cluster can exert the effect after being fully exposed from the ribosome. We concluded that as few as two to three residues of positively charged amino acids can slow the movement of the nascent polypeptide chain across the endoplasmic reticulum membrane. This effect provides a fundamental basis of the topogenic function of positively charged amino acids. PMID:25093244

  3. Dynamics of the formation of a hydrogel by a pathogenic amyloid peptide: islet amyloid polypeptide.

    PubMed

    Jean, Létitia; Lee, Chiu Fan; Hodder, Peter; Hawkins, Nick; Vaux, David J

    2016-01-01

    Many chronic degenerative diseases result from aggregation of misfolded polypeptides to form amyloids. Many amyloidogenic polypeptides are surfactants and their assembly can be catalysed by hydrophobic-hydrophilic interfaces (an air-water interface in-vitro or membranes in-vivo). We recently demonstrated the specificity of surface-induced amyloidogenesis but the mechanisms of amyloidogenesis and more specifically of adsorption at hydrophobic-hydrophilic interfaces remain poorly understood. Thus, it is critical to determine how amyloidogenic polypeptides behave at interfaces. Here we used surface tensiometry, rheology and electron microscopy to demonstrate the complex dynamics of gelation by full-length human islet amyloid polypeptide (involved in type II diabetes) both in the bulk solution and at hydrophobic-hydrophilic interfaces (air-water interface and phospholipids). We show that the hydrogel consists of a 3D supramolecular network of fibrils. We also assessed the role of solvation and dissected the evolution over time of the assembly processes. Amyloid gelation could have important pathological consequences for membrane integrity and cellular functions. PMID:27535008

  4. Comparison of the polypeptide composition of Escherichia coli outer membranes prepared by two methods.

    PubMed Central

    Chopra, I; Shales, S W

    1980-01-01

    Escherichia coli outer membranes were prepared by centrifugation to equilibrium in sucrose gradients and then treated with Sarkosyl in the presence of ethylenediaminetetraacetate. The polypeptide profiles of the two outer membrane preparations were compared by two-dimensional polyacrylamide gel electrophoresis. The patterns obtained were not identical, and Sarkosyl removed several minor proteins from the outer membrane. Images PMID:6998960

  5. Use of Rigid Liquid Crystalline Polypeptides as Alignment Matrices for Organic Nonlinear Optical Molecules.

    NASA Astrophysics Data System (ADS)

    Tokarski, Zbigniew

    The orientation of nonlinear optical (NLO) organic molecules is crucial for the existence of high values for the macroscopic susceptibilities. The orientation and interaction of several smaller NLO active molecules with an easily alignable polypeptide host was investigated to determine which functional groups and molecular shapes would produce the largest orientation with the host material; these parameters included aromatic vs aliphatic, polar vs nonpolar, saturate vs unsaturated hydrocarbons and the length of the guest molecule. The host materials were either poly ( gamma-benzyl-l-glutamate) (PBLG) or poly ( gamma-ethyl-l-glutamate) (PELG) lyotropic liquid crystals. These host polymers formed pseudo-hexagonal crystalline structures with long rigid alpha -helical backbones. The interstitial alignment of the guest molecules was dictated by the overall alignment of the host polypeptide rigid rods. Within these films many of the guest molecules existed in a metastable state that delayed phase separation for several hours. The rate of phase separation was influenced by the concentration of the guest molecule and on the side chain moiety of the polypeptide. Guest phase separation to a solid or a liquid occurred at a faster rate in PELG films, due to the lack of the side chain induced hindrance, than in PBLG films. An indicator of the occurrence of phase separation was with the onset of opaqueness in the films. The thin polypeptide films containing the aligned guest molecules became optically opaque as the incompatibilities between the side chains of the polypeptides and the guest molecules increased. The nonlinear optical susceptibility measurements were hampered by either the low guest solubility or the low concentration level required to avoid the guest -host incompatibility. Electro-optic and degenerate two and four wave mixing were done and produced signals in solutions but not in the doped films. The semiflexible aromatic guest molecules, such as the derivatives

  6. The polymerization of amino acid adenylates on sodium-montmorillonite with preadsorbed polypeptides

    NASA Astrophysics Data System (ADS)

    Paecht-Horowitz, Mella; Eirich, Frederick R.

    1988-12-01

    We studied the spontaneous polymerization of amino acid adenylates on Na-montmorillonite in dilute, neutral suspension, after polypeptides were adsorbed on the clay. This led to the unexpected finding that the degrees of polymerization (DP's) of the oligo- and poly-peptides obtained depended on the amounts of polypeptides that were preadsorbed. Plotting average molecular weights obtained against c-spacings of the clay platelet aggregates which widened as a result of polypeptide addition and adsorption before the polymerization, does not permit an obvious explanation of these observations. The best correlation assigns a role to the fractional occupation of the individual intercalation layers of the polypeptides, as the adsorption increases towards a first complete mono-interlayer, then to an incipient and eventually to a complete double layer on to a third interlayer, after which the clay stacking breaks up. Spacings which correspond to an intermediate occupation of any of the three successive interlayers favor amino acids self-addition to polymers. The opposite is true for nearly empty or filled intercalation layers. We hypothesize and describe, how a catalytic activity may derive from c-spacings that offer adsorption sites for the reagent amino acid adenylate within the peripheral recesses of irregularly stacked clay platelets by bringing the anhydride bonds and neutral amino groups into favorable reaction distances. Moderately filled intercalation spaces may also act as sinks for the newly formed oligomers and facilitate the freeing of reaction sites for the occupation by fresh reagent. The c-spacings required for these mechanisms are the result of the intercalation of the preadsorbed polymer, but similar conditions prevail when polymers are adsorbed as they are generated during polymerization.

  7. Incorporation of polypeptides into thylakoid membranes of Chlamydomonas reinhardtii. Cyclic variations

    PubMed Central

    1976-01-01

    A purified fraction of unstacked thylakoid membranes (TMF1u) has been obtained from homogenates of Chlamydomonas reinhardtii (wild type 137+) by using repeated centrifugates in sucrose density gradients and low salt treatment. The contaminants of the fraction are reduced to a few mitochondria (approximately 3% of the total mitochondrial population), a few osmiophilic granules, and fragments of chloroplast envelopes. By SDS-polyacrylamide gel electrophoresis the polypeptide components of TMF1u were resolved into at least 30 bands. To determine the relative rates of assembly of newly synthesized polypeptides into thylakoid membranes, synchronized algal cells were doubly labeled in vivo with L- [14C] and L-[3H]arginine--used for long- and short-term labeling, respectively. TMF1u's were isolated from the labeled cells at selected time points during the cycle and the distribution of radioactivity was assayed in the gel electrophoretograms of their solubilized polypeptides. Incorporation of newly synthesized polypeptides into the bands of the gels was found to occur continuously but differentially throughout the cycle. Maximal rates of incorporation for the majority of the polypeptides were detected shortly after cell division (6D-7D; equivalent to early G1 phase). The rates of radioactive labeling decreased gradually to a low level at the end of the dark period and then rose slightly at the beginning of the next light period. The findings suggest that, in addition to the light/dark control postulated in the past, assembly of newly synthesized proteins into thylakoid membranes is activated by signals at work in the early G1 phase. PMID:1262393

  8. Reversible thermal denaturation of a 60-kDa genetically engineered beta-sheet polypeptide.

    PubMed

    Lednev, Igor K; Ermolenkov, Vladimir V; Higashiya, Seiichiro; Popova, Ludmila A; Topilina, Natalya I; Welch, John T

    2006-11-15

    A de novo 687-amino-acid residue polypeptide with a regular 32-amino-acid repeat sequence, (GA)(3)GY(GA)(3)GE(GA)(3)GH(GA)(3)GK, forms large beta-sheet assemblages that exhibit remarkable folding properties and, as well, form fibrillar structures. This construct is an excellent tool to explore the details of beta-sheet formation yielding intimate folding information that is otherwise difficult to obtain and may inform folding studies of naturally occurring materials. The polypeptide assumes a fully folded antiparallel beta-sheet/turn structure at room temperature, and yet is completely and reversibly denatured at 125 degrees C, adopting a predominant polyproline II conformation. Deep ultraviolet Raman spectroscopy indicated that melting/refolding occurred without any spectroscopically distinct intermediates, yet the relaxation kinetics depend on the initial polypeptide state, as would be indicative of a non-two-state process. Thermal denaturation and refolding on cooling appeared to be monoexponential with characteristic times of approximately 1 and approximately 60 min, respectively, indicating no detectable formation of hairpin-type nuclei in the millisecond timescale that could be attributed to nonlocal "nonnative" interactions. The polypeptide folding dynamics agree with a general property of beta-sheet proteins, i.e., initial collapse precedes secondary structure formation. The observed folding is much faster than expected for a protein of this size and could be attributed to a less frustrated free-energy landscape funnel for folding. The polypeptide sequence suggests an important balance between the absence of strong nonnative contacts (salt bridges or hydrophobic collapse) and limited repulsion of charged side chains. PMID:16891363

  9. The rate with which spontaneous mutation alters the electrophoretic mobility of polypeptides.

    PubMed Central

    Neel, J V; Satoh, C; Goriki, K; Fujita, M; Takahashi, N; Asakawa, J; Hazama, R

    1986-01-01

    Studies of a Japanese population, involving a total of 539,170 locus tests distributed over 36 polypeptides, yielded three presumptive spontaneous mutations altering the electrophoretic mobility of the polypeptide. This corresponds to a mutation rate of 0.6 X 10(-5) per locus per generation. The a priori probability that undetected discrepancies between legal and biological parentage might in our test system result in an apparent electrophoretic mutation in this population is calculated to be only 0.3 X 10(-7) per locus per generation. Since electrophoresis only detects about half of the amino acid substitutions due to mutations of nucleotides, the corrected rate for mutations causing amino acid substitutions in polypeptides is 1.2 X 10(-5) per locus per generation. With allowance for synonymous mutations and those resulting in stop codons, the total mutation rate for nucleotide changes in the exons encoding a polypeptide becomes approximately equal to 1.8 X 10(-5) per locus per generation. When the present observations are combined with all of the other available data concerning mutation resulting in electrophoretic variants, the electrophoretic rate drops to 0.3 X 10(-5) per locus per generation, the total locus rate drops to roughly 1.0 X 10(-5), and the nucleotide rate drops to 1 X 10(-8). Even with this lower estimate, given approximately equal to 2 X 10(9) nucleotides in the haploid genome and an average of 10(3) exon nucleotides per polypeptide encoded, the implication, if these exon rates can be generalized, is of approximately equal to 20 nucleotide mutations per gamete per generation. This estimate of the frequency of "point" mutations does not include small duplications, rearrangements, or deletions resulting from unequal crossing-over, transcription errors, etc. PMID:3455776

  10. Synthesis of gluten-forming polypeptides. 1. Biosynthesis of gliadins and glutenin subunits.

    PubMed

    Abonyi, Tibor; Király, István; Tömösközi, Sándor; Baticz, Orsolya; Guóth, Adrienn; Gergely, Szilveszter; Scholz, Eva; Lásztity, Demeter; Lásztity, Radomir

    2007-05-01

    Five winter wheat cultivars--GK Othalom (HMW-GS composition 2*, 7+8, 5+10), Ukrainka (1, 7+8, 5+10), Palotás (2*, 7+9, 5+10), Ködmön (2*, 7+8, 5+10), and Csongrád (2*, 7+9, 2+12)--grown in Hungary and harvested in the year 2005 were studied. The biosynthesis of gluten-forming polypeptides was followed starting at the 12th day after anthesis to the 53rd. Fresh kernel weight, moisture, and dry matter content of fresh kernels and gliadin and glutenin contents were determined. Gliadin components, total amounts of HMW and LMW polypeptides, and individual HMW polypeptides were determined using a RP-HPLC technique. Although considerable quantitative differences were observed concerning the content of total protein, gliadin, glutenin, and individual gluten-forming polypeptides, the character of accumulation of protein components--determined on the basis protein mass/kernel--was the same for the all of the cultivars studied and could be presented by a sigmoid curve. Small quantities of the gliadin and glutenin monomers may be detected in early stages of kernel development, but the bulk of these proteins is synthesized in later stages of development. It is generally suggested by specialists that the formation and accumulation of glutenin polymers starts later than the synthesis of monomers. Experimental data presented in this paper confirm this suggestion and show that in the first phase of protein synthesis the monomers are in "free" form; polymeric glutenin is detected only later. HMW glutenin subunits are synthesized synchronously, and quantitatively the polypeptides coded by chromosomes D and B dominate.

  11. Isolation and characterisation of five neurotoxic and cardiotoxic polypeptides from the sea anemone Anthopleura elegantissima.

    PubMed

    Bruhn, T; Schaller, C; Schulze, C; Sanchez-Rodriguez, J; Dannmeier, C; Ravens, U; Heubach, J F; Eckhardt, K; Schmidtmayer, J; Schmidt, H; Aneiros, A; Wachter, E; Béress, L

    2001-05-01

    Five toxins (APE 1 to APE 5) of the sea anemone species Anthopleura elegantissima (Brandt) have been isolated from a toxic by-product fraction of its concentrated crude watery-methanolic extract, prepared previously for the isolation of a neuropeptide (the head-activator) by Schaller and Bodenmüller (Proc. Natl. Acad. Sci. USA 78 (1981) 7000) from 200kg sea anemones. Toxin purification was performed by desalting of the starting material by dialysis (MWCO 3500) against distilled water, anion exchange chromatography on QAE-Sephadex A25 at pH 8, twice gel filtration on Sephadex G50 m, repeated chromatography on QAE-Sephadex at pH 10 and chromatography on the cation exchanger Fractogel EMD SO(3)(-)-650 M.Final purification of the toxins was achieved by HPLC on MN SP 250/10 Nucleosil 500-5 C(18) PPN and MN SP 250/21 Nucleosil 300-7 C(18). Each toxin was composed of at least two isotoxins of which APE 1-1, APE 1-2, APE 2-1, APE 2-2 and APE 5-3 were isolated in preparative scale. With exception of APE 5-3 the sequences of the isotoxins have been elucidated. They resemble the 47 residue type-I long polypeptide toxins native to Anemonia sulcata (Pennant). All isotoxins paralyse the shore crab (Carcinus maenas) by tetanic contractions after i.m. application. The toxins modify current passing through the fast Na(+) channel in neuroblastoma cells, leading to delayed and incomplete inactivation. APE 1-1, APE 2-1 and APE 5-3 produce a positive inotropic effect in mammalian heart muscle, although they differ in potency. The order of potency is APE 2-1>APE 1-1>APE 5-3 (i.e. threshold concentrations are 1, 10 and 300nM, respectively). In addition, they enhance the spontaneous beating frequency in isolated right atria (guinea pig). The most potent cardiotoxic isotoxin is APE 2-1, its sequence is identical with that of AP-C, a toxin isolated and characterised previously by Norton et al. (Drugs and Foods from the Sea, 1978, University of Oklahoma Press, p. 37-50).LD50 APE 2-1:1 micro

  12. Novel ATPase Cu(2+) transporting beta polypeptide mutations in Chinese families with Wilson's disease.

    PubMed

    Gu, Shaojuan; Yang, Huarong; Qi, Yong; Deng, Xiong; Zhang, Le; Guo, Yi; Huang, Qing; Li, Jing; Shi, Xiaoliu; Song, Zhi; Deng, Hao

    2013-01-01

    Wilson's disease (WD) is an autosomal recessive inherited disorder caused by mutations in the ATPase Cu(2+) transporting beta polypeptide gene (ATP7B). The detailed metabolism of copper-induced pathology in WD is still unknown. Gene mutations as well as the possible pathways involved in the ATP7B deficiency were documented. The ATP7B gene was analyzed for mutations in 18 Chinese Han families with WD by direct sequencing. Cell viability and apoptosis analysis of ATP7B small interfering RNA (siRNA)-treated human liver carcinoma (HepG2) cells were measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and Hoechst 33342 staining. Finally, the expression of B-cell CLL/lymphoma 2 (BCL2), BCL2-associated X protein (BAX), sterol regulatory element binding protein 1 (SREBP1), and minichromosome maintenance protein 7 (MCM7) of ATP7B siRNA-treated cells were tested by real-time polymerase chain reaction (real-time PCR) and Western blot analysis. Twenty different mutations including four novel mutations (p.Val145Phe, p.Glu388X, p.Thr498Ser and p.Gly837X) in the ATP7B gene were identified in our families. Haplotype analysis revealed that founder effects for four mutations (p.Arg778Leu, p.Pro992Leu, p.Ile1148Thr and p.Ala1295Val) existed in these families. Transfection of HepG2 cells with ATP7B siRNA resulted in decreased mRNA expression by 86.3%, 93.1% and 90.8%, and decreased protein levels by 58.5%, 85.5% and 82.1% at 24, 48 and 72 hours, respectively (All P<0.01). In vitro study revealed that the apoptotic, cell cycle and lipid metabolism pathway may be involved in the mechanism of WD. Our results revealed that the genetic cause of 18 Chinese families with WD and ATP7B deficiency-induce apoptosis may result from imbalance in cell cycle and lipid metabolism pathway.

  13. Deletion of the gene encoding the adenovirus 5 early region 1b 21,000-molecular-weight polypeptide leads to degradation of viral and host cell DNA.

    PubMed Central

    Pilder, S; Logan, J; Shenk, T

    1984-01-01

    The adenovirus 5 mutant H5dl337 lacks 146 base pairs within early region 1B. The deletion removes a portion of the region encoding the E1B 21,000-molecular-weight (21K) polypeptide, but does not disturb the E1B-55K/17K coding region. The virus is slightly defective for growth in cultured HeLa cells, in which its final yield is reduced ca. 10-fold compared with wild-type virus. The mutant displays a striking phenotype in HeLa cells. The onset of cytopathic effect is dramatically accelerated, and both host cell and viral DNAs are extensively degraded late after infection. This defect has been described previously for a variety of adenovirus mutants and has been termed a cytocidal (cyt) phenotype. H5dl337 serves to map this defect to the loss of E1B-21K polypeptide function. In addition to its defect in the productive growth cycle, H5dl337 is unable to transform rat cells at normal efficiency. Images PMID:6492257

  14. Characterization of novel, phenol-soluble polypeptides which confer rigidity to the sheath of Methanospirillum hungatei GP1.

    PubMed Central

    Southam, G; Beveridge, T J

    1992-01-01

    Treatment of the Methanospirillum hungatei GP1 sheath with 90% (wt/vol) phenol resulted in the solubilization of a novel phenol-soluble group of polypeptides. These polypeptides were purified by the removal of insoluble material by ultracentrifugation and represented approximately 19% of the mass of the sheath. The phenol-insoluble material resembled untreated sheath but had lost its rigidity and cylindrical form. Recombination of phenol-soluble and phenol-insoluble fractions by dialysis to remove phenol resulted in cylindrical reassembly products. Although bona fide sheath (complete with the 2.8-nm lattice) was not produced, a role for the phenol-soluble polypeptides in the maintenance of sheath rigidity is implied. The phenol-soluble polypeptides have limited surface exposure as detected by antibodies on intact sheath; therefore, they are not responsible for the 2.8-nm repeat occurring on the outer face of the sheath. However, longitudinal and transverse linear labeling by protein A-colloidal gold on the outer and inner faces, respectively, occurred with monoclonal antibodies specific to the phenol-soluble polypeptides. Restricted surface exposure of phenol-soluble polypeptides on the sheath highlighted molecular defects in sheath architecture. These lattice faults may indicate sites of sheath growth to accommodate cell growth or division (longitudinal immunogold label) and filament division (transverse immunogold label). The identification of a second group of polypeptides within the infrastructure of the sheath suggests that the sheath is a trilaminar structure in which phenol-soluble polypeptides are sandwiched between sodium dodecyl sulfate-beta-mercaptoethanol-EDTA-soluble polypeptides (G. Southam and T. J. Beveridge, J. Bacteriol. 173:6213-6222, 1991) (phenol-insoluble material). Images PMID:1732226

  15. Modeling Organic Anion-Transporting Polypeptide 1B1 Inhibition to Elucidate Interaction Risks in Early Drug Design.

    PubMed

    Zamora, Ismael; Winiwarter, Susanne

    2016-10-01

    The importance of transporter proteins for the disposition of drugs has become increasingly apparent during the past decade. A noted drug-drug interaction risk is the inhibition of organic anion-transporting polypeptides (OATPs), key transporters for the liver uptake of the widely used statins. We show here the development of a ligand-based in silico model for interaction with OATP1B1, an important representative of the OATP family. The model is based on a structural overlay of 6 known OATP1B1 inhibitors. A data set of about 150 compounds with published OATP1B1 inhibition data was compared to the resulting "transportophor," and a similarity threshold was defined to distinguish between active and inactive molecules. In addition, using a statistical model based on physicochemical properties of the compounds as prefilter was found to enhance the overall predictivity of the model (final accuracy 0.73, specificity 074, and sensitivity 0.71, based on 126 compounds). The combined model was validated using an in-house data set (accuracy, specificity, and sensitivity were 0.63, 0.59, and 0.78, respectively; 62 compounds). The model gives also a structural overlay to the most similar template enabling visualization of where a change in a given structure might reduce the interaction with the transporter.

  16. Modeling Organic Anion-Transporting Polypeptide 1B1 Inhibition to Elucidate Interaction Risks in Early Drug Design.

    PubMed

    Zamora, Ismael; Winiwarter, Susanne

    2016-10-01

    The importance of transporter proteins for the disposition of drugs has become increasingly apparent during the past decade. A noted drug-drug interaction risk is the inhibition of organic anion-transporting polypeptides (OATPs), key transporters for the liver uptake of the widely used statins. We show here the development of a ligand-based in silico model for interaction with OATP1B1, an important representative of the OATP family. The model is based on a structural overlay of 6 known OATP1B1 inhibitors. A data set of about 150 compounds with published OATP1B1 inhibition data was compared to the resulting "transportophor," and a similarity threshold was defined to distinguish between active and inactive molecules. In addition, using a statistical model based on physicochemical properties of the compounds as prefilter was found to enhance the overall predictivity of the model (final accuracy 0.73, specificity 074, and sensitivity 0.71, based on 126 compounds). The combined model was validated using an in-house data set (accuracy, specificity, and sensitivity were 0.63, 0.59, and 0.78, respectively; 62 compounds). The model gives also a structural overlay to the most similar template enabling visualization of where a change in a given structure might reduce the interaction with the transporter. PMID:27526968

  17. Poly(L-lysine) and Clay Nanocomposite with Desired Matrix Secondary Structure: Effects of Polypeptide Molecular Weight

    SciTech Connect

    Hule,R.; Pochan, D.

    2007-01-01

    Nanocomposites (NC) were formed using cationic poly(L-lysine) (PLL), a semicrystalline polypeptide, that was reinforced by sodium montmorillonite (MMT) clay via solution intercalation technique. By varying solution conditions such as pH, temperature, and polypeptide concentration in the presence of clay platelets, the secondary structure of PLL was controllably altered into {alpha}-helical, {beta}-sheet, and random coil. The high molecular weight polypeptide shows a strong propensity to fold into the {beta}-sheet structure when cast as films, irrespective of the initial secondary structure in solution. Nanocomposite local morphology confirms intercalated MMT platelets with PLL over a wide range of compositions.

  18. Temperature-triggered Phase Separation of a Hydrophilic Resilin-like Polypeptide

    PubMed Central

    Li, Linqing; Luo, Tianzhi

    2015-01-01

    Temperature-triggered phase separation of recombinant proteins has offered substantial opportunities in the design of nanoparticles for a variety of applications. Herein we describe the temperature-triggered phase separation behavior of a recombinant hydrophilic resilin-like polypeptide (RLP). The transition temperature and sizes of RLP-based nanoparticles can be modulated based on variations in polypeptide concentration, salt identity, ionic strength, pH, and denaturing agents, as indicated via UV-Vis spectroscopy and dynamic light scattering (DLS). The irreversible particle formation is coupled with secondary conformational changes from a random coil conformation to a more ordered β-sheet structure. These RLP-based nanoparticles could find potential use as mechanically-responsive components in drug delivery, nanospring, nanotransducer, and biosensor applications. PMID:25424611

  19. Isolation and amino acid sequences of opossum vasoactive intestinal polypeptide and cholecystokinin octapeptide.

    PubMed Central

    Eng, J; Yu, J; Rattan, S; Yalow, R S

    1992-01-01

    Evolutionary history suggests that the marsupials entered South America from North America about 75 million years ago and subsequently dispersed into Australia before the separation between South America and Antarctica-Australia. A question of interest is whether marsupial peptides resemble the corresponding peptides of Old or New World mammals. Previous studies had shown that "little" gastrin of the North American marsupial, the opossum, is identical in length to that of the New World mammals, the guinea pig and chinchilla. In this report, we demonstrate that opossum cholecystokinin octapeptide, like that of the Australian marsupials, the Eastern quoll and the Tamar wallaby, is identical to the cholecystokinin octapeptide of Old World mammals and differs from that of the guinea pig and chinchilla. However, opossum vasoactive intestinal polypeptide differs from the usual Old World mammalian vasoactive intestinal polypeptide in five sites: [sequence; see text]. PMID:1542675

  20. Sequence analysis and protein import studies of an outer chloroplast envelope polypeptide.

    PubMed Central

    Salomon, M; Fischer, K; Flügge, U I; Soll, J

    1990-01-01

    A chloroplast outer envelope membrane protein was cloned and sequenced and from the sequence it was possible to deduce a polypeptide of 6.7 kDa. It has only one membrane-spanning region; the C terminus extends into the cytosol, whereas the N terminus is exposed to the space between the two envelope membranes. The protein was synthesized in an in vitro transcription-translation system to study its routing into isolated chloroplasts. The import studies revealed that the 6.7-kDa protein followed a different and heretofore undescribed translocation pathway in the respect that (i) it does not have a cleavable transit sequence, (ii) it does not require ATP hydrolysis for import, and (iii) protease-sensitive components that are responsible for recognition of precursor proteins destined for the inside of the chloroplasts are not involved in routing the 6.7-kDa polypeptide to the outer chloroplast envelope. Images PMID:2377616

  1. Membrane-associated polypeptides induced in Chlamydomonas by limiting CO sub 2 concentrations

    SciTech Connect

    Spalding, M.H.; Jeffrey, M. )

    1989-01-01

    Chlamydomonas reinhardtii and other unicellular green algae have a high apparent affinity for CO{sub 2}, little O{sub 2} inhibition of photosynthesis, and reduced photorespiration. These characteristics result from operation of a CO{sub 2}-concentrating system. The CO{sub 2}-concentrating system involves active inorganic carbon transport and is under environmental control. Cells grown at limiting CO{sub 2} concentrations have inorganic carbon transport activity, but cells grown at 5% CO{sub 2} do not. Four membrane-associated polypeptides (M{sub r}, 19, 21, 35, and 36 kilodaltons) have been identified which either appear or increase in abundance during adaptation to limiting CO{sub 2} concentrations. The appearance of two of the polypeptides occurs over roughly the same time course as the appearance of the CO{sub 2}-concentrating system activity in response to CO{sub 2} limitation.

  2. Catalytic and reactive polypeptides and methods for their preparation and use

    DOEpatents

    Schultz, Peter

    1994-01-01

    Catalytic and reactive polypeptides include a binding site specific for a reactant or reactive intermediate involved in a chemical reaction of interest. The polypeptides further include at least one active functionality proximate the binding site, where the active functionality is capable of catalyzing or chemically participating in the chemical reaction in such a way that the reaction rate is enhanced. Methods for preparing the catalytic peptides include chemical synthesis, site-directed mutagenesis of antibody and enzyme genes, covalent attachment of the functionalities through particular amino acid side chains, and the like. This invention was made with Government support under Grant Contract No. AI-24695, awarded by the Department of health and Human Services, and under Grant Contract No. N 00014-87-K-0256, awarded by the Office of Naval Research. The Government has certain rights in this invention.

  3. Hierarchical Bionanotubes Formed By the Self Assembly of Microtubules With Cationic Membranes Or Polypeptides

    SciTech Connect

    Raviv, U.; Needleman, D.J.; Ewert, K.K.; Safinya, C.R.

    2009-06-05

    At present there is a surge in interest in biophysical research aimed at elucidating collective interactions between cellular proteins and associated biomolecules leading to supramolecular structures, with the ultimate goal of relating structure to function. The nerve cell cytoskeleton provides a rich example of highly ordered bundles and networks of interacting neurofilaments, microtubules and filamentous actin, where the nature of the interactions, structures and structure-function correlations remain poorly understood. We present synchrotron X-ray diffraction and electron microscopy data, in reconstituted protein systems from the bovine central nervous system, which reveal unexpected structures not predicted by current electrostatic theories. By mixing preassembled microtubules with charged membranes or polypeptides we found hierarchical bionanotubes made of microtubules coated by lipid bilayers or polypeptides, which in turn are coated with a third layer of tubulin oligomers forming rings or spirals.

  4. Self-assembling chimeric polypeptide-doxorubicin conjugate nanoparticles that abolish tumours after a single injection

    NASA Astrophysics Data System (ADS)

    Andrew Mackay, J.; Chen, Mingnan; McDaniel, Jonathan R.; Liu, Wenge; Simnick, Andrew J.; Chilkoti, Ashutosh

    2009-12-01

    New strategies to self-assemble biocompatible materials into nanoscale, drug-loaded packages with improved therapeutic efficacy are needed for nanomedicine. To address this need, we developed artificial recombinant chimeric polypeptides (CPs) that spontaneously self-assemble into sub-100-nm-sized, near-monodisperse nanoparticles on conjugation of diverse hydrophobic molecules, including chemotherapeutics. These CPs consist of a biodegradable polypeptide that is attached to a short Cys-rich segment. Covalent modification of the Cys residues with a structurally diverse set of hydrophobic small molecules, including chemotherapeutics, leads to spontaneous formation of nanoparticles over a range of CP compositions and molecular weights. When used to deliver chemotherapeutics to a murine cancer model, CP nanoparticles have a fourfold higher maximum tolerated dose than free drug, and induce nearly complete tumour regression after a single dose. This simple strategy can promote co-assembly of drugs, imaging agents and targeting moieties into multifunctional nanomedicines.

  5. Properties of pertussis toxin B oligomer assembled in vitro from recombinant polypeptides produced by Escherichia coli.

    PubMed Central

    Burnette, W N; Arciniega, J L; Mar, V L; Burns, D L

    1992-01-01

    The subunits that make up the pentameric B oligomer of pertussis toxin (S2, S3, S4, and S5) were individually synthesized as recombinant polypeptides in Escherichia coli, isolated as insoluble inclusion bodies, and assembled into a multimeric form in vitro by spontaneous association following treatment with a chaotropic agent, reduction, and reoxidation. The recombinant B multimer, purified by fetuin-Sepharose affinity chromatography, contained all four of the individual subunits and possessed the mitogenic and hemagglutinating activities characteristic of the native B oligomer. Immunization of mice with the recombinant B oligomer elicited antibodies that neutralized pertussis toxin in vitro and, moreover, provided protection in vivo against the leukocytosis-promoting activity of the toxin. These results demonstrate the potential for assembly of complex multimeric proteins from recombinant DNA-derived polypeptides and provide a novel means for production of an acellular pertussis vaccine component. Images PMID:1587592

  6. Experimental Milestones in the Discovery of Molecular Chaperones as Polypeptide Unfolding Enzymes.

    PubMed

    Finka, Andrija; Mattoo, Rayees U H; Goloubinoff, Pierre

    2016-06-01

    Molecular chaperones control the cellular folding, assembly, unfolding, disassembly, translocation, activation, inactivation, disaggregation, and degradation of proteins. In 1989, groundbreaking experiments demonstrated that a purified chaperone can bind and prevent the aggregation of artificially unfolded polypeptides and use ATP to dissociate and convert them into native proteins. A decade later, other chaperones were shown to use ATP hydrolysis to unfold and solubilize stable protein aggregates, leading to their native refolding. Presently, the main conserved chaperone families Hsp70, Hsp104, Hsp90, Hsp60, and small heat-shock proteins (sHsps) apparently act as unfolding nanomachines capable of converting functional alternatively folded or toxic misfolded polypeptides into harmless protease-degradable or biologically active native proteins. Being unfoldases, the chaperones can proofread three-dimensional protein structures and thus control protein quality in the cell. Understanding the mechanisms of the cellular unfoldases is central to the design of new therapies against aging, degenerative protein conformational diseases, and specific cancers.

  7. The Effects of Solvent Environment on the Optical Rotatory Dispersion Parameters of Polypeptides

    PubMed Central

    Cassim, Joseph Y.; Taylor, Edwin W.

    1965-01-01

    The constancy of the Moffitt optical rotatory dispersion parameters for polypeptides in different solvents was tested by dispersion measurements on poly-γ-benzyl-L-glutamate in fifty-five solvents and solvent mixtures. b0 was not constant but varied linearly with the refractive index of the solvent according to the equation -b0 = 1701 - 730.3 n8. This variation could not be explained by changes in configuration of the polypeptide. a0 also showed a trend with solvent index but the values were widely scattered. λ0 did not show a statistically significant dependence on solvent index. The variation in b0 can be interpreted as an effect of solvent polarizability on the frequencies of optically active transitions. PMID:5861706

  8. Dual effect of crowders on fibrillation kinetics of polypeptide chains revealed by lattice models

    NASA Astrophysics Data System (ADS)

    Co, Nguyen Truong; Hu, Chin-Kun; Li, Mai Suan

    2013-05-01

    We have developed the lattice model for describing polypeptide chains in the presence of crowders. The influence of crowding confinement on the fibrillation kinetics of polypeptide chains is studied using this model. We observed the non-trivial behavior of the fibril formation time τfib that it decreases with the concentration of crowders if crowder sizes are large enough, but the growth is observed for crowders of small sizes. This allows us to explain the recent experimental observation on the dual effect of crowding particles on fibril growth of proteins that for a fixed crowder concentration the fibrillation kinetics is fastest at intermediate values of total surface of crowders. It becomes slow at either small or large coverages of cosolutes. It is shown that due to competition between the energetics and entropic effects, the dependence of τfib on the size of confined space is described by a parabolic function.

  9. UV Spectrophotometric Method for Estimation of Polypeptide-K in Bulk and Tablet Dosage Forms

    NASA Astrophysics Data System (ADS)

    Kaur, P.; Singh, S. Kumar; Gulati, M.; Vaidya, Y.

    2016-01-01

    An analytical method for estimation of polypeptide-k using UV spectrophotometry has been developed and validated for bulk as well as tablet dosage form. The developed method was validated for linearity, precision, accuracy, specificity, robustness, detection, and quantitation limits. The method has shown good linearity over the range from 100.0 to 300.0 μg/ml with a correlation coefficient of 0.9943. The percentage recovery of 99.88% showed that the method was highly accurate. The precision demonstrated relative standard deviation of less than 2.0%. The LOD and LOQ of the method were found to be 4.4 and 13.33, respectively. The study established that the proposed method is reliable, specific, reproducible, and cost-effective for the determination of polypeptide-k.

  10. Thermodynamic Approach to Enhanced Dispersion and Physical Properties in a Carbon Nanotube/Polypeptide Nanocomposite

    NASA Technical Reports Server (NTRS)

    Lovell, Conrad S.; Wise, Kristopher E.; Kim, Jae-Woo; Lillehei, Peter T.; Harrison, Joycelyn S.; Park, Cheol

    2009-01-01

    A high molecular weight synthetic polypeptide has been designed which exhibits favorable interactions with single wall carbon nanotubes (SWCNTs). The enthalpic and entropic penalties of mixing between these two molecules are reduced due to the polypeptide's aromatic sidechains and helical secondary structure, respectively. These enhanced interactions result in a well dispersed SWCNT/Poly (L-Leucine-ran-L-Phenylalanine) nanocomposite with enhanced mechanical and electrical properties using only shear mixing and sonication. At 0.5 wt% loading of SWCNT filler, the nanocomposite exhibits simultaneous increases in the Young's modulus, failure strain, and toughness of 8%, 120%, and 144%, respectively. At one kHz, the same nanotube loading level also enhances the dielectric constant from 2.95 to 22.81, while increasing the conductivity by four orders of magnitude.

  11. A polarizable force field for computing the infrared spectra of the polypeptide backbone.

    PubMed

    Schultheis, Verena; Reichold, Rudolf; Schropp, Bernhard; Tavan, Paul

    2008-10-01

    The shapes of the amide bands in the infrared (IR) spectra of proteins and peptides are caused by electrostatically coupled vibrations within the polypeptide backbone and code the structures of these biopolymers. A structural decoding of the amide bands has to resort to simplified models because the huge size of these macromolecules prevents the application of accurate quantum mechanical methods such as density functional theory (DFT). Previous models employed transition-dipole coupling methods that are of limited accuracy. Here we propose a concept for the computation of protein IR spectra, which describes the molecular mechanics (MM) of polypeptide backbones by a polarizable force field of "type II". By extending the concepts of conventional polarizable MM force fields, such a PMM/II approach employs field-dependent parameters not only for the electrostatic signatures of the molecular components but also for the local potentials modeling the stiffness of chemical bonds with respect to elongations, angle deformations, and torsions. Using a PMM/II force field, the IR spectra of the polypeptide backbone can be efficiently calculated from the time dependence of the backbone's dipole moment during a short (e.g., 100 ps) MD simulation by Fourier transformation. PMM/II parameters are derived for harmonic bonding potentials of amide groups in polypeptides from a series of DFT calculations on the model molecule N-methylacetamide (NMA) exposed to homogeneous external electric fields. The amide force constants are shown to vary by as much as 20% for relevant field strengths. As a proof of principle, it is shown that the large solvatochromic effects observed in the IR spectra of NMA upon transfer from the gas phase into aqueous solution are not only excellently reproduced by DFT/MM simulations but are also nicely modeled by the PMM/II approach. The tasks remaining for a proof of practice are specified.

  12. Effects of surface hydrophobicity on the conformational changes of polypeptides of different length

    NASA Astrophysics Data System (ADS)

    Mu, Yan

    2011-09-01

    We studied the effects of surface hydrophobicity on the conformational changes of different length polypeptides by calculating the free energy difference between peptide structures using the bias-potential Monte Carlo technique and the probability ratio method. It was found that the hydrophobic surface plays an important role in the stability of secondary structures of the polypeptides with hydrophobic side chains. For short GAAAAG peptides, the hydrophobic surface destabilizes the α helix but stabilizes the β hairpin in the entire temperature region considered in our study. Interestingly, when the surface hydrophobic strength ɛhpsf≥ɛhp, the most stable structure in the low temperature region changes from α helix to β hairpin, and the corresponding phase transition temperature increases slightly. For longer GAAAAAAAAAAG peptides, the effects of the relatively weak hydrophobic surface (ɛhpsf < ɛhp) on α-helical structures may be neglected, while the relatively strongly hydrophobic surface (ɛhpsf≥ɛhp) leads to the obvious partial helicity loss. In contrast, the stability of β structures can be enhanced significantly by the hydrophobic surface, especially by the strongly hydrophobic surface, at low and intermediate temperatures. At high temperatures, in addition to thermal fluctuations, the strongly hydrophobic surface (ɛhpsf>ɛhp) may further disturb the formation of both α-helical and β structures. Moreover, the phase transition temperature between α-helical structures and random coils significantly decreases due to the helicity loss when ɛhpsf>ɛhp. Our findings provide a basic and quantitative picture for understanding the effects of a hydrophobic surface on the conformational changes of the polypeptides with hydrophobic side chains. From an application viewpoint, the present study is helpful in developing alternative strategies of producing high-quality biological fibrillar materials and functional nanoscale devices by the self-assembly of

  13. Specific photoaffinity labeling of two plasma membrane polypeptides with an azido auxin

    NASA Technical Reports Server (NTRS)

    Hicks, G. R.; Rayle, D. L.; Jones, A. M.; Lomax, T. L.

    1989-01-01

    Plasma membrane vesicles were isolated from zucchini (Cucurbita pepo) hypocotyl tissue by aqueous phase partitioning and assessed for homogeneity by the use of membrane-specific enzyme assays. The highly pure (ca. 95%) plasma membrane vesicles maintained a pH differential across the membrane and accumulated a tritiated azido analogue of 3-indoleacetic acid (IAA), 5-azido-[7-3H]IAA ([3H]N3IAA), in a manner similar to the accumulation of [3H]IAA. The association of the [3H]N3IAA with membrane vesicles was saturable and subject to competition by IAA and auxin analogues. Auxin-binding proteins were photoaffinity labeled by addition of [3H]N3IAA to plasma membrane vesicles prior to exposure to UV light (15 sec; 300 nm) and detected by subsequent NaDodSO4/PAGE and fluorography. When the reaction temperature was lowered to -196 degrees C, high-specific-activity labeling of a 40-kDa and a 42-kDa polypeptide was observed. Triton X-100 (0.1%) increased the specific activity of labeling and reduced the background, which suggests that the labeled polypeptides are intrinsic membrane proteins. The labeled polypeptides are of low abundance, as expected for auxin receptors. Further, the addition of IAA and auxin analogues to the photoaffinity reaction mixture resulted in reduced labeling that was qualitatively similar to their effects on the accumulation of radiolabeled IAA in membrane vesicles. Collectively, these results suggest that the radiolabeled polypeptides are auxin receptors. The covalent nature of the label should facilitate purification and further characterization of the receptors.

  14. Folding and self-assembly of polypeptides: Dynamics and thermodynamics from molecular simulation

    NASA Astrophysics Data System (ADS)

    Fluitt, Aaron Michael

    Empowered by their exquisite three-dimensional structures, or "folds," proteins carry out biological tasks with high specificity, efficiency, and fidelity. The fold that optimizes biological function represents a stable configuration of the constituent polypeptide molecule(s) under physiological conditions. Proteins and polypeptides are not static, however: battered by thermal motion, they explore a distribution of folds that is determined by the sequence of amino acids, the presence and identity of other molecules, and the thermodynamic conditions. In this dissertation, we apply molecular simulation techniques to the study of two polypeptides that have unusually diffuse distributions of folds under physiological conditions: polyglutamine (polyQ) and islet amyloid polypeptide (IAPP). Neither polyQ nor IAPP adopts a predominant fold in dilute aqueous solution, but at sufficient concentrations, both are prone to self-assemble into stable, periodic, and highly regular aggregate structures known as amyloid. The appearance of amyloid deposits of polyQ in the brain, and of IAPP in the pancreas, are associated with Huntington's disease and type 2 diabetes, respectively. A molecular view of the mechanism(s) by which polyQ and IAPP fold and self-assemble will enhance our understanding of disease pathogenesis, and it has the potential to accelerate the development of therapeutics that target early-stage aggregates. Using molecular simulations with spatial and temporal resolution on the atomic scale, we present analyses of the structural distributions of polyQ and IAPP under various conditions, both in and out of equilibrium. In particular, we examine amyloid fibers of polyQ, the IAPP dimer in solution, and single IAPP fragments at a lipid bilayer. We also benchmark the molecular models, or "force fields," available for such studies, and we introduce a novel simulation algorithm.

  15. Peptide Map Comparison of Infectious Pancreatic Necrosis Virus-Specific Polypeptides

    PubMed Central

    Dobos, Peter; Rowe, David

    1977-01-01

    An investigation of virus-specific protein synthesis in infectious pancreatic necrosis virus (IPNV)-infected rainbow trout gonad cells was undertaken to find a relationship between the coding capacity of the virus genome (two segments of double-stranded RNA of 2.5 × 106 and 2.3 × 106 molecular weight) and the sizes and relative amounts of virus-specific proteins. Using polyacrylamide slabgel electrophoresis and autoradiography, eight distinct virus-specific polypeptides were detected in infected, [35S]methionine-labeled cells. These proteins may be grouped into three size classes on the basis of molecular weight: (i) large, α (90,000); (ii) medium, β1 (59,000), β2 (58,000), and β3 (57,000); and (iii) small, γ1 (29,000), γ1A (28,000), γ2 (27,000), and γ3 (25,000). The combined molecular weight of these polypetides (373,000) is beyond the coding capacity of the virus genome. Purified IPNV contained polypeptides α, β3, γ1, and γ1A. Pulse-chase experiments and tryptic peptide map comparisons revealed that only four of the eight intracellular proteins were primary gene products, namely, α, β1, γ1, and β2, with a combined molecular weight of 205,000. Of these primary gene products only the α polypeptide was found to be stable, whereas the other three underwent intracellular proteolytic cleavage during virus morphogenesis. Polypeptide β1 was cleaved to generate β2 and β3; γ1 was trimmed to produce γ1A, and the only nonstructural primary gene product, γ2, was found to be a precursor of γ3. These results suggest that IPNV possesses a unique mechanism to synthesize three size classes of proteins using mRNA transcripts from two high-molecular-weight double-stranded RNA genome segments. Images PMID:563479

  16. Differential Proteolysis of Glycinin and beta-Conglycinin Polypeptides during Soybean Germination and Seedling Growth.

    PubMed

    Wilson, K A; Rightmire, B R; Chen, J C; Tan-Wilson, A L

    1986-09-01

    The degradation of the major seed storage globulins of the soybean (Glycine max [L.] Merrill) was examined during the first 12 days of germination and seedling growth. The appearance of glycinin and beta-conglycinin degradation products was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cotyledon extracts followed by electroblotting to nitrocellulose and immunostaining using glycinin and beta-conglycinin specific antibodies. The three subunits of beta-conglycinin were preferentially metabolized. Of the three subunits of beta-conglycinin, the larger alpha and alpha' subunits are rapidly degraded, generating new beta-conglycinin cross-reactive polypeptides of 51,200 molecular weight soon after imbibition of the seed. After 6 days of growth the beta-subunit is also hydrolyzed. At least six polypeptides, ranging from 33,100 to 24,000 molecular weight, appear as apparent degradation products of beta-conglycinin. The metabolism of the glycinin acidic chains begins early in growth. The glycinin acidic chains present at day 3 have already been altered from the native form in the ungerminated seed, as evidenced by their higher mobility in an alkaline-urea polyacrylamide gel electrophoresis system. However, no change in the molecular weight of these chains is detectable by sodium dodecyl sulfate-polyarylamide gel electrophoresis. Examination of the glycinin polypeptide amino-termini by dansylation suggests that this initial modification of the acidic chains involves limited proteolysis at the carboxyl-termini, deamidation, or both. After 3 days of growth the acidic chains are rapidly hydrolyzed to a smaller (21,900 molecular weight) form. The basic polypeptides of glycinin appear to be unaltered during the first 8 days of growth, but are rapidly degraded thereafter to unidentified products. All of the original glycinin basic chains have been destroyed by day 10 of growth.

  17. Wall-associated kinase-like polypeptide mediates nutritional status perception and response

    DOEpatents

    Yang, Zhenbiao; Karr, Stephen

    2014-02-11

    The disclosure relates to methods for modulating plant growth and organogenesis using dominant-negative receptor-like kinases. The disclosure further provides a method for increasing plant yield relative to corresponding wild type plants comprising modulating the expression in a plant of a nucleic acid encoding a Wall-Associated Kinase-like 14 polypeptide or a homolog thereof, and selecting for plants having increased yield or growth on a nutrient deficient substrate.

  18. Hybrid Nanomaterials by Surface Grafting of Synthetic Polypeptides Using N-Carboxyanhydride (NCA) Polymerization.

    PubMed

    Borase, Tushar; Heise, Andreas

    2016-07-01

    The interaction of materials with their environment is largely dictated by interfacial phenomena. Polymers are very versatile materials to modulate material interfaces to provide functionality, stability and compatibility. A class of polymers that can close the gap between fully synthetic and natural macromolecules are polypeptides derived from N-carboxyanhydride (NCA) polymerization. Recent advances in using this technique to create biomimetic interfaces and hybrid materials are highlighted, with special emphasis on nanomaterials.

  19. Antigenic relationships among homologous structural polypeptides of porcine, feline, and canine coronaviruses.

    PubMed Central

    Horzinek, M C; Lutz, H; Pedersen, N C

    1982-01-01

    Transmissible gastroenteritis virus of swine (TGEV), feline infectious peritonitis virus (FIPV), and canine coronavirus were studied with respect to their serological cross-reactivity in homologous and heterologous virus neutralization, immune precipitation of radiolabeled TGEV, electroblotting, and enzyme-linked immunosorbent assay using individual virion polypeptides prepared by polyacrylamide gel electrophoresis. TGEV was neutralized by feline anti-FIPV serum, and the reaction was potentiated by complement; heterologous neutralization involved antibody reacting with the peplomer protein (P), the envelope protein (E), and cellular (glycolipid) components incorporated into the TGEV membrane. Electrophoretic analysis of immune precipitates containing [35S]methionine-labeled disrupted TGEV and feline anti-FIPV antibody confirmed the reaction with the P and E polypeptides and showed the nucleocapsid protein (N) in addition. Electroblotting, followed by incubation with antibody, 125I-labeled protein A, and fluorography, disclosed cross-reactions between the three viruses at the N and E levels and revealed differences in the apparent molecular weights of the latter. Enzyme immunoassays performed with standard amounts of immobilized P, N, and E polypeptides of the three viruses showed recognition of the antigens by homologous and heterologous antibody to comparable degrees. These results indicate a close antigenic relationship between TGEV, FIPV, and canine coronavirus due to common determinants on the three major virion proteins. The taxonomic implications of these findings are discussed. Images PMID:6182101

  20. Cotranslational and posttranslational N-glycosylation of polypeptides by distinct mammalian OST isoforms.

    PubMed

    Ruiz-Canada, Catalina; Kelleher, Daniel J; Gilmore, Reid

    2009-01-23

    Asparagine-linked glycosylation of polypeptides in the lumen of the endoplasmic reticulum is catalyzed by the hetero-oligomeric oligosaccharyltransferase (OST). OST isoforms with different catalytic subunits (STT3A versus STT3B) and distinct enzymatic properties are coexpressed in mammalian cells. Using siRNA to achieve isoform-specific knockdowns, we show that the OST isoforms cooperate and act sequentially to mediate protein N-glycosylation. The STT3A OST isoform is primarily responsible for cotranslational glycosylation of the nascent polypeptide as it enters the lumen of the endoplasmic reticulum. The STT3B isoform is required for efficient cotranslational glycosylation of an acceptor site adjacent to the N-terminal signal sequence of a secreted protein. Unlike STT3A, STT3B efficiently mediates posttranslational glycosylation of a carboxyl-terminal glycosylation site in an unfolded protein. These distinct and complementary roles for the OST isoforms allow sequential scanning of polypeptides for acceptor sites to insure the maximal efficiency of N-glycosylation.

  1. Contractile properties of synthetic cationic polypeptides in guinea-pig isolated trachea.

    PubMed Central

    Spina, D.; Goldie, R. G.

    1994-01-01

    1. The synthetic polypeptides, poly-L-arginine, poly-L-lysine and poly-D-lysine contract guinea-pig isolated trachea in a concentration-dependent, epithelium-independent manner. Indomethacin augmented the contractile response to poly-L-arginine. 2. The contractile response to poly-L-arginine was not significantly inhibited by nicardipine, a selective L-type calcium channel blocker or by the histamine H1-receptor antagonist, mepyramine nor significantly augmented by the neutral endopeptidase inhibitor, phosphoramidon. 3. The contractile response to poly-L-arginine was inhibited in a concentration-dependent manner by prior incubation of guinea-pig tracheal rings with a number of anionic polypeptides including, low molecular weight heparin, poly-L-aspartic acid and bovine serum albumin. 4. In vitro capsaicin-induced desensitization failed to attenuate the contractile response to poly-L-arginine, suggesting little, if any role for sensory neuropeptides in the functional response in the guinea-pig. 5. Synthetic polypeptides induce an epithelium-independent, charge-dependent contraction of guinea-pig isolated trachea. PMID:8012709

  2. Rh polypeptide is a major fatty acid-acylated erythrocyte membrane protein

    SciTech Connect

    de Vetten, M.P.; Agre, P.

    1988-12-05

    The erythrocyte Rh antigens contain an Mr = 32,000 integral protein which is thought to contribute in some way to the organization of surrounding phospholipid. To search for possible fatty acid acylation of the Rh polypeptide, intact human erythrocytes were incubated with (3H)palmitic acid prior to preparation of membranes and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Several membrane proteins were labeled, but none corresponded to the glycophorins or membrane proteins 1-8. An Mr = 32,000 band was prominently labeled on Rh (D)-negative and -positive erythrocytes and could be precipitated from the latter with anti-D. No similar protein was labeled on membranes from Rhmod erythrocytes, a rare phenotype lacking Rh antigens. Labeling of the Rh polypeptide most likely represents palmitic acid acylation through thioester linkages. The 3H label was not extracted with chloroform/methanol, but was quantitatively eluted with hydroxylamine and co-chromatographed with palmitohydroxamate and free palmitate by thin layer chromatography. The fatty acid acylations occurred independent of protein synthesis and were completely reversed by chase with unlabeled palmitate. It is concluded that the Rh polypeptide is fatty acid-acylated, being a major substrate of an acylation-deacylation mechanism associated with the erythrocyte membrane.

  3. Pairwise energies for polypeptide coarse-grained models derived from atomic force fields

    NASA Astrophysics Data System (ADS)

    Betancourt, Marcos R.; Omovie, Sheyore J.

    2009-05-01

    The energy parametrization of geometrically simplified versions of polypeptides, better known as polypeptide or protein coarse-grained models, is obtained from molecular dynamics and statistical methods. Residue pairwise interactions are derived by performing atomic-level simulations in explicit water for all 210 pairs of amino acids, where the amino acids are modified to closer match their structure and charges in polypeptides. Radial density functions are computed from equilibrium simulations for each pair of residues, from which statistical energies are extracted using the Boltzmann inversion method. The resulting models are compared to similar potentials obtained by knowledge based methods and to hydrophobic scales, resulting in significant similarities in spite of the model simplicity. However, it was found that glutamine, asparagine, lysine, and arginine are more attractive to other residues than anticipated, in part, due to their amphiphilic nature. In addition, equally charged residues appear more repulsive than expected. Difficulties in the calculation of knowledge based potentials and hydrophobicity scale for these cases, as well as sensitivity of the force field to polarization effects are suspected to cause this discrepancy. It is also shown that the coarse-grained model can identify native structures in decoy databases nearly as well as more elaborate knowledge based methods, in spite of its resolution limitations. In a test conducted with several proteins and corresponding decoys, the coarse-grained potential was able to identify the native state structure but not the original atomic force field.

  4. Beta-glucosidase enzymatic activity of crystal polypeptide of the Bacillus thuringiensis strain 1.1.

    PubMed

    Papalazaridou, A; Charitidou, L; Sivropoulou, A

    2003-01-01

    The crystals of Bacillus thuringiensis strain 1.1 consist of the 140 kDa delta-endotoxin, which exhibits beta-glucosidase enzymatic activity, based on the following data. (i) Purified crystals exhibit beta-glucosidase enzymatic activity. When the crystals are reacted with specific antibodies directed either against the commercial (almond purified) beta-glucosidase or against the 140 kDa polypeptide, then considerable reduction of enzymatic activity is observed almost at the same level with both antibodies. (ii) Commercial beta-glucosidase and the 140 kDa crystal polypeptide share antigenic similarities; in Western immunoblots, the 140 kDa crystal polypeptide is recognized by anti-beta-glucosidase antibodies, and commercial beta-glucosidase is recognized by anti-140-kDa antibodies. (iii) The enzymatic properties of commercial beta-glucosidase and that resident in the crystals of B. thuringiensis strain 1.1 are very similar. Thus, both enzymes hydrolyze a wide range of substrates (aryl-beta-glucosides, disaccharides with alpha- or beta-linkage polysaccharides) and have an optimum activity at 40 degrees C and pH 5. Both enzymes are relatively thermostable and are resistant to end-product inhibition by glucose. Additionally, they show the same pattern of inhibition or activation by several chemical compounds. (iv) The crystals and commercial beta-glucosidase show almost equivalent levels of insecticidal activity against Drosophila melanogaster larvae and, furthermore, cause reduction in adult flies that emerge from larvae surviving treatment.

  5. Polycarbophil-cysteine conjugates as platforms for oral polypeptide delivery systems.

    PubMed

    Bernkop-Schnürch, A; Thaler, S C

    2000-07-01

    The purpose of the present study was to evaluate the potential of polycarbophil-cysteine conjugates as carrier systems for orally administered peptide and protein drugs. Mediated by a carbodiimide, cysteine was covalently attached to polycarbophil. The properties of resulting conjugates, displaying 35-50 microM thiol groups per gram of polymer, to bind polypeptides and to inhibit pancreatic proteases was evaluated in vitro. Results demonstrated that only some polypeptides are immobilized to the polycarbophil-cysteine conjugate. Due to the covalent attachment of cysteine to polycarbophil, the inhibitory effect of the polymer toward carboxypeptidase A (EC 3.4. 17.1) and carboxypeptidase B (EC 3.4.17.2) could be significantly (p < 0.05) improved. As the zinc binding affinity of polycarbophil could be improved by the covalent attachment of cysteine, the raised inhibitory effect seems to be based on the complexation of this divalent cation from the enzyme structure. Whereas the covalent attachment of cysteine on polycarbophil had no influence on the enzymatic activity of trypsin (EC 3.4.21.4) and elastase (EC 3.4.21. 36), the inhibitory effect of the polymer-cysteine conjugate toward chymotrypsin (EC 3.4.21.1) was significantly (p < 0.05) higher than that of the unmodified polymer. Because of these inhibitory features, polycarbophil-cysteine conjugates seem to be a promising tool in protecting orally administered therapeutic polypeptides, which are not bound to the polymer, from presystemic metabolism in the intestine.

  6. Role of polypeptide growth factors in phenotypic transformation of normal rat kidney cells

    SciTech Connect

    van Zoelen, E.J.J.; van Oostwaard, T.M.J.; de Laat, S.W.

    1988-01-05

    A serum-free assay has been established for studying the role of polypeptide growth factors in inducing loss of density-dependent inhibition of growth of normal rat kidney (NRK) cells. The process has been characterized by measuring the time course of (/sup 3/H)thymidine incorporation into confluent, quiescent NRK cultures stimulated by defined polypeptide growth factors, in combination with cell counting studies, increases in DNA content, and cell cycle analysis by means of a fluorescence-activated cell sorter. It is shown that none of the growth factors tested is able to induce loss of density-dependent inhibition of growth by itself, but strong synergism was observed when combinations of growth factors were tested. None of the above factors was found to be essential, however, since any combination of three of the above four growth factors strongly induced the process. Strong parallels were observed between the growth factor requirements for inducing loss of density-dependent inhibition of growth under serum-free conditions and the requirements for induction of anchorage-independent proliferation under growth factor-defined assay conditions. This indicates that most likely the same cellular processes underlie these two aspects of phenotypic transformation, although data indicate that anchorage-independent proliferation may be a more restricted property of phenotypic transformation than loss of density dependence of proliferation. It is concluded that phenotypic transformation of NRK cells does not require specific polypeptide growth factors, but reflects the ability of these cells to respond to multiple growth factors.

  7. Polypept(o)ides: Hybrid Systems Based on Polypeptides and Polypeptoids.

    PubMed

    Klinker, Kristina; Barz, Matthias

    2015-11-01

    Polypept(o)ides combine the multifunctionality and intrinsic stimuli-responsiveness of synthetic polypeptides with the "stealth"-like properties of the polypeptoid polysarcosine (poly(N-methyl glycine)). This class of block copolymers can be synthesized by sequential ring opening polymerization of α-amino acid N-carboxy-anhydrides (NCAs) and correspondingly of the N-substituted glycine N-carboxyanhydride (NNCA). The resulting block copolymers are characterized by Poisson-like molecular weight distributions, full end group integrity, and dispersities below 1.2. While polysarcosine may be able to tackle the currently arising issues regarding the gold standard PEG, including storage diseases in vivo and immune responses, the polypeptidic block provides the functionalities for a specific task. Additionally, polypeptides are able to form secondary structure motives, e.g., α-helix or β-sheets, which can be used to direct self-assembly in solution. In this feature article, we review the relatively new field of polypept(o)ides with respect to synthesis, characterization, and first data on the application of block copolypept(o)ides in nanomedicine. The summarized data already indicates the great potential of polypept(o)ides. PMID:26398770

  8. Pairwise energies for polypeptide coarse-grained models derived from atomic force fields.

    PubMed

    Betancourt, Marcos R; Omovie, Sheyore J

    2009-05-21

    The energy parametrization of geometrically simplified versions of polypeptides, better known as polypeptide or protein coarse-grained models, is obtained from molecular dynamics and statistical methods. Residue pairwise interactions are derived by performing atomic-level simulations in explicit water for all 210 pairs of amino acids, where the amino acids are modified to closer match their structure and charges in polypeptides. Radial density functions are computed from equilibrium simulations for each pair of residues, from which statistical energies are extracted using the Boltzmann inversion method. The resulting models are compared to similar potentials obtained by knowledge based methods and to hydrophobic scales, resulting in significant similarities in spite of the model simplicity. However, it was found that glutamine, asparagine, lysine, and arginine are more attractive to other residues than anticipated, in part, due to their amphiphilic nature. In addition, equally charged residues appear more repulsive than expected. Difficulties in the calculation of knowledge based potentials and hydrophobicity scale for these cases, as well as sensitivity of the force field to polarization effects are suspected to cause this discrepancy. It is also shown that the coarse-grained model can identify native structures in decoy databases nearly as well as more elaborate knowledge based methods, in spite of its resolution limitations. In a test conducted with several proteins and corresponding decoys, the coarse-grained potential was able to identify the native state structure but not the original atomic force field.

  9. Preparation and Antitumor Activity of CS5931, A Novel Polypeptide from Sea Squirt Ciona Savignyi

    PubMed Central

    Chen, Xiaoshuang; Xu, Huanli; Li, Bo; Wang, Feng; Chen, Xiaoliang; Kong, Dexin; Lin, Xiukun

    2016-01-01

    CS5931 is a novel anticancer agent isolated from the sea squirt Ciona savignyi. However, its content in the species is very low, and developing a novel approach for production of the polypeptide is promising. In the present study, we expressed and purified the polypeptide from E. coli, and the fermentation conditions were studied using response surface methodology. The yield of CS5931 was increased from 2.0 to 7.5 mg/L. The denaturing and renaturation conditions were also studied. Using the optimized renaturation condition, the anticancer activity of refolding CS5931 was increased significantly; the value of IC50 was decreased from 23.2 to 11.6 μM. In vivo study using xenograft nude mice bearing HCT116 cancer cells revealed that CS5931 was able to inhibit the growth of tumor significantly. The study provides a useful approach for obtaining enough amount of CS5931 for further study. This study is also important for developing the polypeptide as a novel anticancer agent. PMID:27007382

  10. Cadherin 23-like polypeptide in hair bundle mechanoreceptors of sea anemones.

    PubMed

    Watson, Glen M; Pham, Lankhanh; Graugnard, Erin M; Mire, Patricia

    2008-09-01

    We investigated hair bundle mechanoreceptors in sea anemones for a homolog of cadherin 23. A candidate sequence was identified from the database for Nematostella vectensis that has a shared lineage with vertebrate cadherin 23s. This cadherin 23-like protein comprises 6,074 residues. It is an integral protein that features three transmembrane alpha-helices and a large extracellular loop with 44 contiguous, cadherin (CAD) domains. In the second half of the polypeptide, the CAD domains occur in a quadruple repeat pattern. Members of the same repeat group (i.e., CAD 18, 22, 26, and so on) share nearly identical amino acid sequences. An affinity-purified antibody was generated to a peptide from the C-terminus of the cadherin 23-like polypeptide. The peptide is expected to lie on the exoplasmic side of the plasma membrane. In LM, the immunolabel produced punctate fluorescence in hair bundles. In TEM, immunogold particles were observed medially and distally on stereocilia of hair bundles. Dilute solutions of the antibody disrupted vibration sensitivity in anemones. We conclude that the cadherin 23-like polypeptide likely contributes to the mechanotransduction apparatus of hair bundle mechanoreceptors of anemones. PMID:18654787

  11. Thermal expansivities of peptides, polypeptides and proteins as measured by pressure perturbation calorimetry.

    PubMed

    Pandharipande, Pranav P; Makhatadze, George I

    2015-04-01

    The main goal of this work was to provide direct experimental evidence that the expansivity of peptides, polypeptides and proteins as measured by pressure perturbation calorimetry (PPC), can serve as a proxy to characterize relative compactness of proteins, especially the denatured state ensemble. This is very important as currently only small angle X-ray scattering (SAXS), intrinsic viscosity and, to a lesser degree, fluorescence resonance transfer (FRET) experiments are capable of reporting on the compactness of denatured state ensembles. We combined the expansivity measurements with other biophysical methods (far-UV circular dichroism spectroscopy, differential scanning calorimetry, and small angle X-ray scattering). Three case studies of the effects of conformational changes on the expansivity of polypeptides in solution are presented. We have shown that expansivity appears to be insensitive to the helix-coil transition, and appears to reflect the changes in hydration of the side-chains. We also observed that the expansivity is sensitive to the global conformation of the polypeptide chain and thus can be potentially used to probe hydration of different collapsed states of denatured or even intrinsically disordered proteins.

  12. Detection of Matrilysin Activity Using Polypeptide Functionalized Reduced Graphene Oxide Field-Effect Transistor Sensor.

    PubMed

    Chen, Hu; Chen, Peng; Huang, Jingfeng; Selegård, Robert; Platt, Mark; Palaniappan, Alagappan; Aili, Daniel; Tok, Alfred Iing Yoong; Liedberg, Bo

    2016-03-15

    A novel approach for rapid and sensitive detection of matrilysin (MMP-7, a biomarker involved in the degradation of various macromolecules) based on a polypeptide (JR2EC) functionalized reduced graphene oxide (rGO) field effect transistor (FET) is reported. MMP-7 specifically digests negatively charged JR2EC immobilized on rGO, thereby modulating the conductance of rGO-FET. The proposed assay enabled detection of MMP-7 at clinically relevant concentrations with a limit of detection (LOD) of 10 ng/mL (400 pM), attributed to the significant reduction of the net charge of JR2EC upon digestion by MMP-7. Quantitative detection of MMP-7 in human plasma was further demonstrated with a LOD of 40 ng/mL, illustrating the potential for the proposed methodology for tumor detection and carcinoma diagnostic (e.g., lung cancer and salivary gland cancer). Additionally, excellent specificity of the proposed assay was demonstrated using matrix metallopeptidase 1 (MMP-1), a protease of the same family. With appropriate selection and modification of polypeptides, the proposed assay could be extended for detection of other enzymes with polypeptide digestion capability. PMID:26887256

  13. Characterization, structure and function of linker polypeptides in phycobilisomes of cyanobacteria and red algae: an overview.

    PubMed

    Liu, Lu-Ning; Chen, Xiu-Lan; Zhang, Yu-Zhong; Zhou, Bai-Cheng

    2005-06-30

    Cyanobacteria and red algae have intricate light-harvesting systems comprised of phycobilisomes that are attached to the outer side of the thylakoid membrane. The phycobilisomes absorb light in the wavelength range of 500-650 nm and transfer energy to the chlorophyll for photosynthesis. Phycobilisomes, which biochemically consist of phycobiliproteins and linker polypeptides, are particularly wonderful subjects for the detailed analysis of structure and function due to their spectral properties and their various components affected by growth conditions. The linker polypeptides are believed to mediate both the assembly of phycobiliproteins into the highly ordered arrays in the phycobilisomes and the interactions between the phycobilisomes and the thylakoid membrane. Functionally, they have been reported to improve energy migration by regulating the spectral characteristics of colored phycobiliproteins. In this review, the progress regarding linker polypeptides research, including separation approaches, structures and interactions with phycobiliproteins, as well as their functions in the phycobilisomes, is presented. In addition, some problems with previous work on linkers are also discussed.

  14. Polypept(o)ides: Hybrid Systems Based on Polypeptides and Polypeptoids.

    PubMed

    Klinker, Kristina; Barz, Matthias

    2015-11-01

    Polypept(o)ides combine the multifunctionality and intrinsic stimuli-responsiveness of synthetic polypeptides with the "stealth"-like properties of the polypeptoid polysarcosine (poly(N-methyl glycine)). This class of block copolymers can be synthesized by sequential ring opening polymerization of α-amino acid N-carboxy-anhydrides (NCAs) and correspondingly of the N-substituted glycine N-carboxyanhydride (NNCA). The resulting block copolymers are characterized by Poisson-like molecular weight distributions, full end group integrity, and dispersities below 1.2. While polysarcosine may be able to tackle the currently arising issues regarding the gold standard PEG, including storage diseases in vivo and immune responses, the polypeptidic block provides the functionalities for a specific task. Additionally, polypeptides are able to form secondary structure motives, e.g., α-helix or β-sheets, which can be used to direct self-assembly in solution. In this feature article, we review the relatively new field of polypept(o)ides with respect to synthesis, characterization, and first data on the application of block copolypept(o)ides in nanomedicine. The summarized data already indicates the great potential of polypept(o)ides.

  15. An altered Q sub B polypeptide as the basis for atrazine resistance in photoautotrophic potato cells

    SciTech Connect

    Smeda, R.J.; Hasegawa, P.M.; Weller, S.C. )

    1990-05-01

    A photoautotrophic potato cell line (variant) was isolated and is capable of sustained growth in media containing the herbicide atrazine at concentrations up to 100-fold greater than the lethal concentration (1.0 {mu}M) for the unselected (wild type) cell line. The basis for atrazine resistance could not be identified by differential uptake or metabolism. Photosynthetic electron transport rates for both intact cell and isolated thylakoid membranes from chloroplasts were unaffected in variant cells at atrazine concentrations up to 100-fold greater than for wild type cells. Photoaffinity labeling of isolated thylakoid membranes from both cell lines with {sup 14}C-azidoatrazine revealed an altered Q{sub B} polypeptide in variant cells resulting in low or no affinity for atrazine. A portion of the chloroplast psbA gene, encoding the Q{sub B} polypeptide, was sequenced for both cell lines. The basis for atrazine resistance in variant cells was identified as a single base change resulting in the alteration of serine to threonine at position 264 of the Q{sub B} polypeptide. In addition to atrazine resistance, variant cells exhibit enhanced tolerance to the herbicides DCMU and metribuzin, but greater sensitivity to bentazon. No reductions in variant cell growth and photosynthetic efficiency in the absence of atrazine were observed.

  16. Kinetics of Internal-Loop Formation in Polypeptide Chains: A Simulation Study

    PubMed Central

    Doucet, Dana; Roitberg, Adrian; Hagen, Stephen J.

    2007-01-01

    The speed of simple diffusional motions, such as the formation of loops in the polypeptide chain, places one physical limit on the speed of protein folding. Many experimental studies have explored the kinetics of formation of end-to-end loops in polypeptide chains; however, protein folding more often requires the formation of contacts between interior points on the chain. One expects that, for loops of fixed contour length, interior loops will form more slowly than end-to-end loops, owing to the additional excluded volume associated with the “tails”. We estimate the magnitude of this effect by generating ensembles of randomly coiled, freely jointed chains, and then using the theory of Szabo, Schulten, and Schulten to calculate the corresponding contact formation rates for these ensembles. Adding just a few residues, to convert an end-to-end loop to an internal loop, sharply decreases the contact rate. Surprisingly, the relative change in rate increases for a longer loop; sufficiently long tails, however, actually reverse the effect and accelerate loop formation slightly. Our results show that excluded volume effects in real, full-length polypeptides may cause the rates of loop formation during folding to depart significantly from the values derived from recent loop-formation experiments on short peptides. PMID:17208979

  17. Doxorubicin-loaded polypeptide nanorods based on electrostatic interactions for cancer therapy.

    PubMed

    Zhang, Longlong; Zhang, Pei; Zhao, Qingyun; Zhang, Yongchun; Cao, Longqiao; Luan, Yuxia

    2016-02-15

    An amphiphilic anionic polypeptide, methoxypolyethylene glycol-poly (glutamic acid) (mPEG-PGA), was synthesized, characterized and evaluated as a nanocarrier for the cationic anticancer drug doxorubicin hydrochloride (DOX·HCl). The complex self-assembled into nanorods in aqueous solutions via electrostatic interactions and exhibited a superior drug loading content (50.8%) and drug loading efficiency (90.2%). The average major axis of the drug-loaded nanorods was approximately 300nm, as determined by transmission electron microscopy. An in vitro release assay showed that drug-loaded nanorods exhibited pH-sensitivity and sustained release. Haemolysis assays demonstrated that the polypeptide was haemocompatible, and the polypeptide drug carrier significantly reduced the haemolysis ratio of DOX·HCl. The pharmacokinetics study showed that DOX-loaded nanorods significantly prolonged the resident time in blood. An in vitro cytotoxicity study and cellular uptake assays demonstrated that the DOX-loaded nanorods resulted in higher cell proliferation inhibition and a higher level of tumour cell uptake in A549 cells than with free DOX·HCl. The prolonged circulation and enhanced antitumor efficacy of DOX-loaded nanorods shows promise for efficient cancer chemotherapy. PMID:26609932

  18. Solution NMR Studies of Antiamoebin, a Membrane Channel-Forming Polypeptide

    PubMed Central

    Galbraith, T. P.; Harris, R.; Driscoll, P. C.; Wallace, B. A.

    2003-01-01

    Antiamoebin I is a membrane-active peptaibol produced by fungi of the species Emericellopsis which is capable of forming ion channels in membranes. Previous structure determinations by x-ray crystallography have shown the molecule is mostly helical, with a deep bend in the center of the polypeptide, and that the backbone structure is independent of the solvent used for crystallization. In this study, the solution structure of antiamoebin was determined by NMR spectroscopy in methanol, a solvent from which one of the crystal structures was determined. The ensemble of structures produced exhibit a right-handed helical C terminus and a left-handed helical conformation toward the N-terminus, in contrast to the completely right-handed helices found in the crystal structures. The NMR results also suggest that a “hinge” region exists, which gives flexibility to the polypeptide in the central region, and which could have functional implications for the membrane insertion process. A model for the membrane insertion and assembly process is proposed based on the antiamoebin solution and crystal structures, and is contrasted with the assembly and insertion mechanism proposed for other ion channel-forming polypeptides. PMID:12524274

  19. Compositions comprising a polypeptide having cellulolytic enhancing activity and a nitrogen-containing compound and uses thereof

    DOEpatents

    Quinlan, Jason; Xu, Feng; Sweeney, Matthew

    2016-05-31

    The present invention relates to compositions comprising: a polypeptide having cellulolytic enhancing activity and a nitrogen-containing compound. The present invention also relates to methods of using the compositions.

  20. UV cross-linking identifies four polypeptides that require the TATA box to bind to the Drosophila hsp70 promoter

    SciTech Connect

    Gilmour, D.S.; Dietz, T.J.; Elgin, S.C. )

    1990-08-01

    A protein fraction that requires the TATA sequence to bind to the hsp70 promoter has been partially purified from nuclear extracts of Drosophila embryos. This TATA factor produces a large DNase I footprint that extends from -44 to +35 on the promoter. A mutation that changes TATA to TATG interferes both with the binding of this complex and with the transcription of the hsp70 promoter in vitro, indicating that this interaction is important for transcriptional activity. Using a highly specific protein-DNA cross-linking assay, we have identified four polypeptides that require the TATA sequence to bind to the hsp70 promoter. Polypeptides of 26 and 42 kilodaltons are in intimate contact with the TATA sequence. Polypeptides of 150 and 60 kilodaltons interact within the region from +24 to +47 in a TATA-dependent manner. Both the extended footprint and the polypeptides identified by UV cross-linking indicate that the Drosophila TATA factor is a multicomponent complex.

  1. Disturbed expression of ribonucleotide reductase and cytokeratin polypeptides in focal epithelial hyperplasia. An immunohistochemical study using monoclonal antibodies.

    PubMed

    Rozell, B; Stenman, G; Magnusson, B; Lekholm, U; Nagle, R B; Hansson, H A

    1986-05-01

    Four cases of focal epithelial hyperplasia (FEH) were studied immunohistochemically, using monoclonal antibodies against the M1 subunit of ribonucleotide reductase and different cytokeratin polypeptides. The FEH lesions showed, compared to normal oral mucosa, extensive alterations in their staining patterns. This included ectopic suprabasal M1 staining and the novel expression of cytokeratin polypeptides differing from those previously reported for other HPV infections. The results are discussed in relation to the causative agent, human papillomavirus, and its expression in focal epithelial hyperplasia.

  2. Biosynthesis and characterization of a non-repetitive polypeptide derived from silk fibroin heavy chain.

    PubMed

    Yang, Gaoqiang; Wu, Mingyang; Yi, Honggen; Wang, Jiannan

    2016-02-01

    Silk fibroin heavy chain is the major protein component of Bombyx mori silk fibroin and is composed of 12 repetitive and 11 non-repetitive regions, with the non-repetitive domain consisting of a hydrophilic polypeptide chain. In order to determine the biomedical function of the non-repetitive domain or potentially use it to modify hydrophobic biomaterials, high-purity isolation is necessary. Previously, we cloned and extended a gene motif (f(1)) encoding the non-repetitive domain. Here, this motif and its multimers are inserted into a glutathione S-transferase (GST)-tagged fusion-protein expression vector. Motif f(1) and multimers f(4) and f(8) were expressed in Escherichia coli BL21 cells following isopropyl β-D-1-thiogalactopyranoside induction, purified by GST-affinity chromatography, and single bands of purified fusion proteins GST-F(1), GST-F(4), and GST-F(8), were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Target polypeptides F(1), F(4), and F(8), were cleaved clearly from the GST-fusion tag following thrombin digestion. Mass spectrometry results indicate that the molecular weights associated with fusion proteins GST-F(1), GST-F(4), and GST-F(8) are 31.5, 43.8, and 59.0kDa, respectively, and with the cleaved polypeptides F(1), F(4), and F(8) are 4.8, 16.8, and 32.8kDa, respectively. The F(1), F(4), and F(8) polypeptide chains are negatively charged with isoelectric points (pI) of 3.3, 3.2, and 3.0, respectively. The molecular weight and pI values of the polypeptide chains are consistent with the predicted values and the amino acid compositions similar to predicted sequences. FTIR and CD results show the molecular conformation of F(1) was mainly random coil, and more stable α-helix structure formed in longer molecular chain. PMID:26652374

  3. Biosynthesis and characterization of a non-repetitive polypeptide derived from silk fibroin heavy chain.

    PubMed

    Yang, Gaoqiang; Wu, Mingyang; Yi, Honggen; Wang, Jiannan

    2016-02-01

    Silk fibroin heavy chain is the major protein component of Bombyx mori silk fibroin and is composed of 12 repetitive and 11 non-repetitive regions, with the non-repetitive domain consisting of a hydrophilic polypeptide chain. In order to determine the biomedical function of the non-repetitive domain or potentially use it to modify hydrophobic biomaterials, high-purity isolation is necessary. Previously, we cloned and extended a gene motif (f(1)) encoding the non-repetitive domain. Here, this motif and its multimers are inserted into a glutathione S-transferase (GST)-tagged fusion-protein expression vector. Motif f(1) and multimers f(4) and f(8) were expressed in Escherichia coli BL21 cells following isopropyl β-D-1-thiogalactopyranoside induction, purified by GST-affinity chromatography, and single bands of purified fusion proteins GST-F(1), GST-F(4), and GST-F(8), were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Target polypeptides F(1), F(4), and F(8), were cleaved clearly from the GST-fusion tag following thrombin digestion. Mass spectrometry results indicate that the molecular weights associated with fusion proteins GST-F(1), GST-F(4), and GST-F(8) are 31.5, 43.8, and 59.0kDa, respectively, and with the cleaved polypeptides F(1), F(4), and F(8) are 4.8, 16.8, and 32.8kDa, respectively. The F(1), F(4), and F(8) polypeptide chains are negatively charged with isoelectric points (pI) of 3.3, 3.2, and 3.0, respectively. The molecular weight and pI values of the polypeptide chains are consistent with the predicted values and the amino acid compositions similar to predicted sequences. FTIR and CD results show the molecular conformation of F(1) was mainly random coil, and more stable α-helix structure formed in longer molecular chain.

  4. Stimuli-Triggered Sol-Gel Transitions of Polypeptides Derived from α-Amino Acid N-Carboxyanhydride (NCA) Polymerizations.

    PubMed

    He, Xun; Fan, Jingwei; Wooley, Karen L

    2016-02-18

    The past decade has witnessed significantly increased interest in the development of smart polypeptide-based organo- and hydrogel systems with stimuli responsiveness, especially those that exhibit sol-gel phase-transition properties, with an anticipation of their utility in the construction of adaptive materials, sensor designs, and controlled release systems, among other applications. Such developments have been facilitated by dramatic progress in controlled polymerizations of α-amino acid N-carboxyanhydrides (NCAs), together with advanced orthogonal functionalization techniques, which have enabled economical and practical syntheses of well-defined polypeptides and peptide hybrid polymeric materials. One-dimensional stacking of polypeptides or peptide aggregations in the forms of certain ordered conformations, such as α helices and β sheets, in combination with further physical or chemical cross-linking, result in the construction of three-dimensional matrices of polypeptide gel systems. The macroscopic sol-gel transitions, resulting from the construction or deconstruction of gel networks and the conformational changes between secondary structures, can be triggered by external stimuli, including environmental factors, electromagnetic fields, and (bio)chemical species. Herein, the most recent advances in polypeptide gel systems are described, covering synthetic strategies, gelation mechanisms, and stimuli-triggered sol-gel transitions, with the aim of demonstrating the relationships between chemical compositions, supramolecular structures, and responsive properties of polypeptide-based organo- and hydrogels.

  5. Comparisons of the immunological properties of two structural polypeptides of type C RNA viruses endogenous to old world monkeys.

    PubMed Central

    Stephenson, J R; Reynolds, R K; Aaronson, S A

    1976-01-01

    Immunologically very closely related type C RNA viruses are endogenous to the domestic cat and to an old world primate, the baboon. In the present studies, radioimmunological techniques have been developed for detection of the 15,000 and 30,000 molecular weight (MW) polypeptides of each virus. The much more pronounced type-specific antigenic determinants of the lower MW polypeptides made it possible to readily differentiate these viruses from each other as well as from a type C virus isolate from a second baboon species. Normal rhesus monkey tissues were partially purified and shown to contain a reactivity with MW and immunological properties similar to that of the baboon virus 30,000 MW polypeptide. Despite a similar degree of purification, antigenic reactivity like that of the baboon virus 15,000 MW polypeptide was undetectable even in the brodest immunological tests available for this polypeptide. The present findings indicate that the immunological properties of two structural polypeptides of closely related viruses endogenous to primate and feline species have undergone different rates of antigenic change in the course of evolution within their respective host cell genome. PMID:56455

  6. Characterization and Mutational Analysis of a Two-Polypeptide Bacteriocin Produced by Citrus Iyo-Derived Lactobacillus brevis 174A.

    PubMed

    Noda, Masafumi; Miyauchi, Rumi; Danshiitsoodol, Narandalai; Higashikawa, Fumiko; Kumagai, Takanori; Matoba, Yasuyuki; Sugiyama, Masanori

    2015-01-01

    In the present study, we isolated a lactic acid bacterium (LAB) from a citrus iyo fruit and identified it as Lactobacillus brevis. This plant-derived LAB strain, designated 174A, produces bacteriocin consisting of two polypeptides designated brevicin 174A-β and 174A-γ. Although each polypeptide itself displays antibacterial activity, the ability is enhanced 100 fold by mixing both polypeptides at a 1 : 1 ratio. Significantly, brevicin 174A inhibits even the growth of several pathogenic bacteria that are more resistant to a lantibiotic bacteriocin, nisin A, which is commonly utilized as a preservative added to foodstuffs. Structural analysis of the 174A bacteriocin using a program that predicts secondary structure suggests that both component polypeptides have a positively charged N-terminal region, as well as two cysteine residues in both the N- and C-terminals. Judging from a mutational analysis of the antibacterial polypeptides, these unique amino acid sequences of 174A-β might be important for the expression of the synergistic activity that occurs in the presence of the two polypeptides combined. PMID:26632181

  7. Two N-myc polypeptides with distinct amino termini encoded by the second and third exons of the gene.

    PubMed Central

    Mäkelä, T P; Saksela, K; Alitalo, K

    1989-01-01

    The N-myc and c-myc genes encode closely related nuclear phosphoproteins. We found that the N-myc protein from human tumor cell lines appears as four closely migrating polypeptide bands (p58 to p64) in sodium dodecyl sulfate-polyacrylamide gels. This and the recent finding that the c-myc protein is synthesized from two translational initiation sites located in the first and second exons of the gene (S. R. Hann, M. W. King, D. L. Bentley, C. W. Anderson, and R. N. Eisenman, Cell 52:185-195, 1988) prompted us to study the molecular basis of the N-myc protein heterogeneity. Dephosphorylation by alkaline phosphatase reduced the four polypeptide bands to a doublet with an electrophoretic mobility corresponding to the two faster-migrating N-myc polypeptides (p58 and p60). When expressed transiently in COS cells, an N-myc deletion construct lacking the first exon produced polypeptides similar to the wild-type N-myc protein, indicating that the first exon of the N-myc gene is noncoding. Furthermore, mutants deleted of up to two thirds of C-terminal coding domains still retained the capacity to produce a doublet of polypeptides, suggesting distinct amino termini for the two N-myc polypeptides. The amino-terminal primary structure of the N-myc protein was studied by site-specific point mutagenesis of the 5' end of the long open reading frame and by N-terminal radiosequencing of the two polypeptides. Our results show that the N-myc polypeptides are initiated from two alternative in-phase AUG codons located 24 base pairs apart at the 5' end of the second exon. Both of these polypeptides are phosphorylated and localized to the nucleus even when expressed separately. Interestingly, DNA rearrangements activating the c-myc gene are often found in the 1.7-kilobase-pair region between the two c-myc translational initiation sites and correlate with the loss of the longer c-myc polypeptide. Thus the close spacing of the two N-myc initiation codons could explain the relative resistance

  8. Cloning and expression of two human p70 S6 kinase polypeptides differing only at their amino termini

    SciTech Connect

    Grove, J.R.; Banerjee, P.; Balasubramanyam, A.; Price, D.J.; Avruch, J. ); Coffer, P.J.; Woodgett, J.R. )

    1991-11-01

    Two classes of human cDNA encoding the insulin/mitogen-activated p70 S6 kinase have been isolated; the two classes differ only in the 5{prime} region, such that the longer polypeptide consists of 525 amino acids, of which the last 502 longer residues are identical in sequence to the entire polypeptide encoded by the second cDNA. Both p70 S6 kinase polypeptides predicted by these cDNAs are present in p70 S6 kinase purified from rat liver, and each is thus expressed in vivo. Moreover, both polypeptides are expressed from a single mRNA transcribed from the (longer) p70 S6 kinase {alpha}I cDNA through the utilization of different translational start sites. Transient expression of p70 {alpha}I and {alpha}II S6 kinase cDNA in COA cells results in a 2.5- to 4-fold increase in overall S6 kinase activity. Transfection with the {alpha}II cDNA yields only the smaller set of bands, while transfection with the {alpha}I cDNA generates both sets of bands. Mutation of Met-24 in the {alpha}I cDNA to Leu or Thr suppresses synthesis of the {alpha}II polypeptides. Only the p70 {alpha}I and {alpha}II polypeptides of slowest mobility on SDS-PAGE comigrate with the 70-and 90-kDa proteins observed in purified rat liver S6 kinase. The recombinant p70 S6 kinase undergoes multiple phosphorylation and partial activation in COS cells. Acquisition of S6 protein kinase catalytic function, however, is apparently restricted to the most extensively phosphorylated recombinant polypeptides.

  9. Fabricating and Characterizing Physical Properties of Electrospun Polypeptide-based Nanofibers

    NASA Astrophysics Data System (ADS)

    Khadka, Dhan Bahadur

    This dissertation has aimed to fabricate polypeptide based biomaterial and characterize physical properties. Electrospinning is used as a tool for the sample fabrication. Project focused on determining the feasibility of electrospinning of certain synthetic polypeptides and certain elastin-like peptides from aqueous feedstocks and to characterize physical properties of polymer aqueous solution, cast film and spun fibers and fiber mats. The research involves peptide design, polymer electrospinning, fibers crosslinking, determining the extent of crosslinking, fibers protease degradation study, fibers stability and self-organization analysis, structure and composition determination by various spectroscopy and microscopy techniques and characterization of mechanical properties of individual suspended fibers. Fiber mats of a synthetic cationic polypeptide poly(L-ornithine) (PLO) and an anionic co-polypeptide of L-glutamic acid and L-tyrosine (PLEY) of defined composition have been produced by electrospinning. Fibers were obtained from polymer aqueous solution at concentrations of 20-45% (w/v) in PLO and at concentrations of 20-60% (w/v) in PLEY. Applied voltage and spinneret-collector distance were also found to influence polymer spinnability and fibers morphology. Oriented fibers were obtained by parallel electrodes geometry. Fiber diameter and morphology was analyzed by scanning electron microscopy (SEM) and atomic force microscopy (AFM). PLO fibers exposed on glutaraldehyde (GTA) vapor rendered fiber mats water-insoluble. A common chemical reagent, carbodiimide was used to crosslink PLEY fibers. Fiber solubility in aqueous solution varied as a function of crosslinking time and crosslinker concentration. Crosslink density has been quantified by a visible-wavelength dye-based method. Degradation of crosslinked fibers by different proteases has been demonstrated. Investigation of crosslinked PLEY fibers has provided insight into the mechanisms of stability at different

  10. ABSINTH: A new continuum solvation model for simulations of polypeptides in aqueous solutions

    PubMed Central

    Vitalis, Andreas; Pappu, Rohit V.

    2009-01-01

    A new implicit solvation model for use in Monte Carlo simulations of polypeptides is introduced. The model is termed ABSINTH for self-Assembly of Biomolecules Studied by an Implicit, Novel, and Tunable Hamiltonian. It is designed primarily for simulating conformational equilibria and oligomerization reactions of intrinsically disordered proteins in aqueous solutions. The paradigm for ABSINTH is conceptually similar to the EEF1 model of Lazaridis and Karplus (Proteins: Struct. Func. Genet., 1999, 35: 133-152). In ABSINTH, the transfer of a polypeptide solute from the gas phase into a continuum solvent is the sum of a direct mean field interaction (DMFI), and a term to model the screening of polar interactions. Polypeptide solutes are decomposed into a set of distinct solvation groups. The DMFI is a sum of contributions from each of the solvation groups, which are analogs of model compounds. Continuum-mediated screening of electrostatic interactions is achieved using a framework similar to the one used for the DMFI. Promising results are shown for a set of test cases. These include the calculation of NMR coupling constants for short peptides, the assessment of the thermal stability of two small proteins, reversible folding of both an alpha-helix and a beta-hairpin forming peptide, and the polymeric properties of intrinsically disordered polyglutamine peptides of varying lengths. The tests reveal that the computational expense for simulations with the ABSINTH implicit solvation model increase by a factor that is in the range of 2.5-5.0 with respect to gas-phase calculations. PMID:18506808

  11. The abundance and organization of polypeptides associated with antigens of the Rh blood group system.

    PubMed

    Gardner, B; Anstee, D J; Mawby, W J; Tanner, M J; von dem Borne, A E

    1991-06-01

    Twelve murine monoclonal antibodies, which react with human red cells of common Rh phenotype but give weak or negative reactions with Rh null erythrocytes, were used in quantitative binding assays and competitive binding assays to investigate the abundance and organization of polypeptides involved in the expression of antigens of the Rh blood group system. Antibodies of the R6A-type (R6A, BRIC-69, BRIC-207) and the 2D10-type (MB-2D10, LA18.18, LA23.40) recognize related structures and 100,000-200,000 molecules of each antibody bind maximally to erythrocytes of common Rh phenotype. Antibodies of the BRIC-125 type (BRICs 32, 122, 125, 126, 168, 211) recognize structures that are unrelated to those recognized by R6A-type and 2D10-type antibodies and between 10,000 and 50,000 antibody molecules bind maximally to erythrocytes of the common Rh phenotype. The binding of antibodies of the R6A-type and the 2D10-type, but not of antibodies of the BRIC-125-type could be partially inhibited by human anti-D antibodies (polyclonal and monoclonal) and a murine anti-e-like antibody. These results are consistent with evidence (Moore & Green 1987; Avent et al., 1988b) that the Rh blood group antigens are associated with a complex that comprises two groups of related polypeptides of M(r) 30,000 and M(r) 35,000-100,000, respectively, and suggest that there are 1-2 x 10(5) copies of this complex per erythrocyte. The polypeptide recognized by antibodies of the BRIC-125 type is likely to be associated with this complex. PMID:9259831

  12. Effects of Protein Structure on Iron–Polypeptide Vibrational Dynamic Coupling in Cytochrome c

    PubMed Central

    2015-01-01

    Cytochrome c (Cyt c) has a heme covalently bound to the polypeptide via a Cys-X-X-Cys-His (CXXCH) linker that is located in the interface region for protein–protein interactions. To determine whether the polypeptide matrix influences iron vibrational dynamics, nuclear resonance vibrational spectroscopy (NRVS) measurements were performed on 57Fe-labeled ferric Hydrogenobacter thermophilus cytochrome c-552, and variants M13V, M13V/K22M, and A7F, which have structural modifications that alter the composition or environment of the CXXCH pentapeptide loop. Simulations of the NRVS data indicate that the 150–325 cm–1 region is dominated by NHis–Fe–SMet axial ligand and polypeptide motions, while the 325–400 cm–1 region shows dominant contributions from ν(Fe–NPyr) (Pyr = pyrrole) and other heme-based modes. Diagnostic spectral signatures that directly relate to structural features of the heme active site are identified using a quantum chemistry-centered normal coordinate analysis (QCC-NCA). In particular, spectral features that directly correlate with CXXCH loop stiffness, the strength of the Fe–His interaction, and the degree of heme distortion are identified. Cumulative results from our investigation suggest that compared to the wild type (wt), variants M13V and M13V/K22M have a more rigid CXXCH pentapeptide segment, a stronger Fe–NHis interaction, and a more ruffled heme. Conversely, the A7F variant has a more planar heme and a weaker Fe–NHis bond. These results are correlated to the observed changes in reduction potential between wt protein and the variants studied here. Implications of these results for Cyt c biogenesis and electron transfer are also discussed. PMID:25531247

  13. The abundance and organization of polypeptides associated with antigens of the Rh blood group system.

    PubMed

    Gardner, B; Anstee, D J; Mawby, W J; Tanner, M J; von dem Borne, A E

    1991-06-01

    Twelve murine monoclonal antibodies, which react with human red cells of common Rh phenotype but give weak or negative reactions with Rh null erythrocytes, were used in quantitative binding assays and competitive binding assays to investigate the abundance and organization of polypeptides involved in the expression of antigens of the Rh blood group system. Antibodies of the R6A-type (R6A, BRIC-69, BRIC-207) and the 2D10-type (MB-2D10, LA18.18, LA23.40) recognize related structures and 100,000-200,000 molecules of each antibody bind maximally to erythrocytes of common Rh phenotype. Antibodies of the BRIC-125 type (BRICs 32, 122, 125, 126, 168, 211) recognize structures that are unrelated to those recognized by R6A-type and 2D10-type antibodies and between 10,000 and 50,000 antibody molecules bind maximally to erythrocytes of the common Rh phenotype. The binding of antibodies of the R6A-type and the 2D10-type, but not of antibodies of the BRIC-125-type could be partially inhibited by human anti-D antibodies (polyclonal and monoclonal) and a murine anti-e-like antibody. These results are consistent with evidence (Moore & Green 1987; Avent et al., 1988b) that the Rh blood group antigens are associated with a complex that comprises two groups of related polypeptides of M(r) 30,000 and M(r) 35,000-100,000, respectively, and suggest that there are 1-2 x 10(5) copies of this complex per erythrocyte. The polypeptide recognized by antibodies of the BRIC-125 type is likely to be associated with this complex.

  14. Control of stability of polypeptide multilayer nanofilms by quantitative control of disulfide bond formation

    NASA Astrophysics Data System (ADS)

    Zhong, Yang; Li, Bingyun; Haynie, Donald T.

    2006-12-01

    The crosslinking of polymers in a polymeric material will alter the mechanical properties of the material. Control over the mechanical properties of polyelectrolyte multilayer films (PEMs) could be useful for applications of the technology in medicine and other areas. Disulfide bonds are 'natural' polypeptide crosslinks found widely in wild-type proteins. Here, we have designed and synthesized three pairs of oppositely charged 32mer polypeptide to have 0, 4, or 8 cysteine (Cys) residues per molecule, and we have characterized physical properties of the peptides in a PEM context. The average linear density of free thiol in the designed peptides was 0, 0.125, or 0.25 per amino acid residue. The peptides were used to make 10-bilayer PEMs by electrostatic layer-by-layer self-assembly (LBL). Cys was included in the peptides to study specific effects of disulfide bond formation on PEM properties. Features of film assembly have been found to depend on the amino acid sequence, as in protein folding. Following polypeptide self-assembly into multilayer films, Cys residues were disulfide-crosslinked under mild oxidizing conditions. The stability of the crosslinked films at acidic pH has been found to depend on the number of Cys residues per peptide for a given crosslinking procedure. Crosslinked and non-crosslinked films have been analysed by ultraviolet spectroscopy (UVS), ellipsometry, and atomic force microscopy (AFM) to characterize film assembly, surface morphology, and disassembly. A selective etching model of the disassembly process at acidic pH is proposed on the basis of the experimental data. In this model, regions of film in which the disulfide bond density is low are etched at a higher rate than regions where the density is high.

  15. Effects of Protein Structure on Iron–Polypeptide Vibrational Dynamic Coupling in Cytochrome c

    DOE PAGESBeta

    Galinato, Mary Grace I.; Bowman, Sarah E. J.; Kleingardner, Jesse G.; Martin, Sherri; Zhao, Jiyong; Sturhahn, Wolfgang; Alp, E. Ercan; Bren, Kara L.; Lehnert, Nicolai

    2014-12-22

    Cytochrome c (Cyt c) has a heme covalently bound to the polypeptide via a Cys-X-X-Cys-His (CXXCH) linker that is located in the interface region for protein–protein interactions. To determine whether the polypeptide matrix influences iron vibrational dynamics, nuclear resonance vibrational spectroscopy (NRVS) measurements were performed on 57Fe-labeled ferric Hydrogenobacter thermophilus cytochrome c-552, and variants M13V, M13V/K22M, and A7F, which have structural modifications that alter the composition or environment of the CXXCH pentapeptide loop. Simulations of the NRVS data indicate that the 150–325 cm–1 region is dominated by NHis–Fe–SMet axial ligand and polypeptide motions, while the 325–400 cm–1 region shows dominantmore » contributions from ν(Fe–NPyr) (Pyr = pyrrole) and other heme-based modes. Diagnostic spectral signatures that directly relate to structural features of the heme active site are identified using a quantum chemistry-centered normal coordinate analysis (QCC-NCA). In particular, spectral features that directly correlate with CXXCH loop stiffness, the strength of the Fe–His interaction, and the degree of heme distortion are identified. Cumulative results from our investigation suggest that compared to the wild type (wt), variants M13V and M13V/K22M have a more rigid CXXCH pentapeptide segment, a stronger Fe–NHis interaction, and a more ruffled heme. Conversely, the A7F variant has a more planar heme and a weaker Fe–NHis bond. These results are correlated to the observed changes in reduction potential between wt protein and the variants studied here. Lastly, we discuss the implications of these results for Cyt c biogenesis and electron transfer.« less

  16. Final Report

    SciTech Connect

    DeTar, Carleton

    2012-12-10

    This document constitutes the Final Report for award DE-FC02-06ER41446 as required by the Office of Science. It summarizes accomplishments and provides copies of scientific publications with significant contribution from this award.

  17. Final Report

    SciTech Connect

    Gurney, Kevin R.

    2015-01-12

    This document constitutes the final report under DOE grant DE-FG-08ER64649. The organization of this document is as follows: first, I will review the original scope of the proposed research. Second, I will present the current draft of a paper nearing submission to Nature Climate Change on the initial results of this funded effort. Finally, I will present the last phase of the research under this grant which has supported a Ph.D. student. To that end, I will present the graduate student’s proposed research, a portion of which is completed and reflected in the paper nearing submission. This final work phase will be completed in the next 12 months. This final workphase will likely result in 1-2 additional publications and we consider the results (as exemplified by the current paper) high quality. The continuing results will acknowledge the funding provided by DOE grant DE-FG-08ER64649.

  18. Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) Signalling Exerts Chondrogenesis Promoting and Protecting Effects: Implication of Calcineurin as a Downstream Target

    PubMed Central

    Juhász, Tamás; Matta, Csaba; Katona, Éva; Somogyi, Csilla; Takács, Roland; Gergely, Pál; Csernoch, László; Panyi, Gyorgy; Tóth, Gábor; Reglődi, Dóra; Tamás, Andrea; Zákány, Róza

    2014-01-01

    Pituitary adenylate cyclase activating polypeptide (PACAP) is an important neurotrophic factor influencing differentiation of neuronal elements and exerting protecting role during traumatic injuries or inflammatory processes of the central nervous system. Although increasing evidence is available on its presence and protecting function in various peripheral tissues, little is known about the role of PACAP in formation of skeletal components. To this end, we aimed to map elements of PACAP signalling in developing cartilage under physiological conditions and during oxidative stress. mRNAs of PACAP and its receptors (PAC1,VPAC1, VPAC2) were detectable during differentiation of chicken limb bud-derived chondrogenic cells in micromass cell cultures. Expression of PAC1 protein showed a peak on days of final commitment of chondrogenic cells. Administration of either the PAC1 receptor agonist PACAP 1-38, or PACAP 6-38 that is generally used as a PAC1 antagonist, augmented cartilage formation, stimulated cell proliferation and enhanced PAC1 and Sox9 protein expression. Both variants of PACAP elevated the protein expression and activity of the Ca-calmodulin dependent Ser/Thr protein phosphatase calcineurin. Application of PACAPs failed to rescue cartilage formation when the activity of calcineurin was pharmacologically inhibited with cyclosporine A. Moreover, exogenous PACAPs prevented diminishing of cartilage formation and decrease of calcineurin activity during oxidative stress. As an unexpected phenomenon, PACAP 6-38 elicited similar effects to those of PACAP 1-38, although to a different extent. On the basis of the above results, we propose calcineurin as a downstream target of PACAP signalling in differentiating chondrocytes either in normal or pathophysiological conditions. Our observations imply the therapeutical perspective that PACAP can be applied as a natural agent that may have protecting effect during joint inflammation and/or may promote cartilage regeneration

  19. A prostate-specific antigen-dependent fusion polypeptide inhibits growth of prostate cancer cells in vitro and in vivo

    PubMed Central

    Zhang, Xiang; Ma, Yueyun; Wei, Hua; Li, Bin; Xiao, Fengjing; Yang, Jing; Yue, Qiaohong; Yang, Angang; Hao, Xiaoke

    2016-01-01

    Polypeptide APP8 is a prostate-specific antigen (PSA)-activated prodrug that was designed to synergize the effects of the Bcl-2 homology domain 3 (BH3) peptide, K237 and the DG2 peptide. The aim of this study is to evaluate its biodistribution and anticancer effect in vitro and in vivo. In this study, APP8 and each component peptide were synthesized. The biodistribution was identified using con-focal microscopyin both PSA+ cell line and PSA- cell line in vitro. Then cell cycle, MTT and in-cell western blot were accessed to analyze the effect mechanisms. Finally, xenografts were used to confirm the anticancer effect in vivo. Here, it was shown that APP8 was hydrolyzed and BH3 was released into the nucleus, while K237 and DG2 were located predominantly in the cytoplasm, only in LNCaP cells (PSA+), but not PC3 cells (PSA-). K237 and DG2 could induce cell apoptosis through decreasing the phosphorylation of ERK-2 and Flk-1. APP8 also caused the death of LNCaP cells, and was predominantly dependent on BH3 in vitro. In addition, It was noted that as the tumor grew in vivo, APP8 could inhibit the tumor volume to 77.3%, mainly depending on K237 and DG2 via inhibition of the growth of vascular endothelial cells. Our results suggested that APP8 could promote prostate cancer cell death and stop prostate cancer growth via synergizing apoptosis induction of tumor cell and inhibition of the growth of vascular endothelial cells. It provides a novel candidate prodrug for specific therapy of prostate cancer. PMID:27293998

  20. The generation of hydrophilic polypeptide-siloxane conjugates via n-carboxyanhydride polymerisation.

    PubMed

    Thornton, Paul D; Brannigan, Ruairi; Podporska, Joanna; Quilty, Brid; Heise, Andreas

    2012-01-01

    A novel methodology to create covalently linked polypeptide-siloxane hybrid materials by controlled n-carboxyanhydride ring opening polymerisation is disclosed. Poly-L-glutamic acid and poly-L-lysine conjugated products were formed that possessed excellent surface wettability. In addition, the poly-L-lysine-siloxane hybrids formed demonstrated bactericidal attributes against gram-positive Staphylococcus aureus and gram-negative Escherichia coli. It is anticipated that these materials may be of significance for the generation of hydrophilic siloxane-containing polymers that are commonly employed in contemporary medical devices.

  1. Atomic structure of the cross-[beta] spine of islet amyloid polypeptide (amylin)

    SciTech Connect

    Wiltzius, J.J.; Sievers, S.A.; Sawaya, M.R.; Cascio, D.; Popov, D.; Riekel, C.; Eisenberg, D.

    2009-03-27

    Human islet amyloid polypeptide (IAPP or amylin) is a 37-residue hormone found as fibrillar deposits in pancreatic extracts of nearly all type II diabetics. Although the cellular toxicity of IAPP has been established, the structure of the fibrillar form found in these deposits is unknown. Here we have crystallized two segments from IAPP, which themselves form amyloid-like fibrils. The atomic structures of these two segments, NNFGAIL and SSTNVG, were determined, and form the basis of a model for the most commonly observed, full-length IAPP polymorph.

  2. On the fragmentation of biomolecules: Fragmentation of alanine dipeptide along the polypeptide chain

    SciTech Connect

    Solov'yov, I. A. Yakubovich, A. V.; Solov'yov, A. V.; Greiner, W.

    2006-09-15

    The interaction potential between amino acids in alanine dipeptide has been studied for the first time taking into account exact molecular geometry. Ab initio calculation has been performed in the framework of density functional theory taking into account all electrons in the system. The fragmentation of dipeptide along the polypeptide chain, as well as the interaction between alanines, has been considered. The energy of the system has been analyzed as a function of the distance between fragments for all possible dipeptide fragmentation channels. Analysis of the energy barriers makes it possible to estimate the characteristic fragmentation times and to determine the degree of applicability of classical electrodynamics for describing the system energy.

  3. Localization of glucose-dependent insulinotropic polypeptide (GIP) to a gene cluster on chromosome 17q

    SciTech Connect

    Lewis, T.B.; Saenz, M.; O'Connell, P.; Leach, R.J. )

    1994-02-01

    Glucose-dependent insulinotropic polypeptide (GIP) has been regionally localized to a gene cluster on human chromosome 17q. Genetic mapping through CEPH reference families demonstrated that GIP was tightly linked to NME1 and PPY and fully linked to HOXB6 and NGFR. High-resolution radiation hybrid mapping resolved the gene order as cen-PPY-HOXB6-NGFR-GIP-NME1-tel. GIP maps distal to NGFR with an estimated distance of 250 kb. 12 refs., 1 fig., 1 fig.

  4. Free energies of solvation for peptides and polypeptides using SCRF methods

    NASA Astrophysics Data System (ADS)

    Alemán, Carlos; Ishiki, Hamilton Mitsugu; Armelin, Elaine A.; Abrahão Junior, Odonírio; Galembeck, Sergio E.

    1998-07-01

    The effects of the aqueous solvent in the conformational preferences of peptides and homopeptides have been investigated using two different and widely used self-consistent reaction-field models. The free energies of solvation were predicted using the polarizable continuum model developed by Tomasi and co-workers and adapted to semi-empirical hamiltonians by Orozco and Luque, and the solvation model developed by Cramer and Truhlar. The set of compounds investigated is constituted by five dipeptides with different chemical nature and structural properties as well as by two homopeptides in which the size of the polypeptidic chain was varied. Results provided by the different methods are compared and discussed.

  5. Polypeptide micelles with dual pH activatable dyes for sensing cells and cancer imaging

    NASA Astrophysics Data System (ADS)

    Gong, Ping; Yang, Yueting; Yi, Huqiang; Fang, Shengtao; Zhang, Pengfei; Sheng, Zonghai; Gao, Guanhui; Gao, Duyang; Cai, Lintao

    2014-04-01

    pH is an important control parameter for maintenance of cell viability and tissue functions. pH monitoring provides valuable information on cell metabolic processes and the living environment. In this study, we prepared dual pH-sensitive, fluorescent dye-loaded polypeptide nanoparticles (DPNs) for ratiometric sensing of pH changes in living cells. DPNs contain two types of dyes: N-(rhodamine B) lactam cystamine (RBLC), an acid activatable fluorescent dye with increased fluorescence in an acidic environment, and fluorescein isothiocyanate (FITC), a base activatable fluorescent dye with enhanced fluorescence in an alkaline environment. Hence, DPNs exhibited a dual response signal with strong red fluorescence and weak green fluorescence under acidic conditions; in contrast, they showed strong green fluorescence and almost no red fluorescence under alkaline and neutral conditions. The favorable inverse pH responses of the two fluorescent dyes resulted in ratiometric pH determination for DPNs with an optimized pH-sensitive range of pH 4.5-7.5. Quantitative analysis of the intracellular pH of intact MCF-7 cells has been successfully demonstrated with our nanosensor. Moreover, single acid activatable fluorescent dye doped polypeptide nanoparticles that only contained RBLC can distinguish tumor tissue from normal tissue by monitoring the acidic extracellular environment.pH is an important control parameter for maintenance of cell viability and tissue functions. pH monitoring provides valuable information on cell metabolic processes and the living environment. In this study, we prepared dual pH-sensitive, fluorescent dye-loaded polypeptide nanoparticles (DPNs) for ratiometric sensing of pH changes in living cells. DPNs contain two types of dyes: N-(rhodamine B) lactam cystamine (RBLC), an acid activatable fluorescent dye with increased fluorescence in an acidic environment, and fluorescein isothiocyanate (FITC), a base activatable fluorescent dye with enhanced fluorescence

  6. Recognition of subsets of the mammalian A/B-type core heterogeneous nuclear ribonucleoprotein polypeptides by novel autoantibodies.

    PubMed Central

    Dangli, A; Plomaritoglou, A; Boutou, E; Vassiliadou, N; Moutsopoulos, H M; Guialis, A

    1996-01-01

    The structurally related A/B-type core heterogeneous nuclear ribonucleoprotein (hnRNP) polypeptides of 34-39 kDa (A1, A2, B1 and B2) belong to a family of RNA-binding proteins that are major components of 40 S hnRNP complexes. By two-dimensional gel electrophoresis and peptide mapping analysis we compared each member of the A/B-type core proteins in the human and rat liver cells. This comparison revealed the unique presence in rat cells of major protein species, referred to as mBx polypeptides, that appeared as three charge isoforms at a position corresponding to the minor HeLa B1b protein spot. In addition, clear differences in the ratios of the A1 polypeptide to the A1b isoform were observed. The detection, in sera of patients with rheumatic autoimmune diseases, of two novel autoantibody specificities, one recognizing solely B2 protein and the second both the B2 and mBx polypeptides, helped to identify mBx proteins as new A/B-type hnRNP components, immunologically related to B2 protein. A common immunoreactive V8 protease peptide of approx. 17 kDa has been identified in B2 and mBx hnRNP polypeptides. mBx protein species are identified in cells of murine origin, and have a ubiquitous tissue distribution and developmental appearance. PMID:9003360

  7. In Vitro and in Vivo Phosphorylation of Polypeptides in Plasma Membrane and Tonoplast-Enriched Fractions from Barley Roots 1

    PubMed Central

    Garbarino, Joan E.; Hurkman, William J.; Tanaka, Charlene K.; DuPont, Frances M.

    1991-01-01

    Phosphorylation of polypeptides in membrane fractions from barley (Hordeum vulgare L. cv CM 72) roots was compared in in vitro and in vivo assays to assess the potential role of protein kinases in modification of membrane transport. Membrane fractions enriched in endoplasmic reticulum, tonoplast, and plasma membrane were isolated using sucrose gradients and the membrane polypeptides separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis. When the membrane fractions were incubated with γ-[32P]ATP, phosphorylation occurred almost exclusively in the plasma membrane fraction. Phosphorylation of a band at 38 kilodaltons increased as the concentration of Mg2+ was decreased from millimolar to micromolar levels. Phosphorylation of bands at 125, 86, 58, 46, and 28 kilodaltons required millimolar Mg2+ concentrations and was greatly enhanced by Ca2+. When roots of intact plants were labeled with [32P]orthophosphate, polypeptides at approximately 135, 116, 90, 46 to 53, 32, 28, and 19 kilodaltons were labeled in the plasma membrane fraction and polypeptides at approximately 73, 66, and 48 kilodaltons were labeled in the tonoplast fraction. Treatment of the roots of intact plants with 150 millimolar NaCl resulted in increased phosphorylation of some polypeptides while treatment with 100 mm NaCl had no effect. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 PMID:16668115

  8. N-terminus conservation in the anchor polypeptide of a prokaryotic and eukaryotic alga. [Nostoc; Porphydium cruentum

    SciTech Connect

    Gantt, E.; Lipschultz, C.A.; Cunningham, F.X. Jr.; Mimuro, M.

    1987-04-01

    Energy flow between the extrinsic phycobilisomes and the photosystems within thylakoids, is probably mediated by a blue anchor polypeptide. Polypeptides in the 94 kD range, purified by LiDS-PAGE from phycobilisomes of Nostoc and Porphyrdium cruentum, crossreacted with anti-Nostoc-94 (although weakly with the latter). Though rich in ASP and GLU, the polypeptides were very hydrophobic, and low in MET, CYS, and HIS. Partial sequence of the N-terminus shows considerable homology 1 - 5 - 10 - 15 - 20 N: (S)-V-K-A-S-G-G-S-S-V-A-(R)-P-Q-L-Y-Q-(G)-L-(A)-V- P: V-()-K-A-S-G-G-S-P-V-V-K-P-Q-L-Y-(K)-()-A-(S)- between the species. There is a lack of homology when compared with ..cap alpha.. and ..beta.. polypeptides of allophycocyanin with rod linkers of phycobilisomes and other phycobiliproteins. Polypeptides of 94 and 92 kD from thylakoids of Nostoc, also immunoreactive with anti-94, were blocked at the N-terminus.

  9. Temperature-dependent morphology of hybrid nanoflowers from elastin-like polypeptides

    SciTech Connect

    Ghosh, Koushik; Balog, Eva Rose M.; Sista, Prakash; Williams, Darrick J.; Martinez, Jennifer S. E-mail: rcrocha@lanl.gov; Rocha, Reginaldo C. E-mail: rcrocha@lanl.gov; Kelly, Daniel

    2014-02-01

    We report a method for creating hybrid organic-inorganic “nanoflowers” using calcium or copper ions as the inorganic component and a recombinantly expressed elastin-like polypeptide (ELP) as the organic component. Polypeptides provide binding sites for the dynamic coordination with metal ions, and then such noncovalent complexes become nucleation sites for primary crystals of metal phosphates. We have shown that the interaction between the stimuli-responsive ELP and Ca{sup 2+} or Cu{sup 2+}, in the presence of phosphate, leads to the growth of micrometer-sized particles featuring nanoscale patterns shaped like flower petals. The morphology of these flower-like composite structures is dependent upon the temperature of growth and has been characterized by scanning electron microscopy. The composition of nanoflowers has also been analyzed by energy-dispersive X-ray spectroscopy, X-ray photoelectron spectroscopy, and X-ray diffraction. The temperature-dependent morphologies of these hybrid nanostructures, which arise from the controllable phase transition of ELPs, hold potential for morphological control of biomaterials in emerging applications such as tissue engineering and biocatalysis.

  10. Cytotype regulation of P transposable elements in Drosophila melanogaster: repressor polypeptides or piRNAs?

    PubMed

    Jensen, Philip A; Stuart, Jeremy R; Goodpaster, Michael P; Goodman, Joseph W; Simmons, Michael J

    2008-08-01

    The telomeric P elements TP5 and TP6 are associated with the P cytotype, a maternally inherited condition that represses P-element-induced hybrid dysgenesis in the Drosophila germ line. To see if cytotype repression by TP5 and TP6 might be mediated by the polypeptides they could encode, hobo transgenes carrying these elements were tested for expression of mRNA in the female germ line and for repression of hybrid dysgenesis. The TP5 and TP6 transgenes expressed more germ-line mRNA than the native telomeric P elements, but they were decidedly inferior to the native elements in their ability to repress hybrid dysgenesis. These paradoxical results are inconsistent with the repressor polypeptide model of cytotype. An alternative model based on the destruction of P transposase mRNA by Piwi-interacting (pi) RNAs was supported by finding reduced P mRNA levels in flies that carried the native telomeric P elements, which are inserted in a known major piRNA locus. PMID:18579507

  11. Crotamine, a small basic polypeptide myotoxin from rattlesnake venom with cell-penetrating properties.

    PubMed

    Rádis-Baptista, Gandhi; Kerkis, Irina

    2011-12-01

    Crotamine, a low molecular weight cationic polypeptide from the venom of the South American rattlesnake Crotalus durissus terrificus is a natural cell-penetrating peptide with functional versatility. The presence of nine lysine residues and three disulfide bonds renders crotamine highly compact, stable and positively charged. Topologically, crotamine adopts an ancient β-defensin fold that is found in diverse families of endogenous and venom polypeptides dedicated to host defense. Crotamine is unique among several classes of bioactive peptides because it possesses both cell penetrating and antimicrobial activities and selective biological action toward some cell types at a given cell cycle phase. Because it can rapidly and efficiently translocate into actively proliferating cells, crotamine is being investigated for labeling highly replicating cells and for use as a chemotherapeutic adjuvant. Peptides derived from crotamine, nucleolar targeting peptides (NrTPs), have been designed and are being studied. NrTPs retain some crotamine properties, such as efficient cellular uptake and preferential nuclear localization whereas they improve upon other properties. For example, NrTPs are smaller than crotamine, show higher preferential nucleolar localization, and better facilitate ZIP-code localization of therapeutic proteins. PMID:22204433

  12. Oxidation of methionine residues in polypeptide ions via gas-phase ion/ion chemistry.

    PubMed

    Pilo, Alice L; McLuckey, Scott A

    2014-06-01

    The gas-phase oxidation of methionine residues is demonstrated here using ion/ion reactions with periodate anions. Periodate anions are observed to attach in varying degrees to all polypeptide ions irrespective of amino acid composition. Direct proton transfer yielding a charge-reduced peptide ion is also observed. In the case of methionine and, to a much lesser degree, tryptophan-containing peptide ions, collisional activation of the complex ion generated by periodate attachment yields an oxidized peptide product (i.e., [M + H + O](+)), in addition to periodic acid detachment. Detachment of periodic acid takes place exclusively for peptides that do not contain either a methionine or tryptophan side chain. In the case of methionine-containing peptides, the [M + H + O](+) product is observed at a much greater abundance than the proton transfer product (viz., [M + H](+)). Collisional activation of oxidized Met-containing peptides yields a signature loss of 64 Da from the precursor and/or product ions. This unique loss corresponds to the ejection of methanesulfenic acid from the oxidized methionine side chain and is commonly used in solution-phase proteomics studies to determine the presence of oxidized methionine residues. The present work shows that periodate anions can be used to 'label' methionine residues in polypeptides in the gas phase. The selectivity of the periodate anion for the methionine side chain suggests several applications including identification and location of methionine residues in sequencing applications.

  13. An immunohistochemical observation of polypeptides and monoamines in the nucleus preopticus medianus of the rat.

    PubMed

    Kawano, H; Chiba, T; Masuko, S

    1989-07-17

    A detailed regional distribution of nerve cells and terminals immunoreactive to polypeptides or monoamines was examined in the 5 subdivisions (rostral, mid-dorsal, mid-ventral, caudo-dorsal and caudo-ventral parts) of the nucleus preopticus medianus (POMe) of the rat. In general, immunoreactive nerve cells and terminals are more numerous in the ventral parts of the middle and caudal POMe. Nerve cells immunoreactive to neurotensin (NT), Met-enkephalin-Arg6-Gly7-Leu8 (mENK8) or cholecystokinin-octapeptide (CCK8) are distributed throughout the POMe, while those immunoreactive to luteinizing hormone-releasing hormone (LHRH) are found in the rostral and middle POMe. Nerve cells immunoreactive to substance P (SP) are seen in the middle and caudal POMe and those immunoreactive to somatostatin (SRIF) are scattered in the middle part of the nucleus. The densities of nerve terminals immunoreactive to neuropeptide tyrosine, mENK8, SP or noradrenaline are high throughout the POMe, while nerve terminals immunoreactive to CCK8, LHRH, NT, SRIF or vasoactive intestinal polypeptide are moderate and those immunoreactive to calcitonin gene-related peptide, serotonin or dopamine are sparse. This varied distributional pattern of immunoreactive nerve cells and terminals suggests regional differences in function within the POMe.

  14. Bacteriocin AS-48, a microbial cyclic polypeptide structurally and functionally related to mammalian NK-lysin.

    PubMed

    González, C; Langdon, G M; Bruix, M; Gálvez, A; Valdivia, E; Maqueda, M; Rico, M

    2000-10-10

    The solution structure of bacteriocin AS-48, a 70-residue cyclic polypeptide from Enterococcus faecalis, consists of a globular arrangement of five alpha-helices enclosing a compact hydrophobic core. The head-to-tail union lies in the middle of helix 5, a fact that is shown to have a pronounced effect on the stability of the three-dimensional structure. Positive charges in the side chains of residues in helix 4 and in the turn linking helix 4 to helix 5 form a cluster that most probably determine its antibacterial activity by promoting pore formation in cell membranes. A similar five-helix structural motif has been found in the antimicrobial NK-lysin, an effector polypeptide of T and natural killer (NK) cells. Bacteriocin AS-48 lacks the three disulfide bridges characteristic of the saposin fold present in NK-lysin, and has no sequence homology with it. Nevertheless, the similar molecular architecture and high positive charge strongly suggest a common mechanism of antibacterial action.

  15. Purification of recombinant protein by cold-coacervation of fusion constructs incorporating resilin-inspired polypeptides.

    PubMed

    Lyons, Russell E; Elvin, Christopher M; Taylor, Karin; Lekieffre, Nicolas; Ramshaw, John A M

    2012-12-01

    Polypeptides containing between 4 and 32 repeats of a resilin-inspired sequence AQTPSSYGAP, derived from the mosquito Anopheles gambiae, have been used as tags on recombinant fusion proteins. These repeating polypeptides were inspired by the repeating structures that are found in resilins and sequence-related proteins from various insects. Unexpectedly, an aqueous solution of a recombinant resilin protein displays an upper critical solution temperature (cold-coacervation) when held on ice, leading to a separation into a protein rich phase, typically exceeding 200 mg/mL, and a protein-poor phase. We show that purification of recombinant proteins by cold-coacervation can be performed when engineered as a fusion partner to a resilin-inspired repeat sequence. In this study, we demonstrate the process by the recombinant expression and purification of enhanced Green fluorescent protein (EGFP) in E. coli. This facile purification system can produce high purity, concentrated protein solutions without the need for affinity chromatography or other time-consuming or expensive purification steps, and that it can be used with other bulk purification steps such as low concentration ammonium sulfate precipitation. Protein purification by cold-coacervation also minimizes the exposure of the target protein to enhanced proteolysis at higher temperature.

  16. Effects of hydrophobic and dipole-dipole interactions on the conformational transitions of a model polypeptide

    NASA Astrophysics Data System (ADS)

    Mu, Yan; Gao, Yi Qin

    2007-09-01

    We studied the effects of hydrophobicity and dipole-dipole interactions between the nearest-neighbor amide planes on the secondary structures of a model polypeptide by calculating the free energy differences between different peptide structures. The free energy calculations were performed with low computational costs using the accelerated Monte Carlo simulation (umbrella sampling) method, with a bias-potential method used earlier in our accelerated molecular dynamics simulations. It was found that the hydrophobic interaction enhances the stability of α helices at both low and high temperatures but stabilizes β structures only at high temperatures at which α helices are not stable. The nearest-neighbor dipole-dipole interaction stabilizes β structures under all conditions, especially in the low temperature region where α helices are the stable structures. Our results indicate clearly that the dipole-dipole interaction between the nearest neighboring amide planes plays an important role in determining the peptide structures. Current research provides a more unified and quantitative picture for understanding the effects of different forms of interactions on polypeptide structures. In addition, the present model can be extended to describe DNA/RNA, polymer, copolymer, and other chain systems.

  17. Primary structure of pancreatic polypeptide from four species of Perissodactyla (Przewalski's horse, zebra, rhino, tapir).

    PubMed

    Henry, J S; Lance, V A; Conlon, J M

    1991-12-01

    Pancreatic polypeptide (PP) has been purified from extracts of the pancreas of four species of odd-toed ungulates (Perissodactyla): Przewalski's horse, mountain zebra, white rhinoceros, and mountain tapir. The amino acid sequence of Przewalski's horse pancreatic polypeptide was established as Ala-Pro-Met-Glu-Pro-Val-Tyr-Pro-Gly-Asp10-Asn- Ala-Thr-Pro-Glu-Gln-Met-Ala-Gln-Tyr20-Ala-Ala-Glu-Leu-Arg-Arg-Tyr- Ile-Asn-Met30 - Leu-Thr-Arg-Pro-Arg-Tyr.NH2. Zebra PP was identical to Przewalski's horse PP, rhinoceros PP contained three substitutions relative to the horse (Ser for Ala1, Leu for Met3, and Glu for Gln16), and tapir PP contained one substitution relative to the horse (Leu for Met3). On the basis of morphological characteristics and the fossil record, the rhinocerotids are classified with the tapirids in the suborder Ceratomorpha, whereas the horse and zebra belong to a separate suborder, Hippomorpha. On the basis of structural similarity of the PP molecules, however, it would appear that the tapir is more closely related to the horse than to the rhinoceros. These observations provide a further example of the need for extreme caution when inferring taxonomic or phylogenetic relationships between species from the structures of homologous peptides.

  18. Direct targeting of Arabidopsis cysteine synthase complexes with synthetic polypeptides to selectively deregulate cysteine synthesis.

    PubMed

    Wawrzyńska, Anna; Kurzyk, Agata; Mierzwińska, Monika; Płochocka, Danuta; Wieczorek, Grzegorz; Sirko, Agnieszka

    2013-06-01

    Biosynthesis of cysteine is one of the fundamental processes in plants providing the reduced sulfur for cell metabolism. It is accomplished by the sequential action of two enzymes, serine acetyltransferase (SAT) and O-acetylserine (thiol) lyase (OAS-TL). Together they constitute the hetero-oligomeric cysteine synthase (CS) complex through specific protein-protein interactions influencing the rate of cysteine production. The aim of our studies was to deregulate the CS complex formation in order to investigate its function in the control of sulfur homeostasis and optimize cysteine synthesis. Computational modeling was used to build a model of the Arabidopsis thaliana mitochondrial CS complex. Several polypeptides based on OAS-TL C amino-acid sequence found at SAT-OASTL interaction sites were designed as probable competitors for SAT3 binding. After verification of the binding in a yeast two-hybrid assay, the most strongly interacting polypeptide was introduced to different cellular compartments of Arabidopsis cell via genetic transformation. Moderate increase in total SAT and OAS-TL activities, but not thiols content, was observed dependent on the transgenic line and sulfur availability in the hydroponic medium. Though our studies demonstrate the proof of principle, they also suggest more complex interaction of both enzymes underlying the mechanism of their reciprocal regulation. PMID:23602110

  19. Quantitative model of the phase behavior of recombinant pH-responsive elastin-like polypeptides.

    PubMed

    Mackay, J Andrew; Callahan, Daniel J; Fitzgerald, Kelly N; Chilkoti, Ashutosh

    2010-11-01

    Quantitative models are required to engineer biomaterials with environmentally responsive properties. With this goal in mind, we developed a model that describes the pH-dependent phase behavior of a class of stimulus responsive elastin-like polypeptides (ELPs) that undergo reversible phase separation in response to their solution environment. Under isothermal conditions, charged ELPs can undergo phase separation when their charge is neutralized. Optimization of this behavior has been challenging because the pH at which they phase separate, pHt, depends on their composition, molecular weight, concentration, and temperature. To address this problem, we developed a quantitative model to describe the phase behavior of charged ELPs that uses the Henderson-Hasselbalch relationship to describe the effect of side-chain ionization on the phase-transition temperature of an ELP. The model was validated with pH-responsive ELPs that contained either acidic (Glu) or basic (His) residues. The phase separation of both ELPs fit this model across a range of pH. These results have important implications for applications of pH-responsive ELPs because they provide a quantitative model for the rational design of pH-responsive polypeptides whose transition can be triggered at a specified pH.

  20. Polypeptide and antigenic variability among strains of Mycoplasma ovipneumoniae demonstrated by SDS-PAGE and immunoblotting.

    PubMed

    Thirkell, D; Spooner, R K; Jones, G E; Russell, W C

    1990-01-01

    Comparison of the polypeptide patterns of 22 isolates of M. ovipneumoniae by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a marked degree of heterogeneity with only limited groupings identifiable. Of the 50 major polypeptides identified in one strain (956/2), 35 were shown to be antigenic using immunoblotting with a homologous polyclonal serum. Radioimmune precipitation of 125I-surface-labelled proteins and phase partition using Triton X-114 detergent indicated that these were membrane associated. Cross-reactivity between the isolates was examined by immunoblotting using one polyclonal serum and four monoclonal antibodies (MAbs), all raised against strain 956/2. The polyclonal serum revealed considerable antigenic heterogeneity, but at least nine major antigens were conserved across all isolates. Two MAbs cross-reacted with all 22 strains, but the other two MAbs allowed some differentiation of the strains. One (MO/3) divided the isolates into groups of 16 and 6 based on the presence of absence of a 26-kDa antigen. All strains isolated from sheep with pulmonary adenomatosis fell into the smaller group and did not possess the 26-kDa antigen.

  1. Colostrinin: a proline-rich polypeptide complex of potential therapeutic interest.

    PubMed

    Janusz, M; Zabłocka, A

    2013-01-01

    A proline-rich polypeptide complex (PRP) subsequently known as ColostrininTM was found for the first time in ovine colostrum as a fraction accompanying colostral IgG2. Subsequently, similar polypeptides were found in human, bovine and caprine colostrum. PRP is a complex of peptides of molecular masses from 500 to 3000 Da. It contains 25% proline residues and 40% hydrophobic amino acids. It is not species specific, and is active both in vivo and in vitro. PRP possesses immunoregulatory properties, including effects on humoral and cellular immune responses, shows regulatory activity in Th1 and Th2 cytokine induction, and has the ability to inhibit the overproduction of reactive oxygen species and nitric oxide. PRP has also shown psychotropic properties. Both immunoregulatory and psychotropic properties suggest potential clinical use of PRP for neurodegenerative disorders. Beneficial effects of PRP/Colostrinin in the case of Alzheimer's disease were shown in double-blind placebo-controlled trials, in long-term open-label studies and in multicenter clinical trials. A very important property of PRP/Colostrinin and one of its components, a nonapeptide (NP), is the prevention of Aβ aggregation and the disruption of aggregates already formed. Moreover, PRP has been found to modulate neurite outgrowth, suppress uncontrolled activation of cells, and reduce 4-HNE-mediated cellular damage. Biological response modifying activity of PRP/Colostrinin can play an important role in its use in the treatment of Alzheimer's disease and suggests its application beyond neurodegenerative disorders. PMID:24200016

  2. Cystosolic chaperonin subunits have a conserved ATPase domain but diverged polypeptide-binding domains.

    PubMed

    Kim, S; Willison, K R; Horwich, A L

    1994-12-01

    CCT (also called the TCP-1 complex or TriC) is a chaperonin found in the eukaryotic cytosol, and has unique structural and functional features. Unlike homo-oligomeric chaperonins, CCT comprises at least eight different subunits, and appears to have a limited range of physiological substrates. We have analysed CCT sequences in light of the recent determination of the crystal structure and mutational identification of the functional domains of the bacterial chaperonin GroEL. A high level of identity among all chaperonin subunits is observed in those regions that correspond to the ATP-binding site of GroEL. By contrast, no significant identity is shared in the region corresponding to the polypeptide-binding region of GroEL, either between CCT subunits or between CCT subunits and GroEL. This suggests that the polypeptide-binding sites of CCT subunits have diverged both from each other and from GroEL, which may explain the apparently different range of substrates recognized by CCT.

  3. cDNA encoding a polypeptide including a hev ein sequence

    DOEpatents

    Raikhel, Natasha V.; Broekaert, Willem F.; Chua, Nam-Hai; Kush, Anil

    2000-07-04

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74-79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.

  4. Specific photoaffinity labeling of two plasma membrane polypeptides with an azido auxin

    SciTech Connect

    Hicks, G.R.; Rayle, D.L.; Jones, A.M.; Lomax, T.L. )

    1989-07-01

    Plasma membrane vesicles were isolated from zucchini (Cucurbita pepo) hypocotyl tissue by aqueous phase partitioning and assessed for homogeneity by the use of membrane-specific enzyme assays. The highly pure plasma membrane vesicles maintained a pH differential across the membrane and accumulated a tritiated azido analogue of 3-indoleacetic acid (IAA), 5-azido-(7-{sup 3}H)IAA(({sup 3}H)N{sub 3}IAA), in a manner similar to the accumulation of ({sup 3}H)IAA. The association of the ({sup 3}H)N{sub 3}IAA with membrane vesicles was saturable and subject to competition by IAA and auxin analogues. Auxin-binding proteins were photoaffinity labeled by addition of ({sup 3}H)N{sub 3}IAA to plasma membrane vesicles prior to exposure to UV light and detected by subsequent NaDodSO{sub 4}/PAGE and fluorography. When the reaction temperature was lowered to {minus}196{degree}C, high-specific-activity labeling of a 40-kDa and a 42-kDa polypeptide was observed. Collectively, these results suggest that the radiolabeled polypeptides are auxin receptors. The covalent nature of the label should facilitate purification and further characterization of the receptors.

  5. Effect of explosive noise on gastrointestinal transit and plasma levels of polypeptide hormones

    PubMed Central

    Mu, Zhen-Bin; Huang, Yu-Xin; Zhao, Bao-Min; Liu, Zhen-Xiong; Zhang, Bing-Hua; Wang, Qing-Li

    2006-01-01

    AIM: To investigate the effect of firing noise on gastrointestinal transit and probe its mechanism by measuring the levels of plasma polypeptide hormones. METHODS: A total of 64 SD rats were randomly divided into a control group and three stimulating groups. Firing noise of different intensity by sub-machine guns was used as inflicting factor. The effect of firing noise on liquid substance gastrointestinal transit and solid substance gastrointestinal transit was observed by measuring the ratio of carbon powder suspension transmitting and barium sticks transmitting respectively. Plasma levels of polypeptide hormones were measured by radio-immunoassay. RESULTS: The noise accelerated gastrointestinal transit of solid food by more than 80 db;and accelerated gastrointestinal transit of liquid food significantly by more than 120 db. Meantime, plasma levels of plasma motilin (MTL)(157.47±16.08; 151.90±17.08), somatostatin (SS)(513.97±88.77; 458.25±104.30), substance P (SP)(115.52±20.70; 110.28±19.96) and vasoactive intestinal peptide (VIP) (214.21±63.17; 251.76±97.24) remarkably changed also. CONCLUSION: Within a certain intensity range, the firing noise changes the levels of rat plasma gastrointestinal hormones, but the gastrointestinal transit is still normal. Beyond the range, the noise induces plasma hormone levels disturbance and gastrointestinal transit disorder. PMID:16610038

  6. A novel giant secretion polypeptide in Chironomus salivary glands: implications for another Balbiani ring gene

    PubMed Central

    1985-01-01

    Chironomus salivary glands contain a family of high Mr (approximately 1,000 X 10(3)) secretion polypeptides thought to consist of three components: sp-Ia, sp-Ib, and sp-Ic. The use of a new extraction protocol revealed a novel high Mr component, sp-Id. Results of a survey of individual salivary glands indicated that sp-Id was widespread in more than a dozen strains of C. tentans and C. pallidivittatus. Sp-Id was phosphorylated at Ser residues, and a comparison of cyanogen bromide and tryptic peptide maps of 32P-labeled polypeptides suggested that sp-Ia, sp-Ib, and sp-Id are comprised of similar but nonidentical tandemly repeated amino acid sequences. We concluded that sp-Id is encoded by an mRNA whose size and nucleotide sequence organization are similar to Balbiani ring (BR) mRNAs that code for the other sp-I components. Furthermore, parallel repression of sp-Ib and sp-Id synthesis by galactose led us to hypothesize that both of their genes exist within Balbiani ring 2. PMID:4030890

  7. Oxidation of Methionine Residues in Polypeptide Ions Via Gas-Phase Ion/Ion Chemistry

    NASA Astrophysics Data System (ADS)

    Pilo, Alice L.; McLuckey, Scott A.

    2014-06-01

    The gas-phase oxidation of methionine residues is demonstrated here using ion/ion reactions with periodate anions. Periodate anions are observed to attach in varying degrees to all polypeptide ions irrespective of amino acid composition. Direct proton transfer yielding a charge-reduced peptide ion is also observed. In the case of methionine and, to a much lesser degree, tryptophan-containing peptide ions, collisional activation of the complex ion generated by periodate attachment yields an oxidized peptide product (i.e., [M + H + O]+), in addition to periodic acid detachment. Detachment of periodic acid takes place exclusively for peptides that do not contain either a methionine or tryptophan side chain. In the case of methionine-containing peptides, the [M + H + O]+ product is observed at a much greater abundance than the proton transfer product (viz., [M + H]+). Collisional activation of oxidized Met-containing peptides yields a signature loss of 64 Da from the precursor and/or product ions. This unique loss corresponds to the ejection of methanesulfenic acid from the oxidized methionine side chain and is commonly used in solution-phase proteomics studies to determine the presence of oxidized methionine residues. The present work shows that periodate anions can be used to `label' methionine residues in polypeptides in the gas phase. The selectivity of the periodate anion for the methionine side chain suggests several applications including identification and location of methionine residues in sequencing applications.

  8. Antifreeze proteins from the sea raven, Hemitripterus americanus. Further evidence for diversity among fish polypeptide antifreezes

    SciTech Connect

    Slaughter, D.; Fletcher, G.L.; Ananthanarayanan, V.S.; Hew, C.L.

    1981-02-25

    The antifreeze proteins of the sea raven, Hemitripterus americanus, were isolated and compared with other fish antifreeze proteins. The sea raven contains one major protein of molecular weight 14,000-16,000 with little or no carbohydrate. Except for its similar seasonal appearance, the sea raven antifreeze protein differs from other polypeptide antifreeze in its amino acid composition, secondary structure, and immunological specificity. Amino acid analysis of sea raven antifreeze showed that it contains a high amount of half-cystine, hydrophilic amino acids, and only an average amount of alanine. In contrast, all other fish antifreeze proteins contain approximately 60% alanine and no half-cystine residues. Furthermore, the sea raven antifreeze protein is sensitive to sulfhydryl reagents. The antifreeze activity was decreased by 67% in the presence of 0.01 M dithiothreitol. Circular dichroism studies indicated the absence of significant amounts of ..cap alpha..-helix and the possible presence of ..beta..-structure. Antibodies raised against the antifreeze protein did not cross-react with the known polypeptide antifreeze from the winter flounder and shorthorn sculpin (Hew, C.L., Fletcher, G.L., and Ananthanarayanan, V.S. (1980) Can. J. Biochem. 58, 377-383). A specific radioimmunoassay was developed for the sea raven antifreeze protein and was used to quantitate the protein concentration in the fish. The seasonal profile obtained by radioimmunoassay was compatible with the antifreeze activity determined with a freezing point osmometer.

  9. Polypeptide micelles with dual pH activatable dyes for sensing cells and cancer imaging.

    PubMed

    Gong, Ping; Yang, Yueting; Yi, Huqiang; Fang, Shengtao; Zhang, Pengfei; Sheng, Zonghai; Gao, Guanhui; Gao, Duyang; Cai, Lintao

    2014-05-21

    pH is an important control parameter for maintenance of cell viability and tissue functions. pH monitoring provides valuable information on cell metabolic processes and the living environment. In this study, we prepared dual pH-sensitive, fluorescent dye-loaded polypeptide nanoparticles (DPNs) for ratiometric sensing of pH changes in living cells. DPNs contain two types of dyes: N-(rhodamine B) lactam cystamine (RBLC), an acid activatable fluorescent dye with increased fluorescence in an acidic environment, and fluorescein isothiocyanate (FITC), a base activatable fluorescent dye with enhanced fluorescence in an alkaline environment. Hence, DPNs exhibited a dual response signal with strong red fluorescence and weak green fluorescence under acidic conditions; in contrast, they showed strong green fluorescence and almost no red fluorescence under alkaline and neutral conditions. The favorable inverse pH responses of the two fluorescent dyes resulted in ratiometric pH determination for DPNs with an optimized pH-sensitive range of pH 4.5-7.5. Quantitative analysis of the intracellular pH of intact MCF-7 cells has been successfully demonstrated with our nanosensor. Moreover, single acid activatable fluorescent dye doped polypeptide nanoparticles that only contained RBLC can distinguish tumor tissue from normal tissue by monitoring the acidic extracellular environment.

  10. Estimation of N-H...O=C intramolecular hydrogen bond energy in polypeptides.

    PubMed

    Deshmukh, Milind M; Gadre, Shridhar R

    2009-07-01

    The previously proposed molecular tailoring approached (MTA) [Deshmukh, M. M.; Gadre, S. R.; Bartolotti, L. J. J. Phys. Chem. A 2006, 110, 12519] for the estimation of intramolecular O-H...O hydrogen bond energy is extended to that for the N-H...O=C bond within polypeptides. The methodology is initially tested on a tetrapeptide containing two types of N-H...O=C hydrogen bonds and is found to distinguish between them. The estimated values are in good agreement with the trends predicted by the geometrical parameters. Furthermore, this methodology is applied to partially as well as fully substituted, capped polyglycines that contain five glycine residues (acetyl-(gly)(5)-NH(2)) to check the effect of substituents on the energetics of hydrogen bonds. The estimated N-H...O=C bond energy values lie in the range of 4-6 kcal/mol. These estimated values are not only in concurrence with the geometric parameters but also able to reflect the subtle effects of substituents for the substituted polypeptides studied in the present work.

  11. Pancreatic spasmolytic polypeptide protects the gastric mucosa but does not inhibit acid secretion or motility.

    PubMed

    McKenzie, C; Marchbank, T; Playford, R J; Otto, W; Thim, L; Parsons, M E

    1997-07-01

    The objectives of these studies were to examine whether the trefoil peptide porcine pancreatic spasmolytic polypeptide (PSP) had gastric mucosal protectant properties similar to its human equivalent human spasmolytic polypeptide (hSP) and to confirm the antisecretory and antimotility action of the peptide. PSP and recombinant hSP reduced gastric mucosal damage caused by a combination of subcutaneous indomethacin and restraint stress in the conscious rat. At a dose of 500 micrograms/kg bolus plus 500 micrograms.kg-1.h-1 sc, PSP significantly reduced the total area of damage by 58%. PSP at a dose of 150 micrograms/kg iv had no inhibitory effect on pentagastrin-stimulated gastric acid secretion in the perfused stomachs of anesthetized rats. This lack of antisecretory activity was confirmed in vitro using an isolated stomach preparation from the immature rat. PSP and hSP at concentrations up to 800 nM did not inhibit electrically or chemically evoked contractions of the guinea pig ileum and duodenum in vitro. Thus antisecretory and antimotility actions do not underlie the mucosal protectant properties of PSP. PSP did, however, stimulate cell migration, and this may, at least in part, account for its protectant properties.

  12. Schistosoma mansoni polypeptides immunogenic in mice vaccinated with radiation-attenuated cercariae

    SciTech Connect

    Dalton, J.P.; Strand, M.

    1987-10-01

    We compared the humoral immune response of mice protected against Schistosoma mansoni by vaccination with radiation-attenuated cercariae to that of patently infected mice, and we identified antigens that elicit a greater, or unique, immune response in the vaccinated mice. These comparisons were based upon radioimmunoprecipitations and immunodepletion of (/sup 35/S)methionine-labeled schistosomular and adult worm polypeptides, followed by one- and two-dimensional polyacrylamide gel analyses. The humoral responses of patently infected mice and of mice vaccinated once were remarkably similar and were directed against schistosome glycoproteins ranging in molecular size from greater than 300 to less than 10 kDa. Exposing mice to a second vaccination resulted in a marked change in the immune response, to one predominantly directed toward high molecular size glycoproteins. Sequential immunodepletion techniques identified five schistosomular and seven adult worm antigens that showed a greater or unique immunogenicity in vaccinated mice as compared with patently infected mice. These adult worm antigens were purified by preparative sequential immunoaffinity chromatography and used to prepare a polyclonal antiserum, anti-irradiated vaccine. This antiserum bound to the surface of live newly transformed and lung-stage schistosomula, as assessed by immunofluorescence assays, and was reactive with a number of /sup 125/I-labeled schistosomular surface polypeptides, including a doublet of 150 kDa that was also recognized by sera of vaccinated mice but not by sera of patently infected mice.

  13. Wet-spinnability and crosslinked fibre properties of two collagen polypeptides with varied molecular weight.

    PubMed

    Tronci, Giuseppe; Kanuparti, Ramya Sri; Arafat, M Tarik; Yin, Jie; Wood, David J; Russell, Stephen J

    2015-11-01

    The formation of naturally derived materials with wet stable fibrous architectures is paramount in order to mimic the features of tissues at the molecular and microscopic scale. Here, we investigated the formation of wet-spun fibres based on collagen-derived polypeptides with comparable chemical composition and varied molecular weight. Gelatin and hydrolysed fish collagen (HFC) were selected as widely available linear amino-acidic chains of high and low molecular weight, respectively, and functionalised in the wet-spun fibre state in order to preserve the material geometry in physiological conditions. Wet-spun fibre diameter and morphology were dramatically affected depending on the polypeptide molecular weight, wet-spinning solvent (i.e. 2,2,2-trifluoroethanol and dimethyl sulfoxide) and coagulating medium (i.e. acetone and ethanol), resulting in either bulky or porous internal geometry. Dry-state tensile moduli were significantly enhanced in gelatin and HFC samples following covalent crosslinking with activated 1,3-phenylenediacetic acid (Ph) (E: 726±43-844±85MPa), compared to samples crosslinked via intramolecular carbodiimide-mediated condensation reaction (E: 588±38MPa). Resulting fibres displayed a dry diameter in the range of 238±18-355±28μm and proved to be mechanically stable (E: 230kPa) following equilibration with PBS, whilst a nearly complete degradation was observed after 5-day incubation in physiological conditions.

  14. High-resolution polypeptide structure and dynamics in anisotropic environments: The gramicidin channel

    SciTech Connect

    Cross, T.A.; Lee, K.C.; Ketchem, R.R.; Hu, W.; Lazo, N.D.; Huo, S.

    1994-12-01

    To understand the details of macromolecular function, high-resolution structural and dynamic detail is essential. The polypeptide fold of the gramicidin channel has been effectively modeled for the past 20 years, yet the functional changes in conductance and channel lifetime associated with amino acid substitutions cannot be predicted. To accomplish this goal, high-resolution electrostatic modeling and the precise orientation of all dipoles are required. Furthermore, an enhanced knowledge of the complex molecular environment of this membrane-bound peptide is needed. An aqueous environment is relatively uniform and achiral. The membrane environment is very heterogenous and chiral. A knowledge of the interactions, specific and nonspecific, between peptide and lipid will aid in developing a better understanding of this environment. To accomplish this goal, it is necessary to study the peptide in an extended lipid bilayer, rather than in a vesicular or micellar form. These latter environments are likely to possess increased dynamics, increased water penetration, and distorted interactions between the polypeptide and membrane surface. To perform NMR studies on bilayer bound peptides, solid state NMR methods are required, and for specific site information, isotopic labels are incorporated using solid phase peptide synthesis.

  15. Bacteriocin AS-48, a microbial cyclic polypeptide structurally and functionally related to mammalian NK-lysin

    PubMed Central

    González, Carlos; Langdon, Grant M.; Bruix, Marta; Gálvez, Antonio; Valdivia, Eva; Maqueda, Mercedes; Rico, Manuel

    2000-01-01

    The solution structure of bacteriocin AS-48, a 70-residue cyclic polypeptide from Enterococcus faecalis, consists of a globular arrangement of five α-helices enclosing a compact hydrophobic core. The head-to-tail union lies in the middle of helix 5, a fact that is shown to have a pronounced effect on the stability of the three-dimensional structure. Positive charges in the side chains of residues in helix 4 and in the turn linking helix 4 to helix 5 form a cluster that most probably determine its antibacterial activity by promoting pore formation in cell membranes. A similar five-helix structural motif has been found in the antimicrobial NK-lysin, an effector polypeptide of T and natural killer (NK) cells. Bacteriocin AS-48 lacks the three disulfide bridges characteristic of the saposin fold present in NK-lysin, and has no sequence homology with it. Nevertheless, the similar molecular architecture and high positive charge strongly suggest a common mechanism of antibacterial action. PMID:11005847

  16. De novo prediction of polypeptide conformations using dihedral probability grid Monte Carlo methodology.

    PubMed Central

    Evans, J. S.; Mathiowetz, A. M.; Chan, S. I.; Goddard, W. A.

    1995-01-01

    We tested the dihedral probability grid Monte Carlo (DPG-MC) methodology to determine optimal conformations of polypeptides by applying it to predict the low energy ensemble for two peptides whose solution NMR structures are known: integrin receptor peptide (YGRGDSP, Type II beta-turn) and S3 alpha-helical peptide (YMSEDEL KAAEAAFKRHGPT). DPG-MC involves importance sampling, local random stepping in the vicinity of a current local minima, and Metropolis sampling criteria for acceptance or rejection of new structures. Internal coordinate values are based on side-chain-specific dihedral angle probability distributions (from analysis of high-resolution protein crystal structures). Important features of DPG-MC are: (1) Each DPG-MC step selects the torsion angles (phi, psi, chi) from a discrete grid that are then applied directly to the structure. The torsion angle increments can be taken as S = 60, 30, 15, 10, or 5 degrees, depending on the application. (2) DPG-MC utilizes a temperature-dependent probability function (P) in conjunction with Metropolis sampling to accept or reject new structures. For each peptide, we found close agreement with the known structure for the low energy conformational ensemble located with DPG-MC. This suggests that DPG-MC will be useful for predicting conformations of other polypeptides. PMID:7549884

  17. Architecture effects on multivalent interactions by polypeptide-based multivalent ligands

    NASA Astrophysics Data System (ADS)

    Liu, Shuang

    protein materials, including structural as well as functional proteins. Therefore, polypeptide-based multivalent scaffolds are used to display ligands to assess the contribution of different architectural parameters to the multivalent binding events. In this work, a family of alanine-rich alpha-helical glycopolypeptides was designed and synthesized by a combination of protein engineering and chemical coupling, to display two types of saccharide ligands for two different multivalent binding systems. The valencies, chain length and spacing between adjacent ligands of these multivalent ligands were designed in order to study architecture effects on multivalent interactions. The polypeptides and their glycoconjugates were characterized via various methods, including SDS-PAGE, NMR, HPLC, amino acid analysis (AAA), MALDI, circular dichroism (CD) and GPC. In the first multivalent binding system, cholera toxin B pentamer (CT B5) was chosen to be the protein receptor due to its well-characterized structure, lack of significant steric interference of binding to multiple binding sites, and requirement of only simple monosaccharide as ligands. Galactopyranoside was incorporated into polypeptide scaffolds through amine-carboxylic acid coupling to the side chains of glutamic acid residues. The inhibition and binding to CT B5 of these glycopolypeptide ligands were evaluated by direct enzyme-linked assay (DELA). As a complement method, weak affinity chromatography (WAC) was also used to evaluate glycopolypeptides binding to a CT B5 immobilized column. The architecture effects on CT B 5 inhibition are discussed. In the second system, cell surface receptor L-selectin was targeted by polypeptide-based multivalent ligands containing disulfated galactopyranoside ligands, due to its important roles in various immunological activities. The effects of glycopolypeptide architectural variables L-selectin shedding were evaluated via ELISA-based assays. These polypeptide-based multivalent ligands

  18. Architecture effects on multivalent interactions by polypeptide-based multivalent ligands

    NASA Astrophysics Data System (ADS)

    Liu, Shuang

    protein materials, including structural as well as functional proteins. Therefore, polypeptide-based multivalent scaffolds are used to display ligands to assess the contribution of different architectural parameters to the multivalent binding events. In this work, a family of alanine-rich alpha-helical glycopolypeptides was designed and synthesized by a combination of protein engineering and chemical coupling, to display two types of saccharide ligands for two different multivalent binding systems. The valencies, chain length and spacing between adjacent ligands of these multivalent ligands were designed in order to study architecture effects on multivalent interactions. The polypeptides and their glycoconjugates were characterized via various methods, including SDS-PAGE, NMR, HPLC, amino acid analysis (AAA), MALDI, circular dichroism (CD) and GPC. In the first multivalent binding system, cholera toxin B pentamer (CT B5) was chosen to be the protein receptor due to its well-characterized structure, lack of significant steric interference of binding to multiple binding sites, and requirement of only simple monosaccharide as ligands. Galactopyranoside was incorporated into polypeptide scaffolds through amine-carboxylic acid coupling to the side chains of glutamic acid residues. The inhibition and binding to CT B5 of these glycopolypeptide ligands were evaluated by direct enzyme-linked assay (DELA). As a complement method, weak affinity chromatography (WAC) was also used to evaluate glycopolypeptides binding to a CT B5 immobilized column. The architecture effects on CT B 5 inhibition are discussed. In the second system, cell surface receptor L-selectin was targeted by polypeptide-based multivalent ligands containing disulfated galactopyranoside ligands, due to its important roles in various immunological activities. The effects of glycopolypeptide architectural variables L-selectin shedding were evaluated via ELISA-based assays. These polypeptide-based multivalent ligands

  19. Virus-Specific Messenger RNA and Nascent Polypeptides in Polyribosomes of Cells Replicating Murine Sarcoma-Leukemia Viruses

    PubMed Central

    Vecchio, G.; Tsuchida, N.; Shanmugam, G.; Green, M.

    1973-01-01

    We present evidence that virus-specific RNA is present in polyribosomes of transformed cells replicating the murine sarcoma-leukemia virus complex and that it serves as messenger RNA for the synthesis of viral-coded proteins. Both virus-specific RNA (detected by hybridization with the [3H]DNA product of the viral RNA-directed DNA polymerase) and nascent viral polypeptides (measured by precipitation with antiserum to purified virus) were found in membrane-bound and free polyribosomes. Membrane-bound polyribosomes contained a higher content of both virus-specific RNA and nascent viral polypeptides. From 60 to 70% of viral RNA sequences were released from polyribosomes with EDTA, consistent with a function as messenger RNA. Maximum amounts of both virus-specific RNA and nascent viral polypeptides were found in the polyribosome region sedimenting at about 350 S. PMID:4352969

  20. The Relationship between Albumin-Binding Capacity of Recombinant Polypeptide and Changes in the Structure of Albumin-Binding Domain.

    PubMed

    Bormotova, E A; Gupalova, T V

    2015-07-01

    Many bacteria express surface proteins interacting with human serum albumin (HSA). One of these proteins, PAB from anaerobic bacteria, contains an albumin-binding domain consisting of 45 amino acid residues known as GA domain. GA domains are also found in G proteins isolated from human streptococcal strains (groups C and G) and of albumin-binding protein isolated from group G streptococcal strains of animal origin. The GA domain is a left-handed three-helix bundle structure in which amino acid residues of the second and third helixes are involved in albumin binding. We studied the relationship between HSA-binding activity of the recombinant polypeptide isolated from group G streptococcus of animal origin and structure of the GA domain is studied. Structural changes in GA domain significantly attenuated HAS-binding capacity of the recombinant polypeptide. Hence, affinity HSA-binding polypeptide depends on stability of GA domain structure.

  1. Final Words

    ERIC Educational Resources Information Center

    Bers, Trudy

    2012-01-01

    This final chapter provides observations about institutional research in community colleges derived from the preceding chapters and the issue editors' own experiences. Taken as a whole, the chapters in this issue, as well as the editors' experiences, suggest several observations about institutional research in community colleges. These include the…

  2. Biosynthesis of intestinal microvillar proteins. Translational evidence in vitro that aminopeptidase N is synthesized as a Mr-115000 polypeptide.

    PubMed Central

    Danielsen, E M; Norén, O; Sjöström, H

    1982-01-01

    A crude RNA fraction, prepared from pig small intestine, was found to be more efficient than a fraction enriched in polyadenylated RNA in directing translation of polypeptides with Mr greater than 100000 in a rabbit reticulocyte lysate system. Aminopeptidase N (EC 3.4.11.2) synthesized in vitro was immunopurified from the translation mixture and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. It was found to have an apparent Mr of 115000 regardless of whether the translation was performed in the absence or presence of proteinase inhibitors. This result contradicts the possibility of aminopeptidase N being synthesized as a large single-chain precursor polypeptide. Images Fig. 1. PMID:6180737

  3. Cloning, characterization and expression of the polypeptide release factor gene, eRF1, of Blepharisma japonicum.

    PubMed

    Wang, Wei; Chai, Bao-Feng; Heckmann, Klaus; Liang, Ai-Hua

    2004-06-01

    The polypeptide release factor gene, eRF1, of Blepharisma japonicum (Bj-eRF1) was cloned and sequenced. Its coding region was 1314 base pairs and encodes a protein of 437 amino acids. The cloned gene was expressed in Escherichia coli and the recombinant Bj-eRF1 polypeptide was purified by Ni2+-nitrilotriacetic acid agarose and Superose12 chromatography. Pull-down analysis showed that the recombinant Bj-eRF1 interacts with the heterologously-expressed release factor, eRF3C, of Euplotes octocarinatus.

  4. Growing up in a dangerous environment: a network of multiple targeting and folding pathways for nascent polypeptides in the cytosol.

    PubMed

    Bukau, B; Hesterkamp, T; Luirink, J

    1996-12-01

    The first events in the lives of proteins are the most hazardous. Starting at the ribosome, nascent polypeptides undergo complex folding processes endangered by aggregation reactions. Proteins with organellar destinations require correct targeting to the translocation machineries and prevention from premature folding. The high precision and speed of these processes is ensured by a cystosolic system consisting of molecular chaperones, folding catalysts and targeting factors. This review focuses on the interactions of this system with nascent polypeptides and discusses new concepts for protein folding in the cytosol. It is proposed that folding and targeting are promoted by a flexible network of multiple unassisted and assisted pathways.

  5. Self-assembling Polypeptide Nanoparticles: Design, Synthesis, Biophysical Characterization and Biomedical Applications

    NASA Astrophysics Data System (ADS)

    Araujo Pereira Falcao Pimentel, Tais de

    Inspired by the architecture of icosahedral viruses, self-assembling polypeptide nanoparticles (SAPN) with icosahedral symmetry were developed. The building block for the SAPN was a single polypeptide chain. Similarly, the capsid of quite a few small viruses are built from one single peptide chain. The polypeptide chain of the SAPN consists of a pentameric coiled-coil domain at the N-terminus joined by a short linker segment to a trimeric coiled-coil domain at the C-terminus. Here we have studied factors governing self-assembly of the SAPN such as linker constitution and trimer length. The interdomain linker 2i88 afforded the most homogenous nanoparticles as verified by TEM and DLS. Furthermore, AUC and STEM analyses suggest that the nanoparticles formed using the linker 2i88 have a T=3-like architecture confirming computer modeling predictions. As for trimer length, we have shown that it is possible to synthesize SAPN with a trimer that is as short as only 17 amino acids. Given that the N-terminus and C-terminus of the SAPN can be extended to include epitopes and give rise to a repetitive antigen display system, vaccine applications of the SAPN were also investigated here. We grafted parts of the SARS virus' spike protein onto our SAPN to repetitively display this B-cell epitope. Biophysical characterization showed that single nanoparticles of the expected size range were formed. Immunization experiments in mice at University of Colorado Denver revealed that the antibodies elicited were conformation-specific. Moreover, the antibodies significantly inhibited SARS virus infection of Vero E6 cells. SAPN were also functionalized at the C-terminus with a B-cell epitope from the circumsporozoite protein (CSP) of the malaria parasite Plasmodium falciparum and at the N-terminus with CTL epitopes from CSP. The trimeric coiled-coil domains of these malaria SAPN were modified to include a HTL epitope. Even will all these modifications, self-assembly occurred as confirmed by

  6. Final Report

    SciTech Connect

    Marchant, Gary E.

    2013-04-23

    This is the final report of a two year project entitled "Governing Nanotechnology Risks and Benefits in the Transition to Regulation: Innovative Public and Private Approaches." This project examined the role of new governance or "soft law" mechanisms such as codes of conduct, voluntary programs and partnership agreements to manage the risks of emerging technologies such as nanotechnology. A series of published or in publication papers and book chapters are attached.

  7. Final Report

    SciTech Connect

    R. Paul Drake

    2001-11-30

    This final report describes work involving 22 investigators from 11 institutions to explore the dynamics present in supernova explosions by means of experiments on the Omega laser. The specific experiments emphasized involved the unstable expansion of a spherical capsule and the coupling of perturbations at a first interface to a second interface by means of a strong shock. Both effects are present in supernovae. The experiments were performed at Omega and the computer simulations were undertaken at several institutions. B139

  8. Assignment of the gastric inhibitory polypeptide receptor gene (GIPR) to chromosome bands 19q13.2-q13.3 by fluorescence in situ hybridization

    SciTech Connect

    Stoffel, M.; Fernald, A.A.; Bell, G.I.; Le Beau, M.M.

    1995-08-10

    The gastric inhibitory polypeptide receptor gene (GIPR) was localized, using fluorescence in situ hybridization (FISH), to human chromosome bands 19q13.2-q13.3. Gastric inhibitory polypeptide (GIP) is a potent stimulator of insulin secretion and mutations in the GIPR gene may be related to non-insulin-dependent diabetes mellitus (NIDDM). 13 refs., 1 fig.

  9. Expression of a Deschampsia antarctica Desv. Polypeptide with Lipase Activity in a Pichia pastoris Vector

    PubMed Central

    Rabert, Claudia; Gutiérrez-Moraga, Ana; Navarrete-Gallegos, Alejandro; Navarrete-Campos, Darío; Bravo, León A.; Gidekel, Manuel

    2014-01-01

    The current study isolated and characterized the Lip3F9 polypeptide sequence of Deschampsia antarctica Desv. (GeneBank Accession Number JX846628), which was found to be comprised of 291 base pairs and was, moreover, expressed in Pichia pastoris X-33 cells. The enzyme was secreted after 24 h of P. pastoris culture incubation and through induction with methanol. The expressed protein showed maximum lipase activity (35 U/L) with an optimal temperature of 37 °C. The lipase-expressed enzyme lost 50% of its specific activity at 25 °C, a behavior characteristic of a psychrotolerant enzyme. Recombinant enzyme activity was measured in the presence of ionic and non-ionic detergents, and a decrease in enzyme activity was detected for all concentrations of ionic and non-ionic detergents assessed. PMID:24514564

  10. The genetic code and its optimization for kinetic energy conservation in polypeptide chains.

    PubMed

    Guilloux, Antonin; Jestin, Jean-Luc

    2012-08-01

    Why is the genetic code the way it is? Concepts from fields as diverse as molecular evolution, classical chemistry, biochemistry and metabolism have been used to define selection pressures most likely to be involved in the shaping of the genetic code. Here minimization of kinetic energy disturbances during protein evolution by mutation allows an optimization of the genetic code to be highlighted. The quadratic forms corresponding to the kinetic energy term are considered over the field of rational numbers. Arguments are given to support the introduction of notions from basic number theory within this context. The observations found to be consistent with this minimization are statistically significant. The genetic code may well have been optimized according to energetic criteria so as to improve folding and dynamic properties of polypeptide chains.

  11. Molecular dynamics of an α-helical polypeptide: Temperature dependence and deviation from harmonic behavior

    PubMed Central

    Levy, Ronald M.; Perahia, David; Karplus, Martin

    1982-01-01

    The mean square amplitudes of atomic fluctuations for a polypeptide (decaglycine) α-helix evaluated from molecular dynamics simulations at seven temperatures between 5 and 300 K are compared with analytic harmonic results and with experimental values. Above 100 K the harmonic approximation significantly underestimates the amplitudes of the displacements. Analysis of the time dependence of the fluctuations shows that low-frequency modes (<75 cm-1) dominate the atomic fluctuations and that there is a contribution with a very long relaxation time (>10 ps). Quantum corrections to the amplitude of the fluctuations are found to be small above 50 K. The mean square amplitudes obtained from the molecular dynamics simulations are compared with the values derived from x-ray temperature (Debye-Waller) factors for metmyoglobin (80, 250, and 300 K) and ferrocytochrome c (300 K). PMID:16593164

  12. Islet amyloid polypeptide and high hydrostatic pressure: towards an understanding of the fibrillization process

    NASA Astrophysics Data System (ADS)

    Lopes, D. H. J.; Smirnovas, V.; Winter, R.

    2008-07-01

    Type II Diabetes Mellitus is a disease which is characterized by peripheral insulin resistance coupled with a progressive loss of insulin secretion that is associated with a decrease in pancreatic islet β-cell mass and the deposition of amyloid in the extracellular matrix of β-cells, which lead to islet cell death. The principal component of the islet amyloid is a pancreatic hormone called islet amyloid polypeptide (IAPP). High-pressure coupled with FT-IR, CD, ThT fluorescence spectroscopic and AFM studies were carried out to reveal information on the aggregation pathway as well as the aggregate structure of IAPP. Our data indicate that IAPP pre-formed fibrils exhibit a strong polymorphism with heterogeneous structures very sensitive to high hydrostatic pressure, indicating a high percentage of ionic and hydrophobic interactions being responsible for the stability the IAPP fibrils.

  13. Comparative assessment of the polypeptide profiles from lateral and primary roots of Phaseolus vulgaris L

    NASA Technical Reports Server (NTRS)

    Westberg, J.; Odom, W. R.; Guikema, J. A.; Spooner, B. S. (Principal Investigator)

    1994-01-01

    In Phaseolus vulgaris, primary roots show gravitational sensitivity soon after emerging from the seed. In contrast, lateral roots are agravitropic during early development, and become gravitropic after several cm growth. Primary and lateral root tissues were examined by polyacrylamide gel electrophoresis, coupled with western blotting techniques, to compare proteins which may contribute to the acquisition of gravitational sensitivity. Root tips and zones of cell elongation were compared for each root type, using immunological probes for calmodulin, alpha-actin, alpha-tubulin, and proteins of the plastid envelope. Lateral roots contained qualitatively less calmodulin, and showed a slightly different pattern of actin-related epitope proteins, than did primary root tissues, suggesting that polypeptide differences may contribute to the gravitational sensitivity which these root types express.

  14. Rational engineering of single-chain polypeptides into protein-only, BBB-targeted nanoparticles.

    PubMed

    Serna, Naroa; Céspedes, María Virtudes; Saccardo, Paolo; Xu, Zhikun; Unzueta, Ugutz; Álamo, Patricia; Pesarrodona, Mireia; Sánchez-Chardi, Alejandro; Roldán, Mónica; Mangues, Ramón; Vázquez, Esther; Villaverde, Antonio; Ferrer-Miralles, Neus

    2016-07-01

    A single chain polypeptide containing the low density lipoprotein receptor (LDLR) ligand Seq-1 with blood-brain barrier (BBB) crossing activity has been successfully modified by conventional genetic engineering to self-assemble into stable protein-only nanoparticles of 30nm. The nanoparticulate presentation dramatically enhances in vitro, LDLR-dependent cell penetrability compared to the parental monomeric version, but the assembled protein does not show any enhanced brain targeting upon systemic administration. While the presentation of protein drugs in form of nanoparticles is in general advantageous regarding correct biodistribution, this principle might not apply to brain targeting that is hampered by particular bio-physical barriers. Irrespective of this fact, which is highly relevant to the nanomedicine of central nervous system, engineering the cationic character of defined protein stretches is revealed here as a promising and generic approach to promote the controlled oligomerization of biologically active protein species as still functional, regular nanoparticles. PMID:26949165

  15. An engineered polypeptide around nano-sized manganese-calcium oxide: copying plants for water oxidation.

    PubMed

    Najafpour, Mohammad Mahdi; Ghobadi, Mohadeseh Zarei; Sarvi, Bahram; Haghighi, Behzad

    2015-09-14

    Synthesis of new efficient catalysts inspired by Nature is a key goal in the production of clean fuel. Different compounds based on manganese oxide have been investigated in order to find their water-oxidation activity. Herein, we introduce a novel engineered polypeptide containing tyrosine around nano-sized manganese-calcium oxide, which was shown to be a highly active catalyst toward water oxidation at low overpotential (240 mV), with high turnover frequency of 1.5 × 10(-2) s(-1) at pH = 6.3 in the Mn(III)/Mn(IV) oxidation range. The compound is a novel structural and efficient functional model for the water-oxidizing complex in Photosystem II. A new proposed clever strategy used by Nature in water oxidation is also discussed. The new model of the water-oxidizing complex opens a new perspective for synthesis of efficient water-oxidation catalysts.

  16. The neuroendocrine polypeptide 7B2 is an endogenous inhibitor of prohormone convertase PC2.

    PubMed Central

    Martens, G J; Braks, J A; Eib, D W; Zhou, Y; Lindberg, I

    1994-01-01

    The subtilisin-like prohormone convertase PC2 and the polypeptide 7B2 (an intracellularly cleaved protein of unknown function) are both selectively present in the regulated secretory pathway of neurons and endocrine cells. Here we demonstrate that intact recombinant 7B2 is a potent inhibitor of PC2 and prevents proPC2 cleavage in vitro, whereas the 7B2 cleavage product is virtually inactive. The PC2-related proteinase PC1/PC3 is not inhibited by 7B2. Furthermore, the carboxyl-terminal half of the 7B2 protein sequence is distantly related to the so-called potato inhibitor I family (which includes subtilisin inhibitors). Our findings indicate that 7B2 is a physiological inhibitor of PC2 and may provide alternative avenues for the manipulation of peptide hormone levels. Images PMID:8016065

  17. A Placental Polypeptide Activator of a Membranous Protein Kinase and Its Relation to Histone 1

    NASA Astrophysics Data System (ADS)

    Abdel-Ghany, M.; Riegler, C.; Racker, E.

    1984-12-01

    Crude transforming growth factor preparations of placenta contain a polypeptide that is required for the activity of a protein kinase that has been purified from plasma membrane preparations of Ehrlich ascites tumor cells. The kinase activator has been separated from transforming growth factor β by reversed-phase HPLC and affinity chromatography. Like the transforming growth factor, it is heat stable and trypsin labile, but it is not inactivated by dithiothreitol. In sodium dodecyl sulfate/polyacrylamide gel electrophoresis the purified preparation shows a major double band at about 31,000 daltons. Comparisons of electrophoretic mobility, protein kinase stimulatory activity, and cross-reactivity with an antibody against histone 1 suggest that the placental activator is identical with histone 1.

  18. Central and peripheral effects of sculpin pancreatic polypeptide and anglerfish peptide Y in rats.

    PubMed

    Balasubramaniam, A; Rigel, D F; Chance, W T; Fischer, J E

    1992-01-01

    Sculpin pancreatic polypeptide (PP) and anglerfish peptide Y (aPY-amide) are 36-residue peptide amides isolated from the pancreas of the respective species of fish. They are 86% homologous, and exhibit about 65% homology to porcine neuropeptide Y (NPY) and peptide YY (PYY). This homology to mammalian peptides suggests that the fish peptides may constitute a good model system for structure-activity investigations. We therefore synthesized sculpin PP and aPY-amide by the solid phase method and investigated their central and peripheral effects on feeding and blood pressure, respectively. These investigations revealed that both peptides, like NPY, increased blood pressure and induced feeding in rats, presumably by interacting with receptors of NPY. Although there were comparable responses to both peptides on feeding, aPY-amide exhibited a more potent pressor effect than sculpin PP. These observations suggest that the central and peripheral effects of NPY may be mediated by different subclasses of NPY receptors. PMID:1581637

  19. Two genes encoding midgut-specific maltase-like polypeptides from Anopheles gambiae.

    PubMed

    Zheng, L; Whang, L H; Kumar, V; Kafatos, F C

    1995-11-01

    Full-length cDNA clones of two genes have been isolated from the African malaria vector mosquito, Anopheles gambiae. These genes, designated Agm1 and Agm2, encode maltase-like polypeptides of 498 and 599 residues, respectively. Deduced amino acid sequences contain a putative signal peptide sequence and four potential glycosylation sites. Agm1 and Agm2 show highest similarities to the Mal1 gene from Aedes aegypti and three clustered maltase genes from Drosophila melanogaster. Both genes are located at position 46D, in the terminal division of the left arm of the third chromosome. Agm2 has very strict tissue and temporal specificity, being expressed exclusively in the adult midgut. The specificity of Agm1 is similar but appears slightly broader; transcripts of this gene are detected at a low level in the pupae, and occasionally in the adult carcass after removal of the midgut.

  20. Breakout character of islet amyloid polypeptide hydrophobic mutations at the onset of type-2 diabetes

    NASA Astrophysics Data System (ADS)

    Frigori, Rafael B.

    2014-11-01

    Toxic fibrillar aggregates of islet amyloid polypeptide (IAPP) appear as the physical outcome of a peptidic phase transition signaling the onset of type-2 diabetes mellitus in different mammalian species. In particular, experimentally verified mutations on the amyloidogenic segment 20-29 in humans, cats, and rats are highly correlated with the molecular aggregation propensities. Through a microcanonical analysis of the aggregation of IAPP20 -29 isoforms, we show that a minimalist one-bead hydrophobic-polar continuum model for protein interactions properly quantifies those propensities from free-energy barriers. Our results highlight the central role of sequence-dependent hydrophobic mutations on hot spots for stabilization, and thus for the engineering, of such biological peptides.

  1. Isolation and structures of alligator gar (Lepisosteus spatula) insulin and pancreatic polypeptide.

    PubMed

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

    1987-09-01

    Insulin and a 36-residue peptide with homology to pancreatic polypeptide (PP) were isolated from the endocrine pancreas of the alligator gar (Lepisosteus spatula), a ganoid fish, by gel filtration and HPLC. Heterologous radioimmunoassays were used to detect insulin-like and PP-like immunoreactivities during purification of the two peptides. The sequence of the 36-amino acid peptide containing a C-terminal tyrosinamide was identical at 31 of 36 positions to porcine neuropeptide Y (NPY). The amino acid sequence of this peptide is YPPKPENPGEDAPPEELAKYYSALRHYINLITRQRY-NH2. The second peptide, gar insulin, contains 52 amino acid residues and is composed of a 21-residue A chain and a 31-residue B chain. The sequence of the A chain is GIVEQCCHKPCTIYELENYCN. The sequence of the B chain is AANQHLCGSHLVEALYLVCGEKGFFYNPNKV. PMID:3311873

  2. Isolation and structures of alligator gar (Lepisosteus spatula) insulin and pancreatic polypeptide.

    PubMed

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

    1987-09-01

    Insulin and a 36-residue peptide with homology to pancreatic polypeptide (PP) were isolated from the endocrine pancreas of the alligator gar (Lepisosteus spatula), a ganoid fish, by gel filtration and HPLC. Heterologous radioimmunoassays were used to detect insulin-like and PP-like immunoreactivities during purification of the two peptides. The sequence of the 36-amino acid peptide containing a C-terminal tyrosinamide was identical at 31 of 36 positions to porcine neuropeptide Y (NPY). The amino acid sequence of this peptide is YPPKPENPGEDAPPEELAKYYSALRHYINLITRQRY-NH2. The second peptide, gar insulin, contains 52 amino acid residues and is composed of a 21-residue A chain and a 31-residue B chain. The sequence of the A chain is GIVEQCCHKPCTIYELENYCN. The sequence of the B chain is AANQHLCGSHLVEALYLVCGEKGFFYNPNKV.

  3. Apoptosis induced by islet amyloid polypeptide soluble oligomers is neutralized by diabetes-associated specific antibodies

    PubMed Central

    Bram, Yaron; Frydman-Marom, Anat; Yanai, Inbal; Gilead, Sharon; Shaltiel-Karyo, Ronit; Amdursky, Nadav; Gazit, Ehud

    2014-01-01

    Soluble oligomeric assemblies of amyloidal proteins appear to act as major pathological agents in several degenerative disorders. Isolation and characterization of these oligomers is a pivotal step towards determination of their pathological relevance. Here we describe the isolation of Type 2 diabetes-associated islet amyloid polypeptide soluble cytotoxic oligomers; these oligomers induced apoptosis in cultured pancreatic cells, permeated model lipid vesicles and interacted with cell membranes following complete internalization. Moreover, antibodies which specifically recognized these assemblies, but not monomers or amyloid fibrils, were exclusively identified in diabetic patients and were shown to neutralize the apoptotic effect induced by these oligomers. Our findings support the notion that human IAPP peptide can form highly toxic oligomers. The presence of antibodies identified in the serum of diabetic patients confirms the pathological relevance of the oligomers. In addition, the newly identified structural epitopes may also provide new mechanistic insights and a molecular target for future therapy. PMID:24589570

  4. Dithiol amino acids can structurally shape and enhance the ligand-binding properties of polypeptides

    NASA Astrophysics Data System (ADS)

    Chen, Shiyu; Gopalakrishnan, Ranganath; Schaer, Tifany; Marger, Fabrice; Hovius, Ruud; Bertrand, Daniel; Pojer, Florence; Heinis, Christian

    2014-11-01

    The disulfide bonds that form between two cysteine residues are important in defining and rigidifying the structures of proteins and peptides. In polypeptides containing multiple cysteine residues, disulfide isomerization can lead to multiple products with different biological activities. Here, we describe the development of a dithiol amino acid (Dtaa) that can form two disulfide bridges at a single amino acid site. Application of Dtaas to a serine protease inhibitor and a nicotinic acetylcholine receptor inhibitor that contain disulfide constraints enhanced their inhibitory activities 40- and 7.6-fold, respectively. X-ray crystallographic and NMR structure analysis show that the peptide ligands containing Dtaas have retained their native tertiary structures. We furthermore show that replacement of two cysteines by Dtaas can avoid the formation of disulfide bond isomers. With these properties, Dtaas are likely to have broad application in the rational design or directed evolution of peptides and proteins with high activity and stability.

  5. Rational engineering of single-chain polypeptides into protein-only, BBB-targeted nanoparticles.

    PubMed

    Serna, Naroa; Céspedes, María Virtudes; Saccardo, Paolo; Xu, Zhikun; Unzueta, Ugutz; Álamo, Patricia; Pesarrodona, Mireia; Sánchez-Chardi, Alejandro; Roldán, Mónica; Mangues, Ramón; Vázquez, Esther; Villaverde, Antonio; Ferrer-Miralles, Neus

    2016-07-01

    A single chain polypeptide containing the low density lipoprotein receptor (LDLR) ligand Seq-1 with blood-brain barrier (BBB) crossing activity has been successfully modified by conventional genetic engineering to self-assemble into stable protein-only nanoparticles of 30nm. The nanoparticulate presentation dramatically enhances in vitro, LDLR-dependent cell penetrability compared to the parental monomeric version, but the assembled protein does not show any enhanced brain targeting upon systemic administration. While the presentation of protein drugs in form of nanoparticles is in general advantageous regarding correct biodistribution, this principle might not apply to brain targeting that is hampered by particular bio-physical barriers. Irrespective of this fact, which is highly relevant to the nanomedicine of central nervous system, engineering the cationic character of defined protein stretches is revealed here as a promising and generic approach to promote the controlled oligomerization of biologically active protein species as still functional, regular nanoparticles.

  6. Selachian tooth development: II. Immunolocalization of amelogenin polypeptides in epithelium during secretory amelogenesis in Squalus acanthias.

    PubMed

    Slavkin, H C; Samuel, N; Bringas, P; Nanci, A; Santos, V

    1983-01-01

    We have determined the distribution of amelogenin polypeptides in an order of elasmobranchs using indirect immunofluorescence with rabbit polyclonal antibodies prepared to purified murine amelogenins. We find that amelogenins are definitely present within the inner enamel epithelium prior to the production of the extracellular matrix component termed "enameloid" (row II developing tooth organs). During subsequent stages of selachian tooth development (row III tooth organs), immunofluorescence staining data indicated localization of amelogenin antigens within epithelium as well as the enameloid extracellular matrix. The results from these immunohistochemical studies suggest that the 16-20 kdalton amelogenins, which are characteristic of murine inner enamel epithelial cells undergoing terminal biochemical differentiation into secretory ameloblasts, may also be regarded as molecular markers for amelogenesis in developing teeth in the spiny dogfish, Squalus acanthias.

  7. Pharmacological Actions of Glucagon-Like Peptide-1, Gastric Inhibitory Polypeptide, and Glucagon.

    PubMed

    Sekar, R; Singh, K; Arokiaraj, A W R; Chow, B K C

    2016-01-01

    Glucagon family of peptide hormones is a group of structurally related brain-gut peptides that exert their pleiotropic actions through interactions with unique members of class B1 G protein-coupled receptors (GPCRs). They are key regulators of hormonal homeostasis and are important drug targets for metabolic disorders such as type-2 diabetes mellitus (T2DM), obesity, and dysregulations of the nervous systems such as migraine, anxiety, depression, neurodegeneration, psychiatric disorders, and cardiovascular diseases. The current review aims to provide a detailed overview of the current understanding of the pharmacological actions and therapeutic advances of three members within this family including glucagon-like peptide-1 (GLP-1), gastric inhibitory polypeptide (GIP), and glucagon. PMID:27572131

  8. Longitudinal orientation of cross-linked polypeptide γ chains in fibrin fibrils.

    PubMed

    Rosenfeld, M A; Leonova, V B; Bychkova, A V; Kostanova, E A; Biryukova, M I

    2015-01-01

    The crosslinking of fibrin γ-polypeptide chains under the influence of the plasma fibrin-stabilizing factor (FXIIIa), which causes their conversion to γ-γ dimers, is the major enzyme reaction of covalent fibrin stabilization. We studied the self-assembly of soluble cross-linked fibrin oligomers. The results of analytical ultracentrifugation as well as elastic and dynamic light scattering showed that the double-stranded fibrin oligomers formed under the influence of moderate concentrations of urea are cross-linked only due to formation of γ-γ dimers, which can dissociate into single-stranded structure when the concentration of urea increases. This fact proves that γ-γ dimers are formed in the end-to-end manner. PMID:26518549

  9. The Expression and Function of Organic Anion Transporting Polypeptides in Normal Tissues and in Cancer

    PubMed Central

    Obaidat, Amanda; Roth, Megan; Hagenbuch, Bruno

    2011-01-01

    Organic anion transporting polypeptides (OATPs) are members of the SLCO gene superfamily of proteins. The 11 human OATPs are classified in 6 families and subfamilies on the basis of their amino acid sequence similarities. OATPs are expressed in several epithelial tissues throughout the body and transport mainly amphipathic molecules with molecular weights of more than 300 kDa. Members of the OATP1 and OATP2 families are functionally the best-characterized OATPs. Among these are the multispecific OATP1A2, OATP1B1, OATP1B3, and OATP2B1. They transport various endo- and xenobiotics, including hormones and their conjugates as well as numerous drugs such as several anticancer agents. Recent reports demonstrate that some OATPs are up- or downregulated in several cancers and that OATP expression might affect cancer development. On the basis of the findings summarized in this review, we propose that OATPs could be valuable targets for anticancer therapy. PMID:21854228

  10. IgG-subclass-specific antibody reactivity to respiratory syncytial virus polypeptides investigated by western blot.

    PubMed

    Jankowski, M; Hornsleth, A; Olsen, P G

    1990-01-01

    IgG1 and IgG3 subclass-specific antibody reactivity (ABR) in serum samples obtained from infants and children in relation to acute lower respiratory disease caused by respiratory syncytial virus (RSV) infection was investigated by Western blot. IgG1 ABR was directed against the nucleocapsid polypeptides VPN and VPP as well as against the glycoproteins GP48 (F1) and GP90. IgG3 ABR was directed only against VPN and VPP. In infants, a low IgG1 reactivity against glycoproteins was observed. When serum samples obtained in the early convalescent phase were tested, ABR against GP48 and GP90 as well as against VPP differed with respect to RSV subtypes A and B. IgG1 ABR increased in the late convalescent phase, while IgG3 ABR decreased during this phase when serum samples from primary infections were tested.

  11. An engineered polypeptide around nano-sized manganese-calcium oxide: copying plants for water oxidation.

    PubMed

    Najafpour, Mohammad Mahdi; Ghobadi, Mohadeseh Zarei; Sarvi, Bahram; Haghighi, Behzad

    2015-09-14

    Synthesis of new efficient catalysts inspired by Nature is a key goal in the production of clean fuel. Different compounds based on manganese oxide have been investigated in order to find their water-oxidation activity. Herein, we introduce a novel engineered polypeptide containing tyrosine around nano-sized manganese-calcium oxide, which was shown to be a highly active catalyst toward water oxidation at low overpotential (240 mV), with high turnover frequency of 1.5 × 10(-2) s(-1) at pH = 6.3 in the Mn(III)/Mn(IV) oxidation range. The compound is a novel structural and efficient functional model for the water-oxidizing complex in Photosystem II. A new proposed clever strategy used by Nature in water oxidation is also discussed. The new model of the water-oxidizing complex opens a new perspective for synthesis of efficient water-oxidation catalysts. PMID:26017548

  12. Improved Identification and Analysis of Small Open Reading Frame Encoded Polypeptides.

    PubMed

    Ma, Jiao; Diedrich, Jolene K; Jungreis, Irwin; Donaldson, Cynthia; Vaughan, Joan; Kellis, Manolis; Yates, John R; Saghatelian, Alan

    2016-04-01

    Computational, genomic, and proteomic approaches have been used to discover nonannotated protein-coding small open reading frames (smORFs). Some novel smORFs have crucial biological roles in cells and organisms, which motivates the search for additional smORFs. Proteomic smORF discovery methods are advantageous because they detect smORF-encoded polypeptides (SEPs) to validate smORF translation and SEP stability. Because SEPs are shorter and less abundant than average proteins, SEP detection using proteomics faces unique challenges. Here, we optimize several steps in the SEP discovery workflow to improve SEP isolation and identification. These changes have led to the detection of several new human SEPs (novel human genes), improved confidence in the SEP assignments, and enabled quantification of SEPs under different cellular conditions. These improvements will allow faster detection and characterization of new SEPs and smORFs. PMID:27010111

  13. Molecular Dynamics of an α -helical Polypeptide: Temperature Dependence and Deviation from Harmonic Behavior

    NASA Astrophysics Data System (ADS)

    Levy, Ronald M.; Perahia, David; Karplus, Martin

    1982-02-01

    The mean square amplitudes of atomic fluctuations for a polypeptide (decaglycine) α -helix evaluated from molecular dynamics simulations at seven temperatures between 5 and 300 K are compared with analytic harmonic results and with experimental values. Above 100 K the harmonic approximation significantly underestimates the amplitudes of the displacements. Analysis of the time dependence of the fluctuations shows that low-frequency modes (<75 cm-1) dominate the atomic fluctuations and that there is a contribution with a very long relaxation time (>10ps). Quantum corrections to the amplitude of the fluctuations are found to be small above 50 K. The mean square amplitudes obtained from the molecular dynamics simulations are compared with the values derived from x-ray temperature (Debye-Waller) factors for metmyoglobin (80, 250, and 300 K) and ferrocytochrome c (300 K).

  14. Polypeptide N-acetylgalactosaminyltransferase 6 disrupts mammary acinar morphogenesis through O-glycosylation of fibronectin.

    PubMed

    Park, Jae-Hyun; Katagiri, Toyomasa; Chung, Suyoun; Kijima, Kyoko; Nakamura, Yusuke

    2011-04-01

    A high expression of short and immature O-glycans is one of the prominent features of breast cancer cells, which would be attributed to the upregulated expression of glycosyltransferases. Therefore, a detailed elucidation of glycosyltransferases and their substrate(s) may improve our understandings for their roles in mammary carcinogenesis. Here we report that overexpression of polypeptide N-acetylgalactosaminyltransferase 6 (GALNT6), a glycosyltransferase involved in the initial step of O-glycosylation, has transformational potentials through disruptive acinar morphogenesis and cellular changes similar to epithelial-to-mesenchymal transition in normal mammary epithelial cell, MCF10A. As one of the critical O-glycan substrates, we identified fibronectin that was O-glycosylated in vivo and thereby stabilized by GALNT6. Because knockdown of fibronectin abrogated the disruptive proliferation caused by introduction of GALNT6 into epithelial cells, our findings suggest that GALNT6-fibronectin pathway should be a critical component for breast cancer development and progression.

  15. Reverse transcriptase of RNA tumor viruses. V. In vitro proteolysis of reverse transcriptase from avian myeloblastosis virus and isolation of a polypeptide manifesting only RNase H activity.

    PubMed Central

    Lai, M H; Verma, I M

    1978-01-01

    Purified avian myeloblastosis virus reverse transcriptase contains two subunits that are structurally related. The large subunit, beta (molecular weight, 95,000), was converted in vitro by chymotrypsin into a polypeptide of molecular weight 63,000. This polypeptide was indistinguishable from the small subunit, alpha (molecular weight, 65,000), in its chromatographic behavior on the phosphocellulose column and its tryptic peptide composition. During this proteolytic conversion, a polypeptide of molecular weight 32,000 (fragment B) was obtained. It was composed of tryptic peptides unique to beta and appeared to be derived from the portion of the beta subunit that was cleaved off during the conversion of beta into alpha. Upon continued proteolysis, a smaller polypeptide of molecular weight 24,000 (fragment A) was generated. This polypeptide manifested only RNase H activity and shared common amino acid sequences with beta and alpha subunits. Fragment A did not share any amino acid sequence homology with fragment B. Images PMID:75271

  16. Polypeptide substrate specificity of PsLSMT. A set domain protein methyltransferase.

    PubMed

    Magnani, Roberta; Nayak, Nihar R; Mazarei, Mitra; Dirk, Lynnette M A; Houtz, Robert L

    2007-09-21

    Rubisco large subunit methyltransferase (PsLSMT) is a SET domain protein responsible for the trimethylation of Lys-14 in the large subunit of Rubisco. The polypeptide substrate specificity determinants for pea Rubisco large subunit methyltransferase were investigated using a fusion protein construct between the first 23 amino acids from the large subunit of Rubisco and human carbonic anhydrase II. A total of 40 conservative and non-conservative amino acid substitutions flanking the target Lys-14 methylation site (positions P(-3) to P(+3)) were engineered in the fusion protein. The catalytic efficiency (k(cat)/K(m)) of PsLSMT was determined using each of the substitutions and a polypeptide consensus recognition sequence deduced from the results. The consensus sequence, represented by X-(Gly/Ser)-(Phe/Tyr)-Lys-(Ala/Lys/Arg)-(Gly/Ser)-pi, where X is any residue, Lys is the methylation site, and pi is any aromatic or hydrophobic residue, was used to predict potential alternative substrates for PsLSMT. Four chloroplast-localized proteins were identified including gamma-tocopherol methyltransferase (gamma-TMT). In vitro methylation assays using PsLSMT and a bacterially expressed form of gamma-TMT from Perilla frutescens confirmed recognition and methylation of gamma-TMT by PsLSMT in vitro. RNA interference-mediated knockdown of the PsLSMT homologue (NtLSMT) in transgenic tobacco plants resulted in a 2-fold decrease of alpha-tocopherol, the product of gamma-TMT. The results demonstrate the efficacy of consensus sequence-driven identification of alternative substrates for PsLSMT as well as identification of functional attributes of protein methylation catalyzed by LSMT.

  17. Selective release of the Treponema pallidum outer membrane and associated polypeptides with Triton X-114.

    PubMed

    Cunningham, T M; Walker, E M; Miller, J N; Lovett, M A

    1988-12-01

    The effects of the nonionic detergent Triton X-114 on the ultrastructure of Treponema pallidum subsp. pallidum are presented in this study. Treatment of Percoll-purified motile T. pallidum with a 1% concentration of Triton X-114 resulted in cell surface blebbing followed by lysis of blebs and a decrease in diameter from 0.25-0.35 micron to 0.1-0.15 micron. Examination of thin sections of untreated Percoll-purified T. pallidum showed integrity of outer and cytoplasmic membranes. In contrast, thin sections of Triton X-114-treated treponemes showed integrity of the cytoplasmic membrane but loss of the outer membrane. The cytoplasmic cylinders generated by detergent treatment retained their periplasmic flagella, as judged by electron microscopy and immunoblotting. Recently identified T. pallidum penicillin-binding proteins also remained associated with the cytoplasmic cylinders. Proteins released by Triton X-114 at 4 degrees C were divided into aqueous and hydrophobic phases after incubation at 37 degrees C. The hydrophobic phase had major polypeptide constituents of 57, 47, 38, 33-35, 23, 16, and 14 kilodaltons (kDa) which were reactive with syphilitic serum. The 47-kDa polypeptide was reactive with a monoclonal antibody which has been previously shown to identify a surface-associated T. pallidum antigen. The aqueous phase contained the 190-kDa ordered ring molecule, 4D, which has been associated with the surface of the organisms. Full release of the 47- and 190-kDa molecules was dependent on the presence of a reducing agent. These results indicate that 1% Triton X-114 selectively solubilizes the T. pallidum outer membrane and associated proteins of likely outer membrane location.

  18. Chemical synthesis and characterization of elastin-like polypeptides (ELPs) with variable guest residues.

    PubMed

    Aladini, Firouzeh; Araman, Can; Becker, Christian F W

    2016-05-01

    The properties of elastin-like polypeptides (ELPs), specifically the fact that they are soluble in aqueous buffers below and aggregate reversibly above a well-defined transition temperature, are extensively used for protein purification, enzyme recycling, and more recently, for in vivo applications such as drug delivery and tissue engineering. ELPs are artificial but biocompatible polypeptides composed of pentameric repeats (Val-Pro-Gly-Xaa-Gly) containing different guest residues Xaa, derived from mammalian elastin. The temperature-dependent aggregation and desaggregation of ELPs is controlled by composition of the pentameric repeats as well as the number of repetitive units within the ELP. External parameters such as ELP concentration, pH, and most importantly, salt effects heavily influence the transition temperature. Here, we explore the chemical synthesis of a series of 51mer peptides consisting of 10 pentameric ELP repeats with hydrophobic as well as charged guest residues such as isoleucine, leucine, alanine, lysine, and/or glutamate all prepared by Boc-based solid phase peptide synthesis. These guest residues expand the available toolbox of synthetic ELPs and provide ELPs that can be chemically modified and tuned to specific environments. An N-terminal cysteine is added allowing disulfide-based crosslinking of ELPs and to link synthetic ELPs to a recombinantly produced protein using native chemical ligation. Transition temperatures of all synthetic ELPs and the fusion construct were determined by measuring turbidity in solution and spanned a large temperature range between 25 and 70 °C, providing synthetically accessible ELPs with transition temperatures suitable for in vitro and in vivo applications. Cycling between their soluble and aggregate state has been observed at least 6 times without significant loss of material for all synthetic ELPs. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. PMID:27005861

  19. Temperature Triggered Self-Assembly of Polypeptides into Multivalent Spherical Micelles

    PubMed Central

    Dreher, Matthew R.; Simnick, Andrew J.; Fischer, Karl; Smith, Richard J.; Patel, Anand; Schmidt, Manfred; Chilkoti, Ashutosh

    2010-01-01

    We report herein thermally responsive elastin-like polypeptides (ELPs) in a linear AB diblock architecture with an N-terminal peptide ligand that self-assemble into spherical micelles when heated slightly above body temperature. A series of 10 ELP block copolymers (ELPBCs) with different molecular weights and hydrophilic-to-hydrophobic block ratios were genetically synthesized by recursive directional ligation. The self-assembly of these polymers from unimers into micelles was investigated by light scattering, fluorescence spectroscopy and cryo-TEM. These ELPBCs undergo two phase transitions as a function of solution temperature: a unimer to spherical micelle transition at an intermediate temperature, and a micelle to bulk aggregate transition at a higher temperature when the hydrophilic-to-hydrophobic block ratio is between 1:2 and 2:1. The critical micelle temperature is controlled by the length of the hydrophobic block and the size of the micelle is controlled by both the total ELPBC length and hydrophilic-to-hydrophobic block ratio. These polypeptide micelles display a critical micelle concentration in the range of 4-8 μM demonstrating high stability of these structures. These studies have also identified a subset of ELPBCs bearing terminal peptide ligands that are capable of forming multivalent spherical micelles that present multiple copies of the ligand on their corona in the clinically relevant temperature range of 37-42 °C and target cancer cells. These ELPBCs may be useful for drug targeting by thermally triggered multivalency. More broadly, the design rules uncovered by this study should be applicable to the design of other thermally reversible nanoparticles for diverse applications in medicine and biology. PMID:18085778

  20. Characterization of the 70 kDa polypeptide of the Na/Ca exchanger.

    PubMed Central

    Saba, R I; Bollen, A; Herchuelz, A

    1999-01-01

    The Na/Ca exchanger is associated with 160, 120 and 70 kDa polypeptides whose nature is poorly understood. We have purified and characterized the Na/Ca exchanger from bovine cardiac sarcolemmal vesicles (SLVs) by using ion-exchange and affinity chromatographies. The Na/Ca exchanger-enriched fraction was reconstituted into asolectin liposomes [lipid to protein ratio 10:1 (w/w)] that showed Na/Ca exchange activity. Under non-reducing conditions, SDS/PAGE showed a single 70 kDa polypeptide, which was further characterized by immunoblots with different antibodies: SWant, raised against the purified exchanger protein; NH2-terminus, residues 1-21; NCX1, residues 393-406; and Exon F, residues 622-644. Immunoblots under reducing conditions with SWant, NH2-terminus and NCX1 showed three bands migrating at 160, 120 and 70 kDa for SLV preparations, whereas Exon F reacted only with the 160 and 120 kDa bands. Under non-reducing conditions, immunoblots with purified reconstituted Na/Ca exchanger showed a single band at 70 kDa reacting with SWant, NH2-terminus and NCX1 but not with Exon F. We conclude that the 70 kDa protein is associated with Na/Ca exchange activity, has the same N-terminal sequence as the cloned bovine cardiac exchanger, and has its length decreased by at least 35% from its C-terminal portion as compared with that of the wild-type exchanger. PMID:9931309